A HBMOGREGARINE AND A LEUCOCYTOZOON FROM GADUS ATGLEFINUS.’

By HERBERT HENRY, M.D., B.S.(Lond.).

From the Dqiartmant of PathoZogy, University of Shefield.

(PLATES XV1II.-XIX.)

GADUS BCJLLEPINUS, the common haddock, has been found to harbour a trypanosome, a spirochaete, a hremogregarine, and a leucocytozoon, the two last-mentioned parasites being the subject of the present communication.

Infected fish were taken in the month of July 1910, by otter trawl from a depth of about fifty fathoms in the vicinity of Rhona and Sulisker, two barren uninhabited islands situated in the Atlantic about sixty miles N. by E. of Stornoway. Eight months previously this fishing ground had proved to be a very prolific one, but on the occasion of this voyage the only fish obtained in numbers were Ammo- dytes lanceolatus and immature specimens of haddock, the latter being quite unmarketable.

The hamogregarine occurred in but five out of fifty-three fish examined, i .e . , in 9.4 per cent., and even in individual fish infected corpuscles occur very scantily indeed. The parasite is always intra- cellular, and only one individual is present in a red blood corpuscle. In the case of smaller, i.e., of immature forms, either no obvious effect is produced on the host cell, or there is slight mechanical displacement of the nucleus to one side. With the larger forms the host corpuscle may be considerably enlarged from the actual bulk of the contained parasite. In certain instances, in addition to corpuscle enlargement, the protoplasm and nucleus of the host cell are degenerated. The parasite is not encapsulated, neither is there any appearance suggestive of the formation of a “ cytocyst.”

The forms in which the hcemogregarine occurs may be con- veniently classified into three main types.

TYPE I. (Plate XVIII. Figs. 1-1 O).-These are straight, slightly bent, or comma-shaped forms, which show at times one extremity slightly

1 [Received August 1, 1913.1 Communicated to the Pathological Society of Great Britain and Ireland, June 27-28, 1913,

BLOOD PARASITES OP GADUS WGLEFINUS. 233

more pointed than the other. A few show the presence of a recurved limb springing from this more pointed extremity (Plate XVIII. Fig. 10). The length of these thin types varies from 8.5 to 10.5 p, the average being 9.4 p ; and the width of these forms varies from 2 to 3 p, with an average of 2.2 p.

The protoplasm stains a full blue with Leishman, and is seen to be faintly reticular. At times there are scattered throughout it numerous basophilic granules. I n addition, there are found occasionally a t one extremity one, two, or more deeply staining granules which look like chromatin (Plate XVIII. Figs. 2-4, 6-8). The nucleus is situated as a rule about the middle third of the body, and occupies the whole width of the parasite. Or again, the nucleus may be pushed towards one extremity (Plate XVIII. Figs. 2 and 7). The nuclear chromatin granules are fairly large and tightly bunched together, so as to give a compact nucleus.

TYPE 11. (Plate XVIII. Figs. 18-3 O).-These are long, thick, slightly curved forms, which are frequently reniform. They vary from 11 to 1 5 CL in length with an average of 12.? P, and have a breadth of from 3 to 4 p, the average being 3% p. The protoplasm of these forms stains more deeply than does that of Type I., and is often occupied by innumerable fine basophilic granules. The more pointed extremity frequently shows the beginning of a secondary recurved limb (Plate XVIII. Figs. 26-30). The deeply staining polar granules described as occurring in forms of Type I. are here absent, their place being taken by a pale pink rounded body (Plate XVIII. Figs. 19, 20, 22). The nucleus is represented by a large rounded or oval mass, which does not occupy the whole width of the body, and which is apparently composed of very large chromatin granules loosely bunched together (Plate XVIII. Figs. 18-21, 23-30).

