HAEf.;;:ATOZOA FRm,: SOME CDrv~MON P.. r.IPEIBI.4.NS OF Q.UEBEC

by

Jessie Ahmad Shah

A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfilm.ent of the requirements for the degree of Master of Science.

Department of Parasitology, McGill University, Montreal. August 1959 ACKNOT:JLEDG:MENr s

My thanks are due to Dr. Marshall Laird for his very thoughtful help and criticism throughout this study, and to Dr. T.~v ..M. C~meron and the National Research Council for making it financially possible. I also wish to acknowledge the help given by Mr. Robert Verney for constructing the containers for the frogs and toads, to Mr. Felick Druzbaki and Mr. Mack Riddel for the tedious job of preparing the nwmbered aluminum dises used as tags. Many young people contributed amphibia and it is impossible to mention all of their names. Raymond Demers brought in large quantities as did Margaret and Heather Oliver. My sons, Damon and Julius Shah, accompanied me on .many collection trips and helped to handle the frogs in the laboratory. Table of Contents

Page No. INTRODUCTION ...... • . • • • . . . • 1

~.JETEOD3 AND ~&T:ffiiALS • • . . . 6 ...... 11 Trynanosoma rotatoriwm (Mayer, 1843) • . • . • • 11 Rexamita intestinalis Dujardin, 1841 . 15

Lankesterella minima (Naussat, 1850) 20 catesbiana Stebb ins , 1903 • . . 24 ,.. Cytamoaha bacterifera Labbe, 1894 . 30 PlasmodilL.ïl-like artifacts ...... 36 Pirhemocyton chattoni n. sp ...... 38 Morphology of chromatin bodies, multiple nuclei, refractile bodies and vacuoles 41

-~teration of cells • ...... 44 Blood counts . . . . 46 Transmission experiments 48 Discussion • . . . . 49

MITOSIS Ir~ CIRCUlA TING BLOOD OF R. CATESBLUJA . 52 ...... 56 PLATES • • • • . . 58 BIBLIOGRàPEY ...... ?4 List of Illustrations

Page No. Text Figure 1 Lankesterella minima {Naussat, 1850) from R. catesbiana {Phase-contrast, x 2550) 22 Text Figure 2 Pirhemocyton chattoni n. sp. refractile bodies. (Phase-contrast, x 2550) . . 43 Text Figure 3 Late prophase in erythroblast of R. catesbiana. (Phase-contrast, x 2550) . . 54

Plate I Maps indicating collection a reas ...... 58 Plate II Dia gram of an aquarium arrange d for trapping njght flyiqs insects 59 Plate III Trypanosoma rotatorium (Mayer, 1843) and Hexamita lntestinalis Duj ardin, 1841. (Dried films, x 1600) • . . . . 60 Plate IV Pirhemocyton chat toni n. sp. in erythrocytes and erythroblasts of R. ca tesbiana. ( Giemsa, x lôOO) • . . . . . • . 61 Plate V P. chattoni drawn from living m.aterial. (Phase-contrast, x 2550) . . 62 Plate VI L. minima and Haemogregarina catesbiana. Stebbins, 1903. (Giemsa, x 1600) .•..•. . . 63 Plate VII R. catesbiana, -like artifacts and Cytamoeba bacterifera Labbé, 1894. (Giemsa, x 1600) • • • . • . . . 64 Page No. Plate VIII c. bacterifera drawn from living material. (Phase-contrast, x 2550) ....• 65 Plate IX Mi tosis of .erythroblasts from blood of R. catesbiana convalescing from P. chattoni. (Giemsa, x 1600) ...••.. 66 Plate X Key to Hi stograms ...... • 6? Plate XI count (normal erythrocytes, and erythroblasts, parasitized erythrocytes and erythroblasts) from R. catesbiana (#6?-A} with an acute infection of P. cha ttoni • ...... 68 Plate XII Granulocyte count from R. catesbiana (d67-A) . 69 Plate XIII Change in relative size of red blood cells (L'.V) from R. ca te s biana ( 7~6? -A) • • • • • • ?0 Plate XIV Red blood cell count of R. cates­ bi ana (J l24) convalescing from a natural infection of P. cha ttoni. ?1

