3. _Trypanoplasma_, Laveran and Mesnil, 1901, with a kinetic

nucleus and undulating membrane. Of these genera _Prowazekia_ must be discussed. _Bodo_ does not occur in man. Species of _Trypanoplasma_ occur in the blood and in the gut of various fishes, in the seminal receptacle of certain snails, in the gut and genitalia of a flatworm (_Dendrocœlum lacteum_) and in the vagina of a leech. Closely allied to _Trypanoplasma_ is the genus _Trypanophis_, parasitic in the cœlenteric cavity of Siphonophores. Genus. *Prowazekia*, Hartmann and Chagas, 1910. The genus was founded for a flagellate parasite, _Prowazekia cruzi_, discovered in a culture of human fæces in Brazil. Various other species have been referred thereto. The genus is separated from _Bodo_ by the possession of a second nucleus, the so-called kinetonucleus or blepharoplast. It differs from _Trypanoplasma_ in the absence of an undulating membrane. It is heteromastigote, that is, it possesses two dissimilar flagella, one anteriorly directed and the other lateral and trailing. The principal species are: *Prowazekia urinaria*, Hassall, 1859. Syn.: _Bodo urinarius_, Hassall, 1859; _Trichomonas irregularis_, Salisbury, 1868; _Cystomonas urinaria_, Blanchard, 1885; _Plagiomonas urinaria_, Braun, 1895. Hassall[47] in 1859 first found Bodo-like flagellates in human urine. He examined fifty samples of urine from patients suffering from albuminuria and from cholera. The reaction of the urine was alkaline or sometimes only feebly acid. The flagellates were only seen after the urine had been standing for several days. Hassall named the organism _Bodo urinarius_, and gave a very good description of it with illustrations. The flagellate, which was round or oval, measured 14 µ by 8 µ. The organism had “one, usually two, and sometimes three lashes or cilia.” In 1868 Salisbury described a similar flagellate in the urine under the name _Trichomonas irregularis_. Künstler in 1883 described the latter parasite under the name _B. urinarius_. In 1885 Blanchard, considering Künstler’s organism a different parasite from Hassall’s, called it _Cystomonas urinaria_. Braun, in 1895, gave the name _Plagiomonas urinaria_. Barrois (1894) considered Künstler’s and Hassall’s organisms to be identical and not to be true parasites of man. Sinton,[48] in 1912, found the flagellate in the deposit, after centrifuging, of a 24-hour old specimen of alkaline urine from a Mexican sailor in the Royal Southern Hospital, Liverpool. Sinton found a kinetic nucleus or blepharoplast in the organism, and therefore placed it in the genus _Prowazekia_. [47] _Lancet_, 1859, ii, p. 503. [48] _Annals Trop. Med. and Parasitology_, vi, p. 245. [Illustration: FIG. 24.--Types of _Prowazekia urinaria_. (_a_) sausage-shaped; (_b_) round; (_c_) carrot-shaped form. (After Sinton.)] The flagellate stage (fig. 24) of the organism is polymorphic, and may be either (_a_) sausage-shaped, 10 µ to 25 µ in length by 2·5 µ to 6 µ in breadth; (_b_) round or oval, varying from 4 µ in diameter to oval forms 15 µ by 10 µ; (_c_) a carrot-shaped form, of varying size up to 25 µ by 4 µ. The kinetic nucleus is large and pear-shaped. Near it are basal granules, closely applied to one another, from which the flagella arise. There is a small cytostome near the roots of the flagella. There is a well-marked karyosome in the nucleus. The movement is jerky. The shorter, anterior flagellum may be used in food-capture. In life, bacteria have been seen to be ingested. Food-vacuoles tend to accumulate at the posterior (aflagellar) end. A contractile vacuole may be present, near the base of the cytostome, and may really be the dilated fundus of the latter. Division occurs by binary fission. The organism can encyst (fig. 25, _a_), when the flagella are lost, and round or oval cysts are found, 5 µ to 7 µ in diameter. After a time flagella are formed inside the cyst, and the organism emerges therefrom in its typical flagellate form (fig. 25, _b_-_f_). Sinton’s case is interesting. He obtained the flagellate only twice from the same patient, a Mexican then in hospital in Liverpool. The flagellate was not found in the patient’s fæces, nor was it found in the urine on later occasions when taken aseptically. [Illustration: FIG. 25.--_Prowazekia urinaria_. Flagellate emerging from cyst. (After Sinton.)] In cultures _Prowazekia urinaria_ was always found in association with bacteria. The cultures died at a temperature of 37° C., but grew well at 20° C. Various media were useful at the lower temperature, such as urine, salt agar, nutrient agar, serum agar, blood agar, peptone salt solution, and diluted blood serum. The flagellate was, then, considered to be an accidental contamination and not a true parasite of human urine. *Prowazekia asiatica*, Castellani and Chalmers, 1910. The flagellate was found by the discoverers in the stools of patients suffering from ankylostomiasis and diarrhœa in Ceylon. It was referred by them to the genus _Bodo_, but in 1911 Whitmore[49] further studied it and placed it in the genus _Prowazekia_. In the stools the flagellate is found either as a long, slender form measuring 10 µ to 16 µ by 5 µ to 8 µ or as a rounded form 8 µ to 10 µ in diameter. Its cytoplasm is alveolar. A rhizoplast connects the basal granules to the kinetic nucleus. There is multiplication and cyst formation as before. The organism is easily cultivated, especially in the condensation water of nutrose agar and maltose agar. The pathogenicity is stated to be nil. [49] _Arch. f. Protistenk._ xxii, p. 370. *Prowazekia javanensis*, Flu, 1912. Found in agar cultures from the motions of patients at Weltevreden, Dutch East Indies.[50] The flagellates are 12 µ long and 5 µ broad. The lateral flagellum is stated to be attached to the cell body for a short distance. Regarding the karyosome in the nucleus, the author states that the smaller the karyosome the more chromatin is deposited on the nuclear membrane. Flu mentions that the specific name _javanensis_ is a temporary one, as in the course of time it may be shown that there is only one species of _Prowazekia_. [50] _Geneesk. Tijdschr. v. Nederl. Ind._, lii, p. 659; _Med. v. d. Burg. Geneesk. d. Nederl. Ind._, iii, p. 1. *Prowazekia cruzi*, Hartmann and Chagas, 1910. Found in a culture from human fæces on an agar plate in Brazil, and considered to be a free-living form.[51] The organism is oval or pear-shaped, 8 µ to 12 µ long and 5 µ to 6 µ broad. In human stools at Tsingtau, China, a _Prowazekia_ has been found by Martini which he thinks is the same as _Prowazekia cruzi_. He considers it to be a cause of human diarrhœa and intestinal catarrh. [51] _Mem. Inst. Osw. Cruz._, ii, p. 64. *Prowazekia weinbergi*, Mathis and Léger, 1910. This species was found in the fæces of men, both healthy and diarrhœic, in Tonkin.[52] It is pear-shaped, 8 µ to 15 µ long by 4 µ to 6·5 µ broad. The flagella occur at the broad end. [52] _Bull. Soc. Med. Chir. Indo-Chine_, i, p. 471. The discoverers think that _Prowazekia weinbergi_ is an intestinal inhabitant, but non-pathogenic, since it was found to occur in the fæces even when obtained with aseptic precautions. *Prowazekia parva*, Nägler, 1910. A free-living form found in the slime on the stones at the biological station at Lunz. Another _Prowazekia_ was found in 1914 in tap-water in Calcutta. Family. *Trypanosomidæ*, Doflein. The Trypanosomidæ, broadly considered, are uniflagellate organisms, the flagellum being at the anterior end. The flagellum arises near the blepharoplast (kinetic nucleus), which lies anterior, near or posterior to the nucleus. The following genera will be considered:-- _Trypanosoma_--with an undulating membrane along the length of the body. _Crithidia_--with a less well-developed undulating membrane anteriorly (see fig. 49). _Herpetomonas_--including the so-called _Leptomonas_, with anterior free flagellum only, and no undulating membrane. _Leishmania_--non-flagellate forms in mammalian blood, flagellate herpetomonad stages in culture, probably occurring naturally in Arthropods. Genus. *Trypanosoma*, Gruby, 1843. The members of the genus possess a single flagellum, which arises posteriorly, adjacent to a blepharoplast or kinetic nucleus. The flagellum forms a margin to an undulating membrane, and may or may not be continued beyond the body as a free flagellum. Many species are parasitic in vertebrate blood and in the digestive tracts of insects. HISTORICAL. The history of blood flagellates goes back to the year 1841, in which Valentin discovered in the blood of a brook-trout (_Salmo fario_ L.) minute bodies, from 7 µ to 13 µ in length, with active movements and presenting marked changes in form. Valentin considered the parasite a new species of the old genus _Proteus_ or _Amœba_, Ehrbg. This announcement led Gluge (1842) to publish a similar discovery he had made in frog’s blood. The latter forms were called by Mayer (1843) _Amœba rotatoria_, _Paramœcium loricatum_ and _P. costatum_, while Gruby (1843) called them _Trypanosoma sanguinis_.[53] Later it was discovered that similar organisms occurred also in the blood of birds (Wedl (1850), Danilewsky) and of mammals. Gros (1845) found them in the mouse and mole, Chaussat (1850) in the house rat, Lewis (1879) in the Indian rat, Wittich (1881) in the hamster. Danilewsky (1886–89) and Chalachnikow (1888) investigated the structure and division of trypanosomes. [53] Gruby’s generic name is generally accepted. Still others have been used, _e.g._, _Undulina_, Ray _Lankester_, _Globularia_ Wedl, _Paramecioides_ Grassi, _Trypanomonas_ Danilewsky, _Hæmatomonas_ Mitrophanow. In the case of all these forms, there was no discussion as to a pathogenic influence on the host. Opinion, however, as to the action of trypanosomes changed when, in 1880, Evans found flagellates in the blood of horses in India that suffered from a disease endemic there called “surra,” and associated the parasites with the disease. Steel and Evans were successful in transmitting the parasites--first known as _Spirochæta evansi_, Steel, then as _Trichomonas evansi_, Crookshank, and finally as _Trypanosoma evansi_--to dogs, mules and horses. They recognized that the above mentioned flagellates in the blood of the experimental animals were the causal agents of the disease. From that time there was a considerable increase in the literature, the contents of which have been summarized by Laveran and Blanchard. In 1894 Rouget discovered trypanosomes in the blood of African horses that suffer from “stallion’s disease” (dourine). In 1894 Bruce found similar forms (_T. brucei_) in the blood of South African mammals suffering from “nagana,” and in consequence attention was drawn to the part which the much dreaded tsetse-fly played in the transmission of “nagana.” In 1901 Elmassian discovered trypanosomes in the blood of horses that were stricken with “mal de caderas,” which is very common in the Argentine. The disease in cattle named “galziekte” (gall-sickness), occurring in the Transvaal, was also at one time attributed to a trypanosome remarkable for its great size, and like some other species, bearing the name of its discoverer (_T. theileri_). The study of the species hitherto known has been carried on partly by the above mentioned authors and in part by others, _e.g._, Rabinowitsch and Kempner, Laveran and Mesnil, Wasiliewski, Senn. It was greatly advanced by the method of double staining (with alkaline methylene blue and eosin) introduced by Romanowsky (1891) and elaborated by Ziemann, Leishman, Giemsa and others. By this means the presence of a terminal flagellum and of an undulating membrane at the side of the flattened and extended body was demonstrated. Laveran and Mesnil (1901) discovered allied flagellates in the blood of the fish, _Scardinius erythrophthalmus_. These flagellates, now placed in the genus _Trypanoplasma_, had a second free flagellum in addition to the one bordering the undulating membrane. Trypanoplasms have since been found in both freshwater and marine fishes. The transmission of trypanoplasms of freshwater fishes is effected by leeches. _Trypanoplasma varium_ from _Cobitis_ is transmitted by _Hemiclepsis marginata_ according to Léger, while the Trypanoplasmata of _Cyprinus carpio_ and _Abramis brama_ reach new hosts by the agency of _Piscicola_ according to Keysselitz. Another ally of the Trypanosomidæ, _Trypanophis_, lives in the cœlenteric cavity of Siphonophores. It has also an extra terminal flagellum (Poche, Keysselitz). [_Trypanoplasma_ and _Trypanophis_ belong to the _Bodonidæ_, see p. 63]. Finally it was shown that Trypanosomes occurred in human beings. Although Nepveu’s early report of trypanosomes in the blood of malarial patients may be doubtful, subsequent researches by Forde and Dutton demonstrated trypanosomes (fig. 28) in the blood of a European, apparently suffering from malaria, living in the Gambia. Dutton (1902) called the human trypanosome, _T. gambiense_. The expedition despatched by the Liverpool School of Tropical Medicine (1902) to Senegambia found trypanosome infections in six cases among a thousand inhabitants examined. About the same time attention was devoted to the disease of West African negroes known for a century as “sleeping sickness.” Castellani (1903) was the first to succeed in demonstrating the presence of trypanosomes (at first called _T. ugandense_) in centrifugalized cerebro-spinal fluid obtained by puncture from cases of sleeping sickness in Uganda. Similar discoveries were made by Bruce, who also found trypanosomes in the blood of those attacked with sleeping sickness. Sambon regarded a species of _Glossina_ as the transmitter. From consideration of the geographical distribution of the disease Christy regarded _Glossina palpalis_ as the transmitter. Brumpt first thought it was _G. morsitans_, but, later, supported the view of _G. palpalis_. Bruce, Nabarro and Greig also named the same insect as the transmitter, not only for geographical reasons but also because healthy apes became infected by the bite of certain _G. palpalis_. The inoculation of cerebro-spinal fluid from subjects of sleeping sickness into the spinal canal of apes (_Macacus_) had the same result. Just as the discovery of the malarial parasites called forth a whole flood of research memoirs which were followed by a second series on the relation of the mosquitoes to malaria, so a similar outpouring occurred after the discovery of the pathogenic trypanosomes of mammals and men. In both cases the inquiry was not limited to the stages in man and other vertebrate hosts, but the fate of the parasites in the intermediate (invertebrate) hosts was investigated, and allied species were obtained from many different hosts. Novy and MacNeal (1903) were the first to cultivate trypanosomes in artificial media (blood-agar). In 1910 Stephens and Fantham recorded the presence of another human trypanosome, _T. rhodesiense_, from a case of sleeping sickness in Rhodesia, where _G. palpalis_ was absent. Kinghorn has since demonstrated that _T. rhodesiense_ is transmitted by _G. morsitans_. Kinghorn and Yorke believe that big game (_e.g._, antelope) is the reservoir of _T. rhodesiense_. The output of literature on trypanosomiasis in men and animals is enormous. To cope with it the Sleeping _Sickness Bureau Bulletin_ was founded in 1908, and it is now (since November 1912) continued as a section of the _Tropical Diseases Bulletin_, wherein current literature is reviewed. GENERAL. Trypanosomes occur in the blood of representatives of all the vertebrate classes. Often the trypanosomes occur so scantily in the blood that they are overlooked on examination. A useful aid in detecting the flagellates in such cases consists in the use of cultures of the blood of the host on artificial media. Stimulated by the medium multiplication occurs, and hence the parasites are more easily detected. [For the composition of such culture media see Appendix.] There is a periodicity in the appearance of the trypanosomes in the peripheral blood of the host, due to alternating phases of multiplication and of rest on the part of the parasites. Such periodicity has been established both by biological and enumerative methods. Again, a seasonal variation has been observed in the occurrence of certain trypanosomes in the peripheral circulation of the hosts; for example, some trypanosomes (_e.g._, _T. noctuæ_ in birds) are found only in the summer in the blood, while in the winter they occur in the internal organs. Recent cultural researches have established that trypanosomes, _e.g._, _T. americanum_, may be present in very small numbers in hosts, such as cattle, which are quite unharmed by them, and in which the presence of these flagellates formerly was never suspected (“cryptic trypanosomiasis.”) However, the majority of the trypanosomes occurring in domestic animals are usually deleterious or even lethal to their hosts. Many wild animals, such as various species of antelope, harbour trypanosomes without being injured thereby. In such cases it is probable that the vertebrate hosts have been so long parasitized in the past, that they have become tolerant and immune to the effects of the flagellates. Should such trypanosomes of wild animals be transmitted to domesticated stock or man, they may re-acquire their initial virulence and become pathogenic to the new host. As a general statement, the newer a parasite is to its host the greater is its virulence. For example, _T. gambiense_, _T. rhodesiense_ and _T. brucei_ are innocuous to big game in Africa, but are pathogenic to man and domestic animals respectively. Pathogenic trypanosomes appear to have a wider range of hosts, that is, to be less limited to one specific host than non-pathogenic forms. Thus, _T. rhodesiense_ is pathogenic to man and all laboratory animals, while it is non-pathogenic to antelopes and their kind. _Morphology._ [Illustration: FIG. 26.--_Trypanosoma brucei_ in division. _n_, nucleus; _bl_, blepharoplast; _fl_, flagellum. × 2,000. (After Laveran and Mesnil.)] The general structure of the various trypanosomes shows much uniformity, though variations in size and shape occur. Typically the body is elongate and sinuous. The flagellar end tapers gradually to a point, the aflagellar extremity usually being rounded or more blunt. In some trypanosomes there is much diversity in size, the organisms varying from long, slender forms to short, stumpy ones; in other species relative constancy of size is maintained. The former are known as polymorphic trypanosomes, the latter as monomorphic forms. Two nuclei are present. The main or principal nucleus, sometimes termed the trophic nucleus, is often situated towards the centre of the body; it is frequently of the vesicular type, containing a karyosome. The blepharoplast or kinetic nucleus is posterior to the nucleus, and usually is rod-like. The flagellum arises close to the blepharoplast, and forms an edge to the undulating membrane. It may or may not extend beyond the limits of the undulating membrane. If it does so, the unattached part is known as the free flagellum. Sometimes a small granule is found at the origin of the flagellum. This is the basal granule, and is considered by some to function as the centriole of the kinetic nucleus. The undulating membrane is a lateral extension of the ectoplasm or periplast, and is the main agent in locomotion. It is edged by the flagellum, which forms a deeply stainable border to it. Within the membrane substance, often arranged parallel with its edge, are a number of fine contractile elements, the myonemes. These contractile elements may also occur on the body of the trypanosome. They are easily seen in some large trypanosomes, but are difficult of demonstration in others, owing to their great fineness. [Illustration: FIG. 27.--_Trypanosoma lewisi_. Multiplication rosettes. × 1,000. (After Laveran and Mesnil.)] Multiplication of trypanosomes in the blood is brought about by binary longitudinal fission (fig. 26). Division is initiated by that of the blepharoplast and nucleus. The division may be equal or subequal, whereby differences in size of individuals partly arise. Multiple division by repeated binary fission, without complete separation of the daughter forms, is known in some trypanosomes (_e.g._, _T. lewisi_), and rosettes of parasites thereby are produced (fig. 27). The classification of trypanosomes is very difficult. Laveran (1911)[54] has suggested the examination of the relative length of the flagellum as a diagnostic character, and so arranged these flagellates in mammals in three groups. The first group included those trypanosomes always having part of the flagellum free (_e.g._, _T. evansi_, _T. vivax_); the second group comprised forms without a part of the flagellum free (_e.g._, _T. congolense_), while the third group included forms some members of which have free flagella, while others have not (_e.g._, _T. gambiense_). Bruce[55] (1914) and Yorke and Blacklock[56] (1914) have also devised classifications. [54] _Ann. Inst. Pasteur_, xxv, p. 497. [55] _Trans. Soc. Trop. Med. & Hyg._, viii, p. 1. [56] _Annals Trop. Med. and Parasitol._, viii, p. 1. Resting stages of some trypanosomes have been found in the internal organs of their vertebrate hosts. The formation of these oval, Leishmania-like bodies will be noted in individual cases later. Similar small oval bodies form an important phase in the life-history of _T. cruzi_, which multiplies normally by multiple fission or schizogony into these oval, daughter elements, and not by binary longitudinal fission in the circulating blood. Polymorphism in trypanosomes (_e.g._, _T. gambiense_, _T. rhodesiense_) is now interpreted as a phenomenon resulting from growth and division.[57] Long, thin forms are those about to divide. Fully mature forms are shorter and broader. Various intermediate types occur and represent growth forms. Formerly, polymorphism was interpreted in terms of sex, thin forms being regarded as males, broad forms as females, while the intermediate types were termed indifferent. Conjugation was not observed, and there is no evidence in support of the sexual interpretation. [57] Robertson (1912), _Proc. Roy. Soc._, B, lxxxv, p. 527. The transmission of trypanosomes from one vertebrate host to another is usually accomplished by the intermediation of some biting arthropod in the case of terrestrial animals, while leeches are usually considered to act as transmitters in the case of the trypanosomes occurring in aquatic animals. Developmental phases of the life-histories of trypanosomes occur in the invertebrate transmitters, and will be considered in individual cases. *Trypanosoma gambiense*, Dutton, 1902. Syn.: _Trypanosoma hominis_, Manson, 1903. _Trypanosoma nepveui_, Sambon, 1903. _Trypanosoma castellanii_, Kruse, 1903. _Trypanosoma ugandense_, Castellani, 1903. _Trypanosoma fordii_, Maxwell Adams. In vertebrate blood _Trypanosoma gambiense_ is polymorphic, for long, thin forms may be seen in contrast with short, stumpy forms, as well as intermediate forms (fig. 29, _a_--_c_). This polymorphism has been interpreted in terms of sex, especially by German investigators, following Schaudinn (see above). However, there is no evidence of conjugation, and the polymorphic forms are more easily interpreted in terms of growth and division, for the long thin forms are potential dividing organisms, and the stumpy or short parasites, with little or no free flagellum, are the adult individuals. _Morphology of T. gambiense in the Circulating Blood._ _T. gambiense_ varies from 13 µ to 36 µ in length, its average length being 24·8 µ, as was determined in 1913 by exact biometrical methods by Stephens and Fantham.[58] Three forms of parasite occur. According to Miss Robertson,[59] the relatively short forms from 13 µ to 21 µ long may be regarded as the mature or “adult” type of parasite in the blood. They carry on the cycle in the vertebrate. From them intermediate forms, which are longer than the “adult” but at first have the same breadth, arise by growth. They possess a free flagellum. The intermediate forms grow into long individuals, which are those about to divide. The products of division give rise, directly or indirectly, to the adult forms. [58] _Annals Trop. Med. and Parasitol._, vii, p. 27. [59] _Phil. Trans._, B (1913), cciii, pp. 161–184. [Illustration: FIG. 28.--_Trypanosoma gambiense_. × 1,700. (After Dutton.)] [Illustration: FIG. 29.--_Trypanosoma gambiense_. Development in vertebrate host. _a_, long, slender, _b_, intermediate and _c_, short, stumpy forms, found in the blood; _d_, _e_, _f_, non-flagellate, latent forms from internal organs. × 2,000. (Original. From preparations by Fantham.)] The organism has an elongate body with an anterior or flagellar end and a blunter posterior or non-flagellar end. The protoplasm is finely granular, large inclusions being rare. The central nucleus is oval and large, often containing most of its chromatin concentrated as a karyosome, with small granules only scattered near or on the fine nuclear membrane. The blepharoplast is either rounded or rod-shaped. The undulating membrane is thrown into folds and is bordered by the flagellum. A small basal granule may be present near, or at the actual origin of the flagellum. _Multiplication_ in the vertebrate is brought about by longitudinal division. According to the recent account of division by Miss Robertson, the blepharoplast doubles, then the flagellum splits for the greater part of its length, and the daughter flagella separate, one being shorter than the parent flagellum. The nucleus often shows two well marked dark granules on the membrane at opposite poles, and these appear to act as centrosomes. Nuclear constriction occurs and the halves gradually separate. Finally the two daughter organisms become free, the aflagellar end splitting last. The products of division may be equal or unequal. Repeated division goes on in the general circulation until the blood swarms with parasites. Then the trypanosomes gradually disappear, and a period occurs when it is practically impossible to demonstrate the parasite in the blood. At such a period, trypanosomes can be obtained by puncture of the enlarged lymphatic glands or of the spinal canal, or can be found in the internal organs, more particularly in the spleen, lungs, liver and bone-marrow. In the latter organs, latent bodies are produced (fig. 29, _d_--_f_) which are capable of again becoming flagellates and entering the general circulation. Their formation was described by Fantham (1911).[60] The parasite contracts, the blepharoplast migrates towards the nucleus, a very thin coat differentiates around the two nuclei and a certain amount of cytoplasm, and the parts exterior to the coat disintegrate, leaving a small, oval body behind. Fuller details are given in connection with _T. rhodesiense_. Laveran (1911)[61] considers that latent bodies are “involution” forms, but acknowledges that they can flagellate and become infective in fresh blood. [60] _Proc. Roy. Soc._, B, lxxxiii, p. 212. [61] _C. R. Acad. Sci._, 153, p. 649. No multiplication of the trypanosomes within the cells of the lung, liver or spleen of infected monkeys was found by Miss Robertson in her recent researches. There appear to be negative periods in infected monkeys, since, although trypanosomes may occur in their blood at such times, they are not infective to _Glossina_. _Development in Glossina palpalis._--The principal accounts are those by Sir D. Bruce and his colleagues (1911),[62] and by Miss Robertson[63] (1912), whose results will be followed. According to the latter investigator _T. gambiense_ never enters the body cells of the fly (_G. palpalis_), nor does it penetrate the gut wall into the body cavity. Practically no crithidial stage occurs in the fly’s main gut, but a trypanosome facies is retained therein. [62] _Proc. Roy. Soc._, B, lxxxiii, p. 513. [63] _Proc. Roy. Soc._, B, lxxxvi, p. 66. After the trypanosomes are ingested by the fly during a meal of infected blood, sooner or later multiplication occurs. This development usually begins in the middle or posterior part of the mid gut, and trypanosomes of varying sizes are produced. After the tenth or twelfth day, many long, slender trypanosomes (fig. 30, _a_) are found, which gradually move forwards into the proventriculus. Such long, slender forms represent the limit of development in the lumen of the main gut. The proventricular type, developed about the eighth to the eighteenth or twentieth day, is not infective; it may occur in the crop, but is not to be found permanently there. Between the tenth and the fifteenth days multinucleate forms of trypanosomes are found, and may be styled multiple forms (fig. 30, _b_). Some of these latter may be degenerative. [Illustration: FIG. 30.--_Trypanosoma gambiense_. Development in the fly, _Glossina palpalis_. _a_, slender, proventricular form; _b_, multinucleate form; _c_, _d_, crithidial forms; _e_, infective type of trypanosome found in salivary gland. × 2,500. (After Robertson.)] _Invasion of the Salivary Glands of the Fly._--Long, slender trypanosomes from the proventriculus pass forward into the hypopharynx. They then pass back along the salivary ducts, about sixteen to thirty days after the fly’s feed. The trypanosomes reach the salivary glands as long, slender forms. In the glands they become shorter and broader, attach themselves to the surrounding structures, and assume the crithidial facies (fig. 30, _c_, _d_). As crithidial forms they remain attached to the wall and multiply in the glands. These crithidial stages differentiate into the short, broad trypanosome forms, capable of swimming freely (fig. 30, _e_). Miss Robertson considers the development in the main gut to be indifferent multiplication, and that salivary fluid seems necessary to stimulate trypanosomes to the apparently essential reversion to the crithidial type. The second development in the salivary gland is the essential feature. The short, stumpy forms of trypanosomes (fig. 30, _e_) finally produced in the salivary glands are alone infective. No conjugation of trypanosomes occurs in the fly. Only about 5 per cent. of captive tsetse flies fed on trypanosome-infected blood become infective, but they probably remain infective for the rest of their lives. J. G. Thomson and Sinton (1912)[64] have obtained in cultures the various trypanosome forms of _T. gambiense_ seen in the fly’s main gut. [64] _Annals Trop. Med. and Parasitol._, vi, p. 331. Duke (1912)[65] found _T. gambiense_ in a species of antelope, the situtunga (_Tragelaphus spekei_), on Damba Island in Victoria Nyanza. Wild _G. palpalis_ could be infected therefrom. The antelope may then act as a sleeping sickness reservoir in that district, but men are apparently the chief reservoir. [65] _Proc. Roy. Soc._, B, lxxxv, pp. 156, 483. *Trypanosoma nigeriense*, Macfie, 1913.[66] Macfie has recently (August, 1913) described a human trypanosome from the Eket district of Southern Nigeria. It is common in young people. The disease produced does not seem to be of a virulent type in Nigeria, and does not occur in epidemic form. In the early stages the glands of the neck are enlarged. In the later stages--cases of which are rarer--lethargy appears. The parasite is a polymorphic trypanosome, morphologically almost indistinguishable from _T. gambiense_, though it may be slightly shorter. Macfie recorded the occurrence in his preparations of a few trypanosomes appearing to have a flagellum free during their whole length. Some of the parasites, as seen in a sub-inoculated guinea-pig, are very small (8 µ long). Other trypanosomes have their nuclei displaced somewhat anteriorly. This parasite may only be a variety of _T. gambiense_. The parasite is perhaps spread by _Glossina tachinoides_. [66] _Annals Trop. Med. and Parasitol._, vii, p. 339; viii, p. 379. *Trypanosoma rhodesiense*, Stephens and Fantham, 1910. The parasite was found in the blood of a young Englishman who had contracted sleeping sickness in the Luangwa Valley, North-eastern Rhodesia, in the autumn of 1909. The patient had never been in an area infested with _Glossina palpalis_. (1) _Morphology._--The morphology of the parasite in man and sub-inoculated rats was studied by Stephens and Fantham in 1910.[67] They pointed out a morphological peculiarity in the presence of certain trypanosomes with posterior nuclei in sub-inoculated animals, that is, parasites in which the nucleus (trophonucleus) was situated towards the posterior or aflagellar end, close up to or even beyond the blepharoplast or kinetic nucleus (fig. 31, _4_, _5_). When the nucleus was beside the blepharoplast, the former was seen to be kidney-shaped (fig. 31, _4_). The posterior nuclear forms were of the stout and stumpy variety, and about 6 per cent. of the stumpy forms were found to have their nuclei displaced from the centre. The anterior or flagellar end of these trypanosomes often contained chromatoid granules. _T. rhodesiense_ varies in length from 12 µ to 39 µ[68]; short stumpy forms vary from 13 µ to 21 µ, intermediate forms from 21 µ to 24 µ, and long, slender forms from 25 µ onwards. The average length is 24·1 µ. [67] _Proc. Roy. Soc._, B, lxxxiii, p. 28. [68] Stephens and Fantham (1912–13), _Proc. Roy. Soc._, B, lxxxv, p. 223, and _Annals Trop. Med. and Parasitol._, vii, p. 27. [Illustration: FIG. 31.--_Trypanosoma rhodesiense._ 1, Long narrow form; 2–4, nucleus passing to posterior (aflagellar) end; 5, nucleus quite posterior. × 1,800. (After Stephens and Fantham.)] Certain regular periods occur in the course of the trypanosomiasis when few or no flagellate trypanosomes are found in the peripheral blood of the patient or of the sub-inoculated animal. These periods can be explained in terms of morphology, for the trypanosomes are capable of assuming a non-flagellate form in the internal organs of the host, particularly in the lungs and in the spleen. Such forms are known as “latent” or “resting” forms. The term “latent body” was first used by Moore and Breinl in 1907[69] in connection with _T. gambiense_. Fantham[70] (1911) has described the process of formation of latent from motile forms and the reconversion of the latent bodies into active flagellates. Fresh preparations of splenic blood or lung blood containing trypanosomes were made. A trypanosome gradually withdrew or cast off its flagellum, concentrated its cytoplasm, and became more or less elongate oval. Nucleus and blepharoplast approached one another and came to lie more or less side by side. Then an opaque line often made its appearance around the nuclear area and differentiated as a slight envelope or covering, the cytoplasm external to this merely degenerating. The small, oval, refractile body (fig. 29, _d_--_f_) thus formed was a non-flagellate latent body, 2 µ to 4 µ in diameter, like _Leishmania_ or the non-flagellate, multiplicative forms of _T. cruzi_ (fig. 34), and remains temporarily inactive in the internal organs of the host. After this period of inactivity, the non-flagellate body, recuperated by its rest, begins to elongate again. The nuclei separate. From a small vacuole-like portion the flagellum differentiates and forces out the ectoplasm, which assumes the form of the undulating membrane with its flagellar border. Subsequent growth results in the production of the typical trypanosome form, which re-enters the circulating blood and multiplies by longitudinal binary fission. Division of the parasite prior to the formation of a latent body may occur and division of the latent forms themselves is known, though less common. Consequently latent bodies, like the flagellate forms themselves, show diversity in size. The blepharoplast of the latent bodies is sometimes less well marked than in _Leishmania_ (see fig. 29, _d_-_f_). Laveran’s views on these bodies have already been given on p. 74. [69] _Annals Trop. Med. and Parasitol._, i, p. 441. [70] _Proc. Roy. Soc._, B, lxxxiii, p. 212. (2) _Animal Reactions._--The posterior nuclear trypanosomes were found in all sub-inoculated animals, such as rats, guinea-pigs, dogs, mice, Macacus, rabbits and horses, but were not seen in the human patient, as few trypanosomes occurred in his peripheral blood. R. Ross and D. Thomson[71] found a periodic, cyclical variation in the number of the parasites in the patient’s blood from day to day, the cyclical period being about a week (fig. 32). Fantham and J. G. Thomson[72] (1911) found a similar periodic, cyclical variation in the trypanosomes in the blood of sub-inoculated rats, guinea-pigs and rabbits. On counting the parasites in the blood of similar animals inoculated with _T. gambiense_, they established, by enumerative methods, that _T. rhodesiense_ was more virulent than _T. gambiense_, while Yorke also showed this marked virulence of _T. rhodesiense_ in practically all laboratory animals. In other words the duration of infection in the case of _T. rhodesiense_ was shorter. It was also found that _T. rhodesiense_ was resistant to atoxyl. The patient, from whom the original strain was obtained, died about nine months after the probable date of infection. Some patients infected with _T. rhodesiense_ have died in an even shorter period, such as four or five months. [71] _Proc. Roy. Soc._, B, lxxxii, p. 411. [72] _Annals Trop. Med. and Parasitol._, iv, p. 417. In sheep and goats _T. rhodesiense_ causes an acute disease, marked by high fever, œdema of the face, and keratitis, as shown by Bevan and others, death resulting after a relatively short period. _T. gambiense_ gives rise, in these animals, to no symptoms except fever, which may be overlooked. _T. rhodesiense_ produces keratitis in dogs. [Illustration: FIG. 32.--Chart showing daily counts of number of trypanosomes per cubic millimetre of peripheral blood from a case of Rhodesian sleeping sickness. (After R. Ross and D. Thomson.)] Stannus and Yorke (1911) observed _T. rhodesiense_ in animals inoculated from a case of sleeping sickness in Nyasaland. Sir D. Bruce and his colleagues[73] have shown (1912) that _T. rhodesiense_ is the parasite usually found in man and in animals sub-inoculated from cases of sleeping sickness in Nyasaland. It has since been found in German East Africa and Portuguese East Africa, while Ellacombe has described a case from North-western Rhodesia. [73] _Proc. Roy. Soc._, B, lxxxv, p. 423. (3) _Serum Reactions._--Interesting experiments on this subject were performed during 1911 and 1912 by various French investigators. (_a_) _Action of Immune Serum_ (Mesnil and Ringenbach)[74]: (1) A goat was infected with _T. rhodesiense_. Twenty-two days later its serum mixed with _T. rhodesiense_ was injected into a mouse. Result: Protection. (2) The serum mixed with _T. gambiense_ was injected into a mouse. Result: Infection. [74] _C.R. Soc. Biol._, lxxii, p. 58. (_b_) _Action of Baboon Serum._--Contrary to _T. gambiense_, _T. rhodesiense_ is very susceptible to human and baboon sera. Mesnil and Ringenbach[75] showed that a dose of 1 c.c. of baboon (_Papio anubis_) serum cured mice infected with _T. rhodesiense_. In the same dose it acted very feebly on _T. gambiense_. [75] _C.R. Acad. Sci._, 153, p. 1,097. (_c_) _Action of Human Serum._--_1 c.c._ of human serum cured _T. rhodesiense_ mice in three out of four cases; on _T. gambiense_ mice there was no appreciable effect. Laveran and Nattan-Larrier[76] have shown the same, namely, that human sera act on _T. rhodesiense_, but are quite without action on _T. gambiense_. [76] _C.R. Acad. Sci._, 154, p. 18. (_d_) _Trypanolytic Reactions._--Mesnil and Ringenbach[77] have also shown that the sera of animals (man, monkey and guinea-pig) infected with _T. gambiense_ are trypanolytic for the homologous trypanosome, that is, _T. gambiense_, but have no action on the heterologous trypanosome, that is, _T. rhodesiense_. [77] _C.R. Soc. Biol._, lxxi, p. 609. (4) _Cross Immunity Experiments._--(_a_) Mesnil and Ringenbach[78] immunized a monkey (_Macacus rhesus_) against _T. gambiense_. It was inoculated with _T. rhodesiense_ on June 7, 1911; on June 27 trypanosomes appeared, the infection being slight; on July 4 it died. A control died in ten and a half days. [78] _C.R. Soc. Biol._, lxxi, p. 271. (_b_) Laveran[79] immunized a goat and mice against _T. gambiense_. When they had acquired a solid immunity, they were inoculated with _T. rhodesiense_. They became infected like the controls. [79] _Bull. Soc. Path. Exot._, v, pp. 26, 241. (_c_) Laveran and Nattan-Larrier[80] immunized a ram against _T. brucei_, it subsequently became infected with _T. rhodesiense_. [80] _C.R. Acad. Sci._, 154, p. 18. (_d_) Laveran[81] immunized a ram and a sheep against different strains of T_. brucei_. Inoculated with _T. rhodesiense_ they both acquired acute infections and died. Conclusion: _T. rhodesiense_ is not _T. brucei_. [81] _Bull. Soc. Path. Exot._, v, p. 101. When the converse set of experiments is tried, namely, immunizing an animal against _T. rhodesiense_, and then inoculating with _T. gambiense_, the difficulty immediately arises that it is impossible to immunize an animal against _T. rhodesiense_, owing to its virulence. But a partial and transitory immunity to _T. rhodesiense_ can be obtained by treating the infected animal with drugs, such as arsenophenylglycin. The results, so far as they go, seem to show that an animal immunized against _T. rhodesiense_ is immune not only to _T. rhodesiense_, but also to _T. gambiense_, a fact which, according to Mesnil and Léger, does not invalidate the specificity of _T. rhodesiense_, but tends to show that the two trypanosomes are closely related. (5) _Mode of Transmission and Reservoir._--Kinghorn has shown that _T. rhodesiense_ is transmitted by _Glossina morsitans_ in which it undergoes development. Kinghorn and Yorke[82] found that about 16 per cent. of the wild game examined in Northern Rhodesia was naturally infected with _T. rhodesiense_. The wild game examined included waterbuck, hartebeest, mpala, bushbuck and warthogs. One native dog near the Nyasaland border was found infected, but not domestic stock. Taute doubts whether _T. rhodesiense_ really occurs in wild game. Approximately 3·5 per cent. of the tsetse flies fed on infected animals may become permanently infected with _T. rhodesiense_, and capable of infecting clean animals. Furthermore, a tsetse fly when once infective probably remains infective for the rest of its life. [82] _Annals Trop. Med. and Parasitol._, vii, p. 183. Kinghorn and Yorke, however, have shown that climatic conditions, namely, those of temperature, also affect the infectivity of the tsetse fly, as the ratio of flies capable of transmitting _T. rhodesiense_ to those incapable of transmitting the virus is 1 : 534 in hot valley districts (_e.g._, Nawalia, Luangwa Valley, temperature 75° to 85° F.), while on elevated plateaux (_e.g._, Ngoa, on the Congo-Zambesi watershed, temperature 60° to 70° F.) the ratio falls to 1 : 1312. Mechanical transmission by the tsetse fly does not occur, if a period of twenty-four hours has elapsed since the infecting meal. _Developmental Cycle in the Fly._--The period which elapses between the infecting feed of the flies and the date on which they become infective varies from eleven to twenty-five days in the Luangwa Valley, according to Kinghorn and Yorke. Attempts carried out at laboratory temperature on the Congo-Zambesi plateau, during the cold season, to transmit _T. rhodesiense_ by means of _G. morsitans_ were always unsuccessful. The developmental cycle of the trypanosome in the fly is influenced by the temperature to which the flies are subjected (as stated above). The first portion of the developmental cycle proceeds at the lower temperatures (60° to 70° F.), but higher temperatures are necessary for the completion of the development of the trypanosome. Kinghorn and Yorke found that the trypanosomes may persist in the fly, at an incomplete stage of their development, for at least sixty days when the climatic conditions were unfavourable. The first portion of the developmental cycle of the trypanosome takes place in the gut of the fly. Invasion of the salivary glands of the tsetse is secondary to that of the intestine, but is necessary for the infectivity of the fly. A relatively high mean temperature, 75° to 85° F., is essential for the passage of the trypanosomes into the salivary glands and the completion of their development therein. Kinghorn and Yorke[83] state that the predominant type of trypanosome in the intestine of infected _G. morsitans_ was a large broad form, quite different from that which is most common in the salivary glands. The trypanosome in the glands resembles the short form seen in the blood of the vertebrate host. The authors quoted state that both the intestinal and salivary gland forms of infective _G. morsitans_ are virulent when inoculated into healthy animals. [83] _Annals Trop. Med. and Parasitol._, vii, p. 281. Bruce and colleagues[84] have quite recently (June, 1914) published an account of their investigations of _T. rhodesiense_ in _G. morsitans_ in Nyasaland. (Incidentally it may be remarked that Bruce considers _T. rhodesiense_ to be identical with a polymorphic strain of _T. brucei_--see pp. 83, 94). The development of _T. rhodesiense_ takes place in the alimentary canal and salivary glands, not in the proboscis, of the tsetse fly. In feeding experiments with laboratory bred flies, as well as with a few wild flies, fed on infected dogs or monkeys, only 8 per cent. of the flies were found to be infected on dissection. Of such infected flies, however, only some allow of the complete development of the trypanosomes within them, in other words only about 1 per cent of the flies become _infective_. The length of time which elapses before a fly becomes infective varies from fourteen to thirty-one days, averaging twenty-three days, when kept at 84° F. (29° C.). The dominant intestinal type of flagellate in the fly is that seen in the proventriculus, which contains many long, slender trypanosomes. These proventricular forms find their way to the salivary glands, wherein crithidial and encysted forms are seen. They change into “blood forms,” which are short, stumpy trypanosomes and are infective. “The infective type of trypanosome in the salivary glands--corresponding to the final stage of the cycle of development--is similar to the short and stumpy form found in the blood of the vertebrate host.” The cycle is thus very similar to that of _T. gambiense_ in _G. palpalis_ (fig. 30). [84] _Proc. Roy. Soc._, B, lxxxvii, p. 516. CULTURE.