I.—Notes on Recent Researches

Appendix on Protozoology,

Comprising Notes on Recent Researches, Formulæ of some Culture Media, and Brief Notes on General Protozoological Technique.

BY

H. B. FANTHAM, M.A., D.Sc.

Since the foregoing section on Protozoology was sent to press, certain interesting observations and results have been published. Brief notes on such, and some references thereto, are now added.

It is necessary, however, to remark that sometimes it is impossible to give a precise or rigid definition to a genus of Protozoa, owing to differences of opinion, to differences regarding nomenclature or to incompleteness of knowledge. Such a lack of definition, while inconvenient for the time being, is not unhopeful, as it directs attention to the necessity for further work, which is inevitable in such a relatively new and wide subject as protozoology. Thus, it may be noted in illustration that Minchin, in 1912, in his text-book regarding the genus Entamœba writes: “The entozoic amœbæ are commonly placed in a distinct genus, Entamœba, distinguished from the free-living forms by little, however, except their habitat and the general (but not invariable) absence of a contractile vacuole.”

Differences between Entamœba histolytica and E. coli.—In continuation of the remarks on pp. and , it may be added that Lugol’s solution (iodine in aqueous potassium iodide solution) in fresh specimens shows by brownish staining the presence of glycogen in the vacuoles of Entamœba coli. Such a reaction is rarely or never given by E. histolytica.

Phagedænic Amœbæ.—Carini and others record cases in which the skin around an operation wound in connection with liver abscess became gangrenous. Amœbæ, possibly Entamœba histolytica, were found therein and may have been responsible for the gangreno-phagedænic action.

Endamœba gingivalis (see pp. , 44).—Smith and Barrett, after analysing the early literature, state (June, 1915) that Endamœba gingivalis, Gros, 1849, is the correct name for the following organisms: E. buccalis, Prowazek, 1904 (see p. ); Amœba gingivalis, Gros, 1849; Amœba buccalis, Steinberg, 1862, and Amœba dentalis, Grassi, 1879. They conclude that E. gingivalis is the causal agent of pyorrhœa alveolaris, and that this disease responds to treatment with emetine.

Entamœba kartulisi (see p. ), synonym E. maxillaris, Kartulis, is considered to be E. gingivalis.

Smith and Barrett adopt the generic name Endamœba, Leidy, 1879 (see footnote on p. , also p. ). Leidy worked on Endamœba blattæ.

Craigia and Craigiasis (see p. ).—Barlow (May, 1915) found Craigia (Paramœba) hominis in cases of chronic diarrhœa and mild dysentery in Honduras. He also described a new species of Craigia under the name of C. migrans. Fifty-six cases were studied, five of which were due to Craigia hominis, the remainder to C. migrans. In C. migrans, each flagellate, on attaining full development, becomes an amœba without dividing. Each amœba encysts and produces a number of flagellates which are somewhat like cercomonads. On the other hand, in C. hominis the flagellate form produces, by longitudinal fission, several generations of flagellates before entering upon the amœbic stage. The cysts of C. migrans contain fewer “swarmers” (flagellulæ) than those of C. hominis, but the “swarmers” are somewhat larger, namely, 5 µ instead of 3 µ in diameter. Further, there is no accessory nuclear body in C. migrans, but its flagellum stains more deeply than that of C. hominis and has a peculiar banded appearance.

Human Trichomoniasis (see pp. –56).—Lynch (April and May, 1915), working in Charleston, seems to favour the view that the trichomonads found in the vagina, urethra, mouth, lungs and alimentary tract are one and the same organism, and that these flagellates may further excite already existing inflammatory conditions. He gives detailed histories of cases of (a) infection of the vagina and gums, and (b) intestinal infection manifested as intermittent attacks of diarrhœa. The flagellates were found in catarrhal vaginal discharge, in blood-stained scrapings from the gums (together with Endamœba buccalis), and in stools after a purge of magnesium sulphate. The parasites were tetratrichomonads (see footnote, p. ), that is, each possessed four flagella anteriorly as well as an undulating membrane. Lynch successfully infected rabbits from the cases and from cultures of the parasite. Encysted trichomonads were seen in a patient’s stools, in rabbits infected therefrom and in cultures. The culture medium used was bouillon acidified with about 0·05 per cent. acetic acid and the cultures were maintained at 30° C.