TYPE 111. (Plate XVIII. Figs. 11-1 'i).-These forms are inter- mediate in size between those of Types I. and II., and present features in common with both. The cytoplasm is not so deeply stained as in individuals of Type TI. On the other hand, the basophilic granulation is more constant than it is in phase 1. Both the deeply staining granules (Plate XVIII. Figs. 15, 17) and the pale globular body (Plate XVIII. Figs. 12-14) above described are not infrequently present. Tn none of the forms is there to be seen the deeply staining polar mass which proved to be such a constant feature in the case of Emmopgarinn anarrhichadis (1 9 1 2 ".

There is no reason for assuming that the types above described represent different hzemogregarines. Rather are they to be interpreted as phases in the life-history of one and the same parasite. Further, the long thin forms of Type I. may be taken to be of male character, the broad stout forms of Type 11. to be of female character, whereas the intermediate forms, i.e., the forms grouped under Type III., may be looked on as being of indifferent nature. This last indifferent form,

234 HERBERT HENRY.

then, would be none other than the ordinary individual destined to propagate the species in the viscera of the vertebrate host by a simple asexual multiplication or schizogony ; while, on the other hand, the first two types would be responsible for the initiation of the sexual cycle of developinent in some intermediate invertebrate host, probably a leech.

If, indeed, the long thin type and the broad type are sexual phases, then they must represent development along one or other of two lines -either they are schizonts, i.e., micro- and macro-schizonts, or they are micro- aud macro-garnetocytes. If the first of these interpreta- tions be accepted, then one must assume that this hzniogregarine exhibits sexual dimorphism right through a schizogonic cycle, as do certain of the coccidia, and as does Wenyon's Htemogregarina graeilis (1908 7). If, on the other hand, the second hypothesis be correct, then the appearance of sexual dimorphism would be found only in the last generation of a normal asexual schizogony-a condition which is manifest in certain coccidia and in all hmnosporidia.

Had Types I. and 11. only been present in this instance, one might have ventured to suggest a developinent such as that presented by H. gracilis, but the association of Type 111. with Types 1. and 11. tends to negative such a suggestion. One is inclined, therefore, to believe that the sexual dimorphism of this individual hmogregarine becomes evident only in the last stages of an ordinary schizogonic cycle. Such an opinion is, however, merely a matter of conjecture, for the point is one which could be determined only by the examination of the visceral phases in a series of infected fish, the observations being carried out a t repeated intervals and over prolonged periods. Further, in consider- ing the question of sexual diinorpliisrn in the case of the hzemogregar- ineti, one must look upon it as not improbable that the whole series of hmnogregarines may show what one may term a graduated specialisa- tion in sexual development, skirting with the more primitive types which show no apparent sexual difYerentiation and passing through the hiernosporidian type, where sporont formation appears as the last phase of schizogony, to a still higher type in which sexual dimorphism is, as it were, thrown further Lack and becomes obvious from the beginning of the schizogonic cycle.

The first piscine hzmogregarine described as showing forms sug- gestive of sexual differentiation is H. rovignensis, a parasite found by Minchin and WoodcocB (1910 ">, in speciniens of Trigla lineata taken froni the Adriatic. The second haeuogregarine showing similar features is H. anarrhiehadis (1 9 1 2 3, obtained in specimens of Anar- r.hici~as l ~ q m s , obtained in the vicinity of Cape Wrath, Scotland. The haddock hzmogregarine, for which I suggest the name Hternogreyarina qleJini, then, would be the third of the series.

In the same series of specimens of haddock in which there occurred the hmnogregarine just described, there was found a leucocy tozooii. (The term leucocytozoon is here used loosely, just as in the case of