--J. G. Thomson (1912),[85] and subsequently Thomson and Sinton, succeeded in cultivating _T. rhodesiense_ in a modified Novy-MacNeal medium. The development obtained resembled that of the trypanosome in the intestine of _Glossina_. [85] _Annals Trop. Med. and Parasitol._, vi, pp. 103, 331. GENERAL NOTE ON TRYPANOSOMES WITH POSTERIOR NUCLEI. Posteriorly placed nuclei have been found to occur not only in _T. rhodesiense_ by Stephens and Fantham (1910), but also in _T. pecaudi_ by Wenyon (1912), in _T. brucei_ by Blacklock (1912), and in _T. equiperdum_ by Yorke and Blacklock (1912). Recently Stephens and Blacklock (1913)[86] have shown that two trypanosomes, different morphologically, have been confused under the name _T. brucei_. One of these is polymorphic (_i.e._, it exhibits long and slender as well as short and stumpy forms) and came from Uganda, while the other is monomorphic and is the original Zululand strain described by Bruce from cattle suffering from “nagana.” Bruce (1914) considers that morphological change has occurred in _T. brucei_ in its passage through laboratory animals, and thus explains the diversity of views. The posterior nuclear forms described by Blacklock occurred in the Uganda strain of _T. brucei_. (See p. 95.) Similarly, a posterior nuclear form, _T. equi_, has been separated from _T. equiperdum_. (See p. 98.) [86] _Proc. Roy. Soc._, B, lxxxvi, p. 187. Again, Bruce and his colleagues on the Royal Society Commission investigating sleeping sickness in Nyasaland, have stated (April, 1913) that “evidence is accumulating that _T. rhodesiense_ and _T. brucei_ (Plimmer and Bradford) are identical.” The exact identity of trypanosomes showing posterior nuclei is, then, far from settled, although Laveran by cross immunity tests has declared that _T. brucei_ is distinct from _T. rhodesiense_. No one has yet seen posterior nuclei in _T. gambiense_. *Trypanosoma cruzi*, Chagas, 1909. Syn.: _Schizotrypanum cruzi_, Chagas, 1909. The trypanosome was discovered by Chagas[87] in the intestine of the bug, _Triatoma_ (_Conorhinus_) _megista_, in Brazil, and then in the blood of a small monkey bitten by the bug. A little later it was found in the blood of a child, aged two years, suffering from irregular fever, extreme anæmia and enlarged glands in the State of Minas Geraes, Brazil. Chagas found that he was able to infect many of the usual laboratory animals with the trypanosome, by allowing the bug to bite them. He was also able to culture the parasite on blood agar. [87] _Mem. Inst. Oswaldo Cruz._, i, p. 159. Chagas found the Reduviid bug, _Triatoma megista_, in the houses of the poorer inhabitants of the Brazilian mining State, and that it attacked the people, more especially the children, at night, biting the face. On this account the insect is called “barbeiro” by the inhabitants. The bite is somewhat painful. The disease has since been found in other parts of Brazil, _e.g._, Matta de São João in Bahia province, Goyaz, Matto Grosso and São Paulo provinces, as well as in Minas Geraes. _Morphology._--The trypanosome has a large blepharoplast or kinetic nucleus. It is stated to occur both free and in the red blood corpuscles in the peripheral blood. It is about 20 µ long, on an average. Two forms of the parasite (fig. 33, _6_, _7_) are described in the human blood. In one free form there is a large egg-shaped blepharoplast and the posterior (aflagellar) end of the parasite is drawn out. The blepharoplast (kinetic nucleus) may have a chromatin appendage. The nucleus is oval or band-like, containing a karyosome. The flagellum, starting close to the blepharoplast or its appendage, has a free portion of variable length. The other free form in the blood has a more or less round, terminal blepharoplast, smaller than in the first form, without a chromatin appendage as a rule. The body of this second form is decidedly broader than that of the first mentioned. [Illustration: FIG. 33.--_Trypanosoma cruzi_. Schizogony. _1_, merozoite in red blood corpuscle; _2_, parasite totally enclosed in red cell, no flagellum or undulating membrane; _3_-_5_, parasites partially enclosed in red cell; _6_, _7_, parasites in human blood; _8_-_11_, parasites in lungs of the monkey, _Callithrix_; _12_, _13_, initial forms of schizogony; _14_, _15_, schizogony in the lungs of _Callithrix_. (After Chagas.)] The dimorphism has been interpreted sexually, the first mentioned forms being termed males, the second ones females. The correctness of this interpretation is very doubtful. No sign of longitudinal division was ever seen in the peripheral blood or in the internal organs. The “endocorpuscular” forms may be completely or partially enclosed in the red cell or only attached thereto (fig. 33, _1_-_5_). At the beginning of infection the endocorpuscular forms are the more numerous. Some authorities, however, doubt these stages. _Life-history in the Vertebrate Host._--Chagas found fluctuations in the number of the parasites in the peripheral blood. He believes the increase of the parasites to be periodic. The investigations of Chagas and of Hartmann have revealed two types of multiplication which take place in the internal organs of the vertebrate host. (_a_) The first type--which possibly belongs to another organism, _Pneumocystis carinii_, see p. 90--occurs in the capillaries of the lungs. The flagellate parasite entering the lung capillaries loses its flagellum and undulating membrane. Its body becomes curved, and the two ends fuse, and so an oval mass is formed (fig. 33, _8_-_11_). In some cases the blepharoplast disappears, in other cases it blends or fuses with the nucleus. The nucleus of the rounded parasite then divides into eight by successive divisions (fig. 33, _12_-_15_). Next the body, which is surrounded by its own periplast, also divides, giving rise to eight tiny daughter individuals or merozoites (fig. 33, _15_). The merozoites lie inside the periplast, which acts as a sort of “cyst wall.” The merozoites are said to exhibit dimorphism, and Chagas has interpreted the dimorphism in terms of sex. The daughter forms, produced by the parent trypanosomes which kept their blepharoplasts, themselves have blepharoplasts as well as nuclei, and have been termed “males” or “microgametes.” The merozoites, arising from parent trypanosomes which lost their blepharoplasts, have themselves only nuclei, and have been called “females” or “macrogametes.” In the case of the so-called “female” forms the single nucleus divides into two unequal parts, of which the smaller becomes the blepharoplast, and a flagellum is formed later. The so-called “males” possess early a rudiment of a flagellum. Both kinds of merozoites escape from the parent periplast wall, and enter red blood corpuscles. They grow into flagellates within the corpuscles, and then become free as adult trypanosomes in the blood-stream. [Illustration: FIG. 34.--_Trypanosoma cruzi_. Transverse section of a striated muscle containing rounded forms of the parasite in the central portion. × 1,000 approx. (After Vianna.)] (_b_) The second mode of multiplication is one of asexual reproduction (schizogony or agamogony). It was first described by Hartmann from hypertrophied endothelial cells of the lungs. It has since been found in the cardiac muscle, in the neuroglia of the central nervous system, and in striped muscle (fig. 34). In laboratory animals it has also been found in the testicle and suprarenal capsules. In these tissues the parasite is intracellular, appearing as a small rounded body with nucleus and blepharoplast, without flagellum or undulating membrane. In other words the parasite is _Leishmania_-like in the body tissues, and recalls the organism of kala-azar. Chagas considers this second mode of multiplication to be strictly asexual. By this means the number of parasites in the vertebrate host is increased, and symptoms are produced. On the other hand the first mode of multiplication, seen in the lung capillaries, is considered by Chagas to be a process of gametogony, in which sexual forms are differentiated. He finds that (1) the adult trypanosomes exhibit a dimorphism in human blood rarely seen in artificially infected guinea-pigs. In these guinea-pigs (infected from guinea-pigs) the so-called gametogony in the lungs is seldom seen. (2) The intermediate host, _Triatoma_ (_Conorhinus_), becomes infective if fed directly on infected human blood, but very rarely so if fed on guinea-pigs. Chagas is led to believe that the occurrence of sexual forms constantly in the blood of man implies a greater resistance to infection on the part of man than on the part of guinea-pigs or other animals, assuming the general hypothesis that the formation of gametes represents a reaction of the Protozoön to unfavourable conditions. In human infection the number of parasites is always less than in laboratory animals, and their presence in the blood is transitory, lasting from fifteen to thirty days in acute cases. In many cases examination of the tissues at death has shown the presence of parasites in patients who did not exhibit them in the general circulation. [Illustration: FIG. 35.--_Trypanosoma cruzi_. Development in _Triatoma megista_. _1_-_6_, forms found in the mid gut of _Triatoma_; _7_ flagellate forms found in the posterior part of the gut of _Triatoma_. (After Chagas.)] _Life History in the Invertebrate Host._--About six hours after the ingestion of infected blood by the bug (_Triatoma megista_), the kinetic nucleus of the trypanosome moves towards the nucleus, and the flagellum is usually lost (fig. 35, _1_-_5_). The parasite becomes rounded and _Leishmania_-like (fig. 35, _3_-_5_), and multiplies rapidly by division. After a time, multiplication having ceased, the rounded forms become pear-shaped and develop a flagellum at the more pointed end. Crithidial forms (fig. 35, 7) are thus produced and pass into the intestine, where they multiply and may be seen in about twenty-five hours after the ingestion of blood. The crithidial forms may also be found in the rectum and fæces. The last stage in the invertebrate is a small, trypanosome-like type, long and thin with a band-like nucleus and conspicuous kinetic nucleus. These parasites are found in the hind gut and in the body cavity. They find their way into the salivary glands, and are the forms (fig. 36) which are transmissible to a new vertebrate host. The development in the bug takes about eight days altogether, after which time the bugs are infective. There are thus three principal phases in the development of _T. cruzi_ in _Triatoma megista_: (1) A multiplicative phase (_Leishmania_-like) in the stomach of the bug, (2) a crithidial phase, which is also multiplicative, in the hind-gut, and (3) a trypanosome phase, which is “propagative,” and apparently passes through the wall of the alimentary canal into the body cavity and so into the salivary glands. [Illustration: FIG. 36.--_Trypanosoma cruzi_. Forms found in the salivary glands of _Triatoma megista_. (After Chagas.)] Brumpt found that _T. cruzi_ could live in _Cimex lectularius_, _C. boueti_, and _Ornithodorus moubata_. The _Cimex_ fæces may be infective. Blacklock found multiplication of the parasite in _C. lectularius_. _Culture._--The trypanosome can be cultivated on Novy-MacNeal’s blood agar, and the cultural forms resemble those described in the bug. _Possible Reservoir._--Chagas thinks that probably the armadillo or “tatu” (_Dasypus novemcinctus_) may be the reservoir of _T. cruzi_. He also thinks that _Triatoma geniculata_ is a transmitter; it lives in the burrows of the armadillo. Other carriers may be _Triatoma infestans_ and _T. sordida_. _Clinical Features._--The trypanosomiasis of Brazil, produced by _T. cruzi_ and spread by _Triatoma_ spp. has received various names, such as oppilação, canguary, parasitic thyroiditis, and coreotrypanosis. It is also known as the human trypanosomiasis of Brazil, South American trypanosomiasis, and Chagas’ disease. Chagas[88] reports two principal forms--acute and chronic. The _acute infection_ is rare, and is characterized by increase in the volume of the thyroid gland, pyrexia, a sensation of crackling in the skin, enlarged lymphatic glands in the neck, axilla, etc., while the liver and spleen are increased in volume. Sclerosis of the thyroid gland is found at autopsy and fatty degeneration of the liver. During an attack of fever, trypanosomes are found in the blood. The acute form was only observed in children. [88] _Brazil Medico_, Nov. 15, 1910. Longer account in _Mem. Inst. Oswaldo Cruz_, iii, pp. 219–275. See _Sleep. Sick. Bull._, Nos. 35 and 40. _In the chronic form_ Chagas reports several varieties: (_a_) A pseudo-myxœdematous form, occurring in most cases, especially up to the age of 15. There is hypertrophy of the thyroid gland or at least signs of hypothyroidism, general hypertrophy of glands, disturbance of heart rhythm, and nervous symptoms. (_b_) The myxœdematous form is characterized by similar symptoms, especially by considerable swelling of the thyroid body, and myxœdema of the subcutaneous cellular tissue; sometimes there is a true pachydermic cachexia. (_c_) In the nervous form there are motor disturbances, aphasia, disturbances of intelligence or signs of infantilism, athetosis of the extremities and idiocy. There are also paralytic symptoms of bulbar origin, disturbances of mastication, phonation and deglutition, and in some cases convulsive attacks. (_d_) The cardiac form, characterized by disturbance of the heart rhythm. In all these forms the parasite is found at autopsy in the nervous substance, brain, bulb and heart. Vianna (1911)[89] has studied the histopathology of the disease. Some of the chief points are: in the heart muscle destruction of the sarcoplasm, followed by interstitial myocarditis; in the central nervous system invasion of the neuroglia cells and inflammatory reaction; in the suprarenal capsule invasion of medulla or cortex; inflammatory reaction can also be seen in the kidneys, the hypophysis and thyroid gland. [89] _Mem. Inst. Oswaldo Cruz_, iii, p. 276. Recently Chagas states[90] that “schizotrypanosomiasis” has been found in a child 15 to 20 days old, and that _Trypanosoma cruzi_ has also been found in a fœtus--the mother being infected with the trypanosome. The trypanosomiasis can, then, be transmitted hereditarily. [90] _Rev. Med. S. Paulo_ (1912), xv, p. 337. *Trypanosoma lewisi*, Kent, 1881. The trypanosome has a nucleus somewhat displaced anteriorly, about one-third of the way from the anterior (flagellar) end of the body, a relatively straight edge to the undulating membrane, and a rod-shaped blepharoplast (fig. 37, A). It averages about 25 µ long and 1·5 µ broad. Much attention has been devoted in recent years to the elucidation of the life history of the rat parasite, _Trypanosoma lewisi_. It is usually non-pathogenic to its host. It has been shown that the trypanosome can be transmitted from rat to rat by the rat-flea, _Ceratophyllus fasciatus_, and by _Ctenocephalus canis_ (the so-called dog-flea). (See also p. 92). The flagellate may also persist, but doubtfully develop, in the rat-louse, _Hæmatopinus spinulosus_. These researches may now be summarized. [Illustration: FIG. 37.--_Trypanosoma lewisi_, from rat’s blood. A, ordinary form; B, small form; C, D, stages in equal binary fission; E, elongate form (_longocaudense_ type), resulting from division as seen in D; F, unequal binary fission; G, H, multiple fission into four and eight; I, small form; J, binary fission of small form; K, division rosette. × 2,000. (After Minchin and Thomson.)] _Life Cycle in the Vertebrate Host._--After infection of a rat, the trypanosomes usually appear in the animal’s blood in five to seven days. This incubation period applies either to a natural or an artificial infection. The trypanosomes first observed in the rat’s blood are diverse in form (fig. 37), being small, medium and large in size. This diversity is explained by the rapid multiplication taking place. A trypanosome may divide by equal longitudinal fission (fig. 37, C, D), but more commonly multiple fission occurs (fig. 37, G, H), and is unequal. Rosette forms are produced, in which the parent form can be recognized by its long flagellum (fig. 37, H) and attached to it are daughter individuals, smaller in size, from which flagella are growing. Minchin and J. D. Thomson (1912) find that the daughter forms may be set free sometimes with a crithidia-like facies (fig. 37, I), the blepharoplast being anterior but near to the nucleus. The daughter forms, when set free, may themselves divide by binary or multiple fission, in the latter case forming rosettes (fig. 37, K). Rosette forms were described by Moore, Breinl and Hindle in 1908. Lingard, some years ago, described as a distinct species, _T. longocaudense_, certain forms with markedly elongate posterior ends (fig. 37, E). According to Minchin, “these forms appear to arise by binary fission” (fig. 37, D). These long drawn-out forms “are of constant occurrence and very numerous at a certain stage of the multiplication period.” It is about the eighth or tenth day after infection that the multiplication of _T. lewisi_ is at its maximum in the rat’s blood. About the twelfth or thirteenth day the trypanosomes seen in the blood appear uniform. According to Minchin (1912)[91] the rat “gets rid of its infection entirely sooner or later, without having suffered, apparently, any marked inconvenience from it, and is then immune against a fresh infection with this species of trypanosome.” There is, then, a cycle of development in the vertebrate host. Minchin notes that the records of the pathogenicity of _T. lewisi_ in rats, causing their death, need further investigation. [91] “Protozoa,” p. 294. _T. lewisi_ inoculated into dormice (_Myoxus nitela_) and jerboas may become pathogenic thereto. Carini found cysts in the lungs of rats infected with _T. lewisi_. He thought the cysts were schizogonic stages of the trypanosome, comparable with those found in the lungs of animals sub-inoculated with _T. cruzi_. Delanoë (1912)[92] has found, however, that such cysts, containing eight vermicules, occurred in rats uninfected with _T. lewisi_. Delanoë concludes that the pneumocysts are independent of _T. lewisi_, and represent a new parasite, _Pneumocystis carinii_. The pneumocysts may be allied to the Coccidia, and must be considered when investigating the life-cycle of a trypanosome in a vertebrate host. Some of the stages of _T. cruzi_ may possibly be of this nature. [92] _C. R. Acad. Sci._, clv, p. 658. _Life-cycle in the Invertebrate Host._--This occurs in fleas, and has been investigated in considerable detail by Minchin and Thomson in _Ceratophyllus fasciatus_, and by Nöller in _Ctenocephalus canis_ and _Ctenopsylla musculi_. When infected rat’s blood is taken up by the flea, the parasites pass with the ingested blood direct to the mid-gut of the Siphonapteran. In the flea’s stomach they multiply in a somewhat remarkable manner, namely, by penetration of the cells of the lining epithelium, and division inside the epithelial cells. Inside these lining cells the trypanosomes first grow to a large size and then form large spherical bodies, within which nuclear multiplication occurs (fig. 38, A-F). Any one of these large spherical bodies contains at first a number of nuclei, blepharoplasts and developing flagella, the original flagellum still remaining attached for a time. The cytoplasm then divides into daughter trypanosomes which are contained within an envelope, formed by the periplast of the parent parasite. Inside the periplast envelope are a number of daughter trypanosomes “wriggling very actively; the envelope becomes more and more tense, and finally bursts with explosive suddenness, setting free the flagellates, usually about eight in number, within the host-cell” (fig. 38, F). The daughter forms escaping from the host cell into the stomach lumen of the flea are fully formed, long trypanosomes. [Illustration: FIG. 38.--_Trypanosoma lewisi_. Developmental stages from stomach of rat flea. O, ordinary blood type; A-F, stages occurring in gut-epithelium of flea, when the trypanosome becomes rounded and undergoes multiplication, forming in F eight daughter trypanosomes; G, type of trypanosome resulting from such division which passes back to the rectum. × 2,000. (After Minchin.)] The trypanosomes (fig. 38, G) pass into the flea’s rectum. The next phase is a crithidial one. The parasites become pear-shaped, in which the blepharoplast (kinetic nucleus) has travelled anteriorly past the nucleus towards the flagellum (fig. 39). The crithidial forms attach themselves to the wall of the rectum, and multiply by binary fission (fig. 39, D). A stock of parasites is thus formed which, according to Minchin and Thomson, “persist for a long time in the flea--probably under favourable conditions, for the whole life of the insect” (fig. 39, A-I). From the crithidial forms of the rectum, according to Minchin, small infective trypanosomes arise by modification morphologically (fig. 39, J--M). The flagellum grows longer and draws out more the anterior part of the body, the blepharoplast migrates posteriorly, behind the nucleus, and carries with it the flagellar origin. These trypanosomes are small, but broad and stumpy (fig. 39, N), and can infect a rat. Minchin and Thomson formerly considered that the small, stumpy, infective trypanosomes pass forwards from the rectum into the stomach, and “appear to be regurgitated into the rat’s blood when the flea feeds.” However, the small infective trypanosomes were previously described by Swellengrebel and Strickland.[93] They may be found in the flea’s fæces. Nöller (1912)[94] has found that the development of _T. lewisi_ proceeds quite well in the dog flea (_Ctenocephalus canis_) in Germany. Wenyon confirms this, and states that the human flea, _Pulex irritans_, and the Indian rat-flea, _Xenopsylla cheopis_, are also able to serve as true hosts for _T. lewisi_. [93] _Parasitology_, iii, p. 360. [94] _Arch. f. Protistenkunde_, xxv, p. 386. [Illustration: FIG. 39.--_Trypanosoma lewisi_. Developmental stages from rectum of rat-flea. A, early rectal form; C, D, division of crithidial form; E, group of crithidial forms; F--I, crithidial forms without free flagella, some becoming rounded; J--M, transitional forms to trypanosome type seen in N, which represents the final form in the flea. × 2,000. (After Minchin.)] Nöller stated that rats were not infected with _T. lewisi_ by infective fleas biting them, but by the rats licking up the fæces passed by the fleas while feeding. This is not in agreement with Minchin and Thomson’s earlier views of regurgitation, which, apparently, they have now abandoned.[95] Wenyon (1912) confirms Nöller’s experiments. He took a dog flea, containing infective trypanosomes in its fæces, and allowed it to feed on a clean rat. The fæces of the flea, passed while feeding, were carefully “collected on a cover glass and taken up in culture fluid with a fine glass pipette.” The contents of the pipette were discharged into the mouth of a second clean rat. Injury to the rat’s mouth was carefully avoided. The first rat, on which the infective flea was fed, did not become infected, while the second rat, in whose mouth infective flea fæces were placed, became infected in six days. [95] Report to Advis. Comm. Trop. Dis. Research Fund for 1913, p. 74. When infective forms of _T. lewisi_ have been developed within the gut of a rat flea, they may enter and infect the vertebrate host by[96] (_a_) being crushed and eaten by the rodent; (_b_) the rat may lick its fur on which an infected flea has just passed infective excrement; or (_c_) the rat may lick, and infect with flea excrement, the wound produced by the bite of the flea. [96] Nuttall, _Parasitology_, v, p. 275. The time taken for the full development of _T. lewisi_ in the flea is about six days. The intracellular phase is at its height about the end of the first day; the crithidial phase, in the flea’s rectum, begins during the second day; the stumpy, infective trypanosomes are developed in the rectum about the end of the fifth day. Wenyon[97] writes that, “the fleas, when once infected with _T. lewisi_, remain infected for long periods, for though many small infective trypanosomes are washed out of the gut at each feed, those that remain behind multiply to re-establish the infection of the hind gut. Further, the infection is still maintained even if the flea is nourished on a human being, so that fresh human blood does not appear to be destructive to the infective forms in the flea.” [97] Report to Advis. Comm. Trop. Dis. Research Fund, October, 1912, p. 91. See also _Journ. Lond. Sch. Trop. Med._, ii, p. 119. The best method of controlling fleas during experiments is that due to Nöller. He adopted the method of showmen who exhibit performing fleas, and secure them on very fine silver wire. Of fleas fed on an infected rat only about 20 per cent. become infective. About 80 per cent. are immune. If fleas are examined twenty-four hours after feeding, trypanosomes will be found in all, so that many of the parasites are destined to degenerate. It may be of interest to note that Gonder[98] (1911) has shown that a strain of _T. lewisi_ resistant to arsenophenylglycin loses its resistance after passage through the rat-louse, _Hæmatopinus spinulosus_. These experiments suggest that physiological “acquired characters” may be lost by passage through an invertebrate host. [98] _Centralbl. f. Bakt._, Orig., lxi, p. 102. *Trypanosoma brucei*, Plimmer and Bradford, 1899. _Trypanosoma brucei_ was discovered by Sir D. Bruce in 1894 in cattle in Zululand and was named _T. brucei_ by Plimmer and Bradford in 1899 in honour of its discoverer. This trypanosome is of considerable economic importance, as it is responsible for the fatal tsetse fly disease, or “nagana,” in cattle, horses and dogs. The disease is widely distributed in Africa and is transmitted from host to host by the tsetse, _Glossina morsitans_, and other species of _Glossina_. The virus is maintained in nature in certain big game, such as wildebeest, bushbuck and koodoo, which thus act as living reservoirs of disease from which the tsetse may become infected. These reservoir hosts are not injured, apparently, by the presence of the parasites. _T. brucei_ is rapidly fatal to the small laboratory animals, such as rats and mice. Horses, asses and dogs practically always succumb to its attacks, while a very small number of cattle recover from “nagana.” The disease is characterized by fever, destruction of red blood corpuscles, severe emaciation and by an infiltration of coagulated lymph in the subcutaneous tissue of the neck, abdomen and extremities giving a swollen appearance thereto. The natural reservoirs in which _T. brucei_ has been long acclimatized are unaffected by the trypanosomes, while the newer hosts, such as imported cattle in Africa, are rapidly destroyed by their action. [Illustration: FIG. 40.--_Trypanosoma brucei._ × 2,000. (After Laveran and Mesnil.)] The general morphology and life history in the vertebrate host is that of a typical trypanosome (fig. 40). Its length is from 12 µ to 35 µ, its breadth from 1·5 µ to 4 µ. Multiplication by longitudinal division proceeds in the peripheral blood (fig. 26), while latent, leishmaniform bodies are produced in the internal organs. Bruce and colleagues[99] have quite recently (June, 1914) described the development of a Zululand strain of _T. brucei_ in _G. morsitans_. The tsetse flies were bred out in Nyasaland. In vertebrate blood the _brucei_ strain was polymorphic. The development was like that found for _T. gambiense_ in _G. palpalis_ (fig. 30), and by Bruce and colleagues for _T. rhodesiense_ in _G. morsitans_ in Nyasaland. Long trypanosomes were found in the proventriculus of the tsetse. Crithidial, rounded or encysted, and immature “blood forms” occurred in the salivary glands; and finally infective, stumpy, “blood forms” were differentiated in the salivary glands. The period of development of _T. brucei_ in _G. morsitans_ takes about three weeks, and then the fly becomes infective. Bruce believes that _T. rhodesiense_ of Nyasaland and _T. brucei_ of Zululand are the same, their cycles of development in _G. morsitans_ being “marvellously alike.” (But see Laveran, p. 80.) [99] _Proc. Roy. Soc._, B, lxxxvii, p. 526. _T. brucei_ has been cultivated with difficulty by Novy and MacNeal, using blood agar. The best treatment for nagana is arsenic in some form. It is probable that more than one trypanosome has been confused under the name _T. brucei_, more especially as the occurrence of many species of trypanosomes in various animals in Africa was not suspected until comparatively recent times. It has been shown by Stephens and Blacklock (1913) that the original Zululand strain of _T. brucei_ was monomorphic, while the organism sent from Uganda, and at the time believed by Bruce to be the same as the Zululand trypanosome, has been found to be polymorphic, with morphological resemblances to _T. rhodesiense_. Stephens and Blacklock[100] have suggested the name _T. ugandæ_ for the polymorphic trypanosome, which, however, has marked resemblances with *Trypanosoma pecaudi*, and they are, perhaps, identical. _T. pecaudi_ was the name given by Laveran[101] in 1907 to the causal agent of “baleri” in equines and sheep in the French Sudan. _T. pecaudi_, which is dimorphic, is widely distributed in Africa. An extremely small number of both _T. pecaudi_ and _T. ugandæ_ have been shown to possess posterior nuclei. _T. pecaudi_ is transmitted by various species of _Glossina_, and is said to develop in the gut and proboscis of the fly. [100] _Proc. Roy. Soc._, B, lxxxvi, p. 187. [101] _C.R. Acad. Sci._, cxliv, p. 243. On the other hand, Bruce and colleagues (1914), examining a strain sent from Zululand in 1913, state that _T. brucei_ is polymorphic. Bruce (1914) suggests that passage through laboratory hosts has influenced and altered the morphology of the parasite. *Trypanosoma evansi*, Steel, 1885. Syn.: _Spirochæta evansi_, Steel, 1885; _Hæmatomonas evansi_, Crookshank, 1886; _Trichomonas evansi_, Crookshank, 1886. _Trypanosoma evansi_, first found by Evans in 1880, in India, is the causal agent of the disease known as “surra.” The malady affects more particularly horses, mules, camels and cattle in India and neighbouring countries, such as Burma and Indo-China. It occurs also in Java, the Philippines, Mauritius and North Africa. Elephants may be affected. A serious outbreak among cattle in Mauritius occurred in 1902, the disease being imported into the island. The symptoms are fever, emaciation, œdema, great muscular weakness and paralysis culminating in death. _T. evansi_ varies from 18 µ to 34 µ in length and 1·5 µ to 2 µ in breadth. It has a pointed posterior extremity, and, anteriorly, there is a free portion to the flagellum (fig. 41). It is possibly monomorphic, but a few broad forms occur. The trypanosome multiplies by longitudinal fission in the blood. Rounded leishmaniform stages occur in the spleen of the vertebrate host, which stages Walker[102] (1912) considers to be phases of schizogony. [102] _Philippine Journ. Sc._ (Sect. B), vii, p. 53. The parasite is transmitted in nature by various species of _Tabanus_ and _Stomoxys_, though at present little is known of the life-history within these invertebrate hosts. Dogs are said to contract the disease by feeding on animals dead of surra. A variety of _T. evansi_ is the cause of “mbori” in dromedaries in Africa (Sahara and Sudan). Another possible variety, or closely allied form, is _T. soudanense_, the causal agent of “el debab” in camels and horses in North Africa, especially Algeria and Egypt. [Illustration: FIG. 41.--_Trypanosoma evansi_. × 2,000. (Original. From preparation by Fantham.)] An extraordinary example of the possible infection of a human being with an animal trypanosome is recorded in the case of Professor Lanfranchi, of the Veterinary School, Parma. The Professor became infected with trypanosomes, although only nagana and surra were maintained in his laboratory, and he himself had never visited the tropics. He suffered from irregular attacks of fever and was œdematous, but his mind remained clear. The identification of the trypanosome from Lanfranchi’s blood has been a matter of great difficulty. Apparently Mesnil and Blanchard (1914)[103] consider the strain found in the patient is almost indistinguishable in its reactions from _T. gambiense_, though the parasite is monomorphic. Lanfranchi considers that he was infected with _T. evansi_. [103] _Bull. Soc. Path. Exot._, vii, p. 196. *Trypanosoma equinum*, Voges, 1901. Syn.: _Trypanosoma elmassiani_, Lignières. _Trypanosoma equinum_ was found by Elmassian to be the cause of the fatal disease, “mal de caderas,” of horses and dogs, in South America (Paraguay, Argentine, Bolivia). The name refers to the fact that in the disease, as in other trypanosomiases, the hind quarters become paralysed. Cattle are refractory to inoculation. [Illustration: FIG. 42.--_Trypanosoma equinum_. × 2,000. (After Laveran and Mesnil.)] _T. equinum_ is about 22 µ to 24 µ long and about 1·5 µ broad (fig. 42). Although this trypanosome is very active, yet it is characterized by the blepharoplast (kinetic nucleus) being very minute or even absent, as the granule sometimes seen may be the basal granule of the flagellum. The mode of transmission of _T. equinum_ is not known with absolute certainty. Migone has shown that the parasite causes a fatal disease in the large South American rodent, the capybara (_Hydrochœrus capybara_). This animal appears to be a reservoir of the parasite. Dogs may become infected by eating diseased capybaras, and it is suggested that the infection is spread from the dogs to horses by the agency of fleas. Some authorities consider that _T. equinum_ may be spread by various _Tabanidæ_ and by _Stomoxys_. Neiva (1913)[104] doubts all these modes of transmission in Brazil, and suggests _Chrysops_ or _Triatoma_ as vectors. [104] _Brazil Medico_, xxvii, p. 366. *Trypanosoma equiperdum*, Doflein, 1901. Syn.: _Trypanosoma rougeti_, Laveran and Mesnil. The malady of horses known as “dourine” or “mal du coït” is due to a trypanosome, _T. equiperdum_, discovered by Rouget in 1894. “Dourine”--also known as “stallion disease” or “covering disease”--is found among horses and asses in Europe, India, North Africa and North America. The trypanosome is transmitted by coitus, and so far as is known not by insect agency. [Illustration: FIG. 43.--_Trypanosoma equiperdum._ × 2000 approximately. (Original. From preparation by Fantham.)] The progress of the disease may be considered under three periods. The _period of œdema_, when signs of œdema of the genitalia are seen. The œdema is generally painless and non-inflammatory. This period lasts about a month. It is succeeded by the _period of eruption_, which sets in about two months after infection. Circular œdematous areas (“plaques”), often about the size of a two-shilling piece, appear under the skin of the sides and hind quarters, and also, at times, under the skin of the neck, thighs and shoulders. The eruption is variable, but usually lasts about a week and leaves the animal in an enfeebled condition. Gland enlargement and swelling of the joints and synovia also may occur. The third period of the disease is described as that _of anæmia and paralysis_. The animal becomes very anæmic, emaciation is marked, superficial non-healing abscesses often form, and conjunctivitis and ulcerative keratitis can occur. Paralysis ensues, and in from two to eighteen months the animal dies. In the acute form of the disease the animal may die after the first period from acute paralysis. It is difficult to find the trypanosomes in naturally infected animals, and they are best obtained from the plaques of the eruption. Apparently the parasite occurs more in the lymph than in the blood. Ruminants are said to be refractory to this trypanosome. _T. equiperdum_ is about 25 µ to 28 µ in length on an average, but varies from 16 µ to 35 µ. Its cytoplasm is relatively clear, and does not show chromatic granules (fig. 43). It is stated to be monomorphic. It has been shown recently by Blacklock and Yorke (1913)[105] that there is another trypanosome giving rise to dourine in horses. This trypanosome is dimorphic (resembling _T. pecaudi_ and _T. ugandæ_), and is named _T. equi_. Previously _T. equiperdum_ and _T. equi_ had been confused. [105] _Proc. Roy. Soc._, B, lxxxvii, p. 89. Uhlenhuth, Hübner and Worthe have demonstrated the presence of endotoxins in _T. equiperdum_. These endotoxins may be set free by trypanolysis. *Trypanosoma theileri*, Bruce, 1902. This parasite, 60 µ to 70 µ long, and 4 µ to 5 µ broad, is distinguished for its large size, though it is not so large as _T. ingens_ from Uganda oxen, whose length may be 72 µ to 122 µ, and breadth 7 µ to 10 µ. The posterior end of _T. theileri_ is drawn out. Small forms of the flagellate are known, 25 µ to 53 µ in length. Probably other forms of the parasite have the nucleus posterior, and these flagellates were formerly separated as _T. transvaaliense_ (Laveran, 1902). Myoneme fibrils may be seen on its body. The pathogenicity of this organism is doubtful, it was formerly thought to be the causal agent of “gall-sickness” in cattle in South Africa. _T. theileri_ also occurs in Togoland, German East Africa, and Transcaucasia. Allied or identical parasites occur in cattle in India. [Illustration: FIG. 44.--_Trypanosoma theileri._ × 2,000. (After Laveran and Mesnil.)] _Trypanosoma theileri_, specific to cattle, is perhaps transmitted by the fly _Hippobosca rufipes_ in South Africa. *Trypanosoma hippicum*, Darling, 1910. _Trypanosoma hippicum_ causes the disease of mules known as “murrina.”[106] It was found in mules imported to Panama from the United States. It can live in other equines. The parasite varies from 18 µ to 28 µ in length, and is from 1·5 µ to 3 µ broad. Its undulating membrane is little folded. The trypanosome has a noticeable blepharoplast. It can penetrate mucous membranes, and it is thought that the trypanosome may be transmitted during coitus. It may also be spread mechanically by species of _Musca_, _Sarcophaga_ and _Compsomyia_, sucking the wounds of infected animals and carrying over the trypanosomes to wounds on healthy ones. [106] _Bull. Soc. Path. Exot._, iii, p. 381. *Endotrypanum schaudinni*, Mesnil and Brimont, 1908. This organism was discovered in the blood of a sloth (_Cholœpus didactylus_), in South America (French Guiana).[107] It possesses special interest, in that the best known form of the organism is endoglobular, inhabiting the erythrocytes of the sloth. A free trypanosome in the same animal was considered to be different from the endoglobular form, which was somewhat like a peg-top, and possessed a short flagellum. Darling[108] (November, 1914) has seen the organism in Panama. He describes free crithidial forms in shed blood, but not in the blood-stream of the sloth. [107] _C. R. Soc. Biol._, lxv, p. 581. [108] _Journ. Med. Research_, xxxi, p. 195. *Trypanosoma boylei*, Lafont, 1912. This is a parasite of the Reduviid bug, _Conorhinus rubrofasciatus_. The insect attacks man in Mauritius, Réunion and other places. Lafont infected rats and mice by intraperitoneal injection with the gut-contents of infected bugs. Trypanosomes appeared in the mice. Other flagellate types were assumed by the parasites in the bug. MONOMORPHIC TRYPANOSOMES. A number of trypanosomes, characterized by relative uniformity in size and structure, may be considered under this heading. They occur in cattle, sheep, goats and horses in Africa, especially West Africa. Morphologically, they are characterized by the posterior (aflagellar) part of the body being swollen, while the anterior part narrows. The nucleus is central and situated at the commencement of the narrowing of the body. The blepharoplast is almost terminal, the undulating membrane is narrow and not markedly folded, so that the flagellar border lies close to or along the body. The flagellum may or may not possess a free portion. Some recent workers have considered that _T. brucei_ (Zululand strain) and _T. evansi_ are also monomorphic, but they do not exhibit the general characteristics outlined above. _T. brucei_ and _T. evansi_ have already been considered separately. The monomorphic trypanosomes, as defined above, include:-- *Trypanosoma vivax*, Ziemann, 1905. This trypanosome[109] occurs in cattle, sheep and goats, and was first found in the Cameroons. It is fatal to cattle. Equines are also affected. Antelopes are the possible reservoirs of the trypanosome. It is probably transmitted by _Glossina palpalis_ and other tsetse flies. Its movement is very active. It possesses a free flagellum (fig. 45) and it averages 23 µ to 24 µ in length. _T. cazalboui_ (Laveran, 1906)--the causal agent of “souma” in bovines and equines in the French Sudan--is probably synonymous with _T. vivax_. [109] See Bruce and colleagues (1910), _Proc. Roy. Soc._, B, lxxxiii, p. 15. [Illustration: FIG. 45.--_Trypanosoma vivax_. × 2,000. (Original. From preparation by Fantham.)] *Trypanosoma capræ* (Kleine, 1910) is allied, but is somewhat broader and more massive. It was found in goats in Tanganyika. *Trypanosoma congolense*, Broden, 1904. Probable synonyms.--_Trypanosoma dimorphon_, Laveran and Mesnil, 1904; _Trypanosoma nanum_, Laveran, 1905; _Trypanosoma pecorum_, Bruce, 1910; _Trypanosoma confusum_, Montgomery, 1909. This trypanosome causes disease among horses (_e.g._, Gambia horse sickness), cattle, sheep, goats, pigs, and dogs. It is widely distributed in Central Africa (_e.g._, Gambia, Congo, Uganda, Nyasaland), the strain probably being maintained naturally in big game. It is transmitted by various _Glossinæ_, and perhaps by _Tabanus_ and _Stomoxys_. It is said to develop in the gut and proboscis of _Glossina palpalis_ and _G. morsitans_. The trypanosome averages 13 µ to 14 µ in length and has no free flagellum (fig. 46). It is about 2 µ broad. Formerly _T. nanum_ and _T. pecorum_ were said to differ in their pathogenicity, the former being said not to infect the smaller laboratory animals. Yorke and Blacklock (1913), however, consider that the virulence varies and that these trypanosomes are probably the same. [Illustration: FIG. 46.--_Trypanosoma congolense_. × 2,000. (Original. From preparation by Fantham.)] [Illustration: FIG. 47.--_Trypanosoma uniforme_. × 2,000. (Original. From preparation by Fantham.)] The _T. dimorphon_ originally obtained by Dutton and Todd (1903) in Gambian horse sickness has been shown to be a mixture of _T. vivax_ and _T. congolense_. *Trypanosoma simiae* (_T. ignotum_) is like _T. congolense_. It averages 17·5 µ long. It is virulent to monkeys and pigs. *Trypanosoma uniforme*, Bruce, 1910. This trypanosome was found in oxen in Uganda.[110] It can be inoculated to oxen, goats and sheep, but is refractory to dogs, rats and guinea-pigs. It has been found in antelopes. It resembles _T. vivax_, but is smaller (fig. 47), averaging 16 µ in length. A free flagellum is present. It is transmitted by _Glossinæ_. [110] _Proc. Roy. Soc._, B, lxxxiii, p. 176. [Illustration: FIG. 48.--_Trypanosoma rotatorium_, from blood of a frog. × 1,400. (After Laveran and Mesnil.)] Many other trypanosomes occur in mammals, while birds, reptiles, amphibia (fig. 48) and fish also harbour them. The discussion of these forms does not come within the scope of the present work. They are dealt with in Laveran and Mesnil’s “Trypanosomes et Trypanosomiases,” 2nd edit., 1912. GENERAL NOTE ON DEVELOPMENT OF TRYPANOSOMES IN GLOSSINA. Before concluding the account of trypanosomes, it may be of interest to remark that several African trypanosomes develop in various species of _Glossina_, and are found in different parts of the alimentary tract and in the proboscis. Thus (_a_) _T. vivax_, _T. uniforme_ and _T. capræ_ develop in the fly’s proboscis (labial cavity and hypopharynx) only; (_b_) _T. congolense_, _T. simiæ_ and _T. pecaudi_ develop first in the gut of the fly and then pass forward to its proboscis; and (_c_) _T. gambiense_ and _T. rhodesiense_ develop first in the gut and later invade the salivary glands of the tsetse. The proboscis or the salivary glands in such cases are termed by Duke[111] the _anterior station_ of the trypanosome, wherein it completes its development. [111] _Repts. Sleeping Sickness Commission Roy. Soc._ (1913), xiii, p. 82. ADAPTATION OF TRYPANOSOMES. These flagellates may exhibit power of adaptation to changes of environment, such as those due to the administration of drugs, change of host, etc. A few examples of such mutations may be briefly considered:-- (1) _Blepharoplastless Trypanosomes_.