Plate 1.V Change in relative size of red blood cells (L;i) from ft. cates biana (#124) •... ?2 :Plate 1..\JI Red blood cel l c ount f rom R. catesbiana recovered from infection of P. chattoni. Matur e erythrocytes completely replaced by erythroblasts ...... ?3 INTRODUCTION

Little work bas been done on the blood parasites of amphibiens in Canada - surprisingly little, when it is considered how much has been written on haematozoa from these animals in the neighbouring U.S.A. as well as in more distant countries, and that frogs, well known as a source of trypanosomes and haemogregarines, are among the commonest laboratory animals.

Nevertheless, two earlier surveys in ~uebec resulted in the discovery of two cosmopolitan trypanosomes and a widely distributed species of Lankesterella, and a number of new species of Microfilaria, Trypanosoma, Haemogregarina, , and Plasmodium. Two species of the last-named (not otherwise known from amphibiens) were recorded, (13) although the validity of the finding has been questioned by some recent investigat ors. These facts suggested that further studies of the fauna might prove rewarding. Khaner (20) recorded the followi ng haematozoa from among parasites of amphibiens collected in and near Montreal; PARASITE HOST

IDEN'riTY 1-.T(Thlf.BER IN:B'ECTED

1 Trypanosome rotatorium (Mayer, 1843) Rana catesbiana 1 N Shaw 1

Trypanosome inopinatum (Ed. and Et. Sergent, 1904) Bufo americanus 1 Ho1brook

Lankesterella ranarum (Labbé, 1899) Rana p. pipiens 2 Schreber -3-

As Khaner's work was based on only ten specimens of R. pipiens, ten of R. clamitans, Latereille and two each of R. catesbiana, B. americanus and T. viridescens, Rafinesque her data are inadequate for parasitaemia estima ti ons.

In five years studies of liE terial from Montreal and the Laurentians, Fantham et al (13) examined 225 B. americanus, 300 R. catesbianà, 200 or more R. pipiens, an unstated number of R. clamitans, and seven R. sylvatica. These proved to harbour the blood parasites as shown on the next page. Some of these have yielded blood protozoa elsewhere in North America ( 44, 45), tbe following names being quoted directly from Nalton, 194? to 1951.

HOST PARASITE Ra na catesbians Cztamoeba bacterifera Labbé,l894 " 11 Haemogregarina catesbiana Stebbins,l905 " " Karyolzsus of Brandt " " Lankesterella sp. of Brandt , " Trzpanosona inopinatum " ff " rota t orium " " " sp. of Hegner " clamitans Cytamoeba bac ter ifera PARASITE HOST

IDENTITY NUMBER PER CENT. INFECTED INFECTED Trypanosoma lavalia Fantmm et al.l942 Bufo amer icanus 1 0.44 tl " montrealis Fantham et al~l942 " 3 1.33 " gaumontis Fantham et al,l942 n " 1 0.44 " rotatorium (Mayer,l843) Rana catesbiana Not sta ted " " lt " pipi ens tl If tt ,, " " clamitans " " inopinatum. Ed. and Et. Sergent, " catesbiana "Small number" " 1904 tl 1 !2iEiens ~ " " " " " 1 Lankesterella canadensis Fantham et al, " catesbiana 2 0.99 1942 Haemogregarina sensu lata " Q!Qiens ? 3.5 " " " B. americanus Not stated " " " R. catesbiana 2 0.99 Plasmodium catesbiana Fantham et al,l942 " " 1 0.33 bufonis Fantham et al 1942 B. americanus 1 0.44 " ' Haemoproteus laurentia Fantbam et al 1942 tt 3 1.33 ' " 1ava1ia Fantham 1942 tl 1 0.44 " ~__;...;;...,et al " ,, Fantbam 1942 1 0.44 1anoraiea ...;...... ;..__;.=:;.'et al " " Dacty1esoma sy1vatica Fantham et a1~1942 R. sylvatica 1 14.28