B L O O D PARASITES 03 GADUS AGLEFINUS. 235

leucocytozoa of mammals. It is applied to the parasite merely by reason of its intraleucocytic habitat. Indeed, the organism does not in any way resemble the true leucocytozoa of birds.) I t was present in two out of fifty-three fish examined. Neither of these fish showed the presence of the hemogregarine just described, but in one a try- panosome Miils found. In its youngest phase (Plate XIX. Figs. 1-4) this parasite presents itself as a rounded or slightly oval body, having a diameter of about 3-4 p) and occupying the cytoplasm of a motio- nuclear cell which is either a leucocyte or an endothelial cell. The outline of the parasite is indefinite, for it is not sharply deliminated from the host cell cytoplasm, and it is possessed of a centrally placed and feebly staining nucleus coniposed of loosely bunched chromatin granules. As it grows it becomes a large globular body which has a diameter of about 9 p , and which fills up more or less completely the whole cytoplasm of the enlarged host cell, the nucleus of which becomes swollen and degenerated (Plate XIX. Fig. 6). I n this phase the nucleus of the parasite becomes still less distinct than in the younger forms, and it is represented by irregular strands or granules of chromatin scattered diffusely throughout the cell body. With still further increase in size of the parasite the host cell protoplasm dis- appears altogether, or it is represented by a mere thin shell or cap. Also, the scattered nuclear granules become aggregated into very definite and deeply staining secondary nuclei (Plate XIX. Figs. 7-1 a), a phase which is succeeded by the formation of individual merozoites (Plate XIX. Figs. 13, 14, 16). These niultinucleate forms vary con- siderably in size and number, the largest being 20 p or more in diameter. The secondary nuclei vary too, both in size and number, hhe largest number counted in a single infected corpuscle being fifty- six. With the advent of merozoite formation the host cell is coni- pletely disorganised. The protoplasm disappears altogether. I ts nucleus is swollen, broken up, and even invaded by the developing parasite (Plate XIX. Fig. 16), and when it ruptures there appear in the blood stream free merozoites (Plate XIX. Figs. 1’7, 19), together with multinucleate plasmodia1 masses of varying size (Plate XIX. Figs. 15, IS), destined to break up into still further swarms of inerozoites. The free merozoites (Plate XIX. Fig. 19) measure about 3 p in length and 1.5 to 2 p in breadth. The nucleus is a deeply staining round or oval body situated either near the centre or pushed towards one extremity. At times larger forms are met with showing two or three separate nuclei. The protoplasm is hyaline, there being no obvious reticulation and no granules.

The only parasite resembling that herein described is one dis- covered by Neumann and called by him Globidium mult$dum (1 9 0 9 He thus sunimarises its main features :-

im Mittel 2 4 p lang und 1 9 p breit. ‘ I Allem Anschein nach eine Teilungsform, von bedeutender Grosse,

Es komnien auch E’ornien bis 40p

im Dnrchmesser vor. Der Parasit entwickelt sich im roten Blutkor- perchen, fullt dieselben allmahlich vollstandig aus und lost dabei den Kern vollstandig auf, wie etwa Karyolysus. IEs ist kein Pigment vor- handen. Die Teilprodukte ahneln am meisten deneii von Proteosoma, sind aber vie1 grosser, rundlicli bis dreieckig. Es entstehen im Mittel 40 bis uber 60. Das Chromatinkorn ist sehr gross, nicht selten findet sich daneben noch ein zweiter blepharoblastahn- licher 2. Kern. Die Merozoiten entstehen offenbar in gleicher Weise aus der Protoplasruamasse wie bei Malaria.”