--_T. brucei_ may become resistant to pyronin and oxazine. Accompanying this drug resistance is a change in morphology, namely, the loss of the blepharoplast (Werbitzki).[112] A race or strain of blepharoplastless trypanosomes may be thus produced which retains its characteristic feature after as many as 130 passages (Laveran).[113] Oxazine is the more powerful drug, and it acts directly on the blepharoplast. (Compare the natural blepharoplastless character of _T. equinum_.) [112] _Centralbl. f. Bakt._ (1910), Orig., liii, p. 303. [113] _Bull. Soc. Path. Exot._, iv, p. 233. (2) Reference has been made on p. 93 to the experiments of Gonder, who showed that a strain of _T. lewisi_ rendered resistant to arsenophenylglycin lost its resistance after passage through the rat louse. This is in marked contrast with the retention of drug resistance during passage by inoculation from rat to rat. (3) _T. lewisi_ from the blood of a rat when transferred to a snake seems largely to disappear, as very few flagellates are seen. When blood from the snake is inoculated into a clean rat, then trypanosomes reappear in the rat, but they are not all like those originally inoculated. It seems certain that, in such a case, changes in form and virulence of the trypanosome have occurred. Similar experiments were made with _T. brucei_ from rats to adders and other animals and back to rats. Changes in the form and virulence of _T. brucei_ occurred. These interesting experiments were performed by Wendelstadt and Fellmer.[114] [114] _Zeitschr. f. Immunitatsforschung_, iv, p. 422 (1909), and v, p. 337 (1910). Genus. *Herpetomonas*, Saville Kent, 1881. _Herpetomonas_ is a generic name for certain flagellates possessing a vermiform or snake-like body, a nucleus placed approximately centrally, and a blepharoplast (kinetic nucleus) near the flagellar end. There is no undulating membrane (fig. 49, _a_). The organisms included in this genus certainly possess one flagellum, while according to Prowazek (1904) _Herpetomonas muscæ-domesticæ_, the type species, possesses two flagella united by a membrane. Patton,[115] Porter[116] and others affirm, however, that the biflagellate character of _H. muscæ-domesticæ_ (from the gut of the house-fly) is merely due to precocious division. The matter is further complicated by the generic name _Leptomonas_, given by Kent in 1881, to an uniflagellate organism found by Bütschli in the intestine of the Nematode worm, _Trilobus gracilis_. This parasite, _Leptomonas bütschlii_, has not yet been completely studied. Until these controversial points relating to the identity or separation of _Herpetomonas_ and _Leptomonas_ have been satisfactorily settled, we may retain the better known name _Herpetomonas_ for such uniflagellate, vermiform organisms. However, the name _Leptomonas_, having been used by Kent two pages earlier in his book (“Manual of the Infusoria”) than _Herpetomonas_, would have priority if the two generic names were ultimately shown to be synonymous. [115] _Arch. f. Protist._, xiii, p. 1. [116] _Parasitology_, ii, p. 367. A full discussion of these interesting and important flagellates hardly comes within the purview of the present work; brief mention can only be given here to certain species. The Herpetomonads occur principally in the digestive tracts of insects, such as Diptera and Hemiptera. They are also known in the guts of fleas and lice, but are not confined to blood-sucking insects. One example, _H. ctenocephali_ (Fantham, 1912)[117] occurs in the digestive tracts of dog fleas, _Ctenocephalus canis_, in England, France, Germany, Italy, India, Tunis, etc. It is a natural flagellate of the flea, and might easily be confused with stages of blood parasites in the gut of the dog flea. Dog fleas are stated by Basile to transmit canine kala-azar, which is believed to be the same as human infantile kala-azar. Confusion is further likely to arise since herpetomonads pass through pre-flagellate, flagellate and post-flagellate or encysted stages; pre- and post-flagellate stages being oval or rounded and _Leishmania_-like. The post-flagellate stages are shed in the fæces, and are the cross-infective stages by means of which new hosts are infected by the mouth. The possible presence of such natural flagellates must always be considered when experimenting with fleas, lice, mosquitoes, etc., as possible vectors of pathogenic flagellates like _Leishmania_ and _Trypanosoma_. _H. pediculi_ (Fantham, 1912) occurs in human body lice.[118] See further remarks on pp. 107, 112. [117] _Bull. Path. Exot._, vi, p. 254. [118] _Proc. Roy. Soc._, B, lxxxiv, p. 505. [Illustration: FIG. 49.--_a_, _Herpetomonas_; _b_, _Crithidia_; _c_, _Trypanosoma_. (After Porter.)] Laveran and Franchini (1913–14)[119] have recently succeeded in inoculating _Herpetomonas ctenocephali_, from the gut of the dog flea, intraperitoneally into white mice, and producing an experimental leishmaniasis in the mice. A dog was also infected. They have also succeeded in infecting mice with _H. pattoni_--a natural flagellate of the rat flea--by mixing infected rat fleas with the food of the mice, and by causing them to ingest infected fæces of rat fleas. Further, they have shown that infection with the herpetomonas occurs naturally by this method, that is, by the rodents eating the fleas and not by the insects inoculating the flagellates into the vertebrates when sucking blood. These experiments shed an interesting light on the probable origin of _Leishmania_ and its cultural herpetomonad stage, which were very probably once parasitic flagellates in the gut of an insect. [119] _C. R. Acad. Sci._, clvii, pp. 423, 744. _Ibid._, clviii, pp. 450, 770. _Bull. Soc. Path. Exot._, vii, 605. Fantham and Porter[120] (1914–15) have shown that young mice may be inoculated or fed with _Herpetomonas jaculum_, from the gut of the Hemipteran, _Nepa cinerea_ (the so-called “water-scorpion”), with fatal results. The pathogenic effects are like those of kala-azar. They also showed that the post-flagellate stages of the herpetomonads seemed most capable of developing in the vertebrate. [120] _Proc. Camb. Philosoph. Soc._, xviii, p. 39. A herpetomonad, _H. davidi_, has been found in the latex of species of the plant-genus _Euphorbia_ in Mauritius, India, Portugal, etc. It is apparently transmitted to the plants by _Hemiptera_. The plants sometimes suffer from “flagellosis.” Franchini (1913)[121] has described a new parasite, _Hæmocystozoon brasiliense_, from the blood of a man who had lived in Brazil for many years. It possesses flagellate and rounded stages, and is closely allied to the herpetomonads. [121] _Bull. Soc. Path. Exot._, vi, pp. 156, 333, 377. Genus. *Crithidia*, Léger, 1902, emend. Patton, 1908. _Crithidia_ is the generic name of vermiform flagellates with a central nucleus, a blepharoplast or kinetic nucleus in the neighbourhood of the principal nucleus, and a rudimentary undulating membrane bordered by a flagellum arising from a basal granule, which is the centrosome of the kinetic nucleus (fig. 49_b_). The anterior or flagellar end of the body is attenuated and fades off as the undulating membrane. _Crithidia fasciculata_, the type species, was found by Léger in the alimentary canal of _Anopheles maculipennis_. Crithidia occur in bugs, flies, fleas,[122] and ticks. Some of them are found in the body-fluid of the invertebrate host as well as in the gut. Others may be restricted to the body cavity or intestine respectively. _C. melophagia_ from the sheep-ked, _Melophagus ovinus_, and _C. hyalommæ_ from the hæmocœlic fluid of the tick, _Hyalomma ægyptium_, pass into the ovaries and eggs of their hosts, and the young keds or ticks are born infected. [122] See Porter, _Parasitology_, iv, p. 237. _C. fasciculata_ has been shown by Laveran and Franchini to be inoculable into white mice, producing a sort of experimental leishmaniasis therein. In one case cutaneous lesions were produced like those of Oriental sore. Crithidia are natural flagellates of Arthropoda, with their own pre-flagellate, flagellate and post-flagellate stages, and must not be confused with transitory crithidial stages of trypanosomes. Genus. *Leishmania*, Ross, 1903. With an oval body containing nucleus and blepharoplast (kinetic nucleus) but no flagellum. An intracellular parasite in the vertebrate host. Included in the genus _Leishmania_ are three species, namely:-- (1) _Leishmania donovani_, Laveran and Mesnil, 1903, the parasite of Indian kala-azar, a generalized systemic disease, usually fatal, occurring in subjects of all ages. (2) _Leishmania tropica_, Wright, 1903, the parasite of Delhi boil, Oriental sore, Aleppo button--a localized, cutaneous disease, usually benign. (3) _Leishmania infantum_, Nicolle, 1908, the parasite of infantile kala-azar, occurring in children (and a few adults) around the shores of the Mediterranean. The disease is perhaps a form of Indian kala-azar, and the parasite is probably identical with _L. donovani_. These diseases may be termed collectively leishmaniases. The morphology of the various species is practically identical. *Leishmania donovani*, Laveran and Mesnil, 1903. Syn.: _Piroplasma donovani_, Laveran and Mesnil. The parasite of Indian kala-azar was demonstrated in 1900 by Leishman from a _post-mortem_ examination of a case of “Dum-Dum fever,” but details were not published till May, 1903. In July, 1903, Donovan found similar bodies from cases in Madras. Rogers succeeded in cultivating the parasite in July, 1904.[123] The original centre of the disease was probably Assam; it occurs also in Madras, Ceylon, Burma, Indo-China, China and Syria. A variety of this leishmaniasis is found in the Sudan. The patient becomes emaciated, with a greatly enlarged spleen. There is anæmia and leucopenia. [123] The literature up to 1912, on kala-azar and other leishmaniases is reviewed in the _Kala-azar Bulletin_. Afterwards in the _Tropical Diseases Bulletin_. The parasite, commonly known as the Leishman-Donovan body, is intracellular (fig. 50, 2, 3). It is found in the endothelial cells of the capillaries of the liver, spleen, bone-marrow, lymphatic glands and intestinal mucosa, and in the macrophages of the spleen and bone-marrow. Some host cells may contain many parasites. It is rather rare in the circulating blood, but may be found in the blood from the femoral, portal and hepatic veins. It does not occur in the red blood corpuscles as was formerly thought. The parasites liberated from the endothelial cells are taken up by the mononuclear and polymorphonuclear leucocytes. The Leishman-Donovan body is the resting stage of a flagellate. As found in man it is a small, oval organism, about 2·5 µ to 3·5 µ in length by 2 µ in breadth, and containing two chromatinic bodies, corresponding to the nucleus and kinetic nucleus (blepharoplast) of a flagellate. The latter element is the smaller and more deeply staining, and is usually placed at the periphery, transversely to the longer axis of the oval organism. There is sometimes a very short, slightly curved filament to be seen, which may be a rhizoplast. Multiplication takes place by binary or multiple fission. The presence of the parasite used to be demonstrated by splenic or hepatic puncture; nowadays it can be demonstrated in peripheral blood, _e.g._, of the finger, or by culture of infected blood. [Illustration: FIG. 50.--_Leishmania donovani_. _1_, Free forms, each with nucleus and rod-shaped blepharoplast (after Christophers); _2_, endothelial cell and leucocytes containing parasites (after Christophers); _3_, capillary in the liver showing endothelial cells containing parasites (after Christophers); _4_, two parasites escaping from a leucocyte in the alimentary canal of the bug (after Patton); _5_, further development in bug (after Patton); _6_, young flagellate forms in bug (after Patton); _7_-_11_, culture forms (after Leishman); _7_, _8_, _9_, show development of flagellum.] _L. donovani_ can be cultivated in citrated splenic blood, under aerobic conditions, at 22° to 25° C. This was first accomplished by Rogers (1904). It is not so easily culturable as _L. infantum_ on the Novy-MacNeal-Nicolle medium.[124] _L. donovani_ is inoculable with some difficulty into experimental animals--in India, white rats, white mice, dogs and monkeys (_Macacus spp._), have been inoculated. The Sudan variety, somewhat less virulent, is inoculable to monkeys. Row also produced a local lesion in _Macacus sinicus_ by subcutaneous inoculation of _L. donovani_. Parasites taken from such a local lesion were found to be capable of producing a generalised infection in _Macacus sinicus_ and white mice. [124] For the composition of this medium, see Appendix. In cultures the various species of _Leishmania_ all grow into herpetomonad, uniflagellate organisms (fig. 50, 10), about 12 µ to 20 µ in body length. On this account Rogers[125] and Patton place the Leishman-Donovan body within the genus _Herpetomonas_. The method of culture may be used in diagnosing leishmaniases. [125] _Proc. Roy. Soc._, B, lxxvii, p. 284. Kala-azar is very probably an insect-borne disease. Patton[126] suspects the bed-bug to be the transmitter and finds (fig. 50, _4_-_6_) that the Leishman-Donovan body can develop into the flagellate stage in the digestive tract of the bed-bug. Feeding experiments are unsatisfactory, since there are very few cases in which the parasites occur in sufficient numbers in the peripheral blood to make the infection of the insect possible, or at any rate easy. In examining the alimentary tracts of insects for possible flagellate stages of _Leishmania_, it must be remembered that in many insects natural flagellate parasites, belonging to the genus _Herpetomonas_, may occur therein; such natural insect flagellates may be harmless, and have no connection with the life-cycle of _L. donovani_. Natural herpetomonads are known to occur in the alimentary tracts of flies, mosquitoes, sand-flies, fleas and lice, but not in bed-bugs. Further, if such flagellates are able to be inoculated into and live within vertebrate hosts, producing symptoms like those of leishmaniasis, the origin of kala-azar is indicated (see pp. 104, 112). [126] _Sci. Mem. Govt. India_, Nos. 27, 31 (1907–08). *Leishmania tropica*, Wright, 1903. Syn.: _Helcosoma tropicum_, Wright, 1903; _L. wrighti_, Nicolle, 1908; _Ovoplasma orientale_, Marzinowsky and Bogrow. It is believed by some that the parasite was first described by Cunningham in 1885, and studied by Firth in 1891, being called by him _Sporozoon furunculosum_. If these earlier studies were of the parasite, then its correct name is _L. furunculosa_, Firth, 1891. The benign disease produced by this parasite has received many names, among the best known being Oriental sore, Tropical sore, Delhi boil and Aleppo button. These names, however, are not happy ones, as cutaneous leishmaniasis (_e.g._, on the ear) is now known to occur in the New World, for example in Mexico, Venezuela, Brazil and neighbouring States. However, it may be necessary to subdivide cutaneous leishmaniases later. In the Old World the disease occurs in India, Persia, Arabia and Transcaucasia. It is also known in Algeria, Northern Nigeria, Egypt, Sudan, Crete, Calabria, Sicily and Greece. The boils often occur on the face, and before ulceration the parasites may be found in the cells at the margin and floor of the “button.” In searching for parasites the scab should be removed and scrapings made from the floor and edges. Where lesions occur atrophy of the epidermis takes place, and infiltration of mononuclear cells (_e.g._, plasma cells, lymphoid and endothelial cells) follows. The parasites are intracellular, being found inside mononuclear cells. In non-ulcerating sores, Cardamitis found some free parasites. Non-ulcerating forms are said to occur in the Sudan. In the Old World the sores are often limited to exposed surfaces of the body. Infection of mucous membranes (such as the lip, palate, buccal and nasal membranes) may occur, especially in South America, and are often known there as “Espundia.” Christopherson (1914) has recorded a case in Khartoum. _Leishmania tropica_ is equally well cultivated on Novy-MacNeal-Nicolle medium or on citrated blood. The usual temperature for cultivation is 22° to 28° C., though Marzinowski claims to have cultivated the parasite at 37° C. _L. tropica_ can be inoculated into monkeys and dogs, with the production of local lesions. Material from a human sore or flagellates from a culture may be thus successfully inoculated. Also infected material may be rubbed directly into a scarified surface. The incubation period is long, extending over several months. The duration of the disease may be from twelve to eighteen months. Recovery from one attack of tropical sore confers immunity, and the Jews in Bagdad inoculate their children with the disease on a part of the body which will be covered, and so secure immunity in adult life. The mode of transmission of _L. tropica_ is unknown. Wenyon (1911)[127] has found that the parasite develops into the flagellate stage in the digestive tract of _Stegomyia fasciata_ in Bagdad. Patton (1912)[128] has found similar development in the bed-bug in Cambay. The house-fly, _Phlebotomus_ and _Simulium_ have been suspected as transmitters in different parts of the world. [127] _Parasitology_, iv, p. 387. [128] _Sci. Mem. Govt. India_, No. 50. An interesting announcement has been made recently (May, 1913), that Neligan has found that _L. tropica_ occurs in dogs in Teheran, Persia, producing ulcers on the dogs’ faces (_cf._ natural occurrence of _L. infantum_ in dogs--see p. 110). Yakimoff and Schokhor (1914),[129] have found the disease in dogs in Tashkent. [129] _Bull. Soc. Path. Exot._, vii, p. 186. Gonder[130] (1913) has performed some interesting experiments showing the relation of infantile kala-azar to Oriental sore. Gonder infected mice with _L. infantum_ and with _L. tropica_. He used culture material and injected intraperitoneally or intravenously. In each a general infection resulted, with enlargement of the liver and spleen. Later, however, mice injected with Oriental sore (North African variety) developed peripheral lesions on the feet, tail and head, and the lesions contained _Leishmania_. No such peripheral lesions developed in the case of the mice infected with the kala-azar virus. Gonder suggested that Oriental sore, like kala-azar, is really a general infection overlooked in its earlier stages, and that it is in the later stages that peripheral lesions on the skin are developed. Row (1914)[131] also obtained a general infection in a mouse by the injection of cultures of _L. tropica_ from Oriental sore of Cambay. [130] _Arch. f. Schiffs- u. Trop. Hyg._, xvii, p. 397. [131] _Bull. Soc. Path. Exot._, vii, p. 272. *Leishmania infantum*, Nicolle, 1908.[132] Infantile splenic anæmia has been long known in Italy. It also occurs in Algeria, Tunis, Tripoli, Syria, Greece, Turkey, Crete, Sicily, Malta,[133] Spain and Portugal. This leishmaniasis is, then, distributed along the Mediterranean littoral; also in Russia. Cathoire (1904) in Tunis and Pianese (1905) in Italy were among the first to see the parasite. Nicolle then found the parasite in patients in Tunis, and further found spontaneous infection in dogs. The patients are usually children between the ages of 2 and 5 years. There are a few cases known in which the infantile type of leishmaniasis occurred in youths and adults of the ages of 17 to 19, while one patient in Calabria was 38 years old. The symptoms are like those of Indian kala-azar. Several Italian investigators and others consider that _L. infantum_ is the same as _L. donovani_, and that the latter name should be used for the parasite of Mediterranean leishmaniasis. This view, as to the identity of _L. donovani_ and _L. infantum_, seems coming into general favour. [132] _Arch. Inst. Pasteur Tunis_, i, p. 26. [133] _See_ Wenyon (1914), _Trans. Soc. Trop. Med. and Hyg._, vii, p. 97; also Critien (1911), _Annals Trop. Med. and Parasitol._, v, p. 37. There are, however, differences between the Indian and infantile kala-azars, in addition to the ages of the patients affected, thus: (_a_) As regards cultures, it is found that _L. infantum_ is readily grown on the Novy-MacNeal-Nicolle (“N.N.N.”) medium (saline blood-agar), and that sub-cultures are easily obtained; in citrated blood _L. infantum_ grows with difficulty. The reverse is the case with regard to culture media for _L. donovani_, which grows with difficulty on the N.N.N. medium, but relatively easily in citrated splenic blood. (_b_) Considering inoculability into experimental animals, it is found that _L. donovani_ is inoculated generally with some difficulty into white rats, white mice and monkeys, and with greater difficulty into dogs, while _L. infantum_ can be inoculated into several experimental animals, especially into dogs and monkeys, with ease. (_c_) At present _L. donovani_ is not known to occur spontaneously in animals, but _L. infantum_ is found naturally in dogs in the Mediterranean region, and the disease in dogs is often referred to as canine kala-azar. Kittens have occasionally been found infected. However, these differences must not be emphasized too much. The material for cultivation is obtained from punctures of spleen, liver or bone-marrow of cases infected with _L. infantum_. It is not always easy, however, to infect from cultures, as the cultural flagellates inoculated into the body are often phagocytosed. Similarly, the material for animal inoculation is obtained from emulsions of infected spleen, liver or bone-marrow. Dogs and monkeys are easily inoculated with such material; Nicolle inoculates into the liver or the peritoneal cavity. Mice, white rats, guinea-pigs and rabbits only show slight infections after such inoculations. Dogs infected experimentally with infantile leishmaniasis may show either acute or chronic symptoms. The acute course occurs more often in young dogs, and is usually fatal in three to five months. The chronic course is found more commonly in older dogs, and may last seventeen to eighteen months. In acute forms there is irregular fever, progressive wasting, diarrhœa occasionally, motor disturbances involving the hind quarters, and the animal dies in a comatose condition. In the chronic form the animal may appear well, except for loss of weight. The parasites may be found in the internal organs of these experimental dogs, but are not numerous in the peripheral blood except at times of high fever. Experimental monkeys live about three months. It may be interesting to record the number of dogs found to be infected naturally with leishmaniasis in various countries. In Tunis, Nicolle and Yakimoff found about 2 per cent. infected out of about 500 dogs examined. Sergent in Algiers found 9 infected out of 125 dogs examined. In Italy and Sicily, Basile found about 40 per cent. of the dogs to be infected out of 93 examined at Rome and Bordonaro. Cardamitis found 15 infected out of 184 examined in Athens. In Malta, Critien found 3 infected out of 30 dogs examined. Alvares found 1 infected dog out of 19 examined in Lisbon. Pringault has recently (December, 1913) found an infected dog in Marseilles.[134] Yakimoff and Schokhor found 24 per cent. infected out of 647 dogs examined in Turkestan. [134] _Bull. Soc. Path. Exot._, vii, p. 41. The distribution of the parasites in the body of the human patient is much the same as in the case of Indian kala-azar. Critien records the finding of parasites in the mucous flakes of the stools of a three-year-old Maltese child.[135] Intestinal lesions rarely occur in infantile leishmaniasis. [135] Quoted by Leishman (1911) in his interesting review of Leishmaniasis, _Journ. Roy. Army Med. Corps_, xvii, p. 567, xviii, pp. 1, 125. Also _Quart. Journ. Med._ v, pp. 109–152. _Ætiology._--Infantile leishmaniasis is stated to be transmitted by fleas, especially dog fleas, _Ctenocephalus canis_ (= _Pulex serraticeps_), and by _Pulex irritans_. Children living in contact with infected dogs may be bitten by infected dog fleas, and so contract the disease. Basile (1910–11) and Sangiorgi (1910) state that they found _L. infantum_ parasites in the digestive tract of the dog flea. After searching they found infected dog fleas on the beds, mattresses, and pillows used by children suffering from the disease. Franchini (1912) thinks that _Anopheles maculipennis_ may be concerned in the transmission. Basile[136] tried a number of experiments to show that infantile leishmaniasis is transmitted by fleas, thus:-- [136] Numerous papers in _Rendiconti R. Accad. dei Lincei_ (Rome), xix, xx (1910–11). (1) Fleas were taken from a healthy dog. They were placed in vessels containing infected spleen-pulp and allowed to feed thereon. The fleas were then killed and dissected, and portions of the gut-contents examined for parasites. The remainder of the gut was emulsified and injected into a young puppy, whose bone-marrow had been shown previously to be uninfected. Basile states that the puppy became infected. The parasites are said to increase in number in the flea’s gut. (2) Two healthy pups, each a month old, and born in the laboratory, were placed in a disinfected, flea-proof cage. A few days after, an infected dog was placed in the cage, so that fleas from the infected dog could pass on to the puppies. A month later the two pups became infected, parasites being found in them after liver puncture. A number of control puppies from the same litter remained uninfected and in good health. (3) Basile next used other laboratory-born puppies, a month old. Four of the litter were placed in a disinfected, flea-proof gauze cage in Rome. The cage was isolated from other dogs. Fleas obtained from an infected area in Sicily were placed in the cage. The puppies were examined by hepatic puncture, but were found to be negative for two months. Then two of the puppies showed infection, and six days later the remaining two puppies were found to be infected, and all four died. They showed irregular temperatures, and were getting thin. Control puppies remained healthy. From these experiments Basile concludes that fleas transmit leishmaniasis. However, Basile did not exclude the possible occurrence of natural herpetomonads in the gut of the fleas.[137] _Herpetomonas ctenocephali_ is known to occur in the gut of _Ctenocephalus canis_. A natural _Herpetomonas_ is also known in the gut of _Pulex irritans_, as well as a _Crithidia_ (_C. pulicis_, Porter). These natural flagellates of the fleas pass through non-flagellate stages, like the Leishman-Donovan body. In consequence Wenyon and Patton, among others, have criticized Basile’s results. Further, other investigators, such as Wenyon and Da Silva (1913), have repeated Basile’s flea experiments and been unable to confirm them. [137] See Fantham, _Brit. Med. Journ._, 1912, ii, p. 1196. In feeding and inoculation experiments the incubation period of the parasite may be long, and so it is necessary to wait a long time to see whether the parasite will develop. _Immunity._--Nicolle has tried some experiments with _L. infantum_ and _L. tropica_. He finds that in animals recovery from an attack of the former confers immunity against infection by the latter and vice-versâ. Laveran[138] records that a monkey having an immunity against _L. infantum_ was also immune to _L. donovani_. [138] _Annales Inst. Pasteur_ (1914–15), xxviii, pp. 823, 885; xxix, pp. 1, 71. As mentioned on p. 103, Laveran and Franchini (1913), working in Paris, have succeeded in inoculating _Herpetomonas ctenocephali_, a natural flagellate in the gut of the flea, _Ctenocephalus canis_, into white mice. Leishmaniform stages of the flea flagellate were recovered from the peritoneal exudate, blood and organs of the mice some weeks after inoculation. The parasites may also be conveyed by way of the digestive tract of the vertebrate. Similar experiments have succeeded with _H. pattoni_. These experiments go to show, together with those of Fantham and Porter with _H. jaculum_ (see p. 104), that, in the words of the latter authors, “it may be expected that the various leishmaniases, occurring in different parts of the world, will prove to be insect-borne herpetomoniases.” Genus. *Histoplasma*, Darling, 1906. Under the name _Histoplasma capsulatum_,[139] Darling described small round or oval parasites, enclosed in a refractile capsule, and each containing a single nucleus. The bodies were found in cases of splenomegaly in Panama. They occurred in the endothelial cells of the small blood-vessels of the liver, spleen, lungs, intestine and lymphatic glands, and also within the leucocytes. A few flagellates were stated to occur in the lungs. The parasite has usually been placed near _Leishmania_, but recently Rocha-Lima has stated that _Histoplasma_ is a yeast. [139] _Journ. Amer. Med. Assoc._, xlvi, p. 1283: _Journ. Exptl. Med._ (1909), xi, p. 515. Genus. *Toxoplasma*, Nicolle and Manceaux, 1908. The genus was created for crescentic, oval or reniform parasites, 2·5 µ to 6 µ by 2 µ to 3 µ, possessing a single nucleus and multiplying by binary fission. They occur in mononuclear and polymorphonuclear cells in the blood, spleen, liver, peritoneum etc. (fig. 51). The parasites have been found in the gondi, dog, rabbit, mole, mouse, pigeon and other birds. Although various species names have been given to the parasites in these hosts, it seems probable, from cross infection experiments, that there is but one species with several physiological races. Splendore[140] (1913) has described a flagellate stage. [140] _Bull. Soc. Path. Exot._, vi, p. 318. [Illustration: FIG. 51.--_Toxoplasma gondii_, endocellular or free in the peritoneal exudate of infected mice. 1, 2, mononuclear leucocytes containing toxoplasms. 3, polynuclear, containing parasites. 4, 5, 6, endothelial cells containing toxoplasms, agglomerated in 6. 7, agglomeration forms. 8–11, free forms. 12–13, division stages. × 1,600. (After Laveran and Marullaz.)] [Illustration: FIG. 52.--_Toxoplasma pyrogenes._ 1, body found in blood. 2–7, bodies found] in spleen. [1 is about the size of a red blood corpuscle, as drawn in the figures]. Magnification not stated. (After Castellani.)] Castellani (1913–14)[141] has described similar parasites from a case of splenomegaly, with fever of long standing, in a Sinhalese boy. The bodies were found in the spleen and more rarely in the blood (fig. 52). Castellani has named them _Toxoplasma pyrogenes_. Further researches are needed. [141] _Journ. Trop. Med. and Hyg._, xvii, p. 113. THE SPIROCHÆTES. The Spirochætes are long, narrow, wavy, thread-like organisms, with a firm yet flexible outer covering or periplast. There is a diffuse nucleus internally in the form of bars or rodlets of chromatin distributed along the body. In some forms there is a membrane or crista present (fig. 53), which in the past was compared with the undulating membrane of a trypanosome, but the membrane of a spirochæte does not undulate. Progression is very rapid, corkscrew-like and undulatory movements occurring simultaneously. The genus _Spirochæta_ was founded by Ehrenberg in 1833 for an organism which he discovered in stagnant water in Berlin. Ehrenberg named the organism _Spirochæta plicatilis_. According to Zuelzer (1912) _S. plicatilis_ does not possess a membrane or crista, but an axial filament. _S. gigantea_ has been described by Warming from sea-water. Spirochætes occur in the crystalline style and digestive tract of many bivalve molluscs. The first molluscan spirochæte to be studied was that of the oyster, named by Certes (1882) “_Trypanosoma_” _balbianii_ (fig. 53). Similar spirochætes, probably belonging to the same species, occur in various species of _Tapes_ and in _Pecten_ (the scallop). _S. balbianii_ has rounded ends (fig. 53). Other spirochætes occur in freshwater mussels (_Anodonta_ spp). _S. anodontæ_, studied by Keysselitz (1906) and by Fantham (1907), has pointed ends. Gross (1911) suggested the generic name _Cristispira_ for molluscan spirochætes, because they possess a well-marked membrane or “crista,” which appears to be absent from _S. plicatilis_, according to Zuelzer’s researches. [Illustration: FIG. 53.--_Spirochæta balbianii._ _a_, basal granule or polar cap. _b_, chromatin rodlets. _c_, membrane (“crista”). _d_, myonemes in membrane. (After Fantham and Porter.)] Schaudinn in 1905 founded the genus _Treponema_ for the parasite of syphilis (_T. pallidum_), discovered by him and by Hoffmann. According to Schaudinn the Treponemata have no membrane or crista. The pathogenic agent of yaws or frambœsia, discovered by Castellani, is also placed in the genus _Treponema_, as _T. pertenue_. There remain the blood spirochætes. It is somewhat disputed as to whether these organisms possess a membrane. The present writer considers that they have a slight membrane or crista. The name of the genus in which to place the blood-inhabiting forms is somewhat uncertain and disputed. Various generic names given to them are _Spirochæta_, _Treponema_, _Spiroschaudinnia_ (Sambon) and _Borrelia_ (Swellengrebel). Included in this division are the causal agents of relapsing or recurrent fever. These Protists will be named, for description, Spirochætes without prejudice as to the ultimate correct generic name. It is sometimes made a matter of argument as to whether the spirochætes are Protozoa or Bacteria. Such arguments are somewhat unprofitable. Morphologically the spirochætes are like the Bacteria in possessing a diffuse nucleus. They differ from _Spirillum_, an undoubted bacterial genus, in being flexible and not possessing flagella. Molluscan spirochætes, however, may appear to have flagella if their membrane becomes frayed or ruptured, when the myonemes therein (fig. 53), becoming separated, form apparent threads or flagella (Fantham, 1907–08).[142] [142] _Quart. Journ. Microsc. Sci._, lii, p. 1. Again, the mode of division of spirochætes has been used as a criterion of their bacterial or protozoal affinity. They have been stated to divide transversely, longitudinally, and by “incurvation,” or bending on themselves in the form of a *U*, “a form of transverse fission.” The present writer believes that they divide both transversely and longitudinally, and that there is a periodicity in their mode of division at first longitudinal (when there are few spirochætes in, say, the blood) and then transversely (when spirochætes are numerous in the blood).[143] Some authors consider that longitudinal division is explained by “incurvation.” [143] _Proc. Roy. Soc._, B, lxxxi, p. 500. The spirochætes of relapsing fever show a remarkable periodic increase and decrease in numbers in the blood. They are transmitted by ticks or by lice. They react to drugs (_e.g._, salvarsan or “606”) rather like trypanosomes, and--like Protozoa, but unlike Bacteria--they are cultivated with difficulty. These and other criteria have been used to endeavour to determine whether they are Protozoa or Bacteria. The present writer believes that they are intermediate in character, showing morphological affinities with the Bacteria and physiological and therapeutical affinities with the Protozoa. The group Spirochætacea, as an appendix to the Protozoa, has been created for them by the present writer (Jan., 1908). Others have placed them in the Spirochætoidea of the Bacteria or with the Spirillacea. Doflein (1909) called them Proflagellata. Further discussion is unnecessary, as they are undoubtedly Protista (see p. 29). There is no true conjugation, sex or encystment in spirochætes, but morphological variation may occur.[144] They may agglomerate. [144] Fantham, _Parasitology_, ii, p. 392. The Spirochætes form an interesting chapter in the evolution of parasites. There are free living forms, parasitic forms in the guts of both vertebrates and invertebrates, and blood-inhabiting forms. These probably represent the order of evolution of parasitism. The blood-inhabiting forms are pathogenic to warm-blooded hosts. We must now consider the blood Spirochætes and the Treponemata (organisms of syphilis and of yaws). THE SPIROCHÆTES OF THE BLOOD. There are at least two important human parasites included hereunder:-- (_a_) _Spirochæta recurrentis_ (=_S. obermeieri_), (_b_) _Spirochæta duttoni_. More is known of the life-cycle of _Spirochæta duttoni_, and it will be convenient to consider that first. *Spirochæta duttoni*, Novy and Knapp, 1906. The specific name _duttoni_ was also given, independently, to this parasite in 1906 by Breinl and Kinghorn. _S. duttoni_ is the pathogenic agent of African tick fever in man, prevalent in the Congo State and other parts of Africa. The full-grown organism is about 16 µ to 24 µ long, and has pointed ends. It is 0·25 µ to 0·5 µ broad. P. H. Ross and Nabarro were among the earliest to see a spirochæte in the blood of patients in Uganda. It is transmitted by the tick, _Ornithodorus moubata_. In the blood of the patient some of the spirochætes may show, after staining, lighter and darker portions (chromatin dots) and evidence of the possession of a very narrow membrane (fig. 54). The mode of division has already been discussed. Periodicity in the direction of division was first described by Fantham and Porter,[145] (1909). Just before the crisis in African tick fever, Breinl has stated that _S. duttoni_ becomes thinner in the spleen and bone-marrow and rolls up into skein-like forms, which are surrounded by a thin “cyst” wall (probably the periplast). Such occur in apyrexial periods. Inside the cyst the spirochæte breaks up into granules. Balfour and Sambon have described somewhat similar rolled up forms, breaking into granules, inside the red blood cells of Sudanese fowls in the case of _S. granulosa_ (possibly only a variety of _S. gallinarum_). The intracorpuscular stage is not definitely established. [145] _Proc. Roy. Soc._, B, lxxxi, p. 500. The granule phase, however, is an essential one in the invertebrate transmitter (fig. 54_c_). In 1905,[146] Dutton and Todd proved experimentally that _O. moubata_ transmitted _S. duttoni_. They fed ticks, obtained from Congo native huts in which infected persons lived, on monkeys and the latter became infected. Dutton and Todd also found the offspring of infected ticks to be capable of transmitting the infection to experimental animals. They concluded that _O. moubata_ was a true intermediate host. [146] _Liverpool Sch. Trop. Med._, _Memoir_ xvii; _Lancet_, Nov. 30, 1907, p. 1523. [Illustration: FIG. 54.--_Spirochæta duttoni_. _a_, blood form showing slight membrane; _b_, granules or coccoid bodies clearly formed within the organism; _c_, beginning of extrusion of coccoid bodies in the tick. (After Fantham.)] A little later in 1905, Koch stated that spirochætes from the gut of the tick penetrated the gut wall and tissues and found their way into the eggs in the ovary. Koch figured tangled masses of spirochætes as occurring in the tick eggs. He found ticks infective to the third generation. He thought that the infection was spread by the salivary fluid of the tick, in the act of biting. (This is now known to be incorrect.) Markham Carter (1907) corroborated Koch’s work on the spirochætes in the tick eggs, and they have been seen since by Kleine and Eckard (1913). Sir William Leishman,[147] in 1909–10, found that at ordinary temperatures the salivary glands of infected ticks (_O. moubata_) were not themselves infective, and that the infection occurred by way of the ticks’ excretion. The spirochætes (contained in the ticks’ excrement) found their way into the vertebrate host through the wound made by biting. While feeding, ticks pass large quantities of clear fluid from the coxal glands; in this fluid an anticoagulin occurs. Some of the ticks also pass thick, white Malpighian secretion, that is, excrement, towards the end of the feed. Leishman, using experimental monkeys, showed that if infected ticks were interrupted while feeding, then no infection resulted in the monkeys. If, however, the ticks were allowed to finish their feed, and the Malpighian secretions were passed, then the experimental monkeys became infected. Fantham[148] and Hindle[149] (1911), independently, have repeated the experiments with mice. [147] _Journ. Roy. Army Med. Corps_, xii, p. 123; _Lancet_ (1910), clxxviii, p. 11. [148] _Annals Trop. Med. and Parasitol._, v, p. 479. [149] _Parasitology_, iv, p. 133. Leishman’s methods and results may be summarized thus: Saline emulsions of the organs of infected ticks were made, after the organs had been most carefully dissected out. The ticks were first kept for several days at certain constant temperatures, such as 24° to 25° C. or blood heat, 37° C. The saline emulsions of the organs were inoculated, separately, into experimental animals, and the results recorded:-- At 24° C. At 37° C. Salivary glands Negative Positive Malpighian tubules Positive Positive Gut and contents Positive Positive Excrement Positive Positive Genital organs Positive Positive Coxal fluid is usually negative; thick, white excrement from Malpighian tubes is positive. When the ticks were incubated at 21° to 24° C. no spirochætes, as such, were seen in the organs, except perhaps in the gut, where they often disappeared in a few days. When the ticks were previously incubated at 35° to 37° C. for two to three days, spirochætes, as such, reappear in the gut, organs and hæmocœlic fluid. The infection proceeds, not from the salivary gland, but from the infective excrement, that is, from the thick, white material voided by the tick while feeding, usually towards the end of the meal. This Malpighian excrement passes into the wound caused by the bite, being greatly aided by the clear and more limpid coxa fluid, which bathes the under surface of the tick’s body, and mixes with and carries the infective excrement into the wound. Ticks remain infective for a long time. [Illustration: FIG. 55.--_Spirochæta duttoni_ and its coccoid bodies in the tick (_O. moubata_).--Mononuclear cells of the tick (_O. moubata_) containing (_a_) Spirochæte breaking up into coccoid bodies; (_b_) Similar tick-cell containing coccoid bodies or granules. Such mononuclear cells occur in various organs of ticks and in developing Malpighian tubules. (Original. From preparations by Fantham.)] The spirochætes in the gut of infected ticks divide by a process of multiple transverse fission into granules, which are composed of chromatin (fig. 54). These granules--sometimes known as coccoid bodies--are capable of multiplication. Leishman first found them in clumps inside the cells of the Malpighian tubules (_cf._ fig. 55). To summarize, when spirochætes are ingested by a tick, some of them pass through the gut-wall into the hæmocœlic (body) fluid. They then bore their way into the cells of various organs (fig. 55_a_) and break up into coccoid bodies. In this manner the granules find their way into the ovaries and ova, thus explaining how the young ticks are born infected. Inoculation of these chromatinic granules usually produces infection. Infective granules are also seen in the rudiments of the Malpighian tubules of embryo ticks. Bosanquet and Fantham (1911), independently, have shown that molluscan spirochætes also break up into similar granules or coccoid bodies. Gross has also demonstrated multiple transverse fission in molluscan forms. Marchoux and Couvy (1913) and Wolbach (1914) consider the granules or coccoid bodies to be degeneration products. This is unlikely (see below). Schuberg and Manteufel have found that certain _O. moubata_, perhaps 30 per cent. of the specimens of a given neighbourhood, may acquire a natural active immunity against infection with _S. duttoni_. _S. duttoni_, or a closely allied form (by some termed _S. novyi_), occurs in Colombia, and is spread by the tick _Ornithodorus turicata_. In Panama a similar spirochæte is probably spread by _O. talaje_. *Spirochæta gallinarum*, Stephens and Christophers, 1905 (= *Spirochæta marchouxi*, Nuttall, 1905). This Spirochæte, which occurs in fowls and is pathogenic, is transmitted by the tick _Argas persicus_. It is about 10 µ to 20 µ long. There is a pathogenic spirochæte known to occur in geese, named by Sakharoff (1891) _S. anserina_, and found in Caucasia. This may be the same as _S. gallinarum_, in which case the name _S. anserina_ will have priority. These organisms cause fever, diarrhœa, anæmia and death. The life history of the avian pathogenic spirochætes has been studied by Balfour, by Hindle[150] and by Fantham.