Microfi1aria ranae-sylvaticae Fantham " tl 1 14.28 et a1~1942 -5-

HOST PAR..~SITE Ra na clami tans Haemogregarina clamatae Stebbins ,1905 sp. of Kudo 1922 " " " 1 " " Trypano soma parvurn Kudo ,1922 " " Il rotatorium " " " sp. of Regner tl pipi ens Haemogregarina sp. of Scott " " " sp. of Kudo .1922 " " Trypanosorœ sp. of Drhoklav, l 929 " " " sp. of Nigre11i,1945

T. rotatoriurn parasitizes a diversity af frogs and toads in such wide1y separated places as China, Africa, and the Americas. T. inopinatum has a distribution almost as extensive. Great numbers of amphibians in many parts of the wor1d harbour haemogregarines, but inadequate knowledge of the 1ife history seldom permits these to be assigned to a genus ether than Haemogregarina sensu lato. Blood­ inhabiting sporozoans are of re1ative1y rare occurrence in these animals. An organism (or organisms), of uncertain taxonomie standing, Cytamoeba bac ter ifera, has be en observed in the red ce11s of Bufonidae, Ranidae, and Caudata in Europe, Caœd a and the U.S.A. (3,11,15,24 ,44,45,48}. -6-

METHODS AND MATERIALS

During the sQmmer of 1958, 218 frogs and toads were collected in the vicinity of Ste. Anne de Bellevue, P.Q. •. Ten more were obtained from Laval des Rapides and Plage des Isles. Many of the R. catesbiana and R. clamitans originated from Ile Perret. Collecting stations are indicated on the accompanying maps of the Montreal area (Plate I). Most of the large bullfrogs were caught from slow-moving waters emptying into Lake St. Louis or the Lake of ·rwo Mountains. The reeds and shallows of these expansions of the Ottawa River shelter large numbers of frogs, many of which were seen and heard there, but the difficulty of manoeuvering in the soft mud prevented the capture of more than small numbers of them. A long­ handled net was used in collecting, but the most satis­ factory means of catching the larger examples proved to be a fishing rod and line, an unbarbed hook being baited with a piece of red cloth. The shallow waters of a large reedy pond near the Morgan Arboretum (Plate I) held masses of spawn in the early spring and proved a ready source of R. pipiens. R. crucifer crucifer and R. sylvatica were also found there. -?-

Bufo americanus was fo und in fields, or pudd.l es on dirt roads after rain. The pond at Harpell Press proved to be the most fruitful place for collecting toads.

\~en amphibiens were brought into the laboratory they were examined for ectoparasites, and tagged with small metal dises. An aluminum wire through the web and around one toe held the numbered dise in place. Blood was obtained by slipping the skin of a toe. Dry smears were fixed in methyl alcohol and stained with Giemsa. Net smears were also made, many of them being examined by phase-contrast. When heart blood was taken f rom a dead frog, the organ was exposed and wiped dry with gauze before the incision was made, to prevent contamination by pericardial fluid. The animals were kept alive for as long as possible. Bull frogs (R. catesbiana and R. clamitans} were kept in a deep galvanized tank equipped with a drain and a screened lid. Their canniba listic habits rendered it neces sary to use partitions to separate them according to size. R. pipiens and smaller frogs were kept in glass aquaria. The water in the t anks and aquaria was cbanged every two days, care being taken to ensure that it was always shallow enough to allow the anima ls to r est on tbe bottom. Wooden float s and stones were provided s o that they co uld l eave the -8- water at will. The toads (B. americanus) were kept in a large metal box with an open top. A layer of small stones was placed on the bottom wi th progressively finer layers of gravel over it. This was kept damp so tba t the toads c ould bury themselves. The insatiable appetites of the bullfrogs posed a problem. Small frogs, tadpoles, minnows and earthworms were supplied to them in large qu~ntities. The toads ate worms, garden insects, slugs and flies. The R. pipiens were the most difficult to maintain, as they preferred flying insects. An attempt was made to culture flies for them, but after hatching two generations of these insects it was found that this did not provide a sufficient food supply. A method for trapping night flying insects was the n devis ed (Pla te II) • The frogs were put int o an aquari~~ with a wide meshed wire screen on the top. This was placed in the window as illustra te d, and ga uze was tacked around it on the inside to prevent the insects fro.m entering the room, the light of which was left on during the night. Until terminated by tœ onset of cold weather, this procedure proved thoroughly satisfactory for the feeding of all the smaller frogs and some toads as well. -9-