It will be seen from this description that Neumann’s parasite was met with only in the fully ripe morula form, and that none of the younger phases were seen. Again, the host cell in the case of Globidium is taken to be a red corpuscle, the nucleus of which would seem to undergo a destruction of much more severe grade than that seen in the organism under discussion. Apart from these differences, there are many features which each organism shows in common with the other. Qlobidium was found to occur in two out of fifty-six specimens of Arnoylossus grohmannii, and in one out of forty-six specimens of Gadus minutus. I n the latter instance-and this I take to be a point of very considerable importance-it occurred side by side with a hzmogregarine, H. minula. The possible connection of Globiditma with this hamogregarine is denied by Neumann on the following grounds. In the first place, the schizogony of a piscine hsmogregarine, where it occurs in the circulating corpuscles, always results in the produrtion of a very definite nuniber of merozoites. For instance, in the case of H. biqemina two individuals are produced, with B. puadrigemina four individuals, with H. simondi eight individ- uals, and with H. polypartita sixteen individuals. So that so far there is no recorded case of a piscine hmnogregarine giving rise in the circulating red corpuscles to inore than sixteen merozoites. The finding of the young intraleucocytic phases of the haddock parasite suggesbs, however, that Neumann’s parasite may indeed have the same habitat, in which case it may be looked on as undergoing schizogony, not in a red corpuscle but in a leucocyte. Viewed from this stand- point, therefore, Neurnann’s objection falls to the ground. A second argument brought forward by this observer is that the host cell in the case of H. polypaytita does not undergo any very marked degenerative changes, whercas such changes %re B marked feature with Globidiimn. Such a contention appears to me to be quite invalid, for with H. biywiaina, H. puadrigeminn, H. clavata, and H. sirnondi, of which I have had experience, the host cell always undergoes degeneration and destruction. In fact, the figures given by Neuniann himself of H. clavata illustrate this point very well. In all my own preparations of ha?niogregnrines undergoing schizogony in the circulating corpuscles, the production of adult individuals is invariably accompanied by enlargement of the whole corpuscle, by rapid degeneration both of its cytopltmi and its nucleus, and by its ultimate total destruction. A

Grosse 25 p lang, 1.5 p breit.

BLOOD PARASITES OF GADUS XGLEFINUS. 237

third argument which Neumann has advanced as a distinctive feature of Globidium as contrasted with any known piscine haemogregarine lies in that with Glo6idiurn the division of the parasite would appear to occur suddenly by the formation of a varying number of merozoites from a large plasmodia1 mass, whereas in the case of the piscine haemogregarines division would seem to be what one might term bigeminal, i.e., one individual divides into two, two into four, and so on up to a maximum number of sixteen individuals. The basis of such an argument lies in the supposition that the host cell is a red corpuscle, and I feel certain that Neumann would not have brought it forward if he had considered the possibility of the host cell being a leucocyte or an endothelial cell.

I venture to suggest, therefore, that the occurrence of Neumann's Globidium along with H. minuta, and of the intraleucocytic parasite along with H. cegle$ni is strong presumptive evidence that each is really a phase in the life-history of the hemogregarine with which it is associated. The small free merozoite in each case, then, would be no other than a young hEmogregarine destined to penetrate a red corpuscle and there develop into the adult individual. No appearance suggestive of such a development has been seen in the case of Gudzcs czglefinus, but i t is of interest to note that Neumann figures lying inside a red corpuscle a single merozoite of GlobidiunL (Tafel V. Fig. 35)) which may very well be taken to be a younger phase of H. minuta (Tafel I., Figs. 48, 49). One is aware that such an assumption still further complicates the haemogregarine life-history, but it seems better to suggest such an affinity than to seek to establish either Globidium or the intraleucocytic parasite from Gadus mgle$nus as being separate entities.

What is the signifi- cance of these multin'ucleate forms, and, if they do represent a phase in the life-cycle of a haemogregarine, what is their exact position in this life- history ? It would be extremely difficult to find a satisfactory solution of such a question were i t not that one is helped considerably by the life-history of HBmoprotcus columbm as described by Aragao (1 9 0 8 ') and the Sergents (190'7 ". Hmmoprotcus columba: is an amaboid melanin-producing parasite found in the blood of pigeons, the inter- mediate host being a hippoboscid fly, of the genus Zyachiu. Ripe gametocytes, taken up from an infected pigeon by the fly, give rise in the stomach of the insect to gametes, which after fertilisation produce zygotes and ookinites. Apparently there is no sporozoite formation, and it is the ookinite which is inoculated back into the bird. The changes which result in the ookinite immediately after inoculation are so far unknown. The pigeon shows infection for the first time on the thirteenth or fourteenth day after being bitten, The youngest form of the parasite to be recorded is a small rounded body embedded in a mononuclear leucocyte, which is adherent to the wall of the lung

The next question which presents itself is this.

capillaries. Thc young parasite grows rapidly in this situation and clivitles, so that from twelve to fifteen individuals are found lying inside the greatly hypertrophied host cell. These individuals give rise in lurii to inultinncleate plasuiodial inasses of varying size, each of which divides up still further into a number of merozoites. The much enlarged leucocyte then ruptures and the individual inerozoites are set free in tlie blood stream, where they penetrate red corpuscles and become the youug Iialteridia.