[151] It is essentially similar to that of _S. duttoni_. [150] _Parasitology_, iv, p. 463. [151] _Annals Trop. Med. and Parasitol._ (1911), v, p. 479. Marchoux and Couvy[152] (1913) consider that the “fragmentation of the chromatin” in spirochætes is a process of degeneration. Working with _A. persicus_ and _S. gallinarum_, they state that a large number of the spirochætes ingested by the Argas almost immediately pass through the wall of the alimentary canal and appear in the hæmocœlic fluid. Marchoux and Couvy consider that Leishman’s granules may be found in the Malpighian tubules of various Arachnids. They found spirochætes in the cephalic glands of infected Argas. They consider that spirochætes remain as wavy spirochætes within the tick, if they are to be infective, though the spirochætes may become so thin as to be invisible! The latter argument is obviously weak, and it was never asserted that all granules in the Malpighian tubules of infected ticks were derived from spirochætes. With dark-ground illumination small, refractile spirochætal granules may be seen to grow into spirochætes. The granule phase of spirochætes has recently been discussed by Fantham[153] (1914). [152] _Annales Inst. Pasteur_, xxvii, pp. 450, 620. [153] _Annals Trop. Med. and Parasitol._, viii, p. 471. *Spirochæta recurrentis*, Lebert, 1874. Syn.: _Spirochæta obermeieri_, Cohn, 1875. This organism was discovered by Obermeier (1873) in cases of relapsing fever in Berlin. Short forms 7 µ to 9 µ long, and longer (probably adult) forms, 16 µ to 19 µ, are found in the blood. The width is 0·25 µ. Parasites 12 µ or 13 µ long are often observed. The spirochæte is found in the blood during febrile attacks and relapses, but not during intervening periods. It can be inoculated into monkeys, rats and mice. It can live in the bed-bug, _Cimex lectularius_, and Nuttall has succeeded in transmitting _S. recurrentis_ from mouse to mouse by the bites of the same bug. The French investigators Sergent and Foley (1908–9) in Algeria, and Nicolle, Blaizot and Conseil (1912) in Tunis, have shown experimentally that _S. recurrentis_ (var. _berbera_) is transmitted by lice. The latter workers also demonstrated the method of infection that commonly occurs, namely, by the scratching of the skin and crushing of lice containing spirochætes on the excoriated surface of the body. Lice as transmitting agents for relapsing fever were indicated by Mackie[154] in 1907. An epidemic among Indian school children furnished the materials.[155] It was noted that out of 170 boys, 137 were infected, and the boys were very verminous. Among the girls, 35 out of 114 suffered, and few lice were found on them. Twenty-four per cent. of the lice taken from the boys contained spirochætes as compared with 3 per cent. of those from the girls. As the epidemic died out among the boys, the lice also became fewer, and an increase in the number of cases among the girls coincided with an increase in the number of lice. Spirochætes were found in the gut, Malpighian tubules and genital organs of the lice. Mackie thought that infection of the patients was brought about by the regurgitation of the spirochætes when the lice fed, but proof of this was lacking. [154] _Brit. Med. Journ._, Dec. 14, 1907, p. 1706. [155] See also Nuttall, Herter Lecture on Spirochætosis, _Parasitology_, v, p. 269. In 1912, Nicolle, Blaizot and Conseil,[156] working in Tunis and using chiefly an Algerian strain of relapsing fever spirochætes (sometimes called _S. berbera_), showed by direct experiments that infection by means of the bites of _Pediculus vestimenti_ and _P. capitis_ was untenable. As many as 4,707 infected lice were fed on one man, and 6,515 on another occasion were allowed to bite a man after they had fed on a monkey heavily infected with spirochætes, yet no infection of the man followed. Examination of the lice showed that the spirochætes left the gut soon after they were ingested, and passed into the body cavity, which swarmed with spirochætes. The contents of the alimentary tract and the fæces of the lice alike were uninfective. The spirochætes did not reappear in the gut till eight days after an infective feed, but some persisted as late as the nineteenth day when kept at 28° C. [156] _C.R. Acad. Sci._, cliv, p. 1636; clv, p. 481. It was noted that the irritation due to the lice caused scratching, and that thereby lice became crushed on to the skin. An emulsion was made of two infected lice and rubbed on to the slightly excoriated skin of one of the above workers. Infection followed five days later. A drop of emulsion placed on the conjunctiva of the human eye produced spirochætosis after an incubation of seven days. The body contents of such lice, then, produce infection when they reach the blood by any excoriated or penetrable surface. The stages leading up to infection in nature briefly are: The irritation due to the louse bites causes scratching, and the lice are crushed on to the skin. The slight abrasion is quite sufficient to permit the entry of the parasite. The louse bite alone is harmless. Infection by way of the eye is quite probable in Africa, remembering the constant trouble due to sand, dust, insects, etc., resulting in frequent touching of the eyes. The spirochætes occur in the body fluid of the lice and can pass in it to the adjacent organs. Thus they probably find their way into the genital organs, and into the eggs of the lice. Eggs laid twenty to thirty days after the parent became infected have retained the infection, and the larvæ issuing from such eggs must have contained some form of spirochætes, for an emulsion of either the eggs or the larvæ produced spirochætosis when inoculated into monkeys. Further details regarding the spirochætosis in the eggs of the lice and in the larvæ are needed. Hereditary infection, however, has been demonstrated, but is not very common. Sergent and Foley (1914) state that the spirochæte possesses a very small and virulent form which it assumes during apyrexial periods in man and during a period following an infecting meal in the louse. Nicolle and Blanc (1914) find that the organisms are infective in the louse just before they reappear as spirochætes. Nicolle and Blaizot found that female lice were more susceptible to spirochætes than males, four times as many females as males being infected. Tictin (1897) found _S. recurrentis_ in bugs recently fed on patients, and infected a monkey with the fluids of crushed bugs. Karlinski (1902) found the spirochætes in bed-bugs in infected houses. There is some other evidence to show that bugs may transmit the spirochæte in Nature. Further researches are needed regarding the relationship of bed-bugs and human spirochætosis. Multiplication of _S. recurrentis_ is by longitudinal and transverse division (including so-called “incurvation”), and the organism forms small, ovoid bodies (“coccoid” bodies) in the same way as _S. duttoni_. _S. recurrentis_ is the cause of European relapsing fever, and a number of possible varieties of it are associated with relapsing fevers in other parts of the world. Such spirochætes only differ by biological reactions, such as acquired immunity tests. They include:-- _S. rossii_, the agent of East African relapsing fever; _S. novyi_, the agent of North American relapsing fever; _S. carteri_, the agent of Indian relapsing fever; _S. berbera_, the agent of North African and Egyptian relapsing fever. OTHER HUMAN SPIROCHÆTES are:-- _S. schaudinni._ This organism, according to Prowazek, is the agent of ulcus tropicum. It varies in length from 10 µ to 20 µ. _S. aboriginalis_ has been found in cases of granuloma inguinale in British New Guinea and Western Australia. It also occurs in dogs, and may not be truly parasitic. _S. vincenti._ This spirochæte is 12 µ to 25 µ in length, tapers at both ends and has few coils. It has been associated with angina vincenti. It often occurs in company with fusiform bacilli. _S. bronchialis_, found by Castellani in 1907 in cases of bronchitis in Ceylon. The parasites are delicate, but show morphological variation. This organism is important and has since been found in the West Indies, India, Philippine Islands and various parts of Africa, such as the Anglo-Egyptian Sudan, Uganda and West Africa. It has recently been the subject of research by Chalmers and O’Farrell, Taylor, and Fantham. _S. phagedenis_ was found by Noguchi in a ten days old ulcerated swelling of the labium. The organism shows much variation in size, being 4 µ to 30 µ in length. _S. refringens_ (Schaudinn, 1905) occurs in association with _Treponema pallidum_ in syphilitic lesions, but is non-pathogenic. It is 20 µ to 35 µ long and 0·5 µ to 0·75 µ broad, being larger than _T. pallidum_ and more easily stained. Various spirochætes have also been notified in vomits, chiefly in Australia; others from the human intestinal tract, _e.g._, _S. eurygyrata_; _S. stenogyrata_ (Werner); _S. hachaizæ_ (Kowalski), in cholera motions; _S. buccalis_ (Cohn, 1875) and _S. dentium_ occurring in the human mouth and in carious teeth (_S. dentium_, Koch, 1877, being the smaller); _S. acuminata_ and _S. obtusa_ found by Castellani in open sores in cases of yaws. Animal spirochætes of economic importance include:-- _S. anserina_, highly pathogenic to geese. _S. gallinarum_ (= _S. marchouxi_) in fowls. (See p. 119.) _S. theileri_ in cattle and _S. ovina_ in sheep also occur in Africa; their pathogenicity is not clear. _S. laverani_ (= _S. muris_), occurring in the blood of and pathogenic to mice, is probably the smallest spirochæte from the blood, being only 3 µ to 6 µ long. Numerous spirochætes have been recorded from the guts of various mammals, birds, fishes, amphibia and insects. CULTIVATION OF SPIROCHÆTES.--Cultures of spirochætes have been made with little success or with great difficulty until comparatively recently, when Noguchi (1912) devised a means whereby he has cultivated most of the pathogenic spirochætes as well as some Treponemata. Noguchi has now cultivated _S. duttoni_, _S. recurrentis_, _S. rossii_, _S. novyi_ and _S. gallinarum_ from the blood; _S. phagedenis_[157] from human phagedænic lesions; _S. refringens_[158] and spirochætes from the teeth. [157] _Journ. Exptl. Med._, xvi, p. 261. [158] _Journ. Exptl. Med._, xv, p. 466. His method is as follows:-- A piece of fresh, sterile tissue, usually rabbit kidney, is placed in a sterile test-tube. A few drops of citrated blood from the heart of an infected animal, _e.g._, rat or mouse, is added, and about 15 c.c. of sterile ascitic or hydrocœle fluid is poured quickly into the tube. Some of the tubes are covered with a layer of sterile paraffin oil, others are left uncovered. The tubes are incubated at 37° C. The best results are obtained if the blood is taken from an animal forty-eight to seventy-two hours after it has been inoculated, that is, before the spirochætes reach their maximum multiplicative period in the blood. The presence of some oxygen seems indispensable for these blood spirochætes, and they fail to develop _in vacuo_ or in an atmosphere of hydrogen. For subcultures, 0·5 c.c. of a culture is added to the medium instead of citrated blood, and it is useful to add a little fresh, normal blood, either human or from an animal, such as a rat. Noguchi found that the events in cultures were:-- _S. duttoni_,[159] maximum multiplication on the eighth to ninth day; disintegration beginning on the tenth day, spirochætes disappeared after about the fifteenth day. No diminution of virulence was found at the ninth day. [159] _Journ. Exptl. Med._, xvi, p. 202. _S. rossii_ (= _S. kochi_).[160] Maximum development on the ninth day, after which the virulence diminishes. The incubation period is also prolonged. [160] _Ibid._, p. 205. _S. recurrentis_[161] (= _S. obermeieri_). Maximum growth on the seventh day. [161] _Ibid._, p. 205. _S. novyi._[162]--Maximum development on the seventh day. It is more difficult to grow than the preceding forms. [162] _Ibid._, p. 208. All the above spirochætes showed undoubted longitudinal division and transverse division was observed in part. _S. gallinarum_[163] can be cultivated as above, but transverse division was usual here. Maximum growth occurred in the culture about the fifth day. [163] _Ibid._, p. 620. TREPONEMATA. The genus _Treponema_ (Schaudinn, 1905), includes minute, thread-like organisms, with spirally coiled bodies, the spirals being preformed or fixed. No membrane or crista is present, according to Schaudinn, though a slight one is said by Blanchard to be present in the organism of yaws. The ends of the organisms are tapering and pointed. Multiplication is by longitudinal and transverse division. The most important members of the genus are _T. pallidum_, the agent of syphilis, and _T. pertenue_, which is responsible for frambœsia or yaws. *Treponema pallidum*, Schaudinn, 1905. Syn.: _Spirochæta pallida_. _Treponema pallidum_ was first described by Schaudinn and Hoffmann in 1905 under the name of _Spirochæta pallida_. It has also been described under the names of _Spironema pallida_, _Microspironema pallida_ and _Trypanosoma luis_. Siegel in 1905 described an organism which he called _Cytorhyctes luis_ and considered to be the agent of syphilis. Schaudinn reinvestigated Siegel’s work and found _T. pallidum_, which he considered to be the causal agent of the disease, and pronounced against _Cytorhyctes luis_. It is probable now that both workers were correct, for Balfour (1911) has seen the emission of minute granules or “coccoid” bodies from _T. pallidum_ and these granules probably correspond to the _C. luis_ of Siegel. Recently E. H. Ross, having observed a spirochæte stage in the development of Kurloff bodies, thinks that _T. pallidum_ is a stage in the life-history of a Lymphocytozoon. MacDonagh has also described a complicated and somewhat similar cycle, but these observations require further study and confirmation. [Illustration: FIG. 56.--_Treponema pallidum_. (After Bell, from Castellani and Chalmers.)] _T. pallidum_ varies from 4 µ to 10 µ in length, its average length being 7 µ, while its width is usually about 0·25 µ. Longer individuals of 16 µ to 20 µ have been recorded. The body has from eight to ten spiral turns and forms a tapering process at each end (fig. 56). The organism is most difficult to stain, and its internal structure is little known. It is possibly like that of _Spirochæta duttoni_ or _S. balbianii_, as the “granule shedding” observed by Balfour is strongly suggestive of the formation of resistant bodies by those spirochætes. Hoffmann (1912) has seen the formation of spores in _T. pallidum_. The Treponemata occur in the primary and secondary sores, but are difficult to find in the tertiary eruptions of syphilis. Noguchi and Moore (1913) and Mott[164] (1913) have demonstrated _T. pallidum_ in the brain in cases of general paralysis of the insane. Marie and Levaditi (1914), however, consider that the treponeme found in the brain in such cases is different from _T. pallidum_. [164] _Brit. Med. Journ._, Nov. 15, 1913. p. 1, 271. CULTIVATION _of T. pallidum_.--This has been accomplished successfully by Noguchi,[165] using a modification of his method for spirochæte cultivation, for _T. pallidum_ is much more difficult to grow than spirochætes, being a strict anaerobe. [165] _Journ. Exptl. Med._, xv, p. 90; xvi, p. 211. [Illustration: FIG. 57.--Diagram of apparatus for cultivation of _Treponema pallidum_ by Noguchi’s method. (After Noguchi.)] The apparatus consists of two glass tubes, the upper being connected to the lower by a narrower tube passing through a rubber cork (fig. 57). Both tubes are carefully sterilized. A piece of fresh, sterile rabbit’s kidney is placed in the lower tube, which is filled with ascitic fluid, or ascitic fluid and bouillon mixture. The tube is inoculated with syphilitic material and corked by inserting the upper tube. In the bottom of the upper tube a piece of sterile rabbit’s kidney is placed and syphilitic material poured over it. A mixture of one part ascitic fluid and two parts of slightly alkaline agar is then poured over the tissue and allowed to solidify. When solid, a layer of sterile paraffin oil is poured on top of it, and the top plugged with cotton wool (fig. 57). The whole is then incubated at 37° C. for two or three weeks. The tissue removes traces of oxygen from the lower levels of the medium and also probably provides a special form of nourishment. At first _T. pallidum_ grows in the solid medium, and then when the cultural conditions in the lower fluid portion become favourable, the organisms migrate thither and multiply abundantly. At first the culture is impure, but after several transferences a pure culture is obtained readily. The syphilitic material for culture is prepared by cutting off pieces of tissue from the lesions, washing in sterile salt solution containing 1 per cent. sodium citrate, and then emulsifying the tissue in a mortar with sodium citrate. Good cultures show rapid multiplication, which is invariably by longitudinal division. In his various cultivation experiments Noguchi[166] found morphological and pathogenic variations in _T. pallidum_. Three forms of the organism were found, namely, thicker, average and thinner types. The lesions caused in the testicle of the rabbit differ according to the variety inoculated, but more work is necessary on the subject. [166] _Journ. Exptl. Med._, xv, p. 201. Noguchi[167] has cultivated a separate organism, _T. calligyrum_, from the surface of human genital or anal lesions, either syphilitic or non-syphilitic. It is apparently non-pathogenic, and is 6 µ to 14 µ long. [167] _Journ. Exptl. Med._, xvii, p. 89. Hata (1913)[168] has modified the Noguchi technique for the cultivation of spirochætes and treponemes, with a view to simplification and convenience. Hata substitutes normal horse serum for ascitic fluid and the “buffy coat” of the clot of horse blood in place of the small pieces of rabbit’s kidney. It is unnecessary to place sterile paraffin on the surface of the medium. [168] _Centralbl. f. Bakt._, Orig., lxxii, p. 107. The horse serum is mixed with twice its volume of physiological saline solution. The mixture is placed in tubes which are heated on a water-bath at 58° C., the temperature being raised gradually until it reaches 70° or 71° C. in three hours. The tubes are then heated at 71° C. for half an hour. After cooling, the contents will consist of an opaque semi-coagulated mass. This semi-coagulated serum and saline mixture may be substituted for Noguchi’s ascitic fluid. The buff coagulum is cut into small pieces, about 1 c.c. in volume. They must be forced with a sterile glass rod to the bottom of the semi-coagulated serum and saline mixture. The medium is inoculated with a small quantity of infected blood and kept at 37° C. In the case of _S. recurrentis_, growth of spirochætes is observed on the second day, reaching a maximum in five to seven days. The growth of the organisms proceeds rather more slowly, they live for a longer period and maintain their virulence better than in Noguchi’s medium. *Treponema pertenue*, Castellani, 1905. Syn.: _Spirochæta pertenuis_; _S. pallidula_, Castellani, 1905. Castellani discovered the organism in 1905, in scrapings of yaws pustules. He first described it under the name of _Spirochæta pertenuis_. [Illustration: FIG. 58.--_Treponema pertenue_. (After Castellani and Chalmers.)] _Treponema pertenue_ (fig. 58), though delicate and slender, shows great morphological variation both in length and thickness. It may be short, _e.g._, 7 µ, but can attain 18 µ to 20 µ in length and may be even larger. In cultures made by Noguchi, thick, medium and thin forms were found, each giving rise to a different type of frambœsial lesion when inoculated into the testicles of rabbits, thus suggesting the possibility of the occurrence of varieties of _T. pertenue_. The organism is difficult to stain, but occasionally deeper staining granules are found along its body. They may represent a diffuse nucleus. Granule formation similar to that of _T. pallidum_ has been observed by Ranken, using dark-ground illumination. Many experiments have been made with a view to establishing the identity of the organism of yaws and also of differentiating between the causative agents of yaws and syphilis. Both monkeys and the human subject have been experimentally inoculated with yaws material and have developed the disease. In an early experiment, negroes were inoculated with the secretion from lesions of yaws. All of them developed the disease, nodules appearing, chiefly at the seat of inoculation, in from twelve to twenty days, followed by the usual eruption. Similar results were obtained with thirty-two Chinese prisoners, who were inoculated with yaws, twenty-eight becoming infected. A naturally infected yaws patient when inoculated with syphilis, contracted that infection, thus showing that yaws does not confer immunity to syphilis. This has also been observed naturally, when yaws patients have contracted syphilis. Experiments with monkeys have been successfully performed. The incubation period varies from sixteen to ninety-two days. Lesions appear first at the seat of inoculation, and in some monkeys the eruption is localized to this spot, though the infection is general, _T. pertenue_ occurring in the spleen, lymphatics, etc. Monkeys inoculated with splenic blood of a yaws patient, and also sometimes with blood from the general circulation, have become infected. Castellani and others have shown that monkeys successfully inoculated with syphilis do not become immune to yaws, and vice-versâ. Craig and Ashburn, using the monkey _Cynomolgus philippinensis_, found these animals susceptible to yaws but not to syphilis. The ulcerated lesions of frambœsia are rapidly invaded by numerous bacteria as well as by different spirochætes, of which Castellani has described three distinct species. One is identical with _Spirochæta refringens_, Schaudinn, the other two are thin and delicate. One, _S. obtusa_, has blunt ends; the other _S. acuminata_, has pointed ends. _T. pertenue_ is also present. The reasons for considering _T. pertenue_ to be the specific cause of frambœsia are:-- (1) _T. pertenue_ is the only organism present in non-ulcerated papules, in the spleen and in the lymphatics of yaws patients, or of monkeys artificially infected with the disease. By no method has any other organism been obtained. (2) Extract of frambœsia material, free from all organisms other than _T. pertenue_, reproduces the disease if inoculated. (3) Extract of frambœsia material deprived by filtration of _T. pertenue_ is no longer infective on inoculation. The method of infection is contaminative, by direct contact. Women in Ceylon are frequently infected by their children. Any slight skin abrasion is sufficient to admit the parasite. In some cases, insects may carry the disease from person to person, and even in hospitals, when dressings are removed, it has been noticed that flies greedily suck the secretion from the ulcers. _T. pertenue_ has been recovered from flies that have fed on yaws, and monkeys have contracted the disease when flies were placed and retained on them for a short time, after the insects had fed on yaws material. CULTIVATION.--_T. pertenue_ has been cultivated by Noguchi, who finds three types of parasites in his cultures, as before mentioned. Its multiplication is by longitudinal division. Noguchi[169] (1912), has cultivated species of Treponema from the human mouth, e.g., _T. macrodentium_, _T. microdentium_ and _T. mucosum_, the latter from pyorrhea alveolaris. These parasites in the past may have been confused under the name _Spirochæta dentium_. [169] _Journ. Exptl. Med._, xv, p. 81; xvi, p. 194. Class III. *SPOROZOA*, Leuckart, 1879. The third group of the Protozoa consists entirely of parasitic organisms forming the class known as the Sporozoa or spore-producing animals. The members of this class are characterized by possessing very great powers of multiplication, coupled with a capacity for producing forms that serve for the transference of the organisms to other hosts. These reproductive bodies, whether for increase of numbers within one host or for transmission to another host, are called spores. But, strictly, the term spore should be used only in the latter connection, when a protective or resistant coat known as a sporocyst envelops the body of the spore. The Sporozoa are widely distributed, occurring in various tissues and organs of Annelids, Molluscs, Arthropods, and Vertebrates. Their food, which is fluid, is absorbed osmotically. The life-cycle of a Sporozoön may be completed within one host or may be distributed between two different hosts. The Sporozoa were divided by Schaudinn into two groups or sub-classes, called (1) the *Telosporidia*, and (2) the *Neosporidia*. The Telosporidia are Sporozoa in which the reproductive phase of the parasites is distinct from the growing or trophic phase, and follows after it. The Neosporidia include Sporozoa in which growth and spore-formation go on simultaneously. This classification is not final, for certain exceptions and difficulties are already known with regard to it. It is possible that the class Sporozoa is not a natural entity, but should be replaced by two classes of equal rank, corresponding in most respects with the Telosporidia and Neosporidia. The *Telosporidia* comprise the *Gregarinida*, the *Coccidiidea*, and the *Hæmosporidia*. Doflein combines the two latter orders into one known as the *Coccidiomorpha*. The *Neosporidia* comprise the *Myxosporidia*, the *Microsporidia*, the *Actinomyxidia*, the *Sarcosporidia*, and the *Haplosporidia*. Doflein combines the first three orders into one, the *Cnidosporidia*. Sub-Class. TELOSPORIDIA, Schaudinn. Sporozoa in which the reproductive phases follow completion of growth. Order. *Gregarinida*, Aimé Schneider emend. Doflein. Knowledge of the Gregarinida probably goes back as far as the year 1684, when Redi observed gregarines in the crab, _Cancer pagurus_. Von Cavolini (1787) found them in _Cancer depressus_. The name _Gregarina_ was created by L. Dufour (1828), who observed masses of these organisms in the gut of insects of different orders. Hammerschmidt (1838) and von Siebold found rich infestations in insects, while Dujardin (1835) and Henle described various genera from segmented worms. Henle (1835) also observed cysts containing “navicellæ” in the sperm-sacs of segmented worms, and attention was drawn to his researches by the discovery by von Siebold (1839) of “pseudonavicellæ” in the gut of _Sciara nitidicollis_. Up to this time many workers considered the gregarines to be worms, but Kölliker (1845) investigated many of them and maintained their unicellular nature, while Stein’s work (1848) showed the interrelation of the pseudonavicellæ and the gregarines. The discovery of amœboid germs in the pseudonavicellæ by Lieberkühn (1855) and the demonstration of myonemes further aided in the elucidation of their true systematic position. The entire process of conjugation, of which Dufour had seen one phase, was followed by Giard under the microscope. From 1873 onwards Aimé Schneider made important additions to the knowledge of the morphology, life-history, and systematic position of numerous gregarines. Bütschli (1881) and L. Léger (1892) also contributed much work on the subject. The discoveries of Schaudinn with regard to the life-cycle of Coccidia gave a fresh stimulus to the study of the Gregarines, whereby the life-cycles of numerous forms and the phases thereof have been elucidated. Asexual multiplication is not common among the Gregarines, but is known to occur in the sub-order Schizogregarinea, formerly known as the Amœbosporidia. Although the Gregarinida are not known to be parasitic in man or other vertebrates, they are of great interest, inasmuch as they are among the earliest known Sporozoa, and therefore will be briefly described here. [Illustration: FIG. 59.--_Monocystis agilis_ from seminal vesicles of _Lumbricus_ × 250. (After Stein.)] [Illustration: FIG. 60.--_Gregarina longa_ from larva of crane-fly (_Tipula_). _a_, in epithelial cell of host; _b_, _c_, gradually leaving host-cell; _d_, adhering to host-cell; _e_, fully developed free trophozoite.] The Gregarines are usually elongate, somewhat flattened organisms (figs. 59, 60), whose bodies are enclosed in an elastic and often thick cuticle. The enclosed living substance shows a separation into ectoplasm and endoplasm, as is common among Protozoa. The cuticle is sometimes regarded as the outer portion or epicyte of the ectoplasm. A single, vesicular, spherical, or elliptical, large nucleus, with its chromatin concentrated to form a spherical karyosome, is present. The body of some gregarines may be divided by ingrowing ectoplasmic partitions or septa, and are then said to be “septate” or “polycystid” (fig. 61). Other gregarines remain simple and non-septate, and are termed “monocystid” (fig. 59). The monocystid gregarines occur especially in the body cavity of Chætopoda and Insecta, more rarely in Echinodermata, in the parenchyma of Platyhelminthes, also in the gut of Tunicata and Insecta (fig. 60) and in the seminal vesicles of Annelida. In the polycystid gregarines a single septum only is present as a rule, and thus the body presents two portions: (1) an anterior portion termed the protomerite; (2) a posterior, larger portion, known as the deutomerite, which generally contains the nucleus. The protomerite is often modified anteriorly to form an organ of attachment, termed the epimerite (fig. 61), which is developed from the pointed rostrum of the sporozoite or primary infecting young gregarine. The structure of the epimerite may be complicated, being provided with hooks, spines, knobs, and other appendages. An extension of the polycystid condition is seen in _Tæniocystis mira_ Léger (from the dipteran larva, _Ceratopogon solstitialis_), whose body shows a number of partitions, giving the organism a superficial resemblance to a tapeworm. [Illustration: FIG. 61.--_Xyphorhynchus firmus_ with epimerite in intestinal epithelial cell of host. (After Léger.)] The ectoplasm of a gregarine exhibits three layers: (1) An epicyte (cuticle) externally of which the epimerite is composed; (2) a sarcocyte which forms the septa if present; (3) the deeper myocyte layer containing contractile elements in the form of fibrils or threads termed myonemes (fig. 62). [Illustration: FIG. 62.--_Gregarina munieri_ (from the beetle, _Chrysomela hæmoptera_). Section through surface layers. _Cu_, cuticle; _E_, ectoplasm proper; _G_, gelatinous layer; _My_, myonemes in myocyte layer. × 1500. (After Schewiakoff.)] The endoplasm is fluid and granular, containing many enclosures, which are of the nature of reserve food materials. They consist of fat droplets or of paraglycogen, and give the organisms an opaque appearance. _Lithocystis_ contains crystals of calcium oxalate in its endoplasm. Many gregarines are capable of active movements, though they do not possess obvious locomotor organs. The movement is of a smooth, gliding character and two suggestions have been put forward to explain it. According to Schewiakoff, a gelatinous substance is secreted between the layers of the ectoplasm. This is extruded posteriorly and thus the animal is pushed forward. On the other hand, Crawley considers that the movements are produced by contractions of the myonemes. These two explanations are probably correct as far as each goes, and are to be regarded as supplementary to one another. [Illustration: FIG. 63.--_Monocystis agilis_. Spores from vesicula seminalis of the Earthworm. _a_, Sporoblast with single nucleus, enclosed in sporocyst; _b_, mature spore containing sporozoites; _c_, diagrammatic cross-section of spore, showing eight sporozoites round residual protoplasm. (After Bütschli.)] Occasionally, temporary associations of gregarines are formed by a number of individuals adhering to one another end to end. Such temporary associations are examples of syzygy. Such syzygies must not be confused with true associations which form an essential part of the life-cycle. The life-cycle of a relatively simple gregarine, such as _Monocystis agilis_ (fig. 59), parasitic in earthworms, may now be considered. The gregarines, being members of the Sporozoa, produce spores at one phase of the life-cycle. Each gregarine spore (fig. 63) develops within itself a number of minute, sickle-shaped or vermicular bodies, known as sporozoites or primary infecting germs. Eight sporozoites are often formed within each spore. When absorbed by a new host, the spore softens and the sporozoites issue from it. They are capable of active movement and may or may not enter a cell, such as one of those of the digestive tract, or, as in _Monocystis_, a cell lining the vesicula seminalis which becomes a sperm-cell aggregate (sperm morula). When the sporozoite has reached the place of its choice in the host it ceases active movements and proceeds to feed passively on the fluid substances around it, whether they be those of tissues or body fluids. This passive, growing and feeding form is known as the trophozoite. After a trophic existence of longer or shorter duration, the trophozoite ceases to feed and prepares for reproduction. Two trophozoites associate together, each of them first becoming somewhat rounded. The two trophozoites, now known as sporonts or gametocytes, become invested in a single common envelope or cyst (fig. 64, _a_). The nucleus of each gametocyte then divides by a series of binary fissions (fig. 64, _b_), and the daughter nuclei thus produced arrange themselves at the periphery of the parent cells (fig. 64, _c_). Cytoplasm collects around each of these nuclei, and thus a number of gametes are formed within each gametocyte. The gametes for a time exhibit active movements, and ultimately ripe gametes of different parentage fuse in pairs, that is, conjugation occurs (fig. 64, _d_). In this way zygotes are produced, the nucleus of each zygote being formed by the fusion of two gamete nuclei. [Illustration: FIG. 64.--Schematic figures of conjugation and spore formation in Gregarines. For details see text. (After Calkins and Siedlecki, modified.)] [Illustration: FIG. 65.--_Stylorhynchus oblongatus_. _a_, cyst containing two sporonts or gametocytes, each full of gametes, those in the upper one being male. _b_, ripe male and female gametes. × 1,600. (After L. Léger.)] The zygote grows slightly and becomes oval or elongate, and at this period is often called the sporoblast. It then secretes an external membrane, the sporocyst. Nuclear division occurs inside the sporocyst by a series of three binary fissions (fig. 64, _e_), so that each sporocyst, now usually referred to as a spore, contains eight nuclei. The cytoplasm collects around each nucleus and eight vermicular sporozoites are produced within each spore (fig. 64, _f_), thus completing the life-cycle. It will be noticed that in the above life-cycle no asexual multiplication occurs. These organisms, such as _Monocystis_, are known as the Eugregarines, and include the majority of the gregarines. The remainder, which have introduced schizogony into their life-cycle, are known as the Schizogregarines. [Illustration: FIG. 66.--Spores of various Gregarines. _a_, _Xiphorhynchus_. _b_, _Ancyrophora_. _c_, _Gonospora_. _d_, _Ceratospora_. (After Léger.)] There are variations in the morphology and life-cycle of gregarines besides those that have been mentioned. It is not within the scope of this book to discuss them in detail, but the following may be noted:-- Morphological differentiation of gametes may occur as in _Stylorhynchus oblongatus_ (fig. 65), which differentiation is probably of a sexual nature. The sporocyst really consists of two layers, an epispore and an endospore. Externally the spores of different gregarines show great variety in shape and markings, and spines, or long processes may be present (fig. 66). The resistant spores serve for the transmission of the gregarines from host to host. The mode of infection is contaminative, the spores expelled with the dejecta of one host being absorbed with the food of a new host. The Gregarinida may be classified as follows:-- Sub-order I.--*Eugregarinea*, without schizogony. Tribe 1.--_Acephalina_.--Without an epimerite and non-septate; often “cœlomic” (body-cavity) parasites. _E.g._: _Monocystis_, with several species parasitic in the seminal vesicles of earthworms. Many other genera parasitic in Echinodermata, Tunicata, Arthropoda, etc. Tribe 2.--_Cephalina_.--With an epimerite, either temporarily or permanently, in the trophic phase. Usually septate (except _Doliocystidæ_). Many families, genera and species. Common in the digestive tracts of insects. _E.g._: _Gregarina_, with several species, _Gregarina ovata_ in the earwig, _Gregarina blattarum_ in the cockroach, _Stylorhynchus_ in beetles, _Pterocephalus_ in centipedes, etc. Sub-order II.--*Schizogregarinea*, with schizogony. Tribe 1.--_Endoschiza_.[170]--With schizogony occurring in the intracellular phase, _e.g._, _Selenidium_ (from Annelida and Gephyrea), _Merogregarina_ (from an Ascidian). [170] See Fantham (1908), _Parasitology_, i, p. 369. Tribe 2.--_Ectoschiza_.--In which the schizont is free, and schizogony is extracellular, _e.g._, _Ophryocystis_ (from _Blaps_, a beetle), and _Schizocystis_ (from _Ceratopogon_ larva). Order. *Coccidiidea*. Hake (1839) first saw the organisms now termed Coccidia during his investigations on the so-called coccidial nodules of rabbits. The opinions as to the nature of these peculiar formations were very diverse. The discoverer considered them to be a sort of pus corpuscle; Nasse (1843) took them for epithelial cells of the biliary passages, others for helminthes, especially the ova of trematodes (Dujardin, Küchenmeister, Gubler, etc). Remak (1845) was the first to draw attention to their relation to the Psorospermia (Myxosporidia), and this investigator found them also in the small intestine and vermiform appendix of rabbits. Lieberkühn (1854), who examined not only the coccidia of rabbits, but found similar forms in the kidneys of frogs, likewise called them definitely psorosperms. To differentiate Müller’s psorosperms, which are found in fishes, from those of rabbits, etc., the latter were called egg-shaped psorosperms (Eimer), until R. Leuckart (1879) named them _Coccidia_ and placed them in a group of the Sporozoa analogous to that of the Gregarinida, Myxosporidia, etc. Numerous works confirmed the occurrence of coccidia, not only in all classes of vertebrate animals, but also in invertebrates (Mollusca, Myriapoda, Annelida, etc.). A large number of genera and species have in the course of time been described which inhabit the epithelium of the intestine and its appendages for choice, but are also found in other organs (kidneys, spleen, ovaries, vas deferens, testicles). Some also live in the connective tissue of various organs, more particularly of the intestine. The knowledge of the development of the coccidia was of particular importance in determining their classification. By means of encysted coccidia from the liver of rabbits, Kauffmann (1847) first confirmed the fact that the cyst, which was partly or entirely filled with granular contents, divided into three or four pale bodies (fig. 71) after a long sojourn in water. Lieberkühn observed the same process in the host in the case of the coccidia of the kidney of the frog. Stieda (1865) studied more minutely the changes that occur within the encysted coccidia of the liver of rabbits after the death of the host. He discovered that the bodies now known as “spores” were oval formations pointed at one pole, and surrounded by a delicate membrane, which exhibited a certain thickness at the pointed extremity and enclosed a slightly bent rodlet, expanding at either end into a strongly light-refracting globule; a finely granular globule was present in the middle of the spore. Waldenburg (1862) saw the same phenomenon in coccidia from the epithelium of the villi and Lieberkühn’s glands of the intestine of the rabbit; but the process in this case took place in a much shorter time. According to the discovery of Kloss (1855), the spores of the coccidia of the urinary organ of the garden snail were formed in far greater numbers: the round spores also harboured several (five to six) rodlets, which after the bursting of the spore-envelope became free. Eimer’s researches (1870) afforded information regarding a Coccidium from the intestine of the mouse, which was transformed _in toto_ into a “spore,” containing small sickle-shaped bodies. The fact was, moreover, established that the little bodies left the delicate envelope when in the intestine, made movements of flexion and extension, and were finally transformed into amœboid organisms, which apparently penetrated the epithelial cells; at all events, similar bodies of various sizes were seen in these cells. Taking the immense number of these parasites into account and the lack of any other cause, Eimer attributed the sudden death of his mice to the _Gregarina falciformis_, as the parasite was then called, just in the same way as a few years previously Reincke ascribed the acute and fatal intestinal catarrh of rabbits to the invasion of intestinal coccidia. All that had become known about coccidia up to 1879 was then compiled by Leuckart, and completed by his own observations on the coccidia of the liver of the rabbit. Experimental infections had already been conducted by Waldenburg (1862) with intestinal coccidia of rabbits, and by Rivolta (1869–73) with the coccidia of fowls, which experiments confirmed the importance of the spores, or bodies enclosed in them, in the transmission of the parasites to other animals. Accordingly, it was assumed that after the entry of the spores into the intestine the sporozoites were set free, actively penetrated into the intestinal cells, where they grew into coccidia, and finally became encysted. The further development, _i.e._, the formation of spores, took place outside the host’s body in these cases; in other cases (Kloss, Eimer) it took place within the host. Although much regarding the cycle of development was still hypothetical, the ideas coincided with the observations, and were therefore universally regarded as established. Further research confirmed this view in numerous new forms. L. Pfeiffer’s statements (1891) on the part that certain coccidia or their sporozoites played, or seemed to play, as causes of disease gave a renewed impetus to the investigation of the coccidia. The ingestion of even very numerous spores did not appear to account for the mass infection so frequently observed, even after Balbiani had confirmed the fact that there were two, and not one, sporozoites contained in every spore of the coccidia of rabbits (fig. 72). The hypothesis was therefore advanced that the sporozoites or young coccidia were able to divide once again by sporulating. The question was finally solved quite differently. R. Pfeiffer (1892) first confirmed the fact that in addition to the well-known method of sporulation in the coccidia of the rabbit that causes the infection of fresh hosts (“exogenous sporulation”), an enormous increase may follow in the already infected host in a manner that Eimer first observed in the coccidia of the intestine of the mouse (“endogenous sporulation”). It had hitherto been believed that some of the species of coccidia increased like the rabbit parasite, then known as _Coccidium oviforme_, and others like _Eimeria falciformis_, and this difference had been made the foundation of a classification. R. Pfeiffer was successful in observing the occurrence of both kinds of development in the same species, and expressed the opinion that endogenous sporulation (fig. 73), which takes place within the host, was the cause of the mass-infection that is mostly accompanied by serious consequences (fig. 74). L. Pfeiffer sought, especially, to demonstrate the correctness of this view as regards other species of coccidia and for this purpose he utilized the experiences already published. Coccidia were known to exist in a number of different hosts, and to propagate in some according to the _Coccidium_ type, in others according to the _Eimeria_ type. It therefore was reasoned that in this case it was not a question of two species belonging to different genera living side by side, with a different mode of development, but of one species, in the life of which both forms of development occurred alternately. This interpretation of facts was combated especially by A. Schneider (1892) and by Labbé, but has, nevertheless, proved true, apart from the circumstance that Schuberg succeeded in discovering the hitherto unknown _Coccidium_ form in the intestine of the mouse; and that, moreover, Léger confirmed the fact that there are no Arthropoda in which Eimeria are not found together with coccidia. The question was finally settled by experiments made by Léger with the coccidia of _Scolopendra cingulata_, by Schaudinn and Siedlecki with those of _Lithobius forficatus_, and by Simond with the coccidia of the rabbit. On Simond’s suggestion the sickle-shaped germs corresponding to the sporozoites, which are formed by endogenous sporulation, are generally termed merozoites; and in accordance with Schaudinn’s suggestion, those individuals which form merozoites are termed schizonts, and those which produce spores are called sporonts. In contradistinction to sporogony (exogenous sporulation), the term schizogony (endogenous sporulation) is used. The more minute examination of these processes at last led to the discovery of sexual dimorphism, of copulation and of alternation of generations in the coccidia. Schuberg was the first to consider the possibility of copulation in coccidia; in addition to the formations which now are termed merozoites, he observed very diminutive bodies (“microsporozoites”) in the coccidia of the intestine of the mouse, which were able eventually to copulate. Labbé confirmed this observation in some of the species, and Simond expressed the opinion that bodies termed “chromatozoites” occurred in all coccidia. Copulation itself was then observed by Schaudinn and Siedlecki (1897). The copulating bodies were termed gametes. As, however, they differed considerably one from the other, the males were called microgametes (_i.e._, the microsporozoites of Labbé and the chromatozoites of Simond) and the females macrogametes. After copulation was completed sporogony took place, and in the cycle of development of one species this regularly alternated with schizogony (asexual multiplication). Schaudinn in 1900 described in detail the life-cycle of _Eimeria_ (_Coccidium_) _schubergi_. The recognition of this unsuspected complicated process was bound to effect reforms in the classification of the coccidia; and all the forms that had been regarded as developmental stages (_Eimeria_, etc.) had to be reconsidered. _Occurrence._--The Coccidiidea in their mature condition usually live within the epithelial cells of various organs, and by choice inhabit those of the intestine and of its associated organs. They also occur frequently in the excretory organs of mammals, birds, amphibia, molluscs, arthropods, and may also be found in the testes and vas deferens, but the statement that they live in hen’s eggs, as well as in the oviducts of fowls, has not been confirmed.