Trichomonads occur in the digestive tracts, for example, the cæca of rats and mice (fig. 422). In man allied flagellates can occur in similar situations, as well as in other parts of the intestine.

Other trichomonad-like organisms have been recently described from the fæces of man, more particularly from cases of chronic dysentery in the tropics. Derrieu and Raynaud (July, 1914), working in Algeria, found a flagellate possessing five free flagella anteriorly and an undulating membrane apparently lateral. They named the parasite Hexamastix ardin-delteili, but the generic name Hexamastix is pre-occupied. Chatterjee (January, 1915), working in India, found probably the same flagellate and called it Pentatrichomonas bengalensis.

Chilomastix (Tetramitus) mesnili (see p. ).—Alexeieff (1914) now places the parasite originally called Macrostoma mesnili, by Wenyon (1910), in the genus Chilomastix, Alexeieff. The differential characters of the genera Tetramitus and Chilomastix are not especially well marked. According to Alexeieff, Tetramitus is characterized by four unequal flagella (which he figures anteriorly), a ventral cytostome in the form of a linear cleft and a pulsatile vacuole in front of the anterior nucleus. Chilomastix, according to the same author, has three forwardly directed flagella and a fourth backwardly directed one in the cytostome, which is well developed (fig. 423). Some authors consider that the fourth flagellum forms the edge of an undulating membrane in the cytostome.

Diagrams of Chilomastix mesnili are given in fig. 423.

Giardia (Lamblia) intestinalis (see p. ).—Alexeieff (1914) considers that Lamblia intestinalis, Lambl, should be placed in the genus Giardia, Kunstler, 1882. Bipartition occurs in the encysted state. The axostyles persist in the quadrinucleate cyst.

Cercomonas hominis (see p. ).—This parasite is considered by some authors to be of a doubtful nature, as it is thought to have been mistaken for deformed or incompletely observed Trichomonas or Chilomastix or even Lamblia.

Wenyon (1910) described Cercomonas longicauda from cultures of human fæces. It is considered that the genus is very confused, and the author points out that the tail flagellum has been overlooked. He considers that the genus Cercomonas should include flagellates with an anterior blunt end from which arises a single long flagellum, and a posterior tapering end also with a flagellum, which can be traced over the surface of the body towards the insertion of the anterior flagellum.

Another species, Cercomonas parva, has been found in cultures of human fæces by Hartmann and Chagas (1910). It has a somewhat different structure.

Further researches are necessary on the organisms variously referred to the genus Cercomonas.

Transmissive Phase of Trypanosomes in Vertebrates.—In addition to the general remarks on the morphology of trypanosomes set forth on pp. to 72, it may be noted that Woodcock (November, 1914) states that, in certain cases, there is a definite transmissive phase of a trypanosome in its vertebrate host. He quotes the work of Minchin and himself on T. noctuæ of the little owl, in which the transmissive form is spindle-shaped and occurs in the bird’s peripheral blood during the early summer months (see p. ). A similar phase occurs in T. fringillarum, and Robertson has found that the short, stumpy form of T. gambiense is its transmissive phase in vertebrates.

Trypanosoma lewisi (see p. ).—Brown (1914–15) has published some interesting results on the potential pathogenicity of T. lewisi.