Auother development rnuch resembling this is that described by Gouder (2) in the case of Yheileria parvu. The sporozoites inoculated by the tick penetrate lymphocytes in the spleen and lymphatic glands a d there give rise to large iiiultinucleate plasmodia1 niasses, the resulting inerozoites being “ agarnonts,” which after a succession of generations give rise t o (‘ gamonts.”

It is not unreasonable, therefore, to suppose that the intraleucocytic parasite in Gadus mgle$nu.s is comparable to those just mentioned, in which case it would represent the first schizogoiiy undergone by the inoculated sporozoites of H. cqle3ni.

REFERENCES,

1. ~ E A G A O . . . . . . , “Der Entwickliingsgang und die Ubertragung von Hsmoproteus columbae,” Arch. f. Prot- istenkunde, Jena, 1908, Bd. xii. S. 154.

2, GONDEI~ . . . . . . . (‘Die Ehtwicklung von Theileria parva,” Ibid., 1911, Bd. xxi. S. 143, Bd. xxii. S. 170.

3. HENRY . . . , . . . (‘Hmnoge(jarina anarrhichaclis, from Anar- rhichas l16pus, the Catfish,” Parasitology, Cambridge, 1912, vol. v. p. 190.

4. NINCIIIN AND WOODCOCK . (( Observations on certain Blood Parasites of Fishes occurring at Rovigno,” Quart. Journ. Mzcr. Sc., London, 1910, vol. lv. p. 113.

5. NELJMANN . . . . . . “Studien uber protozoische Parasitcn im Blut von Meeresfischen,” Ztschr. f. Hyq. u. Infectionskrankheiten, Leipzig, 1909, Bd. xiv. Aeft 1, 8. 1.

6. SERGENT AND SERGENT . . ‘‘ 1,es Htimntozoaires d’Oiseaux,” AWL. de Z’lnst. Pasteur, Paris, 1907, vol. xxi. p. 251.

7. WENYON . . . . . . “Report of Travelling Pathologist,” Third Rep. Wellcome Researclh Laboratory, Khar- toum, 1908, p. 121.

DESUBIPTION OF PLATES XV1II.-XlX.

PLATE XVIII.

( x 1500 diameters.) Hcwi~og?cnaii,ra,cgaiiiia ffigZc$i~i from Gadus cuylc$nus, t he haddock. Leishman stain.

FIGS. Frus. lI-l7.-Forms of Type 111. FIGS. 18-50. -Yon i s of Type 11.

1-10.-Long thin forins of Type I.

JOURNAL OF PATHOLOGY.-VOL. XVIII. PLATE XVIII.

JOURNAL OF PATROLOGY.--VOL. XVIII. PLATE XIX.

BLOOD PARASITES O F GADUS BGLEFINUS. 239

PLATE XIX.

The Intraleucocytic Parasite found in Gadus aglefi?azcs.

FIGS. 1 4 - T h e youngest forms of the parasite. FIG. 5.-A leucocyte showing double infection. FIG. 6.-Large parLisite showing diffusely scattered nuclear chromatin. BIss. 7-12.--Large forms showing the formation of secondary nuclei.

FIGS. 13, 14, lG.-Forms showing the beginning of merozoite formation. FIG. 17.-A ruptured cell with free merozoites. FIGS. 15, 18.-Multinucleate plasmodia1 masses showing division into merozoites. FIG. 19.-Free merozoites.

In Pigs. 11 and 12 the host cell cytoplasm has disappeared altogether.