[171] Some species inhabit the nuclei of cells, others live in the connective tissue, but their presence in the latter situation is probably only secondary, that is, they have only reached it from the epithelium of the affected organs. [171] Notwithstanding the progress made during the last decades, the ova of helminthes and more particularly of trematodes, have been mistaken for Coccidia. Thus Poschinger (_Zool. Anz._, 1819, ix, p. 471) and Gebhard (_Virchow’s Arch._, 1897, No. 147, p. 536) mistook the ova of _Distoma turgidum_, Brds., for Coccidia. Podwyssotzki (_Centralbl. f. allg. Path._, 1890, i, p. 135) made a similar error with the ova (and vitelline sacs) of a species of _Prosthogonimus_ (_Distoma ovatum_ of the authors); von Willach (_Arch. f. wiss. u. prakt. Thierheilk._, 1892, xviii, p. 242) mistook the ova of a nematode for Coccidia. The size of the Coccidiidea, corresponding as a rule to the capacity of their habitat, is usually small, but there are said to be species that attain a diameter of 1 mm. Their form[172] is globular, oval, or elliptical. External appendages are lacking, at least during the trophic or vegetative period of their life, which is spent in epithelial cells, within which they increase in size. Frequently one only is present in each cell, but more can occur. The body substance is composed of a more or less finely granular or distinctly alveolar protoplasm which exhibits no differentiation into ecto- and endoplasm. All species possess a nucleus that enlarges with their growth; sometimes it only shows through the cytoplasm as a lighter spot, or may even be quite concealed. It is vesicular, and besides containing very delicate threads of chromatin in the clear nucleoplasm, it contains generally only one large karyosome. [172] The life-cycle given here is based on that of _Eimeria_ (_Coccidium_) _schubergi_, after Schaudinn (1900). See “Untersuchungen über den Generationswechsel bei Coccidien,” _Zool. Jahrb., Abt. f. Anat._, xiii, pp. 197–292, 4 plates. The infected epithelial cells degenerate sooner or later as the parasite feeds on them (fig. 67, II-IV). After their form has been changed by the action of the growing parasite, they finally perish. The cell membrane then alone surrounds the coccidia, which, at least in the species sufficiently known, begin to propagate by an asexual process (schizogony), the parasites themselves becoming schizonts, as the initial stage is usually called. They differ from later stages (sporonts or gametocytes), which resemble them in form, by the absence of granulations in the cytoplasm, as well as by the vesicular nucleus. The form is not always the same, for in some cases, at least, many schizonts assume a globular form. Schizogony (fig. 67, V-VII) commences with a division of the nucleus, which takes place in different ways in the various species. This finally leads to the formation of numerous daughter nuclei which become smaller and smaller, and which collect beneath the surface of the schizonts. In some species the daughter nuclei collect only in one half of the schizont. A part of the protoplasm of the periphery now divides around each daughter nucleus, the remaining part (residual body) being left in the centre or on one side. The segments of the divided cytoplasm, each containing a nucleus, assume a fusiform shape and become merozoites, which very soon become free (fig. 67, VIII) and leave the residual body. They are distinguishable from the very similar sporozoites (fig. 67, I), as the merozoites possess a karyosome. [Illustration: FIG. 67.--Life-cycle of _Eimeria_ (_Coccidium_) _schubergi_, Schaud., from the intestine of _Lithobius_. (After Schaudinn.) The infection is caused by a cyst (XX), containing spores, which reaches the intestine of a _Lithobius_, where it discharges the sporozoites (I). II, A sporozoite invading an intestinal epithelial cell; III, intestinal epithelial cell with young trophozoite; IV, intestinal epithelial cell with a globular schizont; V, nuclear segmentation within the schizont; VI, the daughter nuclei arranging themselves superficially; VII, formation of the merozoites; VIII, merozoites that have become free, and which, penetrating into other epithelial cells of the same intestine, repeat the schizogony (II-VIII); IX and X, merozoites which, likewise invading the epithelial cells of the same intestine, become sexually differentiated; XIa, young macrogametocyte; XIb, older macrogametocyte; XIc, mature macrogametocyte (discharging particles of chromatin); XIIa, young microgametocyte; XIIb, older microgametocyte; XIIc, increase of nuclei in the microgametocyte; XIId, the globular residual body around which numerous microgametes have formed; XIIe, an isolated microgamete; XIII, the mature macrogamete surrounded by numerous microgametes and forming a cone of reception or fertilization prominence; XIV, shows the nucleus of a microgamete that has penetrated and fused with the nucleus of the macrogamete (fertilization)--the latter forms a membrane and becomes an oöcyst; XV, XVI, XVII, nuclear segmentation in the oöcyst; XVIII, oöcyst with four sporoblasts; XIX, the sporoblasts transformed into spores, each containing two sporozoites; XX, the cyst introduced into the intestine and liberating the sporozoites by bursting.] The merozoites move in a manner similar to that of the sporozoites. The movements consist either of slow incurvations with subsequent straightenings, or annular contractions along the entire extent of the body. In addition, there are gliding movements similar to those of many gregarines, and brought about in a like manner by the secretion at the posterior extremity of a gelatinous substance that hardens rapidly. The merozoites do not gain the open in the usual way, but are destined to infect still further the same host by actively penetrating into other epithelial cells of the affected organ. Here they continue their growth and may again and again undergo schizogony. In the Infusoria the repeated segmentations finally cease and are renewed only after a conjugation. This is likewise the case with the Coccidia, with the difference that in the latter the two conjugating individuals (gametes) are differently constituted one from the other, whereas in the Infusoria they are almost always similar. When the schizogony ceases, the merozoites, that had penetrated the epithelial cells and become trophozoites there, consist of two kinds of differently constituted individuals. One kind possesses a clear cytoplasm (fig. 67, XII), the other an opaque, richly granular cytoplasm (fig. 67, XI), while both possess a vesicular nucleus with a karyosome. In order to continue their development, the more granular individuals must be fertilized, and are therefore termed either female gametes or, on account of their size, macrogametes. The male individuals (microgametes) necessary to conjugation, are formed in greater numbers from the less dense microgametocytes or male mother-cells (fig. 67, XIId). They are slender bodies consisting chiefly of nuclear substance, and in most species bear two flagella of unequal length directed backwards, the place of insertion of which varies according to the species (fig. 67, XIIe). While the development of the microgametes is rapidly advancing a change occurs in the nucleus of the female parent forms or macrogametocytes. Parts of the karyosome are extruded (fig. 67, XIc), and the nucleus loses at the same time its vesicular form. One macrogamete results, after nuclear maturation, from one macrogametocyte. By this time the macrogametes are capable of conjugation, and the process takes place within the host, generally, however, outside the affected and degenerated host cells. The microgametes that have now become free from the very large residual body, crowd around the mature macrogametes, which often send out a small prominence (“cone of reception” or fertilization protuberance) for their reception (fig. 67, XIII). As soon as a microgamete comes in contact with this and penetrates into the cytoplasm of the macrogamete, the latter surrounds itself with a membrane which prevents the intrusion of other microgametes. The nucleus of the microgamete that has gained entry unites with the nucleus of the macrogamete, which latter is afterwards capable of forming the well-known spores. The parasite is now called an encysted zygote or oöcyst. The oöcysts of some other members of the Coccidiidea, _e.g._, _Eimeria avium_, can form their walls prior to fertilization. In such cases, a weak spot is left at one place in the cyst wall, forming a micropyle, that permits of the entry of the male, immediately after which the micropyle is closed. The reduced nucleus of the macrogamete elongates on the entry of the microgamete, and becomes a fertilization spindle to which the male pronucleus (from the microgamete) becomes attached (fig. 67, XIV and XV). Thereupon the spindle divides into two daughter nuclei (fig. 67, XVI) which assume a round shape. The protoplasm at this stage may at once divide, or another segmentation of the daughter nuclei may first occur. In the former case the two halves divide again, so that finally four nucleated segments, the sporoblasts, are formed, whereas in the latter case the four sporoblasts appear simultaneously (fig. 67, XVII). In both cases a residual body of varying size is separated from the protoplasm of the oöcyst. As a rule the oöcysts have already been discharged from the body of the host, and in the manner described above, form the sporoblasts after a longer or shorter period of incubation. The sporoblasts are originally naked, but each soon secretes a homogeneous membrane, the sporocyst, in which it becomes enveloped (fig. 67, XVIII). After the segmentation of the nucleus the contents divide into two sickle-shaped sporozoites, in addition to which there is generally also a residual body (fig. 67, XIX). This terminates the development. The spores are intended for the infection of other hosts. If they reach the intestine of suitable hosts, either free or enclosed in the oöcyst wall, the action of the intestinal juices causes them to open and permits the escape of the sporozoites (fig. 67, XX). The latter move exactly like the merozoites and soon make their way into epithelial cells (fig. 67, I), where they become schizonts, and thus repeat the life cycle. Although our knowledge of the development of the coccidia is but of recent date, yet it already extends to a large number of species, which exhibit various deviations from the cycle of development described above. For instance, in addition to differences in the gametocytes, the schizonts of _Adelea_ and _Cyclospora_ also show differentiation and give rise to macromerozoites and micromerozoites, whilst in _Adelea_ and _Klossia_ a precocious association of the gametocytes precedes the true copulation of the ripe gametes. The classification of the Coccidiidea is based chiefly on the number of sporozoites found in each spore, and the number of sporocysts (spores) found in one oöcyst. Léger[173] recognises two great legions, the Eimeridea and the Adeleidea, the former comprising the greater number of genera, including the genus of most economic importance, _Eimeria_. It must be noted that, though a member of this genus may be frequently referred to as _Coccidium_, strictly it should be termed _Eimeria_, that name having priority. The name of the disease resulting from the action of such parasites is, however, established and remains as coccidiosis. [173] _Arch. f. Protistenkunde_ (1911), xxii, p. 71. Certain of the more important of the Coccidiidea may now be considered. Genus. *Eimeria*, Aimé Schneider, 1875. Syn.: _Psorospermium_, Rivolta, 1878; _Cytospermium_, Rivolta, 1878; _Coccidium_, R. Leuckart, 1879; _Pfeifferia_, Labbé, 1894; _Pfeifferella_, Labbé, 1899. The Eimeria belong to Léger’s old family, the Tetrasporocystidæ, which comprises forms producing oöcysts with four sporocysts, each containing two sporozoites. The cysts are spherical or oval, as are also usually the schizonts. The members of the genus are confined chiefly to vertebrate hosts, the more important economically occurring in mammals and birds. From the mammalian hosts very rarely the parasites may reach man. _Eimeria_ (_Coccidium_) _avium_ of wild birds and poultry, and _Eimeria stiedæ_ parasitic in rabbits, may be considered. There is a general similarity in their life-cycles and each is of great practical importance. *Eimeria avium*, Silvestrini and Rivolta. _Eimeria avium_ is responsible for fatal epizoötics among game birds such as grouse, pheasants and partridges, and domestic poultry such as fowls, ducks, pigeons and turkeys, and can pass from any one of these hosts to any of the others with the same effect. The organism is parasitic in the alimentary tract of the host, affecting more especially the small intestine (duodenum) and the cæca, but in some cases penetrating to the liver and multiplying there (as in turkeys), producing necrotic cheesy patches, that ultimately become full of oöcysts. The gut is rendered very frail by the action of the parasites, its mucous membrane is greatly injured, and is often reduced to an almost structureless pulp, riddled with parasites (fig. 68). Infection is conveyed from host to host by the ingestion of food or drink contaminated with the oöcysts voided in the fæces of infected birds. Oval oöcysts from 24 µ to 35 µ long and from 14 µ to 20 µ broad are the means of infection. The oöcysts develop internally four sporocysts or spores, from each of which two sporozoites are produced. The life-history[174] presents two phases: (1) The asexual multiplicative phase, schizogony, for the increase in numbers of the parasites within the same host; (2) the reproductive phase, following the formation of gametes (gametogony), leading to the production of resistant oöcysts, destined for the transference of the parasite to new hosts (sporogony). [174] Fantham, H. B. (1910), “The Morphology and Life History of _Eimeria_ (_Coccidium_) _avium_, a Sporozoön causing a fatal disease among young Grouse,” _Proc. Zool. Soc. Lond._, 1910, pp. 672–691, 4 plates. Also Fantham, H. B. (1911), “Coccidiosis in British Game Birds and Poultry,” _Journ. Econ. Biol._, vi, pp. 75–96. The oöcysts usually reach the duodenum unharmed, with food or drink. Under the influence of the powerful digestive juices (especially the pancreatic) now encountered, the oöcysts soften, as do the sporocysts, and ultimately two sporozoites emerge from each sporocyst. The sporozoites are from 7 µ to 10 µ long, and each is vermicular with a uniform nucleus (fig. 69, A). After a short period of active movement in the gut, each sporozoite penetrates an epithelial cell (figs. 68 _spz_, 69, B), and once within, gradually becomes rounded (fig. 69, B, C). It grows rapidly, feeding on the contents of the host cell and living as a trophozoite (fig. 69, _D_). When the parasite is from 10 µ to 12 µ in diameter, usually multiplication by schizogony (fig. 69, E-H) begins. The nucleus of the parent cell, now called a schizont, divides into a number of portions that become arranged at the periphery (fig. 69, E). Cytoplasm collects around each nucleus (fig. 69, E, F) and gradually a group of daughter individuals (merozoites) is produced (fig. 69, G), the nucleus of each merozoite showing a karyosome. [Illustration: FIG. 68.--Small piece of epithelial lining of gut of heavily infected Grouse chick, showing various stages in life history of the parasite _Eimeria avium_; _par_, parasite (trophozoite); _mz_, merozoite; _sch_, schizont; _spz_, sporozoite; _ooc_, oöcyst; ♂, male gametocyte; ♀, female gametocyte. × 750. (After Fantham.)] The merozoites of _Eimeria avium_ are arranged “en barillet,” like the segments of an orange (figs. 68 _mz_, 69, G), therein differing from those of _E. schubergi_, which are arranged “en rosace.” They separate from one another (fig. 69, H), penetrate other epithelial cells, where they may, in turn, become schizonts. Eight to fourteen merozoites are usually formed by each schizont, twenty have been found, while in cases of intense infection when space has become limited, the number may be only four. After a number of generations of merozoites have been formed, a limit is reached both to the multiplicative capacity of the parasite and to the power of the bird to provide the invader with food. Consequently, resistant forms of the parasite are necessary, and the trophozoites begin to show sexual differentiation instead of forming schizonts, that is, gametogony commences. [Illustration: FIG. 69.--_Eimeria avium_. Diagram of life-cycle. For explanation see text. (After Fantham.)] Certain trophozoites store food and become large and granular. These are macrogametocytes (fig. 69, I, ♀). The microgametocytes (fig. 69, I, ♂) are smaller and far less granular. The macrogametocyte continues to grow, and becomes loaded with chromatoid and plastinoid granules (fig. 69, J, ♀), while the microgametocyte has its nucleus divide to form a number of bent, rod-like portions (fig. 69, J, ♂). The macrogametocyte gives rise to a single macrogamete, which forms a cyst wall for itself, leaving a thin spot (micropyle) for the entry of the male (fig 69, K, ♀). The microgametocyte gives rise to numerous small, biflagellate microgametes (fig. 69, K, ♂) around a large, central residual mass, from which they ultimately break free, and swim away. When a macrogamete is reached, the microgamete enters through the micropyle (fig. 69, L)--which then closes, thus excluding the other males--and applies itself to the female nucleus (fig. 69, M). Nuclear fusion occurs, the oöcyst (encysted zygote) being thus produced. Sporogony then ensues. The oöcyst (fig. 69, N) at first has its contents completely filling it. They then concentrate into a central spherical mass (fig. 69, O) which gradually becomes tetranucleate (fig. 69, P). Cytoplasm collects around each nucleus, and four sporoblasts are thus formed (fig. 69, Q). Each sporoblast becomes oval (fig. 69, R) and produces a sporocyst. Ultimately two sporozoites are formed in each sporocyst or spore, at first lying tête-bêche (fig. 69, S), but finally twisting to assume the position most convenient for emergence (fig. 69, T) when they reach a new host. The period of the life-cycle of _Eimeria avium_ (as well as the details of the life-cycle) was determined by Fantham to be from eight to ten days, of which period schizogony occupies four to five days. The method of infection[175] is contaminative, by way of food or drink. Young birds are especially susceptible to infection. Certain birds, particularly older ones, may act as reservoirs of oöcysts, being continuously infected themselves, without showing any marked ill effects from the parasite, but being highly infectious to other birds. Much moisture retards the development of sporocysts considerably. The duration of vitality of the infective oöcysts has been determined experimentally to extend well over two years, and in certain cases longer. _Eimeria avium_ is the causal agent of “white diarrhœa” or “white scour” in fowls, and of “blackhead” in turkeys. [175] Fantham, H. B. (1910), “Experimental Studies on Avian Coccidiosis, especially in relation to young Grouse, Fowls and Pigeons,” _Proc. Zool. Soc. Lond._, 1910, pp. 722–731, 1 plate. _Eimeria avium_ of birds and _E. stiedæ_ of rabbits closely resemble one another, but are not the same parasite, for _E. avium_ is not infective to rabbits, nor _E. stiedæ_ to poultry. *Eimeria stiedæ*, Lindemann, 1865. Syn.: _Monocystis stiedæ_, Lindemann, 1865; _Psorospermium cuniculi_, Rivolta, 1878; _Cytospermium hominis_, Rivolta, 1878; _Coccidium oviforme_, Leuckart, 1879; _Coccidium perforans_, Leuckart, 1879; _Coccidium cuniculi_. [Illustration: FIG. 70.--_Eimeria stiedæ_. Section through an infected villus of rabbit’s intestine. × 260.] _Eimeria stiedæ_ is parasitic in the gut epithelium (fig. 70), liver, and epithelium of the bile ducts of rabbits, and is usually considered to be the parasite very occasionally found in man. The life-cycle resembles that of _Eimeria avium_ in its general outlines (see fig. 69) and therefore will not be detailed in full here. The oöcysts (fig. 71) are large, elongate-oval, greenish in fresh preparations and vary in size from 24 µ to 49 µ long and 12·8 µ to 28 µ broad, the gut forms being usually smaller than those occurring in the liver, owing to the more confined space in which they are formed. Formerly, the parasites in the liver were described under the name of _Coccidium oviforme_, while those from the intestine were termed _Coccidium perforans_. This distinction has now broken down. [Illustration: FIG. 71.--_Eimeria stiedæ_, from the liver of the rabbit, oöcysts in various stages of development. (After Leuckart.)] [Illustration: FIG. 72.--_a_, _b_, spores of _Eimeria stiedæ_ (Riv.), with two sporozoites and residual bodies; _c_ represents a free sporozoite. (After Balbiani.)] [Illustration: FIG. 73.--So-called swarm cysts (endogenous sporulation or schizogony) of the Coccidium of the rabbit. The daughter forms are called merozoites. (After R. Pfeiffer.)] The oöcysts[176] are thick-walled, somewhat flattened at one pole, where a large micropyle is present. Four egg-shaped spores (sporocysts) are formed within, each about 12 µ to 15 µ long and 7 µ broad (fig. 72). The oöcysts are voided with the fæces. Sporogony takes, in nature, about three days in the excrement. Fæcal contamination of the food of rabbits results, and coccidian oöcysts are swallowed. Under the influence of the pancreatic juice of a new host, the sporozoites (fig. 72, _a_--_c_) are liberated from the spores and proceed to attack the epithelium and multiply within it, as in the case of _Eimeria avium_. From the gut, infection spreads to the liver, where multiplication of the parasite goes on actively, resulting in the formation of the whitish coccidial nodules, which may be very conspicuous (fig. 74). Proliferation of the connective tissue may occur around the coccidial nodules, which then contain large numbers of oöcysts in various stages of development. It is said that the oöcysts in the older nodules do not seem to be capable of further development. Schizogony (fig. 73) and gametogony in all stages can be found in both liver and gut. [176] For an account of the life-cycle of _Eimeria stiedæ_ consult Wasielewski, Th. von (1904), “Studien und Photogramme zur Kenntnis der pathogenen Protozoen,” Heft. 1 (Coccidia), 118 pp., 7 plates, Leipzig: J. A. Barth. Also, Metzner, R. (1903), _Arch. f. Protistenk._, ii, p. 13. Young rabbits often die of intestinal coccidiosis before infection of the liver occurs. The repeated schizogony of _Eimeria stiedæ_ in the gut is sufficient to cause death. [Illustration: FIG. 74.--_Eimeria stiedæ_. Section through coccidian nodule in infected rabbit’s liver. × 55.] The disease of cattle popularly known as “red dysentery” is also ascribed to the action of _Eimeria stiedæ_. The fæces of infected cattle show blood clots of various sizes and in severe cases watery diarrhœa is present. Acute cases end fatally in about two days. Numerous oöcysts, considered to be those of _Eimeria stiedæ_, occur in the fæces, and there is a heavy infection of the gut, especially the large intestine and rectum, all stages of the parasite being found in the epithelium. It is suspected that cattle contract the disease by feeding on fresh grass contaminated with oöcysts. The disease is recorded from Switzerland and from East Africa. As before mentioned, _Eimeria stiedæ_ is considered to be the organism found in a few cases in man, possibly acquired by eating the insufficiently cooked livers of diseased rabbits. These cases may now be described. (_a_) *Human Hepatic Coccidiosis.* (1) Gubler’s Case. A stone-breaker, aged 45, was admitted to a Paris hospital suffering from digestive disturbances and severe anæmia. On examination the liver was found to be enlarged and presented a prominent swelling, which was regarded as being due to Echinococcus. At the autopsy of the man, who succumbed to intercurrent peritonitis, twenty cysts were found averaging 2 to 3 cm. in diameter, and one measuring 12 to 15 cm. The caseous contents consisted of detritus, pus corpuscles, and oval-shelled formations, which were considered to be Distoma eggs, but which, in accordance with Leuckart’s conjecture, proved to be Coccidia.[177] [177] Gubler, A., “Tumeurs du foie déterm. par des œufs d’helm....” _Mem. Soc. Biol._, Paris, 1858, v, 2; and _Gaz. med. de Paris_, 1858, p. 657; Leuckart, R., _Die menschl. Paras._, 1863, 1ST edition, i, pp. 49, 740. (2) Dressler’s Case (Prague). Relates to three cysts, varying from the size of a hemp-seed to that of a pea, and containing Coccidia, found in a man’s liver.[178] [178] Leuckart, R., _Die menschl. Paras._, 1863, 1st edition, i, p. 740. (3) Sattler’s Case (Vienna). Coccidia were in this case observed in the dilated biliary duct of a human liver.[179] [179] Leuckart, R., _Die Paras. d. mensch._, 1879, 2nd edition, p. 281. (4) Perls’ Case (Giessen). Perls discovered Coccidia in an old preparation of Sömmering’s agglomerations.[180] [180] Leuckart, R., _ibid._, p. 282. (5) Silcock’s Case (London).[181] The patient, aged 50, who had fallen ill with serious symptoms, exhibited fever, enlarged liver and spleen, and had a dry, coated tongue. At the autopsy numerous caseous centres, mostly immediately beneath the surface, were found, while the contiguous parts of the liver were inflamed. Microscopical examination demonstrated numerous Coccidia in the hepatic cells as well as in the epithelium of the biliary ducts. A deposit of Coccidia was likewise found in the spleen, which the parasites had probably reached by means of the blood-stream.[182] [181] Silcock, “A Case of Parasit. by Psorospermia,” _Trans. Path. Soc._, London, 1890, xli, p. 320. [182] Pianese has confirmed the fact that Coccidia actually occur in the blood of the hepatic veins of infected rabbits. (_b_) *Human Intestinal Coccidiosis.* In two cadavers at the Pathological Institute in Berlin, Eimer[183] found the epithelium of the intestine permeated by Coccidia. Railliet and Lucet’s case may be traced back to intestinal Coccidia, which were found in the fæces of a woman and her child, who had both suffered for some time from chronic diarrhœa.[184] In other cases (Grassi, Rivolta), where only the existence of Coccidia in the fæces was known, it is doubtful whether the parasites originated in the intestine or in the liver. [183] _Die ei- u. kugelf. Psorosp. d. Wirbelt._, 1870, p. 16. [184] Railliet and Lucet, “Obs. s. quelq. Cocc. intest.,” _C. R. Soc. Biol._, Paris, 1890, p. 660; Railliet, _Trait. Zool. med. et agric._, 2e éd., 1895, p. 140. (_c_) *Doubtful Cases.* To these belong Virchow’s case[185] where, in the liver of an elderly woman, a thick walled tumour measuring 9 to 11 mm. was found. Among the contents of this tumour there were oval formations 56 µ long, surrounded by two membranes and enclosing a number of round substances. Virchow considered these foreign bodies to be eggs of pentastomes in various stages of development, others consider them to be Coccidia. [185] _Arch. f. path. An._, xviii, 1860, p. 523. The Coccidia which Podwyssotzki claims to have seen in the liver of a man, not only in the liver cells, but also in the nuclei, are also problematic.[186] The parasite was called _Caryophagus hominis_. [186] Podwyssotzki, “Ueb. d. Bedeut. d. Coccid. in d. Path. Leber des Menschen,” _Centralbl. f. Bakt._, vi, 1889, p. 41. Again, other explanations can be given to an observation by Thomas, on the occurrence of _Coccidium oviforme_ in a cerebral tumour of a woman aged 40. The growth was as large as a pea and surrounded by a bony substance.[187] [187] Thomas, J., “Case of Bone Formation in the Human Brain, due to the Presence of _Coccidium oviforme_,” _Journal Boston Soc. Med. Sc._, iii, 1899, p. 167; _Centralbl. f. Bakt._ [I] xxviii, 1900, p. 882. Genus. *Isospora*, Aimé Schneider, 1881. Syn.: _Diplospora_, Labbé, 1893. Belonging to the section _Disporea_, that is, forming only two spores, each with four sporozoites. *Isospora bigemina*, Stiles, 1891. Syn.: “_Cytospermium villorum intestinalium canis et felis_,” Rivolta, 1874; “_Coccidium Rivolta_,” Grassi, 1882; _Coccidium bigeminum_, Stiles, 1891. This parasite lives in the intestinal villi of dogs, cats, and the polecat (_Mustela putorius_, L.). According to Stiles,[188] the oöcyst divides into two equal ellipsoidal portions or sporoblasts which become spores and then each forms four sporozoites. The oöcysts of this species vary from 22 µ to 40 µ in length and from 19 µ to 28 µ in breadth. Each spore is 10 µ to 18 µ long and contains four sporozoites. The parasites live and multiply, not only in the gut epithelium, but also in the connective tissue of the intestinal submucosa. Wasielewski has seen merozoites in the gut of the cat. [188] “Notes on Paras.,” No. II, _Journ. of Comp. Med. and Vet. Sci._, 1892, xiii, p. 517. _Isospora bigemina_ (fig. 75) appears to occur also in man, for Virchow published a case which was communicated to him by Kjellberg, and attributed the illness to this parasite.[189] Possibly also it would be more correct to ascribe the observation of Railliet and Lucet, which is mentioned under “Human Intestinal Coccidiosis,” p. 148, to this species, as the Coccidia in that case were distinguished by their diminutive size (length 15 µ, breadth 10 µ). The case communicated by Grunow may also possibly refer to _Isospora bigemina_.[190] Roundish or oval structures of 6 µ to 13 µ in diameter occurred in the mucous membrane of the gut and in the fæces of a case of enteritis. [189] _Arch. f. path. An._, 1860, xviii, p. 527. [190] Grunow, “Ein Fall von Protozoën (Coccidien?) Erkrankung des Darmes,” _Arch. f. exper. Path. und Pharm._, 1901, xlv, p. 262. [Illustration: FIG. 75.--_Isospora bigemina_, Stiles (from the intestine of a dog). _a_, Piece of an intestinal villus beset with Isospora, slightly enlarged; _b_, _Isospora bigemina_ (15 µ in diameter), shortly before division; _c_, divided; _d_, each portion encysted forming two spores; _e_, four sporozoites in each part, on the left seen in optical section, together with a residual body--highly magnified. (After Stiles.)] DOUBTFUL SPECIES. In literature many other statements are found as to the occurrence of Coccidia-like organisms in different diseases of man. In some of the cases the parasites proved to be fungi. This was the case with the parasites of a severe skin disease of man, formerly called _Coccidioides immitis_ and _Coccidioides pyogenes_. Other statements are founded on misapprehensions, or are still much disputed. If reference is here made to “_Eimeria hominis_,” R. Blanchard, 1895, this is done on the authority of the investigator mentioned. The structures in question are nucleated spindle-shaped bodies of very different lengths (18 µ to 100 µ), which either occurred isolated or were enclosed in large globular or oval cysts, alone or with a larger tuberculated body (“residual body”). These formations were found by J. Künstler and A. Pitres in the pleural exudation removed from a man by tapping. The man was employed on the ships plying between Bordeaux and the Senegal River. Blanchard looks upon the fusiform bodies as merozoites and the cysts as schizonts of a Coccidium. On the other hand, Moniez declares the spindle bodies to be the ova and the supposed residual bodies to be “floating ovaries” of an Echinorhynchus. Severi’s “monocystid Gregarines,” which were taken from the lung tissue of a still-born child, are also quite problematical. No less doubtful are the bodies which Perroncito calls _Coccidium jalinum_, and which he found in severe diseases of the intestine in human beings, pigs, and guinea-pigs; Borini also reported another case. Order. *Hæmosporidia*, Danilewsky emend. Schaudinn. The Hæmosporidia are a group of blood parasites, comprising forms differing greatly among themselves. Some of the forms need much further investigation. However, there are certain true Hæmosporidia which present close affinities with the Coccidia, leading Doflein to use the term *Coccidiomorpha* for the two orders conjoined. The Hæmosporidia present the following general characteristics:-- (1) They are parasites of either red or white blood corpuscles of vertebrates during one period of their life-history. (2) They exhibit alternation of generations--asexual phases or schizogony alternating with sexual phases or sporogony--as do the Coccidia. (3) There is also an alternation of hosts in those cases which have so far been completely investigated. The schizogony occurs in the blood or internal organs of some vertebrates while the sporogony occurs in an invertebrate, such as a blood-sucking arthropod or leech. (4) Unlike the Coccidia, resistant spores in sporocysts are not generally produced, such protective phases in the life-cycle being unnecessary, as the Hæmosporidia are contained within either the vertebrate or invertebrate host during the whole of their life. The Hæmosporidia may be considered for convenience under five main types:-- (1) The _Plasmodium_ or _Hæmamœba_ type. This includes the malarial parasites of man and of birds. The asexual multiplicative or schizogonic phases occur inside red blood corpuscles and are amœboid. They produce distinctive, darkish pigment termed melanin or hæmozoin. Infected blood drawn and cooled on a slide may exhibit “exflagellation” of the male gametocytes, _i.e._, the formation of filamentous male gametes. The invertebrate host is a mosquito. The malarial parasites of man are discussed at length on p. 155. Similar pigmented hæmamœboid parasites have been described in antelopes, dogs, and other mammals, and even reptiles. (2) The _Halteridium_ type. The trophozoite stage inside the red blood corpuscle is halter-shaped. Pigment is produced, especially near the ends of the organism. The parasites occur in the blood of birds. The invertebrate host of _H. columbæ_ of pigeons in Europe, Africa, Brazil and India, is a hippoboscid fly, belonging to the genus _Lynchia_. Halteridium parasites are common in the blood of passerine birds, such as pigeons, finches, stone owls, Java sparrows, parrots, etc. The Halteridium embraces or grows around the nucleus of the host red cell without displacing the nucleus. Young forms and multiplicative stages of _H. columbæ_ have been found in leucocytes in the lungs of the pigeon (fig. 76, _8_-_12_). Male and female forms (gametocytes) are seen in the blood (fig. 76, _3a_, _3b_). The cytoplasm of the male gametocytes is pale-staining and the nucleus is elongate, while the cytoplasm of the females is darker and the nucleus is smaller and round. Formation of male gametes from male gametocytes (the so-called process of “exflagellation”) may occur on a slide of drawn infected blood, also fertilization, and formation of the oökinete, as first seen by MacCallum. The correct generic name for Halteridia is, apparently, _Hæmoproteus_. Wasielewski (1913), working on _H. danilewskyi_ (var. _falconis_), in kestrels, finds that the halteridium may be pathogenic to nestlings. The cycle of _H. noctuæ_ described by Schaudinn (1904) lacks confirmation. The account of the life-cycle of _H. columbæ_ given by Aragão (1908) is illustrated in fig. 76. It agrees with the work of Sergent (1906–7) and Gonder (1915). Mrs. Adie (1915) states that the cycle in _Lynchia_ is like that of a _Plasmodium_. [Illustration: FIG. 76--_Hæmoproteus_ (_Halteridium_) _columbæ_. Life-cycle diagram: 1, 2, stages in red blood corpuscle of bird; 3, 4, gametocytes (3_a_ ♂, 3b ♀); 5_a_, formation of microgametes; 6, fertilization (in fly’s gut); 7, oökinete; 8–12, stages in mononuclear leucocytes in lungs. (After Aragão.)] (3) The _Leucocytozoön_ type. The trophozoites and gametocytes occur within mononuclear leucocytes and young red cells (erythroblasts) in the blood of birds. Laveran and França consider that the Leucocytozoa occur in erythrocytes. The host cells are often greatly altered by the parasites, becoming hypertrophied and the ends usually drawn into horn-like processes (fig. 77), though some remain rounded. Leucocytozoa are limited to birds, and very rarely produce pigment. Male and female forms (gametocytes) are distinguishable in the blood (fig. 77), and the formation of male gametes (“exflagellation”) may occur in drawn blood. [Illustration: FIG. 77.--_Leucocytozoön lovati_. _a_, Male parasite (microgametocyte), within host cell, whose ends are drawn out; _b_, female parasite (macrogametocyte) from blood of grouse. × 1,800. (After Fantham.)] The Leucocytozoa were first seen by Danilewsky in 1884. They are usually oval or spherical. It is not easy sometimes to distinguish the altered host cell from the parasite, as the nucleus of the former is pushed to one side by the leucocytozoön. The cytoplasm of the female parasite stains deeply, and the nucleus is rather small, containing a karyosome. In the male the cytoplasm stains lightly and the nucleus is larger, with a loose, granular structure. Many species of Leucocytozoa are recorded, but schizogony has only been described by Fantham (1910)[191] in _L. lovati_ in the spleen of the grouse (_Lagopus scoticus_), and by Moldovan[192] (1913) in _L. ziemanni_ in the internal organs of screech-owls. [191] _Annals Trop. Med. and Parasitol._, iv, p. 255. [192] _Centralbl. f. Bakt._, Orig., lxxi, p. 66. M. and A. Leger[193] (1914) propose to classify Leucocytozoa, provisionally, according as the host cells are fusiform or rounded. [193] _Bull. Soc. Path. Exot._, vii, p. 437. (4) The _Hæmogregarina_ type. Included herein are many parasites of red blood corpuscles, with a few (the leucocytogregarines) parasitic in the white cells of certain mammals and a few birds. They are not amœboid but gregarine-like, vermicular or sausage-shaped (fig. 78). They do not produce pigment. They are widely distributed among the vertebrata, but are most numerous in cold-blooded vertebrates (fishes, amphibia and reptiles). The hæmogregarines of aquatic hosts are transmitted by leeches, those of terrestrial hosts by arthropods. The nucleus of hæmogregarines is usually near the middle of the parasite, but may be situated nearer one end. The body of the parasite may be lodged in a capsule (“cytocyst”). There is much variation in size and appearance among hæmogregarines. Some are small (_Lankesterella_); some attack the nucleus of the host cell (_Karyolysus_); others have full grown vermicules larger than the containing host corpuscle, and so the hæmogregarines bend on themselves in the form of *U* (fig. 78, _b_). Schizogony often occurs in the internal organs of the host, sometimes in the circulating blood. The hæmogregarines occurring in the white cells (mononuclears or polymorphonuclears) of mammals have been referred to a separate genus, _Leucocytogregarina_ (Porter) or _Hepatozoön_ (Miller). Such leucocytogregarines are known in the dog (fig. 79), rat, mouse, palm-squirrel, rabbit, cat, etc. Schizogony of these forms occurs in the internal organs, such as the liver, lung and bone-marrow of the hosts. They are apparently transmitted by ectoparasitic arthropods, such as ticks, mites, and lice. [Illustration: FIG. 78.--Hæmogregarines from lizards, _a_, _H. schaudinni_, var. _africana_, from _Lacerta ocellata_; _b_, _H. nobrei_ from _Lacerta muralis_; _c_, _H. marceaui_ in cytocyst, from _Lacerta muralis_. (After França.)] A few hæmogregarines are known to be parasitic in the red blood corpuscles of mammals. Such are _H. gerbilli_ in the Indian field rat, _Gerbillus indicus_; _H. balfouri_ (_jaculi_) in the jerboa, _Jaculus jaculus_, and a few species briefly described from marsupials. These parasites do not form pigment. Strict leucocytic gregarines have been described from a few birds by Aragão and by Todd. The sporogony of hæmogregarines is only known in a few cases, and in those affinity with the Coccidia is exhibited. In fact, the Hæmogregarines are now classified by some authors with the Coccidia. (5) The _Babesia_ or _Piroplasma_ type. These are small parasites of red blood corpuscles of mammals. They do not produce pigment. They are pear-shaped, round or amœboid in Babesia, bacilliform and oval in other forms referred to this group. Piroplasms are transmitted by ticks. These parasites are described at length on p. 172. [Illustration: FIG. 79.--_Leucocytogregarina canis_. Life-cycle diagram. Constructed from drawings by Christophers. (After Castellani and Chalmers.) Schizogony occurs in the bone-marrow. The parasite is transmitted from dog to dog by the tick, _Rhipicephalus sanguineus_, development in which, so far as known, is shown on the right.] THE MALARIAL PARASITES OF MAN. Malaria, otherwise known as febris intermittens, chill-fever, ague, marsh fever, paludism, etc., is the name given to a disease of man, which begins with fever. It has been known since ancient times and is distributed over almost all the world, although very unevenly, but does not occur in waterless deserts and the Polar regions. In many places, especially in the civilized countries of Central Europe, the disease is extinct or occurs only sporadically, and large tracts of land have become free from malaria. The rhythmical course of the fever is characteristic. It begins apparently suddenly with chilliness or typical shivering, whilst the temperature of the body rises, the pulse becomes low and tense and the number of beats of the pulse increases considerably. After half to two hours the heat stage begins. The patient himself feels the rise of his temperature (shown by feeling of heat, dry tongue, headache, thirst). The temperature may reach 41°C or more. At the same time there is sensitiveness in the region of the spleen and enlargement of that organ. After four to six hours an improvement takes place, and with profuse perspiration the body temperature falls rapidly, not often below normal. After the attack the patient feels languid, but otherwise well until certain prodromal symptoms (heaviness in the body, headache) which were not noticed at first, denote the approach of another attack of fever, which proceeds in the same way. The intervals between the attacks are of varying length which permit of a distinction in the kinds of fever. If the attacks intermit one day, occurring on the first, third and fifth days of the illness and always at the same time of day, it is termed _febris tertiana_; if two days occur between fever days, it is called _febris quartana_. In the case of the fever recurring daily, later writers speak of typical _febris quotidiana_. But a quotidian fever may arise when two tertian fevers differing by about twenty-four hours exist at the same time (_febris tertiana duplex_). The patient has a daily attack, but the fever of the first, third and fifth days differs in some point (hour of occurrence, height of temperature, duration of cold or hot stage) from the fever of the second, fourth and sixth days. Similarly, two or three quartan fevers which differ by about twenty-four hours each may be observed together (_febris quartana duplex_ or _triplex_); in the latter case the result is also a quotidian fever. Two kinds of tertian fever are differentiated--a milder form occurring especially in the spring (spring tertian fever), and a more severe form appearing in the summer and autumn in warmer districts, especially in the tropics (_summer or autumn fever_, _febris æstivo-autumnalis_, _febris tropica_, _febris perniciosa_). The latter often becomes a quotidian fever. All the afore-mentioned infections are termed acute. They are distinguished from the very different _chronic malarial infection_ by the frequent occurrence of relapses, which finally lead to changes of some organs and particularly of the blood. The relapses are then generally marked by an irregular course of fever. The term masked malaria is used when any disturbance of the state of health of a periodic character shows itself and disappears after treatment with quinine.