During the fall and winter months the few remaining speci­ mens were force-fed with ground beef and heart. The twelve Xenopus laevis Daudin used for experimental purposes were obtained from the Institute of Parasitology. They were kept in large glass jars and fed on ground liver and beef heart which they ate voluntarily. Records were kept of the frogs and their blood parasites. As parasites were detected, daily and weekly series of smears were taken, all such smears were fixed in methy alcohol and stained with Giemsa. Subinoculation was accomplisbsd by injecting heart blood from a sacrificed animal into the peritoneal cavity of another. Dead frogs were dissected and heart blood and organ smears were taken. In some instances organs were sectioned at ten ~ and stained with haemotoxylin­ eosin or with Giemsa. Fresh specimens were examined by phase microscopy. Dried films were stained with Giemsa and searched by bright field for no less than one-half hour before being declared negative. An Abbé camera lucide was employed in making illus trations and measurements are based on 50 specimens unless otherwise indicated. Six species of amphibiens featured in the collections, and haematozoans of six genera were encountered. HOST PARASITES Nwnber Trypano- Haemogre- Lankes- Cyt- Hexamita Pirhemo- sorr.a garina terel1a amoeba in b1ood C;iton Cl{, No. % No . 1 No. % No . % No. % No . %

R. catesbiana ?0 60 85 ? 10 24 34 6 8.6 4 5.? 8 11.4

R. cl ami tans 40 6 15 4 10 4 10 2 5.0 None None

R. pi pians 43 5 12.2 None None None Il " 1 l-' 0 B. america nus 71 None " " " " " 1

R. sy1vatica 3 " Tl tl Il " "

Hz1a versico1or 1 " tl Il Il Lecomte " " -11-

The search for Plasmodium was unsuccessful. Findings are dis c us sed in detail in tbe following section.

SYSTENÂTICS

Class: Mastigophora Order: Protomonadina Trypanosoma rotatorium (Mayer, 1843) (Plate III, Figs. 1 to 6)