Blepharoplastless Trypanosomes (see p. ).—Laveran (April, 1915) suggested a practical use of strains of blepharoplastless trypanosomes produced by the action of drugs. He finds that tryposafrol will also produce such strains, and remarks on blepharoplastless strains of T. evansi and T. brucei, which in the former case can undergo 450 passages without reversion, and in the latter 273 passages. He states that if it is desired to inoculate surra or nagana to Capridæ or Bovidæ in order to produce immunity, use should be made of the blepharoplastless races of the respective trypanosomes, which races are a little less virulent than the corresponding normal ones. Also, the immunity which follows from an infection due to blepharoplastless T. evansi or T. brucei is only a little less complete than that following infections from either of the respective normal strains.

The Experimental Introduction of certain Insect Flagellates into various Vertebrates, and its bearing on the Evolution of Leishmaniasis.—In continuation of the remarks on pp. , 104, and , further researches have been conducted on the introduction into vertebrates of flagellates normally parasitic in insects. The vertebrates became infected by inoculation with the flagellates or by being fed on insects containing the protozoa. Fantham and Porter (June, 1915) published the following results. Flagellates from sanguivorous and non-sanguivorous insects were used, and cold-blooded as well as warm-blooded vertebrates as hosts. The introduced protozoa were pathogenic to the mammals, but not markedly so to the cold-blooded vertebrates. Herpetomonas jaculum, H. stratiomyiæ, H. pediculi, and Crithidia gerridis (parasitic in certain waterbugs) proved pathogenic to mice. A puppy was infected by way of the digestive tract with H. ctenocephali. Frogs became infected with H. jaculum and with C. gerridis, toads and grass snakes with H. jaculum, lizards with C. gerridis, and sticklebacks with H. jaculum. Second and third passages of some of the parasites were obtained. The protozoa, whether Herpetomonas or Crithidia, were present in the vertebrate hosts in either the non-flagellate or the flagellate form, or usually both. They were more abundant in the internal organs of the hosts, more particularly in the liver, spleen and bone-marrow. In all experiments in which C. gerridis was used the parasite invariably retained the crithidial facies in the vertebrate host. No transition to a trypanosome was ever seen. Infections in adult animals were not so heavy as in the young ones, and the parasites were more virulent in young hosts, as is the case with Mediterranean kala-azar in children.

The mode of infection of the vertebrate in Nature seems to be contaminative, either by its food or through an already existing abrasion or puncture on the surface of its body. Cases in which the flagellate-infected insects have been allowed to suck the blood of vertebrates have proved negative up to the present. In areas where leishmaniases are endemic, an examination should be made of all insects and other invertebrates likely to come into contact with men or dogs, or rats and mice (see below), in order to ascertain if these invertebrates harbour herpetomonads. Preventive measures should be directed against such invertebrates, especially arthropods. Further, it is likely that certain vertebrates, such as reptiles and amphibia (especially those that are insectivorous), may serve as reservoirs of leishmaniases, or, as they should preferably be termed, herpetomoniases. From such reservoirs the herpetomonads may reach man by the agency of ectoparasites or flies, especially such as are sanguivorous.

That vertebrates in Nature can harbour herpetomonads in their blood has been shown by the work of Dutton and Todd (1903) on the herpetomonads of Gambian mice, while the recently published investigations of Fantham and Porter (June, 1915) on natural herpetomonads in the blood of mice in England have shown that these rodents may be a natural reservoir of herpetomoniasis. The origin of the infection of mice is to be sought in a flagellate of an ectoparasite of the mouse, very probably Herpetomonas pattoni parasitic in various fleas, which protozoön can adapt itself to life in the blood of mice. Herpetomonads were also found naturally in the blood of birds by Sergent (1907). Recently, Fantham and Porter have successfully infected birds with herpetomonads experimentally.