[194] Generally it is a question of neuralgia. [194] Quinine is still almost exclusively the remedy used in the treatment of malaria. It is prepared from the bark of the cinchona tree. This important remedy was introduced into Europe in 1640 from Ecuador by Juan del Vego, physician of the Countess del Cinchon. That intermittent fever was an infectious disease, although not one which was transmitted direct from man to man, had been assumed for a long time. Therefore it was natural, at a time when bacteriology was triumphing, to look for a living agent causing infection in malaria, which search was, seemingly, successful (Klebs, Tomasi-Crudeli, 1879). Hence it was not surprising that the discovery of the real malarial parasites in November, 1880, by the military doctor A. Laveran[195] in Constantine (Algeria), at first met with violent opposition, even after Richard (1882) had confirmed it and Marchiafava, Celli, Grassi and others, had further extended it. Not that the existence of structures found in the blood of malaria patients by Laveran and Richard was denied; on the contrary, the investigations of the opponents furnished many valuable discoveries, but the organisms were differently interpreted and considered to be degeneration products of red blood corpuscles. Only when Marchiafava and Celli (1885) saw movements in the parasites, which Laveran called _Oscillaria malariæ_ and later _Hæmatozoön malariæ_, was their animal nature admitted and the parasites were named _Plasmodium malariæ_. Shortly before this, Gerhardt (1884) had stated that the disease could be transmitted by the injection of the blood of a malarial patient to a healthy person. [195] The discovery of Laveran is in no way lessened by the fact that one investigator or another (according to Blanchard [_Arch. de Paras._, vii, 1903, p. 152], P. F. H. Klencke in 1843) had seen, mentioned and depicted malarial parasites. (_Neue phys. Abhandl. auf. selbständ. Beob. gegr._, Leipzig, 1843, p. 163, fig. 25). In 1847 Meckel had recognized that the dark colour of the organs in persons dead of malaria was due to pigment. Virchow in 1848 stated that this pigment occurred in blood cells. Kelsch in 1875 recognized the frequency of melaniferous leucocytes in the blood of malarial patients. Beauperthuy (1853) noticed that in Guadeloupe there was no malaria at altitudes where there were no “insectes tipulaires,” and suggested that the disease was inoculated by insects. This supplied the starting point for further investigations, which were made not exclusively, but principally, by Italian investigators (Golgi, Marchiafava and Celli, Bignami and Bastianelli, Grassi and Feletti, Mannaberg, Romanowsky, Osier, Thayer and others). In 1885 Golgi described the asexual cycle in the blood, in the case of the quartan parasite. These investigations, after attention had been drawn by Danilewsky (1890) to the occurrence of similar endoglobular parasites in birds, were extended to the latter (Grassi and Feletti, Celli and Sanfelice, Kruse, Labbé and others). The result was as follows: Malaria in man (and birds) is the result of peculiar parasites included in the _Sporozoa_ by Metchnikoff, which parasites live in the erythrocytes, grow in size and finally “sporulate,” that is, separate into a number of “spores” which leave the erythrocytes and infect other blood corpuscles. Morphologically and biologically several species (and respectively several varieties) of malarial parasites may be distinguished, on which the different intermittent forms depend. Transmission of the blood of patients to healthy people produces a malarial affection which corresponds in character to the fever of the patient from whom the inoculation was made. The combined types of fever (tertiana duplex, quartana duplex or triplex) are explained by the fact that the patient harbours two or three groups of parasites which differ in their development by about twenty-four hours, whilst the irregular fevers depend on deviation from the typical course of development of the parasites. In addition to stages of the parasites which could easily be arranged in a developmental series concurrent with the course of the disease, other phases of the parasites also became known, such as spheres, crescents, polymitus forms, which seemed not to be included in the series and, therefore, were very differently interpreted. The decision reached at the beginning of the last decade of the last century, which found expression in comprehensive statements (Mannaberg, Ziemann and others), only concerned a part of the complete development of the malarial parasites. No one could with any degree of certainty demonstrate how man became infected, nor were there reliable hypotheses based on analogy with other parasites concerning the exit of the excitants of malaria from the infected person and their further behaviour. Numerous hypotheses had been advanced, but none was able to elucidate the various observations made from time to time in dealing with malaria. One hypothesis only seemed to have a better foundation. Manson (1894), who knew from his own experience the part played by mosquitoes in the development of Filaria from the blood of man, applied this also to the malarial parasites living in the blood, whereby at least the way was indicated by which the Hæmosporidia could leave man. The parasites were said finally to get into water through mosquitoes which had sucked the blood of malarial patients, and the germ spread thence to men who drank the water. In some cases the parasites were supposed to reach man by the inhaling of the dust of dried marshes. On the other hand, Bignami believed that the mosquitoes were infected in the open air by malarial parasites which occurred there in an unknown stage and the insects transmitted the germs to man when biting. R. Koch combined both hypotheses, without, however, producing positive proof. R. Ross, then (1897–8) an English military doctor in India, was the first to succeed in this. He had been encouraged by Manson to study the fate of malarial _Plasmodia_ which had entered the intestine of mosquitoes with malaria-infected blood, especially in the case of the _Plasmodium_ (_Proteosoma_) living in the blood of birds. He showed that the _Proteosoma_ penetrate the intestinal wall of the mosquitoes, grow and develop into large cysts which produce innumerable rod-like germs, which burst into the body cavity and penetrate the salivary glands. Ross allowed mosquitoes to suck the blood of birds affected by malaria, and some nine days later, let the infected mosquitoes which had been isolated suck healthy birds. After five to nine days _Proteosoma_ were found to occur in the blood of the birds used. The _Proteosoma_ and _Halteridium_ of birds were also further investigated by MacCallum (1897–8), Koch and others, and important results followed. In any case Ross (1898) had clearly established the importance of mosquitoes in the spread of malaria among birds. It was now only a question of proving whether, and how far, mosquitoes were concerned with human malaria. Ross himself worked to this end. Here the experiments of Italian investigators (Bignami, Bastianelli, Grassi)[196] were of importance. These investigators studied the fate of malarial parasites in man, produced malaria in men experimentally by the bites of infected mosquitoes, and established that only mosquitoes belonging to the genus _Anopheles_ were concerned, and not species of _Culex_. These latter are only able to transmit _Proteosoma_ to birds. It is true that _Culex_ can ingest the human malarial parasites, but the latter do not develop in them. Development only occurs in species of _Anopheles_. In _Anopheles_ (and similarly for _Proteosoma_ in _Culex_) sexual reproduction takes place; crescents, spheres and polymitus forms are necessary stages of development in the mosquito. [196] Grassi, B. (1901), “Die Malaria,” 250 pp., 8 plates. G. Fischer, Jena. With these discoveries the campaign against malaria became more definite. It was directed partly against the transmitters, whose biology and life-cycle were more thoroughly investigated, instead of merely against the infection of the adult _Anopheles_. The latter do not, as was believed for some time, transmit the malarial germs to their offspring. They always infect themselves from human beings, whereby the relapses appearing in early summer, and the latent infection, especially of children of natives, play a principal part (Stephens and Christophers, Koch). Further, the crusade was directed against the infection of man by the bites of _Anopheles_. Important results have been obtained in these directions. Low and Sambon in 1900 lived in a mosquito-screened hut in a malarial part of the Roman Campagna for three of the most malarious months and did not contract the disease. In the same year Dr. P. T. Manson was infected with malaria by infected mosquitoes sent from Italy. The rôle of mosquitoes having been proved, it may be hoped that ultimately the eradication of malaria, or at least a considerable restriction of it, will be achieved. It is of importance to record that, although malarial parasites occur in mammals (monkeys, bats, etc.) the human ones are not transmissible to mammals, not even to monkeys. The species, therefore, are specific to the different hosts (Dionisi, Kossel, Ziemann, Vassall). An important work dealing with the modern applications of the mosquito-malaria theory in all parts of the Tropics was published by Sir Ronald Ross in 1911. It is entitled “The Prevention of Malaria” (John Murray, London, 21s.). DEVELOPMENT OF THE MALARIAL PARASITES OF MAN. The commencement of the developmental cycle and of the infection of man, is the sporozoites (fig. 80, _1_) which are passed into the blood of a person by the bite of an infected mosquito. Prior to this the parasites collect in the excretory ducts of the salivary glands (fig. 80, _27_) of the _Anopheles_. The sporozoites are elongate and spindle-shaped, 10 µ to 20 µ long and 1 µ to 2 µ broad, with an oval nucleus situated in the middle. They are able to glide, perform peristaltic contractions, or curve laterally. Schaudinn has studied the penetration of the red blood corpuscles (fig. 80, _2_) by the sporozoites in the case of the living tertian parasite. The process takes forty to sixty minutes in drawn blood. After its entrance the parasite, which is now called a trophozoite, contracts, and becomes an active amœbula (fig. 80, _3_). It develops a food vacuole and grows at the expense of the invaded blood corpuscle (fig. 80, _4_), which is shown by the appearance of pigment granules (transformed hæmoglobin) in it. When the maximum size is attained, multiplication by schizogony (fig. 80, _5_-_7_) begins with a division of the nucleus, which is followed by further divisions of the daughter nuclei, the number of which varies up to 16 or even 32, depending on the species of the parasite. Then the cytoplasm divides into as many portions as there are nuclei, the result being a structure suggestive of the spokes of a wheel or of a daisy, the centre of the resulting rosette being occupied by dark pigment. Finally, the parts separate from one another, leaving behind a residual body containing the pigment, and the daughter forms issue into the blood plasma as merozoites (fig. 80, _7_). They are actively amœboid (fig. 80, _8_) and soon begin to enter other blood corpuscles of their host, for the entry into which thirty to sixty minutes are necessary, according to Schaudinn’s observations.[197] [197] It should be remembered that some authors (Laveran, Argutinsky, Panichi, Serra) argue against the intra-globular position of malarial parasites and state that they only adhere outwardly to the red blood corpuscles. These views have recently been revived by Mary Rowley-Lawson, and she states that the malarial parasite is “extracellular throughout its life-cycle and migrates from red corpuscle to red corpuscle destroying each before it abandons it.” (_Journ. Exper. Med._, 1914, xix, p. 531.) Here they behave like sporozoites which previously entered and again produce merozoites. This process is repeated until the number of parasites is so large that, at the next migration of the merozoites, the body of the person infected reacts with an attack of fever,[198] which is repeated with the occurrence of the next or following generations. [198] The incubation period, that is, the time between infection and the first attack of fever, is ten to fourteen days; with severe infection fewer days (minimum 5 to 6) are needed. [Illustration: FIG. 80.--Life-cycle of the tertian parasite (_Plasmodium vivax_). Figs. 1 to 17, × 1,200; figs. 18 to 27, × 600. (After Lühe, based on figures by Schaudinn and Grassi.) 1, sporozoite; 2, entrance of the sporozoite into a red blood corpuscle; 3, 4, growth of the parasite, now sometimes called a trophozoite; 5, 6, nuclear division in schizont; 7, free merozoites; 8, the merozoites which have developed making their way into blood corpuscles, (arrow pointing to the left) and increase by schizogony (3–7); after some duration of disease the sexual individuals appear; 9_a_-12_a_, macrogametocytes; 9_b_-12_b_, microgametocytes, both still in the blood-vessels of man. If macrogametocytes (12_a_) do not get into the intestine of _Anopheles_ they may perhaps increase parthenogenetically according to Schaudinn (12_a_; 13_c_-17_c_). The merozoites which have arisen (17_c_) become schizonts 3–7. The phases shown underneath the dotted line (13–17) proceed in the stomach of _Anopheles_. 13_b_ and 14_b_, formation of microgametes; 13_a_ and 14_a_, maturation of the macrogametes; 15_b_, microgamete; 16, fertilization; 17, oökinete; 18, oökinete in the walls of the stomach; 19, penetration of the epithelium of the stomach; 20–25, stages of sporogony on the outer surface of the intestinal wall; 26, migration of the sporozoites to the salivary gland; 27, salivary gland with sporozoites.] The growth and schizogony last different times, according to the species of the parasite, about forty-eight hours in the case of the parasite of febris tertiana or tropica, and seventy-two hours for the quartan parasite. The various intermittent forms produced by them depend on this specific difference in the malarial parasites. The schizogony can, however, only be repeated a certain number of times, supposing that the disease has not been checked prematurely by the administration of quinine, which is able to kill the parasites. It appears that after a number of attacks of fever the conditions of existence in man are unfavourable for the malarial parasites, and this brings about the production of other forms which have long been known, but also long misunderstood (spheres, crescents, polymitus). The merozoites in this case no longer grow into schizonts, or at least not all of them, but become sexual individuals called gametocytes (fig. 80, _9_-_12_), which only start their further development when they have reached the intestine of _Anopheles_. This does not take place in every case, nor with all the gametocytes which exist in the blood of patients with intermittent fever. Of those parasites which remain in the human blood the male ones (microgametocytes) soon perish, the females (macrogametocytes) persist for some long time, and perhaps at last acquire the capacity of increasing by schizogony. They might thus form merozoites which behave in the body as if they had proceeded from ordinary schizonts (fig. 80, _13c_-_17c_). If their number increases sufficiently, in course of time the patient, who was apparently recovering, has a new series of fever attacks, or relapses, without there having been a new infection. This is the view of Schaudinn, who from researches of his own concluded that relapses were brought about by a sort of parthenogenetic reproduction of macrogametocytes. R. Ross, on the contrary, believes that in the relatively healthy periods the number of parasites in the blood falls below that necessary to provoke febrile symptoms; relapses then result merely from increase in the numbers of the parasites present in the individual. Ross’s view is now generally accepted. [Illustration: FIG. 81.--Stages of development of pernicious or malignant tertian parasites in the intestine of _Anopheles macultpennis_. (After Grassi.) _a_, macrogametocyte (crescent) still attached to human blood corpuscles; _b_, macrogametocyte (sphere) half an hour after ingestion by the mosquito; _c_, microgametocyte (crescent) attached to the blood corpuscle; _d_, microgametocyte (sphere) half an hour after ingestion; the nucleus has divided several times; _e_, microgametes attached to the residual body (polymitus stage).] [Illustration: FIG. 82.--Oökinete of the malignant tertian parasite in the stomach of _Anopheles maculipennis_, thirty-two hours after ingestion of blood. (After Grassi.)] If the gametocytes, which are globular, or in the pernicious or malignant tertian parasite crescentic (fig. 81), gain access to the intestine of an Anopheline,[199] they mature. The macrogametocytes extrude a part of their nuclear substance (fig. 80, _13a_, _14a_) and thereby become females or macrogametes. The microgametocytes, on the other hand, undergo repeated nuclear division, preparation for this being made apparently whilst in the blood of man. This results in the formation of threadlike bodies which move like flagella and finally detach themselves from the residual body (fig. 80, _13b_, _14b_). These are the males or microgametes[200] (fig. 80, _15b_). [199] Schizonts ingested about the same time perish in the intestine of the mosquito. [200] If the microgametocytes are sufficiently mature the formation of microgametes occurs in the blood of man as soon as it is taken from the blood-vessel and has been cooled and diluted. Such a stage is called a _Polymitus_ form, and the process has been called “exflagellation.” Copulation takes place in the stomach of the Anopheline (fig. 80, _16_). A microgamete penetrates a macrogamete and coalesces with it. The fertilized females elongate very soon and are called oökinetes or “vermicules” (figs. 80, _17_; 82). They penetrate the walls of the stomach, pierce the epithelium (fig. 80, _18_, _19_), and remain lying between it and the superficial stratum (tunica elastico-muscularis). Then they become rounded and gradually develop into cysts which grow larger and are finally visible to the naked eye, being called oöcysts (figs. 80, _20_-_24_; 83). Their size at the beginning is about 5 µ, the maximum that they attain is 60 µ, only exceptionally are they larger. [Illustration: FIG. 83.--Section of the stomach of an _Anopheles_, with cysts (oöcysts) of the malignant tertian parasite. (After Grassi).] The sporulation (figs. 80, _21_-_25_; 84), which now follows, begins with repeated multiple fission of the nucleus. Long before the definitive number of nuclei, which varies with the individual, is attained the protoplasm, according to Grassi, begins to segment around the individual large nuclei but without separating completely into cell areas. According to Schaudinn, however, there is a condensation of the outstanding protoplasmic strands. It is certain that the number of nuclei increases with simultaneous decrease in size. They soon appear on the surface of the strands or sporoblasts, surround themselves with some cytoplasm and then elongate (fig. 84). In this manner the sporozoites are formed and break away from the unused remains of the cytoplasmic strands of the sporoblasts (fig. 80, _26_). The number of the sporozoites in an oöcyst varies from several hundreds to ten thousand. [Illustration: FIG. 84.--Four different sporulation stages of malarial parasites from _Anopheles maculipennis_, much magnified. _a_-_c_, of the malignant tertian parasite; _a_, four to four and a half days after sucking; _b_ and _c_, five to six days after sucking; _d_, of the tertian parasite, eight days after sucking. (After Grassi.)] The sporulation is influenced in its duration by the external temperature (Grassi, Jansci, Schoo). In the tertian parasite it takes place quickest at a temperature of 25° to 30° C. and takes eight to nine days. A temperature a few degrees lower has a retarding effect (eighteen to nineteen days at 18° to 20° C). A still lower one has a restraining or even destructive effect. Temperatures over 35° C. also exercise a harmful effect. The malignant tertian parasite seems to need a somewhat higher temperature and the quartan parasite a lower one. The sporozoites of the various malarial parasites show no specific differences. They were stated by Schaudinn to occur in three forms, and these were described as indifferent (neuter), female and male. There is, however, little or no evidence for this hypothetical differentiation. The last were said to perish prematurely, that is, in the oöcyst. The others after the rupture of the oöcysts enter the body cavity of the Anophelines, whence they are carried along in the course of the blood. Finally they penetrate the salivary glands (fig. 80, _27_) probably by their own activity, break through their epithelia and accumulate in the salivary duct (fig. 80, _27_). At the next bite by the mosquito they are transmitted to the blood-vessels of man. THE SPECIES OF MALARIAL PARASITES OF MAN. In view of the differences in opinion regarding “species” and “varieties,” the dispute whether the malarial parasites of man represent one species with several varieties, or several species is almost superfluous. If necessary two genera may be distinguished. The parasites of the tertian and quartan fever are alike in that their gametocytes have a rounded shape (figs. 80, _12_, _13_), whilst the corresponding stages of the pernicious or malignant tertian parasites are crescentic (figs. 81, 88). These differences are used by some writers as the distinguishing characteristic of two genera: _Plasmodium_, Marchiafava and Celli, 1885, for the first mentioned species; _Laverania_, Grassi and Feletti, 1889, for the pernicious or malignant tertian parasite. Whether there is a genuine quotidian fever and accordingly a special quotidian parasite is still disputed. These parasites are treated in practical detail in Stephens and Christophers’ “Practical Study of Malaria,” 3rd edition, 1908. *Plasmodium vivax*, Grassi and Feletti, 1890. Syn.: _Hæmamœba vivax_, Grassi and Feletti, 1890; _Plasmodium malariæ_ var. _tertianæ_, Celli and Sanfelice, 1891; _Hæmamœba laverani_ var. _tertiana_, Labbé, 1894; *Hæmosporidium tertianum*, Lewkowitz, 1897; _Plasmodium malariæ tertianum_, Labbé, 1899: _Hæmamœba malariæ_ var. _magna_, Laveran, 1900, p.p.; _Hæmamœba malariæ_ var. _tertianæ_, Laveran, 1901. This species, _P. vivax_,[201] is the causal agent of the simple or spring tertian fever and is, therefore, named directly the tertian or benign tertian parasite (figs. 80, _3_-_8_; 85). During the afebrile period in the patient, the young trophozoites or amœbulæ appear on or in the red blood corpuscles as pale bodies of 1·5 µ to 2 µ diameter which at first show only slow amœboid movements. Their nucleus is difficult to recognize in the early stage. Soon the food vacuole is formed and this grows concomitantly with the trophozoite and the parasite has a ring-like appearance. Afterwards the vacuole diminishes, and at this period the first brownish melanin granule appears. From this time the activity and number of the pigment granules increase with continuous growth. When the parasite has grown to about one-third the diameter of the erythrocyte the latter shows characteristic red Schüffner’s dots or “fine stippling,” after staining with Romanowsky’s solution. Later, after about twenty-four hours, the blood corpuscles begin to grow pale, then to increase in size, and after thirty-six hours, that is, about twelve hours before the next attack of fever, schizogony of the parasite is initiated by the division of the nucleus. The parasite at this time occupies half to two-thirds of the enlarged blood corpuscle. The daughter nuclei continue dividing until sixteen, and occasionally twenty-four, daughter nuclei are produced. The pigment which, up till now lies nearer the periphery, moves to the middle, while the nuclei lie nearer the surface. [201] See Schaudinn, F. (1902), _Arb. a. d. kaiserl. Gesundheits._, xix, pp. 169–250, 3 plates. [Illustration: FIG. 85.--Development of the tertian parasite in the red blood corpuscles of man; on the right a “Polymitus.” (After Mannaberg.) See also fig. 80, _3_--_7_.] Around each nucleus a portion of cytoplasm collects and thus young merozoites are produced. These separate from each other and from the little residual masses[202] which contain the melanin and pass from the blood corpuscles, which now can hardly be recognized, to the blood plasma, where they soon attack new erythrocytes. [202] The pigment masses (melanin or hæmozoin) are taken up by the leucocytes, particularly the mononuclear ones, and are carried especially to the spleen, and also to the liver and the bone-marrow. From this circumstance arises the well-known pigmentation of the spleen in persons who have suffered from malaria. The migration of the merozoites initiates a new attack of fever and two groups of tertian parasites in the blood, differing in development by about twenty-four hours, are the conditions for febris tertiana duplex. After a lengthy duration of fever the gametocytes (figs. 80, _9_--_12_) appear. They are uninucleate. The microgametocytes are about the size of fully developed schizonts, the macrogametocytes are somewhat larger. Their further development takes place in Anophelines. The chief distinctive characteristics of the simple tertian parasite, as seen in infected blood, are:--(1) The infected red-cell is usually enlarged; (2) the presence of fine red granules known as Schüffner’s dots in the red blood corpuscles, after Romanowsky staining; (3) the fragile appearance of the parasite compared with other species. Large forms are pigmented, irregular and “flimsy-looking,” sometimes appearing to consist of separate parts. Irregularity of contour is common. Ahmed Emin[203] (1914) has described a small variety of _P. vivax_. [203] _Bull. Soc. Path. Exot._, vii, p. 385. *Plasmodium malariæ*, Laveran. Syn.: _Oscillaria malariæ_, Laveran, p.p., 1883; _Hæmamœba malariæ_, Gr. et Fel., 1890; _Plasmodium malariæ_ var. _quartanæ_, Celli et Sanfel., 1891; _Hæmamœba laverani_ var. _quartana_, Labbé, 1894; _Hæmosporidium quartanæ_, Lewkowitz, 1897; _Plasmodium malariæ quartanum_, Labbé, 1899; _Plasmodium golgii_, Sambon, 1902; _Laverania malariæ_, Jancso, 1905 nec Grassi et Fel. 1890; _Hæmomœba malariæ_ var. _quartanæ_; Lav., 1901. _Plasmodium malariæ_ is the parasite of quartan malaria (fig. 86). The trophozoites of the quartan parasite differ from the corresponding stages of the tertian parasite in that their motility is less and soon ceases. They differ also in their slower growth, by the early disappearance of the food vacuole, by the more marked formation of the dark brown pigment, and by the fact that the red blood corpuscles attacked are not altered either in colour or size. [Illustration: FIG. 86.--Development of the quartan parasite in the red corpuscles of man--asexual stages. (After Manson.)] When the parasites have grown almost to the size of the erythrocytes schizogony occurs. The pigment granules arrange themselves in lines radiating towards the centre and the merozoites are also radially disposed in groups of 6, 8, 10 or even 12, but are often arranged less regularly. The whole development, growth and schizogony, occupies seventy-two hours. The appearance of quartana duplex or triplex is conditional on the presence in the blood of the patient of two or three groups of _Plasmodia_ differing in their development by twenty-four hours. The chief distinctive characters of the quartan parasite are: (1) The erythrocyte is unchanged in size; (2) the rings are compact and show pigment early; in the larger forms the chromatin is dense and relatively plentiful; (3) the pigment, which is relatively well-marked, may be arranged at the periphery. *Laverania malariæ*, Grassi and Feletti, 1890 = *Plasmodium falciparum*, Welch, 1897. Syn.: _Plasmodium malariæ_ var. _quotidianæ_, Celli et Sanf., 1891; _Hæmamœba malariæ præcox_, Gr. et Fel., 1892 (nec _H. præcox_, Gr. et Fel., 1890); _Hæmamœba laverani_, Labbé, 1894; _Hæmatozoön falciparum_, Welch, 1897; _Hæmosporidium undecimanæ_ and _H. sedecimanæ_ and _H. vigesimo-tertianæ_, Lewkowitz, 1897; _Hæmamœba malariæ parva_, Lav., 1900; _Plasmodium præcox_, Dofl., 1901; _Plasmodium immaculatum_, Schaud., 1902; _Plasmodium falciparum_, Blanch., 1905. The names most commonly used for the parasite of malignant tertian malaria are _Plasmodium falciparum_ and _Laverania malariæ_. The summer and autumn fever (_febris æstivo-autumnalis_), also called malignant tertian or sub-tertian, is caused by a malarial parasite which is distinguished by the small size of its schizont, while the gametocytes are crescentic (figs. 81, 88). Most authors identify this kind of fever or the parasites which cause it (_Laverania malariæ_) with the pernicious malaria of the tropics. Ziemann, however, repeatedly has drawn attention to certain small but definite differences between the usual malignant tertian or pernicious parasites which occur in the tropics and the tropical parasites of some malarial districts, particularly of West Africa, and insists that at least two varieties or sub-species occur. Other investigators distinguish from this or these forms a quotidian parasite. On the other hand, the assertion is made that there are no specific differences, but that the malignant or pernicious tertian parasite which normally needs forty-eight hours for its development in the blood of man, can also develop in twenty-four hours. The establishment of the duration of the development is a matter of especial difficulty, because the stages of schizogony are far less numerous in the peripheral blood than in that of the internal organs. It is also stated that the tropical parasite very seldom forms crescentic but rather rounded gametocytes. According to such an observation the organism would belong to _Plasmodium_ and not to _Laverania_. The question whether the tropical fevers are caused by two different parasites does not seem to be definitely settled. The young trophozoite of the malignant, pernicious tertian, or sub-tertian parasite (fig. 87) are but slightly active and are very small, even after the formation of the comparatively large food vacuole, which makes the body appear annular (“signet ring” stage). Often two and even more parasites are found in one blood corpuscle. Fully grown they only attain two-thirds or less of the diameter of the erythrocytes, which display an inclination to shrink and then appear darker than the normal (brass-coloured). In the early stage dots or stippling--sometimes called Maurer’s dots--appear on the blood corpuscles as in those attacked by the ordinary tertian parasite (_Plasmodium vivax_), but the Maurer’s dots are relatively coarse and few, and are not easily stained. These dots were first described by Stephens and Christophers in 1900, and subsequently by Maurer in 1902. About thirty hours after the entrance into the blood corpuscles, the parasites are rarely found in the peripheral blood, but they are present in the internal organs, and especially in the spleen. The schizogony, which now begins in the internal organs, proceeds on the same lines as that of the quartan parasite, that is, usually with the merozoites radially arranged around a central agglomeration of dark brown pigment. [Illustration: FIG. 87.--The pernicious malignant or sub-tertian parasite in the red corpuscles of man, asexual stages. (After Manson.)] The number of merozoites formed is quoted differently, _e.g._, 8 to 24, on an average 12 to 16. However, according to the recent cultural researches of J. G. and D. Thomson[204] (1913) the number of merozoites of _P. falciparum_ is 32. D. Thomson, from examination of spleen smears at autopsy, also concludes that the number of merozoites may reach 32. During their formation the blood corpuscle which is attacked gets paler and disintegrates. [204] _Proc. Roy. Soc._, B, lxxxvii, p. 77. [Illustration: FIG. 88.--The crescents of the malignant tertian parasite. (After Mannaberg.) See also fig. 81.] The gametocytes which finally appear are attenuated, curved bodies, rounded at each end and known as crescents (figs. 81, 88), and are provided with a nucleus and with coarse pigment masses. In the males the pigment is more scattered than in the females, where it is around the nucleus. Their length is 9 µ to 14 µ, and their breadth is 2 µ to 3 µ. At first they are still in the pale blood corpuscles, later they free themselves and are found in numbers in the peripheral blood in cases of pernicious malaria of Southern Europe and the tropics, while, on the other hand, they occur much more rarely in the peripheral blood in West African malignant tertian. Their further development takes place under the same conditions as in the other malarial parasites. [Illustration: FIG. 89.--Section through a tubule of the salivary gland of an _Anopheles_ with sporozoites of the malignant tertian parasites; on the left at the top a single sporozoite greatly magnified. (After Grassi).] D. Thomson (1914),[205] from studies of autopsy smears, has shown that crescents develop chiefly in the bone-marrow and spleen, and take about ten days to grow into the adult state in the internal organs. He believes that crescents are produced from ordinary asexual spores. Quinine, he states, has no direct destructive action on crescents, but it destroys the asexual source of supply. [205] _Annals Trop. Med. and Parasitol._, viii, p. 85. The sporozoites of _Laverania malariæ_ (_P. falciparum_) are represented in fig. 89. The principal distinctive characters of the malignant tertian parasite are: (1) The ring forms are very small, occasionally bacilliform, and may be marginal (“accolé” of Laveran); (2) the larger trophozoites are often ovoid, and about one-third or one-half of the erythrocyte in size; (3) the infected red cells sometimes show coarse stippling (Maurer’s dots); (4) the gametocytes, or sexual forms, are crescentic in shape. J. W. W. Stephens (1914) has described a new malarial parasite of man; it is called _Plasmodium tenue_. It is very amœboid, with scanty cytoplasm and much chromatin, sometimes rod-like or irregular. The parasite was described from a blood-smear of an Indian child. The creation of a new species for this parasite has been criticized by Balfour and Wenyon, and by Craig. *Plasmodium relictum*, Sergent, 1907. Syn.: _Plasmodium præcox_, Grassi and Feletti, 1890; _Plasmodium danilewskyi_, Gr. et Fel., 1890; _Hæmamœba relicta_, Gr. et Fel., 1891; _Proteosoma grassii_, Labbé, 1894. Hæmamœboid, pigment-producing, malarial parasites are often found in birds. Like the human malarial parasites they have been variously named. Labbé created the genus _Proteosoma_ for them, and this name is still often used as a distinctive one unofficially. The correct name is stated to be either _Plasmodium relictum_ or _P. præcox_, or possibly even _P. danilewskyi_, assuming that there is only one species. The nomenclature of the malarial parasites is most confused. The avian malarial parasites are transmitted by Culicine mosquitoes. The organism was discovered by Grassi in the blood of birds in Italy, and causes a fatal disease in partridges in Hungary. Sparrows are affected in India, and it was this Plasmodium in which Ross first traced the development of a malarial parasite in a mosquito. The parasite may be transmitted from bird to bird by blood-inoculation, canaries being very susceptible. The principal stages of the avian plasmodium closely resemble those of the malarial parasites of man. In its earliest stage _P. relictum_ is unpigmented, but soon the trophozoite grows and becomes pigmented, meanwhile displacing the nucleus of the avian red-blood corpuscle, a characteristic feature, distinguishing it from _Halteridium_. Schizonts are formed, each of which gives rise to about nine merozoites in the circulating blood. Sexual forms or gametocytes also occur in the blood. These develop in _Culex fatigans_, _C. pipiens_ and _C. nemorosus_. Oökinetes or vermicules are formed in twelve to fifteen hours in the stomach of the mosquito, and in one to two days well-developed round oöcysts may be seen. In three to four days sporoblasts have formed within the oöcysts and young sporozoites begin to develop. In nine to ten days the oöcysts are mature, being filled with sporozoites. The oöcysts then burst and the sporozoites travel through the thoracic muscles to the salivary glands of the Culicine. Neumann, experimenting with canaries, found that _Stegomyia fasciata_ could transmit the infection, but less efficiently than species of _Culex_. THE CULTIVATION OF MALARIAL PARASITES. The successful cultivation of malarial parasites _in vitro_ was first recorded by C. C. Bass and by Bass and Johns (1912).[206] Since then, J. G. and D. Thomson,[207] and McLellan (1912–13), Ziemann[208] and others have repeated the experiments. [206] _Journ. Exptl. Med._, xvi, p. 567. [207] _Annals Trop. Med. and Parasitol._, vi, p. 449; vii, pp. 153, 509. [208] _Trans. Soc. Trop. Med. and Hyg._, vi, p. 220. DIFFERENTIAL CHARACTERS OF THE HUMAN MALARIAL PARASITES. ======================================================================= | | |_Laverania malariæ_ Character | _Plasmodium |_Plasmodium vivax_| _Plasmodium | malariæ_ | (Benign tertian) | falciparum_ | (Quartan) | |(Malignant tertian) -------------+------------------+------------------+------------------- Schizogony |Complete in |Complete in forty-|Complete in forty- | seventy-two | eight hours | eight hours | hours | | or less -------------+------------------+------------------+------------------- Trophozoite |Smaller than _P. |Young trophozoite |Young trophozoite | vivax_larger than| large. | small | _L. malariæ_ | | |Pseudopodia not |Long pseudopodia | | marked or long | | -------------+------------------+------------------+------------------- Movements |Rather slow in |Active amœboid |Sometimes actively | immature forms | movements | motile -------------+------------------+------------------+------------------- Pigment |Coarse granules, |Fine granules, |Granules fine and | peripherally | with active | scanty, movement | arranged, little | movement | oscillatory | movement | | -------------+------------------+------------------+------------------- Schizont |Smaller than red |Larger than red |Smaller than red | corpuscle | blood corpuscle | corpuscle -------------+------------------+------------------+------------------- Merozoites |6 to 12 forming |15 to 20 regularly|8 to 32 (according | rosette | arranged | to different | | | authors) arranged | | | irregularly -------------+------------------+------------------+------------------- Gametocytes |Spherical |Spherical |Crescentic -------------+------------------+------------------+------------------- Distribution |About equal number|Larger numbers in |Scanty in periph- of | in peripheral and| visceral blood | eral blood com- parasites in| visceral blood | | pared with the vertebrate | | | enormous numbers host | | | in the internal | | | organs. The latter | | | part of the cycle | | | (schizogony) may | | | occur in the in- | | | ternal organs only -------------+------------------+------------------+------------------- Alterations |Almost normal |Pale and |Corpuscle may be in | | hypertrophied. | shrunken and dark, erythrocytes| |Schüffner’s dots | or may be colour- | | seen in deeply | less. Maurer’s | | stained specimens| coarse dots some- | | | times seen -------------+------------------+------------------+------------------- Essentially the method of cultivation, as used by Thomson, is as follows: 10 c.c. of infected blood are drawn from a vein and transferred to a sterile test tube, in which is a thick wire leading to the bottom of the tube. One-tenth of a cubic centimetre of a 50 per cent. aqueous solution of glucose or dextrose is placed in the test tube, preferably before adding the blood. The blood is defibrinated by stirring gently with the wire. When defibrination is complete the wire and the clot are removed, and the glucose-blood is transferred, in portions, to several smaller sterile tubes, each containing a column of blood about one inch in height. The tubes are plugged and capped and then transferred, standing upright, to an incubator kept at a temperature of 37° C. to 41° C. The blood corpuscles soon settle, leaving a column of serum at the top, to the extent of about half an inch in each tube. The leucocytes need not be removed by centrifugalization. J. G. Thomson (1913) and his collaborators did not find it necessary to destroy the complement in the serum, and they found that the malarial parasites developed at all levels in the column of corpuscles, and not merely on the surface layer of the corpuscles as first stated by Bass and Johns. So far only the asexual generation of the malarial parasites has been grown _in vitro_. Thomson rarely observed hæmolysis in the cultures. Clumping of the malignant tertian parasites occurred. In cultures of the benign tertian parasite (_Plasmodium vivax_) clumping was not observed. J. G. and D. Thomson consider that this difference as regards clumping explains why only young forms of malignant tertian are found in peripheral blood, as the clumping tendency of the larger forms causes them to be arrested in the finer capillaries of the internal organs. It also explains the tendency to pernicious symptoms, such as coma, in malignant tertian malaria. Further it was found from cultures that _P. falciparum_ was capable of producing thirty-two spores (merozoites) in maximum segmentation, while _P. vivax_ produced sixteen spores (merozoites) as a rule, though the number might be greater than sixteen. (Quartan parasites produce eight spores or merozoites in schizogony.) It may also be mentioned here that _Babesia_ (_Piroplasma_) _canis_ has been successfully cultivated _in vitro_ by Bass’s method. This has been accomplished by Thomson and Fantham,[209] Ziemann, and Toyoda in 1913. J. G. Thomson and Fantham used the simplified Bass technique recorded above, namely, infected blood and glucose, incubating at 37° C. In one of the _B. canis_ cultures, starting with heart blood of a dog containing corpuscles infected with one, two, or, exceptionally, four piroplasmata, Thomson and Fantham succeeded in obtaining a maximum of thirty-two merozoites in a corpuscle. The cultures are infective to dogs and sub-cultures have been obtained. [209] _Annals Trop. Med. and Parasitol._, vii, p. 621. Family. *Piroplasmidæ*, França. The parasites included in this provisional family or group belong to the Hæmosporidia. They are minute organisms, sometimes amœboid, but usually possessing a definite form. They are endoglobular, being contained within mammalian red blood corpuscles, but they produce no pigment. The true Piroplasmata, belonging to the genus _Babesia_, destroy the host corpuscles, setting free the hæmoglobin, which is excreted by the kidneys of the cow, sheep, horse, dog, etc., acting as host. The disease produced, variously called piroplasmosis or babesiasis, is consequently characterized by a red coloration of the urine known as hæmoglobinuria, or popularly as “red-water.” One of the best known piroplasms is _Piroplasma bigeminum_ or _Babesia bovis_ (probably the latter name is correct), which is the causal agent of “Texas fever” or “red-water” in cattle and is spread by ticks. [Illustration: FIG. 90.