Following Mayer's description of T. rotatorium, several other workers added to the knowledge of this poly­ morphie species and proposed further specifie names for trypanosomes from amphi bians . ( 4, 13, 46 ). A thorough study of frog trypanosomes was made by Nëller . (46). He experi.men ted wi th blood agar cultures, and transmissions by simple injection and through Hemi­ clepsis marginata, a leech already shawn to be the inter­ mediate host. Nëller concluded tha t only two s~ ci es of trypanosomes are valid, T. rotatorium and T. inooinatum. Scorza and Boyer, (38), who made a systematic study of T. rotatorium and T. leptodactyli . Carini, 190? found the Venezuelan leech Glossosiphonia complanata to be an effective intermediate host. From studies of natural -12- infections and cultures, they considered four species, ( T. leptodactyli, T. arcei, T. costatwn and T. borrelli) to be synonyms of T. rotatorium. The extensive polymorphism of T. rotatoriQID is tao often disregarded, new species being described without the thorough life history studies which should precede this step. Noller characterized three main forms: small attenuated ones having a well-developed undulating membrane, a free flagellum, spherical nucleus and a compact kinetoplast; large ones exhibiting a less well­ developed undulating membrane and usually lacking a free flagellum; and leaf-like one s, ha·ving a long flagellum and a well-developed undulating membrane. Scorza and Boyer observed crithidia forms from G. complanata, and flagellated forms (sorne having clearly roarked myonemes) ranging from the long slender types to more rounded one s. Dividing flagellates were observed in cultures. Specimens which I examined exhibited wide polymor­ phism within the accepted range for T. rotatorium. Studies incidental to investigations of the life-history of ~. ' bacterifera revealed extremely small slender try­ panosomes, some of them no longer than a red blood cell (about 20 p) including the free flagellum. Search for -13- these in dry Giemsa stained films was unsuccessful. From observation of many specimens it seemed most convenient to classify the trypanosorres into three groups. These are samewhat arbitrary, though one blood smear usually contained a majority of one type or another. The first group includes those with a we11-developed undulating membrane and a free flagellum. The second, those with no free flagellum but with an undulating membrane, and the third, rounded forms having neither a free flagellum nor an undu1ating membrane. Dimensions given here are from 50 specimens taken from R. catesbiana, R. clamitans and R. pipiens.

1!EAN 1ŒDIAN S . D. MAXIMUM MH~ ll~1UM Gr oup 1 (Plat e III J:t,igs. 2, 5 & 6) Length of flagellum 1?.0 11· 20.0 p. ?.4 }1 30 .0 p. 12 .5 p. Length of body 36.0 3?.4 11.3 52.? 34.5 ;Nidth of body (widest) 24 .6 20.0 13 . 3 29. 2 4 .4 Diameter of nucleus 3.5 4.4 1.6? 6 .9 3 .1 Per cent. of body 1ength nucleus is from anterior 55 65 ?.8 64 41 Distance from center of nucleus to ble- pharopla st 8 .2 6. 2 3 .?4 9.9 4 .4 -14-

MEAN MEDIAN s.n. MAXIMtJivl MINI:MUM Group 2 (Figs. 1 & 3)

Length of body 44.5 fl 43.5 fl 12.4 Jl 65.0 Il 33 .5 Jl Width of body 21.5 20.5 8.4 45.5 8.8 Diameter of nucleus 4.3 4.4 1.06 6.2 3.1 Per cent. of body length nucleus is from anterior 64 65 10.4 84 46 Distance from center of nucleus to ble- pharoplast 6.6 6.2 2.6 14.2 0

Group 3 (Fig. 4)

Length 52.0 5?.0 ? .4 86.0 36.0 Width 44.0 46.0 ?.4 59.0 29.0 Diameter of nucleus 5 .6 5.3 .96 8.8 3.8

Group 1 f1agellates are small and include att enuated forms (Figs. 5 and 6) as we11 as larger ones {Fig. 2). '.rheir nuclei are more often ova1 and the kinetop1ast (23) of the larger leaf-like forms in this group is 1ess compact. Those in Group 2, may or may not have a well-developed undulating membrane. {Compare Figs. 1 and 3). In the rounded forms, f rom heart blood (Group 3) the kinetoplast is usually adjacent t o the nucleus and in ma ny a short exoneme can be distinguished. Several workers have reported these as -15- multiplicative forms ( 46 ). A round or oval endosome can be seen in all well stained specimens. As can be noted from the table the nucleus is usually nearer the posterior than the anterior end. In Groups 1 and 2 it also exhibits a variation in size which is not related to other dimensions. In life the membrane undulates constantly and rapidly. The body is plastic and displays amoeboid characteristics. Because of their close agreement with descriptions of T. rotatorium by other investigators and lacking biological evidence to the contrary, all of these for.ms are considered here to be developmental stages in the life cycle of this species.