The significance of insect flagellates in relation to the evolution of disease has recently been set forth by Fantham (June, 1915). The deductions to be made from the occurrence of a herpetomonad stage in Leishmania, especially in L. tropica, in man himself, and of flagellate stages of the so-called Histoplasma capsulatum in man are fully discussed and correlated. It is pointed out that flagellosis of plants (see p. ) may possibly be connected with leishmaniasis. The evolution of Leishmania from flagellates of invertebrates is thus traced and the way again indicated for preventive measures against leishmaniasis, as first set forth by Dodds Price and Rogers.

Franchini and Mantovani (March, 1915) have successfully infected rats and mice by inoculation or by feeding with Herpetomonas muscæ domesticæ obtained from flies and from cultures.

It is of great interest to note that the recent observations of Ed. and Et. Sergent, Lemaire and Senevet (1914) have demonstrated the presence of a herpetomonad flagellate in cultures of the blood and organs of geckos obtained from areas in Algeria in which Oriental sore, due to L. tropica, is prevalent. Phlebotomus flies, which may harbour a natural herpetomonad, feed on the geckos and on men. Hence animals like geckos may possibly act as reservoirs of leishmaniasis. Lindsay (1914) writes that the parasite of dermo-mucosal leishmaniasis in Paraguay is believed by native sufferers to be conserved in rattlesnakes, and spread by ticks or flies (Simulium) feeding on the reptiles and transferring the parasite to man.

The Transmission of Spirochæta duttoni (see p. ).—It is probable that Ornithodorus savignyi acts as the transmitting agent of S. duttoni in places like Somaliland (Drake-Brockman, 1915).

Spirochæta bronchialis (see p. ).—The morphology and life-history of S. bronchialis have been investigated by Fantham (July, 1915). From researches conducted in the Anglo-Egyptian Sudan, he found that S. bronchialis is an organism presenting marked polymorphism, a feature that has only been determined by the examination of numerous preparations from the deeper bronchial regions of various patients.

S. bronchialis varies in length from 5 µ to 27 µ, and its breadth is about 0·2 µ to 0·6 µ. These variations are due to the processes of growth and division. Many of the parasites measure either 14 µ to 16 µ long, or 7 µ, to 9 µ, the latter resulting from transverse division of the former. The ends show much variation in form, but approach the acuminate type on the whole. The discrepancies in dimensions given by the very few previous workers on the subject are probably the result of the measurement of a limited number of parasites. All such sizes can be found on some occasion during the progress of the disease, when a larger number of spirochætes is examined.

The movements of S. bronchialis are active, but of relatively short duration, when it is removed from the body. The number of coils of the spirochæte is rather an index of its rapidity of motion than a fixed characteristic of the species.

The motile phase of S. bronchialis is succeeded by one of granule formation, the granules or coccoid bodies serving as a resting stage from which new spirochætes are produced. The formation of coccoid bodies and reproduction of spirochætes from them can be observed in life.

S. bronchialis is a species distinct from the spirochætes occurring in the mouth. It differs from them in morphology, pathogenicity and in staining reactions. It is not a developmental form of any bacterium, and is an entity in itself.

The passage from man to man is effected most probably by means of spirochætes, and especially coccoid bodies, that leave the body in the spray with expired air and by way of the nasal secretions. Owing to the fragility and short life of S. bronchialis extracorporeally, the resistant coccoid bodies in air, in dried sputum and dust, and possibly also on the bodies of flies and other insects, are probably instrumental in inducing attacks of bronchial spirochætosis in human beings, especially those having a lowered bodily resistance, such as occurs after a chill. Lurie (December, 1915), has described a case from Serbia.

The Spirochætes of the Human Mouth (see p. ).—Two species of spirochætes were recorded as occurring in the human mouth about forty or fifty years ago. These are Spirochæta buccalis, Steinberg (often ascribed to Cohn, 1875), and S. dentium, Miller (often attributed to Koch, 1877).