--_Nuttallia equi_, life-cycle as seen in red blood corpuscles in stained preparations of peripheral blood. (After Nuttall and Strickland.)] Of recent years, researches on the morphology of these blood parasites has led to their separation into various genera and species. However, our knowledge is still very far from complete. The various genera recognized by França[210] (1909), and placed in a provisional family, Piroplasmidæ, may be listed, though further research may lead to emendations:-- [210] _Arch. Inst. Bact. Camara Pestana_, iii, p. 11. (1) _Babesia_ (Starcovici) or _Piroplasma_ (Patton). Pyriform parasites, dividing by a special form of budding or gemmation with chromatin forking, as well as by direct binary fission. Parasitic in oxen, dogs, sheep, horses, etc. (2) _Theileria_ (Bettencourt, França and Borges). Rod-shaped and oval parasites occurring in cattle and deer. _T. parva_ is the pathogenic agent of African East Coast fever in cattle. (3) _Nuttallia_ (França). Oval or pear-shaped parasites, with multiplication in the form of a cross. _N. equi_[211] (fig. 90) of equine “piroplasmosis” (nuttalliosis). _N. herpestidis_ in a mongoose. [211] _Parasitology_, v (1912), p. 65. (4) _Nicollia_ (Nuttall). Oval or pear-shaped parasites with characteristic nuclear dimorphism, and with quadruple division at first fan-like, then like a four-leaved clover. _N. quadrigemina_ from the gondi. (5) _Smithia_ (França). Pear-shaped, single forms stretching across the blood corpuscle. Multiplication into four in the form of a cross. _S. microti_ from _Microtus arvalis_, _S. talpæ_ from the mole. (6) _Rossiella_ (Nuttall). This belongs to the family Piroplasmidæ of França. It is intracorpuscular and non-pigment forming, occurring singly, in pairs, or occasionally in fours. It is usually round and larger than Babesia. The parasite multiplies by binary fission. _R. rossi_ in the jackal. The genus _Babesia_ is the best known and most important, and will be considered next. Genus. *Babesia*, Starcovici, 1893. Syn.: _Pyrosoma_, Smith and Kilborne, 1893; _Apiosoma_, Wandolleck, 1895; _Piroplasma_, W. H. Patton, 1895; _Amœbosporidium_, Bonome, 1895. The organisms belonging to this genus are pyriform, round or amœboid. The characteristic mode of division is as follows: Just before division the parasite becomes amœboid and irregular in shape, (fig. 91, _1–5_) with a compact nucleus. The latter gives off a nuclear bud. This nuclear bud divides into two by forking (fig. 91, _6_, _7_). The chromatin forks grow towards the surface of the body of the rounded parasite, and then two cytoplasmic buds grow out. The forking nuclear buds, which are *Y*-shaped, pass into the cytoplasmic outgrowths[212] (fig. 91, _8_, _9_). The buds gradually increase in size at the expense of the parent form until they become two pear-shaped parasites joined at their pointed ends. The connecting strand shrinks and the two daughter forms separate (fig. 91, _10–14_). The pyriform parasites after having exhausted the blood corpuscle escape from it (fig. 91, _15_), and seek out fresh host corpuscles, entering by the rounded, blunt end (fig. 91, _1_). It is the pyriform phase of the parasite which penetrates red blood corpuscles, not rounded forms, which die if set free. The pyriform parasite, however, becomes rounded (fig. 91, _2_, _3_), soon after its entry into a fresh host cell. This interesting mode of division by gemmation and chromatin forking has been made diagnostic of the genus _Babesia_ by Nuttall.[213] Rounded forms of _Babesia_ divide by binary fission, and this direct method can also be adopted by the other forms of Babesia. [212] Nuttall and Graham-Smith, _Journ. Hyg._, vii, p. 232. [213] “Piroplasmosis,” Herter Lectures, _Parasitology_, vi, p. 302. [Illustration: FIG. 91.--_Babesia_ (_Piroplasma_) _canis_, life-cycle in stained preparations of infected blood of dog. (After Nuttall and Graham-Smith.)] The distribution of the chromatin in the pear-shaped _Babesia_, as seen in _B. canis_ and _B. bovis_, is interesting. The main nuclear body consists of a karyosome surrounded by a clear area. There is also a loose (chromidial) mass of chromatin representing the remains of the chromatin forks seen during the formation of the parasite as a daughter form by gemmation. Occasionally there is a small dot or point, the so-called “blepharoplast” of Schaudinn and Lühe. This minute dot is not a flagellate blepharoplast, for there is no flagellate stage in the life-history of Babesia. These nuclear phenomena have been described by Nuttall and Graham-Smith and Christophers (1907)[214] for _B. canis_, by Fantham (1907)[215] for _B. bovis_, and by Thomson and Fantham (1913) from glucose-blood cultures of _B. canis_. [214] _Sci. Mems. Govt. India_, No. 29. [215] _Quart. Journ. Microsc. Sci._, li, p. 297. Babesia are tick borne, as was first shown by Smith and Kilborne (1893). The developmental cycle in the tick is incompletely known. The best accounts are those of Christophers (1907)[216] for _B. canis_ and Koch (1906) for _B. bovis_, and these accounts are supplementary. The principal stages, so far as known, may be summarized thus:-- [216] _Sci. Mems. Govt. India_, No. 29. (1) The piroplasms taken by the tick in feeding on blood pass into the tick’s stomach. The pyriform parasites, which alone are capable of further development, are set free from the blood corpuscles. In about twelve to eighteen hours they become amœboid, sending out long, stiff, slender, pointed pseudopodia. The nucleus of each parasite divides unequally into two. Similar forms have been obtained in cultures. These stellate forms may be gametes, and according to Koch fuse in pairs. (2) A spherical stage follows, possibly representing the zygote. This grows, and a uninucleate globular mass results. This form is found in large numbers on the third day, according to the observations of Koch. (3) A club-shaped organism is next formed. This may represent an oökinete stage. The club-shaped bodies are motile and gregarine-like, and are about four times the size of the blood forms. These club-shaped bodies and subsequent stages were described by Christophers in the development of _B. canis_ in the dog-tick, _Rhipicephalus sanguineus_. (4) The club-shaped bodies pass from the gut of the tick into the ovary, and so get into the ova. There they become globular, and later are found in the cells of the developing tick-embryo. The parasites are, then, transmitted hereditarily. Similar globular bodies are found in the tissue cells of the body of tick nymphs which have taken up piroplasms. The globular stage was called the “zygote” by Christophers, but it may correspond to the oöcyst of Plasmodia. (5) The globular body divides into a number of “sporoblasts,” which become scattered through the tissues of the larval or nymphal tick, as the case may be. (6) The sporoblasts themselves divide into a large number of sporozoites, which are small uninucleate bodies, somewhat resembling blood piroplasms. The sporozoites collect in the salivary glands of the tick. They are inoculated into the vertebrate when the tick next feeds. The chief species of _Babesia_ and their pathogenic importance may be listed thus:-- (1) _Babesia bovis_ (Babes) produces infectious hæmoglobinuria of cattle in Europe and North Africa. It is transmitted by _Ixodes ricinus_. A similar parasite also occurs in deer. (2) _Babesia bigemina_ (Smith and Kilborne) produces Texas fever, tristeza, or red-water in cattle in North and South America, South Africa and Australia. It is transmitted by _Boöphilus annulatus_ in North America, by _B. australis_ in Australia, South America, and the Philippines, and by _B. decoloratus_ in South Africa. The parasite is from 2 µ to 4 µ long, and from 1·5 µ to 2 µ broad. _Babesia bigemina_ may be the same parasite as _B. bovis_. (3) _Babesia divergens_ (MacFadyean and Stockman) is a small parasite. It is found in cattle suffering from red-water in Norway, Germany, Russia, Hungary, Ireland, Finland, and France, and is transmitted by _Ixodes ricinus_. (4) _Babesia canis_ (Piana and Galli-Valerio) gives rise to malignant jaundice or infectious icterus in dogs in Southern Europe, India, and other parts of Asia and North Africa, where it is transmitted by _Rhipicephalus sanguineus_. In Africa generally, especially South Africa, the disease is transmitted by _Hæmaphysalis leachi_. _Babesia canis_ varies from 0·7 µ to 5 µ, the size depending partly on the number of parasites within the corpuscle. It averages about 3 µ. It has been cultivated in Bass’ medium (glucose and infected blood), see p. 172. In India _Piroplasma gibsoni_ (Patton) infects hunt dogs and jackals. It is annular or oval in shape. (5) _Babesia ovis_ (Babes) produces “Carceag,” a disease of sheep in Roumania, the Balkan Peninsula, Italy, and Transcaucasia. It varies in size from 1 µ to 3 µ. It is transmitted by _Rhipicephalus bursa_. The parasite has recently been recorded from Rhodesia. (6) _Babesia caballi_ (Nuttall and Strickland) causes “biliary fever” in equines. The parasite occurs in Russia, Roumania, and Transcaucasia. It varies in size from 1 µ to 2 µ. It is transmitted by _Dermacentor reticulatus_. It should be mentioned that _Nuttallia equi_ also causes “piroplasmosis” in equines, with symptoms of hæmoglobinuria and jaundice in Italy, Sardinia, many parts of Africa, Transcaucasia, India, and Brazil. In Africa it is transmitted by _Rhipicephalus evertsi_. It has been shown experimentally that a horse recovered from _Babesia caballi_ was susceptible to the inoculation of _Nuttallia equi_ blood. (7) _Babesia pitheci_ (P. H. Ross) was found in a monkey, _Cercopithecus_ sp., in Uganda. The pear-shaped forms measure 1·5 µ by 2·5 µ. (8) _Babesia muris_ (Fantham)[217] was found in white rats. The pyriform parasites are 2 µ to 3 µ long and 1 µ to 1·5 µ broad; oval forms are 0·5 to 1·5 µ diameter. [217] _Quart. Journ. Microsc. Sci._, 1, p. 493. The usual symptoms of babesiasis (piroplasmosis) are high fever, loss of appetite, hæmoglobinuria, icterus, anæmia, paralysis, and death in about a week in acute cases. In chronic cases there is anæmia, and hæmoglobinuria is less marked. When animals recover, there are still some piroplasms left in the blood. “Recovered” or “salted” animals are not susceptible to reinfection, but ticks feeding on them acquire piroplasms, and are a source of danger to freshly imported animals. _Treatment._--Trypan-blue is the best drug, as shown by Nuttall and Hadwen[218] (1909). It should be administered intravenously in 1 to 1·5 per cent. aqueous solution. A dose of 5 to 10 c.c. is curative for dogs, one of 100 to 150 c.c. for horses and cattle. Unfortunately, the tissues are coloured blue by the drug. The “salted” animals, after trypan-blue treatment, still harbour the parasites in their blood for years. [218] _Parasitology_, ii, p. 156. Genus. *Theileria*, Bettencourt, França and Borges, 1907. The organisms belonging to this genus are rod-like or bacilliform, and coccoid or round. The best known of the species of Theileria is _T. parva_, the pathogenic agent of East Coast fever or Rhodesian fever in cattle in Africa. *Theileria parva*, Theiler, 1903. Syn.: _Piroplasma parvum_. In the blood corpuscles of infected cattle minute rod-like and oval parasites are seen. Some are comma shaped and others are clubbed (fig. 92, _1–12_). The rod-like forms measure 1 µ to 3 µ in length by 0·5 µ in breadth; the oval forms are 0·7 µ to 1·5 µ in diameter. The intracorpuscular parasites are said by R. Gonder (1910) to be gametocytes, the rod-like forms being thought to be males, the oval forms to be females. Free parasites are practically never seen in the blood. It is known that it is impossible to produce the disease in a healthy animal by blood inoculation, but only by intraperitoneal transplantation of large pieces of infected spleen (Meyer). There may be as many as eight parasites in a corpuscle. The chromatin is usually at one end of the organism. In some parasites the appearance of the chromatin suggests division, but such division, if it takes place, must be very slow, as it has not been actually seen in progress. The red blood corpuscles appear merely to act as vehicles for the parasites (Nuttall, Fantham, and Porter).[219] [219] _Parasitology_, ii, p. 325; iii, p. 117. [Illustration: FIG. 92.--_Theileria parva._ 1–12, intracorpuscular parasites, stained. (After Nuttall and Fantham); 13–18, Koch’s blue bodies, from stained spleen smear; 17–18, breaking up of Koch’s body. (After Nuttall.)] In the internal organs, especially the lymphatic glands, spleen and bone-marrow, are found multinucleate bodies known as Koch’s blue bodies (fig. 92, _13–18_). These are schizonts, according to Gonder.[220] The actual Koch’s blue bodies are said to be extracellular, but similar multinucleate bodies, schizonts, occur in lymphocytes. The schizonts divide and the merozoites resulting probably invade the red blood corpuscles in the internal organs. Gonder considers that the sporozoites injected by the tick collect in the spleen and lymphatic glands, penetrate the lymphocytes and give rise to the schizonts. [220] _Zeitschr. f. Infekt. paras. Krankh. u. Hyg. d. Haustiere_, viii, p. 406. Gonder has studied the cycle of _T. parva_ in the tick. He states that the gametocytes leave the host corpuscles and give rise to gametes, then conjugation occurs producing zygotes. The zygotes are then said to become active to form ookinetes, and to enter the salivary glands of the tick. Multiplication is said to occur therein, producing a swarm of sporozoites. This work needs confirmation. _T. parva_ is transmitted by _Rhipicephalus appendiculatus_, _R. simus_, _R. evertsi_, _R. nitens_, and _R. capensis_. The parasites are not hereditarily transmitted in _Rhipicephalus_, but when taken by the transmitter at one stage of its development the tick is infective in its next stage (_e.g._, if the larva becomes infected, then the nymph is infective; if the nymph becomes infected, then the adult is infective). An animal recovered from _Theileria parva_ is incapable of infecting ticks, but few animals recover from East Coast fever. Animals suffering therefrom do not show hæmoglobinuria. *Theileria mutans*, Theiler, 1907· Syn.: _Piroplasma mutans_. This is transmissible experimentally by blood inoculation. It occurs in cattle in South Africa and Madagascar and is apparently non-pathogenic. No Koch’s blue bodies are formed. It is transmitted by ticks. _Theileria annulata_ (Dschunkowsky and Luhs) occurs in cattle in Transcaucasia. A Theileria (_T. stordii_) has been found in a gazelle (França, 1912). Genus. *Anaplasma*, Theiler, 1910. This genus[221] may be mentioned here. The organisms included therein are, according to Theiler, coccus-like, consisting of chromatin, and are devoid of cytoplasm. They occur in the red blood corpuscles of cattle, causing a disease characterized by destruction of red cells, fever and anæmia, but with yellow urine. The disease is tick transmitted. The bodies now called _Anaplasma marginale_ were formerly described as marginal points. They multiply by simple fission. They are said by Theiler to cause gall-sickness in cattle in South Africa. Some authors doubt whether these bodies are organismal. [221] _Bull. Soc. Path. Exot._, iii, p. 135. Genus. *Paraplasma*, Seidelin, 1911. Under this generic name Seidelin described certain bodies found by him in cases of yellow fever in 1909. The type species is _P. flavigenum_,[222] and is claimed by Seidelin to be the causal agent of yellow fever. [222] _Yellow Fever Bulletin_, i, p. 251. _Paraplasma flavigenum_ occurs in the early days of the disease as small chromatin granules with or without a faint trace of cytoplasm. The bodies are usually intracorpuscular. Also, somewhat larger forms, with distinct cytoplasm, are seen in small numbers. During the later days of the disease still larger forms are found, and these occur also in sections of organs (_e.g._, kidney) made post-mortem. Some of these larger forms are perhaps schizonts. In the second period of the disease possible micro- and macro-gametes may be found, some of which are extracorpuscular. Some small free bodies have been seen. Recently schizogony has been stated to occur in the lungs, and it is said that guinea-pigs can be inoculated with _Paraplasma flavigenum_, and show yellow pigment in the spleen. Seidelin places _Paraplasma_ in the _Babesiidæ_, with resemblances more particularly to _Theileria_. V. Schilling-Torgau and Agramonte have criticized these findings; the former considers them to be the resultant of certain blood conditions. _P. subflavigenum_ was found by Seidelin in 1912 in a man suffering from an unclassified fever in Mexico. Further, it is now known that a Paraplasma occurs naturally in guinea-pigs. More researches are needed on these matters, as some writers (_e.g._, Wenyon and Low) claim that the bodies are not organismal. -------------------------------------------------------------------- |_Paraplasma flavigenum._--The Yellow Fever Commission (West Africa) | |in their third report, dated 1915, have come to the conclusion that | |there is no evidence that the bodies termed _Paraplasma flavigenum_ | |are of protozoal nature or that they are the causal agents of yellow| |fever. | -------------------------------------------------------------------- Sub-class. NEOSPORIDIA, Schaudinn. Sporozoa in which growth and spore formation usually go on together. Order. *Myxosporidia*, Bütschli. [Illustration: FIG. 93.--Upper figure, part of a gill of a roach, _Leuciscus rutilus_ (natural size), with two myxosporidia. Lower figures, _a_, _b_, _d_, spores of myxosporidia from a pike, _Esox lucius_. _c_, Spore from _Platystoma fasciatum_. (After J. Müller.)] [Illustration: FIG. 94.--The tailless spore of _Myxobolus mülleri_, with the polar bodies and their nuclei and the sporozoite. (After Bütschli.)] These parasites, which were discovered by Johannes Müller (1841), live principally in fishes, and occasionally cause destructive epizoötics amongst their hosts. Müller first observed them in the form of whitish-yellow pustules on the skin or on the gills of various fishes. These pustules contained masses of small shell-covered bodies with or without tails (“psorosperms,” see fig. 93). Similar bodies were also found in the air bladders of certain fish. Creplin (1842) demonstrated the resemblance of the cysts (“psorosperm tubes”) harbouring the psorosperms to the “pseudonavicella-cysts” of a gregarine, as described by v. Siebold. Dujardin (1845) considered that there was possibly some connection between the protoplasmic “psorosperm tubes” and the spores they contained, and the developmental stages of monocystid gregarines from the vesiculæ seminales of earth-worms. The relationship of the “fish psorosperms” was placed on a firmer basis by Leydig (1851) and Lieberkühn. The former found numerous forms in marine fish, and he discovered in species which live free in the gall bladder of cartilaginous fishes that the psorosperms originated in a manner similar to the gregarines. Lieberkühn (1854) studied the Myxosporidia in the bladder of the pike (fig. 93, _a_, _b_, _d_), and observed their amœboid movements, as well as the formation of the spores, from each of which a small amœboid body escaped, a discovery that was confirmed by Balbiani. The same author also found that spiral filaments were enclosed in the so-called polar body, _i.e._, the polar capsule of the psorosperm spores, and that these could be protruded (fig. 93, _d_, and fig. 95). The term Myxosporidia, which at the present day is universally applied to the “psorosperm tubes,” was introduced by Bütschli in 1881, who studied not only the structure and development of the spores, but also the protoplasmic body of the parasites (fig. 96), and confirmed the occurrence of numerous nuclei. Many authors have made important additions to our knowledge of the Myxosporidia: Perugia, Thélohan, Mingazzini, L. Pfeiffer, L. Cohn, Doflein, Mercier, Schröder and Auerbach; while the presence of this parasite outside the class of fishes has become known through Lutz, Laveran, and others. The species causing disease in fishes have been described by Ludwig, Railliet, Weltner, L. Pfeiffer, Zschokke, Hofer, Doflein, Gurley, Plehn, Schuberg, Fantham and Porter. With regard to classification the works of Thélohan (1895) and Gurley (1894) may be mentioned. [Illustration: FIG. 95.--Schematic representation of a spore of _Myxobolus_. One polar capsule has protruded its filament; two nuclei and a “vacuole” in the sporozoite. (After Doflein.)] [Illustration: FIG. 96.--_Chloromyxum leydigi._ Active trophozoite (parasitic in gall-bladder of skates, rays, dog-fish). _Ect_, ectoplasm; _ps_, pseudopodia; _end_, endoplasm; _y_, yellow globules in endoplasm; _sp_, spores, each with four polar capsules. × 525. (After Thélohan.)] The Myxosporidia live either free on the epithelial surface of hollow organs (gall or urinary bladder, renal tubules, but never in the intestine), or are enclosed in the tissues of their host. The gills and muscular system are their favourite habitat, but other tissues or organs may be attacked. Species of Myxosporidia are also known from Amphibia, Reptilia, and a few invertebrates. The free forms, which are often amœboid (fig. 96), move by the aid of variously shaped pseudopodia, have a constant form, or may exhibit contractions of the body. The tissue parasites often reach a considerable size, so that the integument of the host forms protuberances over them. They are of a roundish or irregular shape. Frequently they are enveloped in a connective tissue covering formed by the host. The protoplasmic body in the trophic phase (fig. 96) shows a distinct ectoplasm which is finely granular or sometimes striated, and an endoplasm which is coarsely granular and contains many nuclei as well as cell inclusions, such as crystals, pigment grains and fat globules. The nuclei originate by division from the primitive nucleus of the amœboid germ that issues from the spore. This amœbula may or may not live intra-cellularly during the early stages of its existence. The multinucleate trophozoite of a Myxosporidian forms spores in its endoplasm practically throughout its whole period of growth (fig. 96). Vegetative reproduction by a process of external budding or plasmotomy may also occur, as in _Myxidium lieberkühni_ from the urinary bladder of the pike. The myxosporidian trophozoite may produce two spores within itself, when it is placed in the sub-order _Disporea_, or it may produce numerous spores, which is characteristic of the sub-order, _Polysporea_. The phenomenon of spore formation is not simple (fig. 97), and the spore itself is surrounded by a bivalved shell or sporocyst and contains polar capsules in addition to the amœboid germ (fig. 97, G, H). The valves of the sporocyst and the polar capsules are really differentiated nucleate cells, so that each spore is an aggregate of cells rather than one cell, though only a single amœbula issues from a spore. The accounts of spore formation vary somewhat according to the different workers. Spore formation is usually very complicated and there are differences of opinion as to the interpretation of various stages, particularly as to whether conjugation occurs therein. The process is initiated by the concentration of cytoplasm around one of the nuclei of the endoplasm, so that a small spherical mass or initial corpuscle is produced, the pansporoblast (Gurley) or primitive sphere (Thélohan). Some authors state that a pansporoblast really results from a conjugation of two initial corpuscles (fig. 97, A-D). Nuclear multiplication occurs within the pansporoblast (fig. 97, E), and sooner or later two multinucleate sporoblasts are formed within it (fig. 97, F). Each sporoblast gives rise to a single spore, which consists of a sporocyst or envelope composed of two valves each secreted by a cell, two polar capsules each secreted by a cell, and the sporoplasm or amœbula which becomes binucleate (fig. 97, G). During the process of spore formation (fig. 97) various vegetative and reduction nuclei may be produced, in addition to those which are essentially involved in spore formation, and the sporocyst cells may be developed early. [Illustration: FIG. 97.--_Myxobolus pfeifferi._ Spore formation. A, reproductive cell from plasmodial trophozoite; B, cell divided unequally into two; C, smaller cell forming envelope to larger one; D, pansporoblast formed by union of two forms like C; E, multinucleate pansporoblast, two of the nuclei being those of the envelope; F, pansporoblast divided into two multinucleate sporoblasts; G, spore differentiation; _p_, two parietal cells forming sporocyst; _bc_, polar capsules; _am_, binucleate amœbula; H, ripe spore in which the two nuclei of the amœbula have fused. (After Keysselitz.)] Each spore contains two (figs. 94, 95) or more polar capsules which are clearly visible in the fresh condition. Each polar capsule is a hollow, more or less pear-shaped body, secreted by a cell and having a well defined contour. Within it, a long, delicate, elastic filament, the polar filament, is formed, and lies spirally coiled in the polar capsule until just before the emergence of the amœbula from the spore (fig. 95). The polar filament is ejected, probably under the influence of the digestive juice, when the spore reaches a new host, and serves to anchor the spore to the tissue with which it is in contact, and thus allow of the emergence of the amœbula in a situation suitable for its development. The polar capsule with its contained polar filament has been compared with the stinging cells or nematocysts of the Cœlentera, but it has a totally different function. The spores fulfil the purpose of effecting transmission to other hosts. Infection occurs by the ingestion of the parasites per os after their escape by some means from their host. Thélohan and others have demonstrated that the valves of the spores soon open under the influence of the digestive juices, thus allowing the young myxosporidia to escape. Their further history is unknown; but it may be surmised that they either travel direct to the organs usually affected (gall bladder, urinary bladder), or are distributed in the body by means of the circulatory or lymphatic systems. The Myxosporidia that invade tissues are often deadly to their hosts. They may be present in a state of “diffuse infiltration” when practically every organ of the body may be infected, as in barbel disease (due to _Myxobolus pfeifferi_). On the other hand, the parasites may be concentrated at one spot, when cysts, either large or small, are produced. Such cysts occur on the gills of many fishes. A few additional important pathogenic forms are _Myxobolus cyprini_, the excitant of “pockenkrankheit” of carp, and _Lentospora cerebralis_, parasitic in the skeleton of Salmonidæ and Gadidæ. The skeletons of the tail, fins and skull particularly are seats of infection, and from the skull the Lentospora can spread to the semicircular canals, resulting in loss of power to maintain its balance on the part of the fish. On this account the malady is termed “drehkrankheit.” Young fish are more particularly infected. _Myxobolus neurobius_ infects the spinal cord and nerves of trout. Myxosporidia are divided into two sub-orders--_Disporea_ and _Polysporea_--according to whether they form only two or several spores during their growth. The former include two genera limited to fishes, which are easily distinguishable by the shape of the spores: _Leptotheca_, Thél., with a rounded spore, and _Ceratomyxa_, Thél., with a very elongate spore. The larger number of genera belong to the _Polysporea_, which are divided into three families: (1) Amœboid germ with a vacuole {(a) With two polar capsules.-- the contents of which do { _Myxidiidæ._ not stain with iodine. {(b) With four polar capsules.-- _Chloromyxidæ._ (2) Amœboid germ with a vacuole stainable with iodine. Spores with two polar capsules.--_Myxobolidæ._ For further subdivisions the differences in the spores are principally utilized. Order. *Microsporidia*, Balbiani. These are the organisms discovered in the stickleback by Gluge in 1834, and in _Coccus hesperidum_ by Leydig in 1853. They have since been found in numerous other arthropods, especially insects. They acquired particular importance when it was discovered that they were the cause of the “pébrine” disease (“gattina” of the Italians) which caused so much destruction amongst silkworms (_Bombyx mori_). Pasteur (1867–70) and especially Balbiani (1866) participated in the researches on _Nosema bombycis_, and it was the latter who classed the “pébrine bodies” or “psorospermia of the arthropoda” amongst the Sporozoa as Microsporidia (1882).[223] The complete life cycle of _N. bombycis_ was described in 1909 by Stempell. The Microsporidia are not confined to insects and arachnoids, they are now known to occur also in crustacea, worms, bryozoa, fishes, amphibians and reptiles. Certain tumours in fishes, similar to those formed by many Myxosporidia, are produced by Microsporidia. Fantham and Porter found that _Nosema apis_ was pathogenic to bees and other insects, and was the causal agent of the so-called “Isle of Wight” disease in bees[224] in Great Britain. [223] _C. R. Acad. Sci._, Paris, xcv, p. 1168. [224] _Annals Trop. Med. and Parasitol._, vi, pp. 145–214, 3 pls. The Microsporidia, as their name implies, form minute spores which usually are oval or pear-shaped. Each spore contains a single polar capsule which is not easily visible in the fresh state (fig. 98, _f_) and a single amœboid germ issues from the spore (fig. 99, _b_). [Illustration: FIG. 98.--_Nosema apis._ Various stages in life-cycle. _a_, planonts or amœbulæ from chyle stomach of bee; _b_, amœboid planont creeping over surface of gut epithelial cell; _c_, uninucleate trophozoite within epithelial cell; _d_, meront with nucleus divided into four, about to form four spores; _e_, epithelial cell crowded with spores; _f_, young spore; _g_, spore showing five nuclei, polar filament ejected, and amœbula, about to issue. × 1,500, _a-e_; × 2,150, _f-g_. (After Fantham and Porter.)] The life cycle of _Nosema apis_, parasitic in bees, may be taken as an example of that of a microsporidian. The infection of the host is initiated by the ingestion of spores of _N. apis_ in food or drink contaminated with the excrement of other infected bees. Under the influence of the digestive juice of the bee the spore-coat (sporocyst) softens, the polar filament is ejected and anchors the spore to the gut epithelium, and the minute amœbula contained in the spore emerges. The amœbula is capable of active amœboid movements (fig. 98, _b_) and so is termed the planont or wandering form (fig. 98, _a_). After a short time each planont penetrates between or into the cells of the epithelium of the gut, a few only passing through into the body cavity. Within the cells the amœbulæ become more or less rounded, lose their power of movement, and after a period of growth of the trophozoite (fig. 98, _c_) commence to divide actively, these dividing forms being known as meronts (fig. 98, _d_). Various forms of fission occur, and during this phase, termed merogony, the numbers of the parasite within the host are greatly increased, with concomitant destruction of the epithelium (fig. 98, _e_). After a time sporogony commences. The full-grown meront becomes successively the pansporoblast and sporoblast. Nuclear multiplication and differentiation ensue and five nuclei are ultimately produced. At the same time a sporocyst is secreted, and two vacuoles are produced within. One is the polar capsule, and within it the polar filament is differentiated; the other forms the posterior vacuole (fig. 98, _g_). Between the two vacuoles the body cytoplasm or sporoplasm forms a girdle-like mass. Of the nuclei, one regulates the polar capsule, two control the secretion of the sporocyst, and two remain in the sporoplasm. The polar capsule and polar filament are not usually visible in the fresh condition, but can be demonstrated by the use of various chemical reagents (fig. 100). The sporoplasm ultimately becomes the amœbula (fig. 98, _g_) which issues from the spore after the ejection of the polar filament. [Illustration: FIG. 99.--_a_, section through the abdominal wall of a silkworm, whose epithelial cells contain Microsporidia (_Nosema bombycis_); _b_, a spore, the contents of which are escaping. (After Balbiani.)] [Illustration: FIG. 100.--_Nosema bombycis_, Naeg. Spores treated with nitric acid, thus rendering the polar capsule perceptible, and the filament has protruded from one of the spores. (After Thélohan.)] A trophozoite (meront) of _N. apis_ becomes a single pansporoblast which gives rise to one sporoblast producing one spore, and this procedure is characteristic of the genus _Nosema_. In other genera the trophozoite may form more than one pansporoblast and each pansporoblast may form a variable number of spores in different cases. Various attempts at classification have been based on these characteristics. It must suffice here to note that in the cases where the trophozoite becomes one pansporoblast, the latter can produce four spores in the genus _Gurleya_, eight spores in _Thélohania_ and many spores in _Pleistophora_. In other cases, where the trophozoites give rise to many pansporoblasts, each of the latter may form many spores, as in the genus _Glugea_. A few pathogenic microsporidian parasites other than _N. apis_ may be mentioned. _N. bombycis_, causing pébrine in silkworms, may infect any or all the tissues of the host (fig. 99). The larvæ of the host, _i.e._, the “silkworms,” may become infected by eating food contaminated with spore-containing excrement of already infected silkworms. In cases of heavy infection the silkworm dies, but should the infection be less intense the larva becomes a pupa in which the parasite persists, so that the moth emerges from the cocoon already infected. Not only is the moth parasitized itself, but the Nosema reaches the generative organs of both sexes and penetrates the ovaries of the female, with the result that the ova are deposited infected. Such infected eggs are capable of developing, so that infection may be transmitted hereditarily as well as by the contaminative method. Infected eggs can be recognized by microscopic examination, as Pasteur showed, and thus preventive measures may be adopted. A microsporidian parasite is known to occur on the roots of the spinal and cranial nerves of _Lophius piscatorius_, the angler fish. This parasite is variously referred to the genera _Nosema_ and _Glugea_. _Thélohania contejeani_, parasitic in the muscles of crayfish, is believed by some to be the causal agent of recent epizoötics among them, though others believe the disease to be really due to a bacillus. It may be that the one organism aids in the entry of the other into the host. Order. *Actinomyxidia*, Stolč. A brief mention may be made of the Actinomyxidia (fig. 101), which were first described by Stolč in 1899 as parasites of Oligochætes. They have also been investigated by Mrazek, and a detailed study of certain species was made by Caullery and Mesnil (1905). The trophozoite is small and amœboid. The spores are large, and exhibit tri-radiate symmetry. Spore formation is complicated and sexual processes occur therein. Many amœbulæ are set free from each spore. [Illustration: FIG. 101.--Spore of _Hexactinomyxon psammoryctis_. At top of figure three polar capsules, one with polar filament extended. × 450. (After Stolč.)] Order. *Sarcosporidia*, Balbiani. The first member of this group was discovered by Miescher in 1843. This author found white filaments running parallel with the direction of the fibres in the voluntary muscles of mice. They were visible to the naked eye, and proved to be cylindrical tubes tapering at each end. They were as long as the muscular fibres, were enveloped in a membrane, and contained innumerable elongate or kidney-shaped bodies and a smaller number of little spherical forms. Th. v. Hessling confirmed (1853) the occurrence of these “Miescher’s tubes” within the muscular fibres, this author having discovered the same structures in the heart muscles of deer, cattle, and sheep. Both investigators considered them to be pathological transformations of the muscles. v. Siebold, from his own experiences, regarded them as fungus-like entophytes. Rainey (1858) discovered similar structures in the muscular system of pigs, and considered them to be early stages of _Cysticercus cellulosæ_, which error Leuckart rectified, simultaneously emphasizing their relationship with Myxosporidia. Both these authors found them in the muscular fibres, and both observed that they possessed a thick striated membrane. Manz (1867) published the results of more minute investigations on the structure and contents of the cylinders. This observer also recognized the disease in rabbits and attempted to cultivate the parasites. He also tried to induce experimental infection in guinea-pigs, rats, and mice, but the result was negative. However, domestic and wild mammals are not the only hosts of Sarcosporidia; these parasites are also harboured by birds. Thus, according to Kühn, they are found in the domestic fowl; according to Rivolta in _Turdus_, _Corvus_, and other birds; according to Stiles in North American birds; while Fantham found Sarcosporidia in the African mouse-bird, _Colius_. Reptiles also are parasitized occasionally. Bertram found them in the gecko, Lühe in the wall-lizard. It was found also that the Sarcosporidia could develop not only in the muscles but also in the connective tissue. This led to the foundation of a new, but provisional, classification by Blanchard, using the generic name _Miescheria_ for the parasites in the muscles and _Balbiania_ for those in the connective tissue. Finally, Sarcosporidia have also been observed in man. The relation of these parasites to certain diseases of domestic animals has been studied by veterinary surgeons. Sarcosporidia may cause fatal epizoötics among sheep. There is still a wide field open for research in regard to the structure and development of these parasites, and the manner in which the hosts become infected. [Illustration: FIG. 102.--Longitudinal section of a muscle of the domestic pig, with _Sarcocystis miescheriana_. × 30. (After Kühn.)] [Illustration: FIG. 103.--Transverse section of the muscle of a pig, with _Sarcocystis miescheriana_. × 38. (After Kühn.)] The Sarcosporidia usually appear as elongate, cylindrical, or fusiform bodies, rounded at both extremities and of various lengths and breadths (fig. 102). In some species they may be from 16 mm. to 50 mm. long, as in the sheep and roebuck. These bodies are the so-called sarcocysts or Miescher’s tubes. They lie in transversely striated muscular fibres which they distend more or less. The forms found in the connective tissue are apparently parasites which originally inhabited the muscular fibres, and only on disintegration of the fibres reached the connective tissue, where they grow to large oval or globular bodies (fig. 105). The mammalian muscles usually infected are those of the œsophagus, larynx, diaphragm, body-wall, and the psoas muscles. The skeletal muscles may be affected in acute cases, as well as those of the tongue and eye. The heart muscles are sometimes parasitized. In fresh material cut into thin slices the parasites are frequently recognizable, even with the naked eye, because of their yellowish-white colour. Under the microscope they appear to be coarsely granular (fig. 103). Beginners may find some difficulty in distinguishing them from other foreign bodies, such as dead and calcified encapsuled Trichinæ, or from Cysticerci that have died and become calcified in the early stages, more particularly as the Sarcosporidia also occasionally may become calcified. The Sarcosporidia are always enveloped in a membrane, which is probably formed at an early stage. In a few cases it remains thin and simple, in other cases a radially striated ectoplasmic layer is present (figs. 104, 108), which has been variously described. From the inner integument, which may be homogeneous or fibrous, thick or thin, membranes or trabeculæ pass into the interior of the body, forming anastomosing partitions, and so producing a system of chambers of various sizes that do not communicate with one another (figs. 104, 108). These chambers are occupied by sickle- or bean-shaped bodies (spores or sporozoites), or various developmental stages of them. The oldest spores are found in the centre of the Miescher’s tubes or trophozoites. If they are not liberated they die there, so that the central chambers of the tube are empty and hollow. [Illustration: FIG. 104.--_Sarcocystis miescheriana_ from pig. Late stage in which body is divided into numerous chambers or alveoli, each containing many spores. (From Wasielewski, after Manz.)] In the youngest Sarcosporidia (40 µ in length) from the muscles of the sheep there occur, according to Bertram, small roundish or oval cells (4 µ to 5 µ), the nuclei of which are half their size, and are embedded in a granular protoplasmic mass. In somewhat larger, and therefore older, cylinders, the investing membrane of which already shows both layers, the cells have become larger (to 7 µ) and are more sharply outlined from each other (fig. 106). These uninucleate cells may be considered as pansporoblasts. In each pansporoblast division of the nucleus occurs (fig. 107), and meanwhile the pansporoblasts become isolated within the chambers, the dividing partitions of which originate from the granular protoplasm which is present between the pansporoblasts. The numerous uninucleate daughter forms produced within the chambers become spores direct (fig. 108). The process commences in the centre of the cylinders or sarcocysts, and then progresses towards the extremities, the parasites meanwhile increasing in size, and new pansporoblasts being continually formed at the extremities (fig. 107). [Illustration: FIG. 105.--Transverse section of _Sarcocystis tenella_, Raill. From the œsophagus of the sheep, _Ovis aries_. × 38. _a_, marginal chambers filled with spores; _b_, connective tissue of the œsophagus; _c_, muscles of the œsophagus.] [Illustration: FIG. 106.--Young _Sarcocystis tenella_ of the sheep, 47 µ in length. (After Bertram.)] [Illustration: FIG. 107.--End of a trophozoite of _Sarcocystis miescheriana_ from the diaphragm of the pig, showing division in pansporoblasts. × 800. (After Bertram.)] [Illustration: FIG. 108.--_Sarcocystis blanchardi_ of the ox. Longitudinal section of sarcocyst or Miescher’s tube. _a_, substance of muscle fibre; _b_, envelope of sarcocyst; _c_, muscle nuclei; _d_, spores in chambers; _e_, ground substance. × 400. (From Wasielewski, after van Eecke.)] The spores (sometimes called Rainey’s corpuscles), vary in shape according to the species, but are also of different form individually. They are mostly kidney-, bean- or sickle-shaped (fig. 109), and of small size, sometimes reaching 14 µ by 3 µ to 5 µ. They are apparently surrounded by a thin membrane, and at one extremity (according to the discovery of L. Pfeiffer, confirmed by van Eecke, Laveran and Mesnil) contain an obliquely striated body (fig. 109) often homologized with the polar capsule, while the greater part of the spore is taken up by the nucleate sporozoite. Several authors state that they have also observed filamentous appendages (polar filaments) at one end of the spores, and have seen two kinds of spores in the same Sarcosporidium. Spores of various species of Sarcosporidia may contain metachromatic granules, often centrally placed (fig. 109). These granules may be metabolic or possibly may contain toxin (see below). [Illustration: FIG. 109.