Order: Polymastigina Suborder: Diplomonadina Family: Hexamatidae Hexamita intestinalis Dujardin, 1841 (Plate III, Figs. 8 to 10)

Duj ardin ( 46) first recorded this pa ra si te but in overlooking one pair of anterior flagella he gave it the misleading name Hexamita. As the protozoan has three pairs of anterior and one pair of caudal flagella, Prowazek's name Octomitus is more descriptive. -16-

H. intestinalis has been identified and described in detail from the intestine of various Amphibie. {9,10,12,28,41). Species of Hexamita are found in a diversity of other anima1s. Fantham (12} named two species from South African frogs, Hexamita ranae Fantham,l933 and H. xenopi Fantbam,1923 from X. 1aevis. Khaner (20) reported H. xenopi from the rectum and caecum of R. catesbiana, R. clamitans and R. pipiens from Q.uebec. Heavy blood infections in an emac:ia ted and in 1aboratory frogs were reported by Dani1ewsky (8). Moribund R. catesbiana in the London Zoo proved to harbour H. intestinalis in the blood (29, 30) and natural1y occurring heavy infections were found in apparently healthy R. esculenta (31). One of these died after several months of captivity, its blood swarming with f1ag­ e11ates. During the summer of 1923 Lavier and Galliard (26) found a toad (Bufo Oalamita) in ostensibly excellent health but with Hexamita in its b1ood stream. Concomitant invasion by intestinal micro-organisms was not found by any of these authors. Hexamita was found in the blocd of ten per cent (7/?0) R. catesbiana caught during the summer of 1959. In no instance were the flage1lates accompanied by other -17- intestinal micro-organisms. Blood was always taken from a digit which had been wiped with 70 per cent alcohol. Additional haematozoa were present in all but one of the hosts

FROG NUMBER OTHER HAEMATOZOA

28 T. rotatorium (very heav~, c. bacterifera and Lankesterella 34 Pirhemocyton (very heavy), H. catesbiana (very heavy} and Lankesterella 58 Lankesterella, T. rotatorium 65 T. rotatorium 70 Lankesterella, T. rotatorium, C. bacterifera 71 Lankesterella, Pirhemocyton (very heavy) 44 No other parasites found.

All of these frogs were caught in June which is still early for R. catesbi ana, as spawning occurs in la te June and earl y July. The heavy parasitaemi a and small size of all but two of them suggests that, though there was no out- ward appearance of emaciation, their general condition was not good. I n all i nstances He xamita wa s present in the blood in small numbe rs only, and this renders it likely tha t still lighter infections were missed. As a ll earlier investi- ga tors have indica ted, this anima l is notahly non- r efract ile. -18-

It is seldom demonstrable in life by bright field micro­ scopy, but a dark field condenser or better yet phase­ contrast equipment, greatly faci1itates its detection. Giemsa stained smears failed to reveal more than an occasional distorted example. (Fig. 8) These same flagellates were found in covers1ip smears of intestinal scrapings from R. catesbiana and X. laevis. They were most abundant a little above the rectum and in the caecum. These flagellates are elongate­ pyriform in life • They have three pair of anterior flagella which are seen to whip rapidly, propelling the animal nuclear end foremost. Two caudal flagella are directed posteriorly and occasionally serve to anchor the organism. Stained specimens from intestinal smears exhibit polymorphism, sorne elongated as in figures 9 and 10, sorne wider and oval, ethers almost circular. Dimen­ sions of stained specimens from the caecum are:

HEXAMITA FROM: 1ŒAN ME DIAN S.D. N.AXDf.illv1 R. catesbiana

Length of body 7.5 ~ 7.5 p. 1.29 Jl 10.0 J.l 4.4 Jl Width of body 2.6 2.5 0.53 3.5 1.6 X. laevis