The most recent work on S. dentium and S. buccalis is that of Fantham (July, 1915), who observed the parasites ascribed to Cohn and to Koch, these being the two common spirochætes seen in the mouths of natives of the Sudan and of Europeans in England, as well as the forms described and cultivated by recent investigators. Some of the mouth spirochætes are not very active, but there is marked corkscrew and boring movement, and they are flexible. Tangles or tomenta of these mouth spirochætes are common. Internal structure is seen with some difficulty, but in some specimens it can be determined, and chromatin granules are then seen. Mühlens (1907) figured stained specimens of S. buccalis and S. dentium, in which chromatin-coloured granules were distributed along the bodies of the organisms.

S. dentium has tapering ends, and varies in length from 4 µ to 10 µ. S. dentium is rather like Treponema pallidum, and has been placed by some workers—for example, Dobell—in the genus Treponema. It has already been mentioned, on p. , that Noguchi cultivated three species of Treponema from the human mouth—namely, T. macrodentium, T. microdentium, and T. mucosum, but they cannot be easily distinguished morphologically, and so may appear to be biological varieties of S. dentium.

S. buccalis has somewhat rounded or bluntly acuminate ends and varies in length from 9 µ to 22 µ. A slight membrane or crest may sometimes be observed. S. buccalis was found to be the predominant spirochæte in the mouths of eight natives examined by Fantham in the Anglo-Egyptian Sudan.

S. buccalis and S. dentium take up stains well and with relative ease. Intracellular stages of the parasites are uncommon. Multiplication by binary fission has also been observed. Coccoid bodies or granule stages of the mouth spirochætes are formed, but appear to be relatively few in number.

J. G. and D. Thomson (1914) have written an interesting paper on various spirochætes occurring in the alimentary tract of man and of some of the lower animals. They have also given a useful list of references, and the work of some of the earlier authors is discussed in the paper.

With regard to the general morphology of spirochætes, it may be noted that the so-called axial fibre of Zuelzer is acknowledged to be homologous with the membrane or crista of molluscan spirochætes.

Coccidia in Cattle.—Regarding the remarks on coccidiosis or “red dysentery” in cattle on p. , it may be added that Schultz (July, 1915) has found the malady among cattle in the Philippine Islands. He states that some irregular or atypical cases of apparent rinderpest are really due to coccidia. As has been pointed out by Montgomery, rinderpest can be transmitted by blood inoculation, while coccidiosis cannot be so transmitted, but may be diagnosed by the microscope. These differences should be remembered as the two diseases are often found to be associated and are difficult to separate clinically. Coccidia have also been found in Australian cattle.

The Hæmosporidia.—It is likely that this order (see p. ) may be soon abolished. Mesnil (April, 1915) considers that the grouping of the three families, Plasmodiidæ (or Hæmamœbidæ), Hæmogregarinidæ and Piroplasmidæ in the order Hæmosporidia is no longer possible, because of the coccidian nature of the Hæmogregarines (see p. ). The Coccidia are divisible into the Adeleidea and the Eimeridea (see p. ). The Hæmogregarinidæ are allied to the former, and the Plasmodiidæ to the latter. The Piroplasmidæ, until more is known of their life-cycle in the invertebrate host, cannot be more definitely placed.

The Leucocytozoa of Birds.—Regarding the statement, on p. , that Laveran and França consider that avian leucocytozoa may inhabit red blood cells, it may be added that França (April, 1915) remarks that the action of the parasites on the red cells is very rapid and very intense. The host cells become so altered that it is difficult to recognize their true nature. He used very young birds in his researches. Two shapes of host cell are considered, namely, those with fusiform prolongations, and those which are rounded and without such prolongations (see p. ). The movements and form of the Leucocytozoa determine the shape of the host cell, as was pointed out by Fantham in 1910.

Schizogony of these parasites has been seen by França (1915) and by Coles (1914), in addition to Fantham (1910), and to Moldovan (1913), mentioned on p. . Schizogony may also take place in the lungs of the host. The genus Leucocytozoön, established by Ziemann in 1898, belongs to the family Hæmamœbidæ.