--Spores of _Sarcocystis tenella_, Raill. _a_, fresh, showing the polar capsule; _b_, stained, showing metachromatic granules and nucleus. × 1,000. (After Laveran and Mesnil.)] The gymnospores of _Sarcocystis muris_, from the mouse, show active boring movements when kept in saline solution warmed to 35° or 37° C. _S. muris_ is very deadly to its host. From their structure the spores do not appear to have great powers of resistance to external conditions. They measure 12 µ by 3 µ to 4 µ or less. Laveran and Mesnil (1899) isolated a toxin from _S. tenella_ of the sheep and called it sarcocystin. This substance is especially pathogenic to experimental rabbits. The duration of life of the Sarcosporidia is a comparatively long one. The affected muscular fibres may remain intact and capable of performing their functions for a long time, but at last they perish, if the host lives long enough. Thus the Sarcosporidia of the muscles are then enveloped only by sarcolemma, and finally, when this likewise disappears, they fall into the intra-muscular connective tissue. In many cases the Sarcosporidia die off within their hosts, this, according to Bertram, being brought about by a disintegration of the spores in the central chambers. In other cases the leucocytes play a part in the destruction of the Sarcosporidia, and sometimes it happens that lime salts are deposited in and around the vacant cylinders. In some places pigs, sheep, mice and rats are infected with sarcosporidiosis to a remarkable extent, in certain cases almost reaching 100 per cent. Young animals also are infected, and perhaps infection only takes place during youth. Although the natural mode of transmission of the Sarcosporidia remains to be determined, yet various experimental researches on the problem are of interest and importance. Theobald Smith (1901) found that mice could be experimentally infected with _S. muris_ by feeding them with the flesh of other infected mice. The incubation period was a long one, namely forty to fifty days. Thus, on the forty-fifth day after feeding young Sarcosporidia were found, and seventy days after feeding spore formation began. Ripe spores were found two and a half to three months after the commencement of these experiments. This mode of infection--a cannibalistic one--hardly seems likely to be the natural method for the infection of sheep and ruminants generally. Smith’s researches have been confirmed. Nègre[225] (1910) found that the fæces of mice fed on infected muscular tissue were infective to other mice when ingested by them. Negri[226] infected guinea-pigs with _S. muris_ by feeding them on infected mouse flesh, and found that the parasite in guinea-pigs showed different characters from those exhibited by it in mice. Darling[227] also succeeded in infecting guinea-pigs with _S. muris_, and Erdmann infected mice with _S. tenella_ (from the sheep). [225] _C. R. Soc. Biol._, lxviii, p. 997. [226] _Centralbl. f. Bakt._, Orig., xlvii, p. 612; see also xlvii, p. 56; lv, p. 373. [227] _Journ. Exptl. Med._, xii, p. 19. According to Erdmann[228] (1910) the Sarcosporidian spore germinates in the intestine of the host, which has recently ingested infected material. The spore liberates its contained toxin--sarcocystin--which acts upon the adjacent intestinal epithelium, whereby the latter is shed, and an amœbula creeps out of the spore. The amœbula is able to penetrate the denuded area and get directly into the lymph-spaces of the submucous coat of the intestine. The first period of development, lasting some twenty-eight to thirty days, is said to be passed in the lymph-spaces of the intestine. Later the amœbula reaches a muscle fibre. Writing in May, 1914, Erdmann[229] records the appearance of small amœboid and schizogony forms six days after infection of the host. Crawley[230] (1913) controverts some of these statements and considers that the Sarcosporidian spore, still sickle-shaped, bores its way into the epithelial cells of the intestine and comes to rest there. The spore then becomes round or elliptical, and peripheral masses of chromatin appear within it, suggesting schizogony. This happens about twelve hours after feeding, and in twenty-four hours the spores appear to have left the intestine. More recently (May, 1914), Crawley[231] considers that there is sexual differentiation among the Sarcosporidian spores, a few hours after their ingestion by the host. [228] _Sitz. Gesell. naturf. Freunde zu Berlin_, p. 377. [229] _Proc. Soc. Exper. Biol. and Med._, xi, p. 152. [230] _Science_, xxxvii, p. 498. [231] _Proc. Acad. Nat. Sci._, Philadelphia, May, 1914, p. 432. Interesting discussions have occurred as to the site of the toxic sarcocystin within the spore. Metachromatic granules occur in the middle of the Sarcosporidian spore (fig. 109), and the toxin may be contained in these grains, as they disappear, according to Erdmann, before the amœbula penetrates the denuded intestinal wall. However, a polar capsule, containing a polar filament, may be present at one end of a Sarcosporidian spore. Laveran and Mesnil described a striated area at the more pointed end of the spore of _S. tenella_, which area they consider to represent a polar capsule. Fantham[232] (1913) found a vacuole-like, polar capsule area in the spores of _S. colii_ from the African mouse-bird. The sarcocystin may be contained in the polar capsule. The nucleus of the spore is generally at the opposite, blunter end. [232] _Proc. Cambr. Philosoph. Soc._, xvii, p. 221. Again, various authors have stated that Sarcosporidian spores may occur in the blood of the host at times. If so, then an intermediate host may be concerned in their transmission. Perrin suggested that Sarcosporidia might be spread by blow-flies and flesh-flies. The classification of the Sarcosporidia as proposed by R. Blanchard, which was based on their various habitats, can no longer hold, because the same species may occur in the muscles as well as in the connective tissue. For the present, the few species that are known may be placed in one genus, _Sarcocystis_, Ray Lankester, 1882. The following species of _Sarcocystis_ are of interest:-- _S. miescheriana_, Kühn, 1865, in the pig. _S. bertrami_, Doflein, 1901, in the horse. _S. tenella_, Railliet, 1886, in sheep. _S. tenella bubali_ in buffaloes in Ceylon and Egypt. _S. blanchardi_, Doflein, 1901, in cattle. _S. muris_, Blanchard, 1885, in the mouse, to which it is lethal. _S. hueti_, Blanchard, 1885, in the seal. _S. colii_, Fantham, 1913, in the African mouse-bird, _Colius erythromelon_. Also various Sarcosporidia from antelopes, monkeys, opossum, birds, the gecko and wall-lizard are known. The spores of _S. muris_, _S. bertrami_, _S. tenella_, and _S. colii_ can multiply by longitudinal fission. SARCOSPORIDIA OBSERVED IN MAN. (1) Lindemann[233] found on the valves and in the myocardium of a person who had died of dropsy certain brownish masses, 3 mm. in length and 1·5 mm. in breadth which he regarded as gregarines. If these were actually independent animal organisms it may be suggested that they were Sarcosporidia. Rivolta (1878) named the species _S. lindemanni_. [233] “Ueb. d. hyg. Bdtg. d. Gregarinen,” _Dtsche. Ztschr. f. Staatsarzneikunde_, 1868, xxvi, p. 326. (2) Rosenberg[234] found a cyst 5 mm. in length and 2 mm. in breadth in a papillary muscle of the mitral valve of a woman, aged 40, who had died from pleuritis and endocarditis. The cyst contained no scolex nor hooklets of tænia. Numerous small refracting bodies, round, oval or kidney-shaped, were found in a daughter cyst, as well as sickle-shaped bodies. The description hardly appears to indicate Sarcosporidia. [234] “Ein Befund von Psorosp. in Herzmusk d. Menschen,” _Ztschr. f. Hygiene_, 1892, xi, p. 435. (3) Kartulis[235] observed Miescher’s cylinders of various sizes in the liver (?) and in the muscular system, of a Sudanese who had succumbed to multiple abscesses of the liver and abdominal muscles. This may be considered as the first actual case of the occurrence of Sarcosporidia in man. Koch in 1887 described a case in Egypt. [235] Kartulis, “Ueb. pathog. Protoz. b. Menschen,” _Ztschr. f. Hyg. u. Inf._, 1893, xiii, p. 1. Compare also Braun, M., _Die Thier. Par. d. Mensch._, 2nd Edit., Wrzbg., 1895, p. 92; Braun, M., “Z. Vork. d. Sarcosp., b. Menschen,” _Centralbl. f. Bakt._ 1895, xviii, p. 13. (4) The case reported by Baraban and St. Remy[236] was at once demonstrated as certain. It related to a man who had been executed, and in the laryngeal muscles of whom Sarcosporidia were found; the length of the parasites varied between 150 µ and 1,600 µ, their breadth between 77 µ and 168 µ. The affected muscular fibres were distended to four times their normal thickness. This species was described by Blanchard as “_Miescheria_” _muris_, but according to Vuillemin, it was more probably _Sarcocystis tenella_ of the sheep. [236] “Sur un cas de Tub. Psorosp. ob. chez l’homme,” _C. R. Soc. Biol._, Paris, 1894 (x), I, p. 201. “Le Parasitisme d. Sarcosp. chez l’homme,” _Bibliogr. Anat._ 1894, p. 79. (5) Vuillemin has also described a case of Sarcosporidia found in the muscles of a man who died from tubercle at Nancy. The author considered that the parasite corresponded to _S. tenella_. (6) Darling[237] (1909) found Sarcosporidia in the biceps of a negro from Barbados. [237] _Arch. Internal Med._, III, p. 183. The Myxosporidia, Microsporidia, Actinomyxidia and possibly the Sarcosporidia may be included within the section *Cnidosporidia* (Doflein), since they possess spores containing polar capsules. Order. *Haplosporidia*, Caullery and Mesnil. The Haplosporidia are a group of organisms having both a simple structure and life-history. The simplicity may represent a primitive condition or may be due to degradation resultant on parasitism, and thus it is possible that the group is not a homogeneous one. The order Haplosporidia was created by Caullery and Mesnil in 1899, and includes parasites of rotifers, annelids (fig. 110), crustacea, fish, prochordates and man. They may be present in the body cavity or alimentary tract, and can also occur in the septum nasi of man, in the nervous system of Cephalodiscus, and in tumours of fish. As the name implies, the spores of the Haplosporidia are simple, without polar capsules, and are uninucleate. In some genera, _e.g._, _Haplosporidium_, _Urosporidium_ (fig. 111) there is a spore-coat or sporocyst which may be elongate or spiny. The developmental cycle of a Haplosporidian, such as _Haplosporidium_ or _Bertramia_, begins with a small, uninucleate cell, often rounded, possessing a cell membrane that may be prolonged into processes. Growth takes place, coupled with an increase in the number of nuclei, so that a multinucleate trophozoite is produced. Later, this multinucleate trophozoite becomes segmented into a number of ovoid or spherical pansporoblasts, which give rise to few (one to four) spores. Such a spore, when set free, begins the life cycle over again. More recently (1905–1907) two important organisms have been described and included in this group, namely, _Neurosporidium cephalodisci_[238] (Ridewood and Fantham) from the nervous system of the prochordate, _Cephalodiscus nigrescens_, and _Rhinosporidium kinealyi_ (or _seeberi_) from the septum nasi of man. In the case of _Rhinosporidium_ and _Neurosporidium_, after the uninucleate spore has grown into a multinucleate trophozoite, the latter segments into uninucleate pansporoblasts, as in the preceding cases. A difference then occurs, for each pansporoblast enlarges, its nucleus divides and a “spore-morula” is formed. Thus a multinucleate pansporoblast or spore-morula, divided into many uninucleate sporoblasts (spore mother cells) is produced, and each sporoblast without further change becomes a uninucleate spore. [238] _Quart. Journ. Microsc. Sci._, li, p. 81. The Haplosporidia have therefore been divided by Ridewood and Fantham (1907)[239] into two sections:-- [239] See Fantham, _Brit. Assoc. Reports_, 1907, p. 553. (1) The _Polysporulea_, wherein the pansporoblast gives rise to a number of spores (nine or more), _e.g._, _Rhinosporidium_, _Neurosporidium_. (2) The _Oligosporulea_, wherein the pansporoblasts give rise each to a few (four) spores or to only a single spore, _e.g._, _Haplosporidium_, _Bertramia_, _Cœlosporidium_, _Ichthyosporidium_. [Illustration: FIG. 110.--_Haplosporidium heterocirri._ Section throughout wall of the Polychæte worm, _Heterocirrus viridis_, showing various developmental stages of the Haplosporidium. × 550. (After Caullery and Mesnil.)] [Illustration: FIG. 111.--Haplosporidian spores. _a_, _b_, _Haplosporidium heterocirri_. _a_, fresh; _b_, after immersion in sea water; _c_, _d_, _Urosporidium fuliginosum_. × 1000. (After Caullery and Mesnil.)] *Rhinosporidium kinealyi*, Minchin and Fantham, 1905. _Rhinosporidium kinealyi_, parasitic in man, must now be considered in greater detail. This organism was found in nasal polypus in India, and has since been recorded from the ear as small nodules in the external auditory meatus. The Indian cases came from the neighbourhoods of Calcutta and Madras, and the parasite has been seen in Ceylon. Similar structures have since been described from the United States and South America. The Rhinosporidium polypus is said not to be particularly painful, though nasal forms must interfere with breathing to some extent. The first nasal polyp reported from India formed a vascular pedunculated growth on the septum nasi and was about the size of a large pea or raspberry. It was compared with a raspberry, being red in colour with a number of small whitish dots upon its surface. When the tumour was cut, a number of similar whitish dots were seen within. These were the cysts of Rhinosporidium. According to Minchin and Fantham[240] (1905), they vary considerably in size and measure up to 200 µ or 250 µ in diameter. Each possesses a cyst wall which varies in thickness in different cysts. Its outer wall is always firm and distinct, the inner limit being less definite at times. Each large cyst is filled with numbers of spherical or oval bodies, showing every gradation between small ones at the periphery and large ones at the centre (fig. 112). Roughly, three zones of parasites can be distinguished in a large cyst, a peripheral set consisting of the youngest parasites, an intermediate group and a central, oldest zone. A large cyst may possess a pore for the egress of its contents. Some of the cysts show polar distribution of the zones. [240] _Quart. Journ. Microsc. Sci._, xlix, p. 521. The youngest forms of Rhinosporidium are difficult to detect. They are small, granular masses, round, ovoid or irregular and at times even amœboid in appearance. These are young trophozoites. They increase in size, but encystment occurs early, the outer layer becoming firm so that the organisms have a definite contour. Each is soon multinucleate and the cytoplasm segments around the nuclei. The cyst thus becomes full of uninucleate pansporoblasts or sporonts, with a peripheral layer of undifferentiated protoplasm. The pansporoblasts grow in size. In the larger cysts the formation of pansporoblasts progresses at the expense of the peripheral layer of protoplasm, which, however, continues to grow, so that the cyst as a whole increases in size. The pansporoblasts at first are uninucleate (fig. 112, _a_), and then undergo nuclear multiplication. This is well seen in the intermediate zone of parasites, where the pansporoblasts show first one, then two, then four or more spores (fig. 112, _b_), while in the oldest centrally placed pansporoblasts, about a dozen or sixteen closely packed spores (fig. 112, _c_), can be seen. The spore is small and rounded, and its nucleus is clear and distinct. The fully formed pansporoblast or spore morula becomes surrounded by a membrane. Certain of the cysts have been found in a ruptured condition, whereby the spores have been liberated into the surrounding tissue. It is almost certain that the spores serve for the auto-infection of the host, for though the tumours of Rhinosporidium seemed to have been removed entirely, it has been found that they recur, some minute fragment of the parasite having probably been left behind. The method whereby the parasite reaches new hosts has not yet been determined, and it would be of interest if its life-history could be more fully investigated. [Illustration: FIG. 112.--_Rhinosporidium kinealyi._ Portion of ripe cyst containing pansporoblasts of various ages. × 480. (After Minchin and Fantham.)] The Asiatic specimens of _R. kinealyi_ were first described in detail by Minchin and Fantham (1905) from material briefly reported to the Laryngological Society of London in 1903, by O’Kinealy. Material obtained by Dr. Nair, of Madras, was described by Beattie[241] in 1906. This material came from Cochin. Castellani and Chalmers have found similar polypi in Ceylon. [241] _Journ. Pathol. and Bacteriol._, xi, p. 270; and _Brit. Med. Journ._, Nov. 16, 1907, p. 1402. Wright[242] has described the parasite from Memphis, Tennessee. Seeber[243] in 1896 described nasal polypi in Buenos Ayres, and in 1900 Wernicke named the parasite therein _Coccidium seeberi_. Seeber’s parasite is a Rhinosporidium, _R. seeberi_, and may ultimately be found to be the same as _R. kinealyi_. Ingram[244] reports Rhinosporidium cysts, with pores in the cyst walls, in conjunctival polypus and in papilloma of the penis in India. Zschokke has reported the presence of _Rhinosporidium_ in horses in South Africa. [242] _New York Med. Journ._, December 21, 1907, p. 1149. [243] _La Ciencia Medica_ (Buenos Ayres), 1912. [244] _Lancet_, September 3, 1910, p. 726. Class IV. *INFUSORIA*, Ledermüller, 1763. The Infusoria (or Heterokaryota, Hickson, or Ciliophora, Doflein) include the Ciliata and the Suctoria. A few authorities, including Braun, raise the Suctoria (or Acinetaria) to separate rank as a class, but this is not widely followed. The body of the Ciliata usually is bilaterally symmetrical and is enveloped in a cuticle which has numerous openings for the protrusion of the cilia. Most kinds have a fixed shape, whilst changes in the form of others are brought about by the contractions of the body substance. The latter exhibits hyaline ectoplasm, in which myonemes, and occasionally also trichocysts (minute spindle-shaped bodies) appear, and granular endoplasm which may contain numerous vacuoles. The cilia, on whose various arrangements the classification is based, are always processes of the ectoplasm. Their form varies; they may be hair-like, or more rarely thorn-like, spur-like, or hook-shaped; undulatory membranes also may occur, which are probably composed of fused cilia. With the exception of some of the parasitic species, an oral cavity, peristome or cytostome, is always present. It is frequently beset with cilia or provided with undulatory membranes, which help to waft the food inwards; sometimes there is an anal aperture (cytopyge) generally placed at the opposite pole of the organism. A cytopharynx fringed with cilia or sometimes with a specialized supporting apparatus is connected with the peristome. Vacuoles form round the ingested food, and in many species a constant rotation goes on in the endoplasm. Often one, and sometimes two contractile vacuoles are present, the frequency of the pulsations of which depends on the surrounding temperature. Sometimes special conducting channels lead to the vacuoles, or there are outlet channels leading to the exterior. There is in almost every case a large nucleus (macronucleus), and lying close up to it a small nucleus (micronucleus). The form of the large nucleus varies according to the species. Numerous nuclei are not very common, but these occur in _Opalina_, which lives in the hind-gut of amphibia, and is also distinguished by the absence of an oral aperture. Reproduction is effected by binary fission; less commonly, after encystment, by multiple division, or by budding. The divisions can be repeated many times, but finally cease, and then the conjugation of two specimens brings about a regeneration, particularly of the nuclei. Numerous examinations (Bütschli, Hertwig, Maupas, Calkins) have demonstrated that after two individuals have associated by homologous parts of the body, the micronucleus separates from the macronucleus, becomes larger and divides twice by mitosis, so that four micronuclei are present in each one of the two individuals forming the couple. Three of these nuclei perish and become absorbed, the fourth gradually passes to the portion of protoplasm connecting the two conjugants, which has originated by absorption of the cuticle at the point of contact of the conjugants. After a further division one micronucleus of each conjugant passes over into the other conjugant, and fusion ensues between the two micronuclei of each individual. Complicated changes and divisions may occur, but only the main principles can be noted here. A new nuclear body is thus formed in each conjugant, and soon divides into two. Of the segments thus produced one becomes a micronucleus, and one or several of the others, as the case may be, form or amalgamate into a new macronucleus, the old macronucleus usually perishing or becoming absorbed during the conjugation. Usually, sooner or later, the two conjugants separate, or may have separated already, and again multiply independently by fission until a series of divisions by simple fission is again followed by conjugation. The theoretical significance of conjugation cannot be dealt with fully here. It may be remarked, however, that the macronucleus plays no part in it, but governs entirely the metabolism of the Infusorian, whereas the micronucleus is essentially a generative nucleus from which macro- and micro-nuclei are again and again produced. Encystment amongst the Infusoria is very general, and is essentially a means of protection when the surrounding medium dries up. Doubtless these cysts are frequently carried long distances by the wind, which explains the wide geographical distribution of most species. Also, multiplication often takes place in the encysted condition. Some Infusoria live a free life, others are sedentary; the latter form colonies in fresh as well as in salt water. Numerous species are parasites of various lower and higher animals,[245] and a few also are parasitic in man. [245] It may be stated that numerous peculiarly shaped species live in the stomach of ruminants, others in the colon of horses. Several species are found in the rectum of frogs and toads; others, again, on the surface of the bodies of fishes; and various other species exist in and on the bodies of invertebrate animals. The Prague zoologist, v. Stein, introduced a classification of the Infusoria that has been almost universally adopted. It is founded on the different position of the cilia on the body. Though, no doubt, artificial, it is a convenient system. Bütschli has compiled a better one.[246] But for our purpose Stein’s system is sufficient:-- [246] Bronn’s _Cl. u. Ordn. d. Thierr._, i, Protozoa, Part 3, Infusoria. Order 1. _Holotricha_, Infusoria with cilia that are evenly distributed over the entire body. Order 2. _Heterotricha_, ciliated all over like the _Holotricha_, but having stouter cilia about the peristome. Order 3. _Hypotricha_, only ciliated on the ventral surface. Order 4. _Peritricha_, with only a ring of spiral cilia, mostly sedentary. The Infusoria observed in man belong to the order _Heterotricha_, with few exceptions. Genus. *Balantidium*, Claparède et Lachmann. Heterotrichous Infusoria of oval or bag-like form and almost circular on transverse section; the anterior extremity narrowed, the posterior end broad and rounded off, or also narrowed; the peristome starting at the anterior end is broadest there and becomes narrower as it gradually obliquely approaches towards the posterior extremity. There are coarse cilia along the entire left border and the anterior part of the right border. Longitudinal striation is distinct and regular. There are two contractile vacuoles on the right, and occasionally also two or more to the left. The anus (cytopyge) is terminal. The macronucleus is usually horse-shoe or kidney-shaped, sometimes oval; the micronucleus contiguous. Reproduction is by binary fission and conjugation, and encystment occurs. The cysts are spherical or oval. These ciliates are parasitic in the large intestine of human beings and pigs, in Amphibia, and in the body cavity of polychæte Annelida. *Balantidium coli*, Malmsten, 1857. Syn.: _Paramæcium coli_, Malmsten, 1857. [Illustration: FIG. 113.--_Balantidium coli._ _a_, nucleus; _b_, vacuole; _c_, peristome; _d_, bolus of food. (After Leuckart.)] [Illustration: FIG. 114.--_Balantidium coli_, free and encysted; _a_, anus or cytopyge; _n_, macronucleus; _b_, bolus of food. (After Casagrandi and Barbagallo.)] The body is oval, 60 µ to 100 µ in length (up to 200 µ according to Janowski), and 50 µ to 70 µ in breadth. The peristome is funnel-shaped or contracted, the anterior end being then broadened or pointed according to the degree of contraction (figs. 113, 114). The ecto- and endo-plasm are distinct, the latter is granular, containing drops of fat and mucus, granules of starch, bacteria, and occasionally also red and white blood corpuscles. There are usually two contractile vacuoles, seldom more. The anus (cytopyge) opens at the posterior extremity. The macronucleus is bean- or kidney-shaped, rarely oval; the micronucleus is spherical. _Balantidium coli_ lives in the large intestine of man, in the rectum of the domestic pig, and has been found in monkeys. It propagates by transverse division, but conjugation and encystment are known to take place.[247] Transmission to other hosts is effected by the cysts of the parasite (fig. 114). [247] According to Gourvitch (“Bal. coli. Darmk. d. Menschen,” _Russ. Arch. f. Path., klin. med. u. Bact._, Petrograd, 1896), the conjugated Balantidia are supposed to fuse with each other and form oval cysts two or three times the size of the free organisms, and to divide into numerous globules within the cystic membrane; the process, however, has hitherto not been confirmed. The supposed Balantidium cysts appeared in two patients who were simultaneously suffering from _Dibothriocephalus latus_, after the administration of anthelminthics. It therefore seems, according to the description, that in reality these forms were actually abnormally large, possibly swollen, young eggs of the tape-worm mentioned. _Balantidium coli_, first seen by Leeuwenhoek, was described by Malmsten in 1857 in a man aged 35 years, who had two years previously suffered from cholera, and since then had been subject to diarrhœa. The examination showed an ulcer in the rectum above the mid sphincter ani, in the sanguineous purulent secretion of which numerous Balantidia were swimming about. Although the ulcer was made to heal, the diarrhœa did not cease and the stools contained numerous Balantidia, the number of which could only be decreased by extensive enemas of hydrochloric acid. The second case related to a woman who was suffering from severe colitis, and who died ten days after admission. The malodorous, watery evacuations contained innumerable Balantidia, in addition to pus, and at the autopsy the anterior portion of the large intestine was found to be infested with them. Subsequently this parasite has often been observed in human beings, and various cases have been recorded. These occurred in Russia, Scandinavia, Finland, Cochin China, Italy, Germany, Serbia, Sunda Islands, Philippine Islands, China, and in other parts of Asia and in America. Other cases were reported by Askanazy, Ehrnroth, Klimenko, Nagel, Koslowsky, Kossler, Waljeff, Strong and Musgrave, Glaessner[248] and others. Sievers found _B. coli_ very common in Finland. [248] _Centralbl. f. Bakt._, Orig., xlvii, p. 351. In the majority of the cases described by Sievers from Finland, and in other cases from Central Europe, the patients suffered from obstinate intestinal catarrh, which did not always cease even after the Balantidia had disappeared. On the other hand, Balantidia have occasionally still been found to persist, though in small numbers, after the catarrh has been cured. Some authors, nevertheless, do not regard Balantidia as the primary cause of the various diseases of the large intestine, which often commence with the development of ulcers, but they consider that they may aggravate these diseases and render them obstinate. According to Solowjew, Askanazy, Klimenko and Strong and Musgrave, however, the parasites penetrate the intestinal wall, and give rise to ulcerations which may extend deeply into the submucosa, and even be found in the blood and lymphatic vessels of the intestinal wall. According to Stokvis, _B. coli_ occurs also in the lung; at all events this author states that he found one living and several dead paramæcia (?) in the sputum of a soldier, returned from the Sunda Islands, who was suffering from a pulmonary abscess. Sievers has shown that _B. coli_ might occur in persons not suffering from intestinal complaints, but E. L. Walker[249] (1913) states that every person parasitised with _B. coli_ is liable sooner or later to develop balantidian dysentery. [249] _Philip. Jl. Sc._, Sec. B, viii, p. 333. Since Leuckart confirmed the frequent presence of _B. coli_ in the rectum of pigs, and corresponding observations were made in other countries, the pig is universally considered to be the means of the transmission of Balantidium to man. The encysted stages only serve for transmission, because, according to all observations, the free parasites have a very small power of resistance. They perish when the fæces have become cool; they cannot live in ordinary, slimy, or salt water. As they are killed by acids even when much diluted, they cannot pass through the normal stomach alive except under the most unusual circumstances. The pigs, in whose intestines the Balantidium appears to cause little or no disturbance, evacuate numerous encysted Balantidia with the fæces, and their occasional transference to man brings about their colonization there, but perhaps only when a disease of the colon already exists. Experimental transmission of the free parasites to animals (per os or per anum) yielded negative results, even in the case of pigs. Casagrandi and Barbagallo (1896), however, had positive, as well as negative, results. They employed healthy young cats, or cats in which catarrhal entero-colitis had been artificially induced (which in other experiments is apt to cause the death of the animals experimented upon in about six or seven days), or finally cats that had dilatation of the rectum with alkaline reaction of the fæces. An attempt to infect three healthy cats by injecting human fæces containing Balantidium into the rectum proved negative, in so far as the fæces of the experimental animals had an acid reaction and contained no Balantidia, but at the autopsy performed eight days after infection a few encysted parasites were found in the mucus of the ileum. In the case of four cats suffering from entero-colitis, into which human fæces containing Balantidia were introduced per os, Balantidium cysts were found in the fæces three days after the last ingestion. Great numbers, moreover, were found in the cæcum and the posterior part of the small intestine at the autopsy of the animals, which died about eight days after the commencement of the experiments. Actual colonization, therefore, was not effected in either series of experiments. Free or encysted Balantidia of pigs were used for further experiments. The experiments proved negative when fæces containing cysts were injected into the rectum of healthy cats (three experiments), or cats (two) suffering from spontaneous intestinal catarrh, or when such material was introduced per os into three healthy cats. In the case of two cats with intestinal catarrh artificially produced, a small number of the active Balantidia injected into the rectum remained alive. Larger quantities of fæces containing encysted Balantidia were introduced into two other cats affected with the same complaint. These, certainly, did not appear in the fæces, but small numbers, free and alive, were found in the cæcum. Similarly, encysted Balantidia were introduced into two cats with dilated rectum, and whose fæces had an alkaline reaction. In these cases no parasites appeared in the fæces, but three and five days later, when the two animals were examined, a very small number were discovered free in the large intestine. Klimenko did not succeed in infection experiments with _B. coli_ on young dogs, whose intestines had been artificially affected by disease. More recent experiments by Brumpt have shown that young sucking pigs can be infected with Balantidium from infected monkeys (_Macacus cynomolgus_) and suffer heavily from the same, whereas the Balantidium of the pig is rarely harmful to its host. This and previous experiments may be thought to suggest that there are perhaps several pathogenic species, and also that harmless strains of Balantidium may occur. At the same time, it must be remembered that a large proportion of the cases recorded of Balantidian colitis occur among swineherds and butchers, that is, among people in frequent contact with pigs. Morphologically, there are practically no differences between the Balantidia found in man, monkeys and pigs, and it is probable that one species only, under slightly different environmental conditions, may be responsible for the colitis observed. In any case, efficient prophylactic measures should be taken against balantidiasis in countries where it may occur, by confining the pigs and not allowing them to run in yards and dwellings. E. L. Walker (1913) has given a good summary of work on balantidiasis. His own researches in the Philippines showed that monkeys could be infected by Balantidia both from pigs and men. Parasites may appear in the stools only at infrequent intervals. He believes that the ciliates are the primary etiologic factor in the symptoms and lesions of balantidian dysentery. Behrenroth (1913) has given an interesting account of _Balantidium coli_ and its pathogenic significance. *Balantidium minutum*, Schaudinn, 1899. The body is of oval form, with the anterior extremity pointed, and posterior extremity broad and rounded (fig. 115). The length is 20 µ to 32 µ, and the breadth is 14 µ to 20 µ. The peristome, which is fissure-like, extends to the centre of the body (fig. 115). The right lateral border of the peristome is beset with cilia the same length as those of the body, the left side terminates in a thin hyaline membrane that extends towards the back, and can pass over to the right side. A row of longer and stronger cilia (cirri) are on the left border of the peristome. The cuticle is refractile, the ectoplasm hyaline and the endoplasm granular, with numerous food vacuoles. [Illustration: FIG. 115.--_Balantidium minutum._ _P_, peristome; _N_, nucleus; _M_, micronucleus; _V_, contractile vacuole. Food vacuoles are represented in the endoplasm. (After Schaudinn.)] A single contractile vacuole lies dorsally and to one side at the posterior extremity. The macronucleus, which is always spherical, is central and is 6 µ to 7 µ in diameter. The micronucleus, close in front of it, only measures 1 µ (fig. 115). The cysts are oval. These parasites were found in numbers in the evacuations of a man aged 30, who was born in Germany and had repeatedly travelled between Hamburg and North America, where he made long stays. The patient came to the Charité in Berlin to seek advice for constipation alternating with diarrhœa accompanied by abdominal pain. A second case (the parasite of which was described as _Colpoda cucullus_ by Schulz) was observed in a patient in the same institution. As, in both cases, the parasites only appeared during the diarrhœa, and disappeared as soon as the fæces had assumed a normal consistency, or could only be demonstrated in a few encysted specimens, it may be assumed that the small intestine or the duodenum is their habitat. Genus. *Nyctotherus*, Leidy, 1849. Flat, heterotrichous Infusoria, kidney- or bean-shaped. The peristome commences at the anterior pole of the body and extends along the concave side to the middle, where the oral aperture is situated. The cytopharynx is oblique and is more or less curved. The cytopyge is at the posterior extremity, where a single contractile vacuole is also situated. The macronucleus is almost in the centre of the parasite. The members of this genus live parasitically in the intestine of amphibia, insects and myriapods, and at least one species is found in man. *Nyctotherus faba*, Schaudinn, 1899. The body is bean-shaped, and a little flattened dorso-ventrally. It is 26 µ to 28 µ long, 16 µ to 18 µ broad, and 10 µ to 12 µ thick (fig. 116). The peristome is on the right border and extends to the middle; at the left there are large adoral cilia, the cilia on the right border not being larger than those on the body. The cytopharynx is short, slightly curved and turned backwards. The contractile vacuole is large, spherical, situated at the posterior extremity, and its contents are voided through the anus at its left. The macronucleus is in the centre of the body; it is globular (6 µ to 7 µ in size), and contains four or five chromatin masses. The micronucleus lies close to it, and is spherical or somewhat elongate measuring 1 µ to 1·5 µ (fig. 116). The cysts are oval. [Illustration: FIG. 116.--_Nyctotherus faba._ _P_, peristome; _N_, nucleus; _M_, micronucleus; _V_, contractile vacuole. (After Schaudinn.)] This species has hitherto only been seen once in the same patient in whom _Balantidium minutum_ was discovered. *Nyctotherus giganteus*, P. Krause, 1906. Under the name _Balantidium giganteum_ n. sp., P. Krause described an Infusorian which was repeatedly observed with _Trichomonas intestinalis_ in the alkaline evacuations of a typhoid patient in Breslau. The body is ovoid, narrower and rounded anteriorly and broader and stunted posteriorly. The peristome lies to one side; the macronucleus is bean-shaped, the micronucleus small and globular; one or two vacuoles are present. The anus is at the farther end. The organism is 90 µ to 400 µ long, 60 µ to 150 µ broad (fig. 117). After a prolonged stay outside the body, it becomes rounded and encystment occurs. In the thermostat the Infusoria remain alive at 37° C. for five weeks. The species, however, hardly belongs to _Balantidium_, but to all appearances is a _Nyctotherus_ and is distinguished from _N. faba_ by the difference in size. [*Nyctotherus*] *africanus*, Castellani, 1905. In the fæces of a native of Uganda who suffered from sleeping sickness and diarrhœa and had in his intestine _Ascaris lumbricoides_, _Trichocephalus trichiurus_ and _Ancylostoma duodenale_, Castellani found a curiously shaped Infusorian, 40 µ to 50 µ long, and 35 µ to 40 µ broad, with spherical macro- and micronucleus and a contractile vacuole (fig. 118). He included the organism in the genus _Nyctotherus_, perhaps wrongly, or the parasite may have been deformed. After the patient’s death the same parasite was found in the intestine and especially in the cæcum. [Illustration: FIG. 117.--_Nyctotherus giganteus._ (After Krause.)] [Illustration: FIG. 118.--_Nyctotherus africanus._ (After Castellani.)] G. Lindner, in Cassel, studied certain peritrichal Infusoria (stalkless Vorticella), and connected them, probably incorrectly, with the most varied diseases of man and domestic animals, even with Sarcosporidia of pigs. It may be mentioned that according to a communication by letter from Schaudinn, Vorticella may be found in freshly evacuated fæces, but always only after the administration of a water enema. In spite of this, several other investigators mention Vorticellæ as intestinal parasites of man. The _Chilodon dentatus_ (Ehrenberg) recorded in 1903 by J. Guiart as a parasite of man, which may be found in all infusions, can hardly have lived in the man from whose fæces it was cultivated, but may represent a chance admixture both in the fæces and the cultivations. _C. uncinatus_ was also found as a chance parasite of man by Manson and Sambon. According to Doflein[250] (1911) certain Chilodon-like organisms have been found by Selenew in prostate secretions in gonorrhœa. Other species of the genus _Chilodon_ are known, but only as ectoparasites (_e.g._, _Chilodon cyprini_, Moroff, 1902, from the skin and gills of diseased carp). [250] _Lehrbuch der Protozoenkunde_, 3rd ed., p. 963. A number of other parasitic Ciliates are known, among which _Ichthyophthirius multifiliis_, destructive to fish, is important. It lives in the skin and the layers immediately below it, forming small whitish pustules which may become confluent. The pustules are most common on the head and fins, but occur also on the eyes and gills of the host. The young parasite, which is one of many formed in a cyst, is very small. At first it is free swimming, but soon attaches itself to the skin of a fish. It bores inwards and becomes surrounded by the irritated skin. There it attains a relatively large size, being 500 µ to 750 µ and occasionally more in diameter. The body has a rounded terminal mouth, short cytopharynx and a number of minute contractile vacuoles. The macronucleus is large and horseshoe-shaped; the small micronucleus is only seen in the very young animal. When full grown, the organism encysts and forces its way to the surface and bursts through, leaving a small, gaping wound behind. The cyst sinks to the bottom of the water, nuclear multiplication occurs and a number of young parasites are produced, which leave the cyst and either attack new hosts or else perish. _Opalina ranarum_, parasitic in the rectum and urinary bladder of frogs and toads, shows great degradation and simplification due to parasitism, possessing no separate micronuclei, no cytostome, cytopharynx or cytopyge. It has many macronuclei, and is a large parasite. During summer and autumn nuclear multiplication followed by division of the body occurs, the process being repeated after the daughter forms have grown to the size of their parent. In spring, the Opalina divide rapidly, but do not grow much before dividing again. Finally, tiny forms, containing three to six nuclei, encyst and pass from the host with the fæces. As these latter are greedily devoured by tadpoles, the _Opalina_ gain new hosts in which they develop. THE CHLAMYDOZOA. The name Chlamydozoa was proposed by Prowazek in 1907 for a number of minute, problematic organisms (fig. 119) believed to be the causal agents of certain diseases in man and animals, such as vaccinia and variola, trachoma, inclusion blenorrhœa in infants, molluscum contagiosum, and bird epithelioma contagiosum. Other diseases possibly due to Chlamydozoa[251] are hydrophobia, measles, scarlet fever, foot-and-mouth disease, the “Gelbsucht” disease of silkworms, and perhaps even typhus (Prowazek, 1913). The subject is difficult and controversial and can only be briefly discussed here. It is known that the viruses in all these diseases can pass through ordinary bacterial filters, that is, they belong to the group of “filterable viruses.” At such periods the organisms are extracellular or free. It is also known that in many of these cases the virus produces definite and characteristic reaction-products or cell-inclusions in the infected cells, during the intracellular phase of the life-history of the organism. As the organisms to be considered are problematic, it will be convenient to summarize their history:-- [251] For a detailed account of the Chlamydozoa see Prowazek’s _Handbuch der Pathogenen Protozoen_, Bd. i (1911–12). Leipzig, J. A. Barth. (1) Cell-inclusions, usually named after their discoverers, have been found in certain diseases, thus: In vaccinia Guarnieri’s bodies, in scarlet fever Mallory’s bodies, in hydrophobia Negri’s bodies, in trachoma Prowazek’s bodies occur. (2) At first these characteristic cell-inclusions were considered to be actual parasitic organisms causing the diseases in question. The bodies received zoological names and attempts were made to work out their supposed development cycles. The supposed parasites of vaccinia and variola were referred to a so-called genus _Cytoryctes_, those of hydrophobia to _Neuroryctes_, of scarlet fever to _Cyclasterium_, while those of molluscum contagiosum were referred to the Coccidia. Calkins in 1904 studied in detail the cell-inclusions of vaccinia and small-pox, calling them _Cytoryctes variolæ_, Guarnieri. Calkins considered that in the stratified cells of the epidermis they passed through two cycles, the one cytoplasmic, the other intranuclear. The first is the vaccinia cycle, the second the pathogenic (intranuclear) variola cycle. It is hardly necessary to follow all Calkins’ stages here. Negri (1909) described a cycle for _Neuroryctes hydrophobiæ_. Calkins refers both _Cytoryctes variolæ_ and _Neuroryctes hydrophobiæ_ to the Rhizopoda. Siegel (1905) described quite different organisms under the name _Cytorhyctes_. He listed several species: _C. vacciniæ_; of vaccinia and small-pox, _C. scarlatinæ_ of scarlet fever, _C. luis_ of syphilis (this is probably the granule stage of _Treponema pallidum_), and _C. aphtharum_ of foot-and-mouth disease. (3) The afore-mentioned views were criticized, and the bodies were not considered to be living organisms but merely reaction products or cell-inclusions due to the effects of the virus on the host cells. Thus Guarnieri’s bodies were stated to consist of extruded nucleolar or plastin material, having no developmental cycle. It was further asserted that infection could be produced by lymph in which Guarnieri’s bodies had been destroyed. Similar assertions have been made regarding the Negri bodies, and others. The _Cytoryctes_, _Neuroryctes_, etc., are considered, according to these views, to be degeneration products of the nucleus or to be of a mucoid nature. (4) More recently a positive belief has gained ground that there are true parasitic organisms causing these diseases, and that the parasites are very minute, being termed Chlamydozoa by Prowazek and Strongyloplasmata by Lipschütz. The Chlamydozoa are characterized by (_a_) their very minute size, smaller than any bacteria, so that they can pass through bacterial filters; (_b_) they pass through intracellular stages, in the cytoplasm or the nucleus of the host cell, producing therein the reaction products or inclusions in the cell already recorded as characteristic or diagnostic of the diseases produced; (_c_) they pass through definite developmental cycles. Such a cycle consists essentially of growth and division. The mode of division of the Chlamydozoa resembles that of the centriole of a cell, by the formation of a dumb-bell-shaped figure. Two dots are observed connected by a fine line or strand which becomes drawn out and finally snaps across the middle. Prowazek and Aragão (1909) working on smallpox in Rio de Janeiro found that the chlamydozoal granules passed through a Berkefeld filter and that the filtrate was virulent. But if an “ultra-filter” were used, _i.e._, one coated with agar, then the granules were retained and the filtrate was no longer virulent. The surface of the ultra-filter was found to contain many granules. The Chlamydozoa are parasites of epiblastic tissues (_e.g._, epidermal cells, nerve cells, conjunctival cells). [Illustration: FIG. 119.--Chlamydozoa. Trachoma bodies in infected epithelial cells of the conjunctiva. (_a_) initial bodies (above) and cluster of elementary bodies (touching the nucleus); (_b_) cluster of granules surrounded by mantles. × 2,000 approx. (Original. From preparation by Fantham.)] The life-history of a Chlamydozoön (fig. 119), such as that of vaccinia, is, according to Prowazek, Hartmann and their school, as follows:--