Length of body 8.0 8.0 1.45 11.2 5 .5 Width of body 2.9 2.8 0.87 4.7 1.9 -19-

Fifty specimens from each host were measured. Two nuclei and a pattern of basal granules are usually seen as a fused horseshoe-shaped area. The granules are arranged as the four corners of a square with the anterior flagella arising from them (10,28,42). Extending to the posterior end are two flexible axostyles which terminate in two basal granules giving rise to the caudal flagella. The flagella are free throughout their length. The cytoplasm may appear homogeneous or granular and is sometimes vacuolated. Dobell (10) stated that the length of H. intestinalis is about ten ~' while Matubayasi

(28) gave the dimensions as 6.2 to 12.3 ~ by 1.5 to 5.5 ~·

Fantham's H. xenopi are 10 to 15.5 ~long and 3.3 to 7.7 ~ wide when fus iform and 8~1 ~ by 5. 6 p. vvhen oval. The axostyles are said not to be so marked as in the European form. In view of the close agreement in both shape- and size-range of Hexamita from the various hosts, and the lack of any distinctive morphological feature separatjng H. xeno oi from H. intestinalis, it is considered that the former name should be regarded as a synonym of the latter. The diplomonad flagellates from the blood and intestine of my R. catesbiana and X. laevis, are thus all referred to a s H. i ntest inalis. -20-

Order: Suborder: Eimeridea Lankesterella minima (Naussat, 1850) (Plate VI, Figs. 42 to 50, Text Fig. 1)

Lankesterella bas been found in frogs from Europe, Africa, China, Siam, Indochina and North America. Records are usually cited as L. minima though sorne authors (36, 46) have expressed uncertainty as to the validity of the specifie determination. Khaner (20) identified L. minima from R. catesbiana, as did Brandt. (44). Fantham et al (13) described L. canadensis from the same host. During his study of blood parasites of frogs, Kudo (22} failed to find L. minima in R. clamitans. Thirty-four per ceht (24/70) of the R. catesbiana caught on the west end of Montreal Island during this project proved to be infected with Lankesterella. Ten per cent

~/40) of R. clamitans were also found to r~rbour this blood parasite. The tiny vermiculer form present in the erythrocytes is a sporozoite. Leeches transfer this infective stage to the blood of a new host, in which it penetrates the endo- thelial cells of organ capillaries. Here schizogony produce a large number of merozoites. These escape into the blood to enter fresh host cells. Some merozoites eventually -21- develop into macro- and micro-gametocytes. Fertilization takes place by anisogony and a zygote is formed. This develops into an oocyst within which sporozoites form. When the hypertrophied endothelial cell bursts, the sporo­ zoites are released into the blood to enter erythrocytes and remain there with very little change in size until taken up by the host. Little is known of the biology of the parasites in the leech. (46). Sporozoites were the only forms found in the course of this study, though sections of liver, , and were exaruined for endothelial stages. Two morphologically distinct rorms were recognized, rounded or crescentia cnes within a capsule of vacuole . (Figs. 42 t o 46), !:'. nd those of less uniform size showing no evidence of a capsule. (Figs. 4? to 50). Fig ures 45 and 46 depict free forms from peripheral blood of healthy frogs. Figures 42 and 43 give the impression that the parasite lies within a capsule of its own rather than a vacuole formed by the erythrocyte. Stebbins , (39)~mistook these two forms, as well as the free vermicule, for stages in the life cycle of H. catesbiana. Measurement s from 50 specimens of each type were made. -22-

MEAN MEDIAN S.D. M/UThlUM LITNI1~CM

~'liTH NO CAPSULE

Length 13.2 ~ 15.0 Jl 1.53 fl 16.9 Jl 4.4 fl Width 3.2 3.1 1.04 5.0 1.3 .HTHIN A CAPSULE

Length 13.5 14.0 1.89 16.9 ?.5 'Nid th 2.8 2.8 0.95 4.4 1.25

In the for.ms within a capsule the nucleus is central, a nuclear membrane is not obvious and chromatin blacks may be somewhat scattered. Two round areas on either side of the nucleus stain a light blue with Giemsa. By phase contrast (Text Fig. 1) these appear distinctly darkened.

b. a..

Text Figure 1. Lankesterella m~n~ma (Naussat, 1950) from R. catesbiana (by phase contrast x 2550) a. Sporozoite lying within an erythrocyte. b. Sporozoite free in the blood plasma. -23-

The non-encapsuled form has a less central nucleus and the blue staining areas are not readily apparent. It is thought that both forms are referable to L. minima, differences in appearance being due to the maturity of the parasite. The size, shape and staining reaction of the host cell is unaltered, though the erythrocyte nucleus may be slightly displaced. In life a vermicule may be seen leaving an erythro­ cyte and enterine or gliding through others. Host cells are left temporarily misshapen but otherwise undama ged. The parasite is usually more pointed at the anterior end. Infections ranged from extremely heavy to very light. As sorne infections were not disclosed until blood slides were searched for a second or even a third half four, it is possible that further light infections were missed. Thes e haematozoans differ from the larger L. canadens is Fant mm et al., 1942, which rœ as ures from

10.6 to 19.2 ~ by 3 to 7.8 r, and all blood stages of which exhibit polar vacuoles. Earlier des criptions as well as lack of host speci­ ficty agree with the information found in this study, therefore this parasite is referred to as L. minima. -24-

Suborder: Adeleidea Family: Haemogregarj_nidae Haemogregarina catesbiana Stebbins, 1903 (Plate VI and VII, Figs. 51 to 60)

The year following Stebbins' description of this parasite from R. catesbiana (39), a further haemogregarine from the same host {and R. clamitans as well) was desig­ nated c1amatae Stebbins, 1904 (40). The second species was held to be justified on the grounds of its larger size, details of schizogony and karyo1ytic effect upon the host cell. This was sixteen years before the life cycle of K. lacertae Danilewsky was e1ucidated by Reichenow ( 21,33). It has long be en known that some members of the genus Haemogregarina sensu lato are strongly karyo1ytic. Sanders (35) and Laveran (25) examined the haemogregarine and the latter transferred Stebbins' second parasite to this genus, designating it H. c1amitae (Stebbins, 1904). Kudo (22) recorded this parasite from R. clamitans and R. pipiens referring to it as K. clamatae. There was a slight difference in size and shape from each host. Plimmer (30,31) identified H. catesbiana from the type host in the London Zoo. Fantham et al. (13) noted small numbers of a large haemogregarine, 20 by 2.2 ~' in -25- one of 300 Canadian R. catesbiana. Their description of greatly hypertrophied polychromatic red cells agrees with da ta on H. ca tesbia na. The complete life cycles of Lankesterella (46), Haemogregarine and Karyolysus (33,4ô) were unknown when · Stebbins published his description. His clear photo- graphie plates place it beyond doubt that he mistook· quite unrelated parasites for stages in the life cycle of H. catesbiana. However, forms which he thought represented o6'cyst forma tian were des cri bed and ill ustrate d as ovoid bodies with enlarged nuclei. It is clear from his illus­ trations that these are early forms of a true haemogregarine. Two of his photographs depict fading and vacuolation of the host cell cytoplasm, another reveals invasion of an erythro­ blast and in another the parasite lies in a red blood cell with a displaced nucleus. The host cell shows distinct karyorrhexis. Stebbins did not state from which host (R. catesbiana or R. clamitans) these preparations were made. Larger and broader vermicules 22.8 ~ by 2.38 to

3.72 ~ were represented as belonging to his other species, K. clamatae. He observed these vermicules as they moved in and out of red blood cells, often piercing and mutilating the host nucleus. Stained slides exhibited pronounced hypertrophy and degeneration of the host cell and its -26- nucleus, as well as an atypical staining reaction. He differentia ted K. clamatae from H. catesbiana on the basis of what he thought to be a capsule found only with the latter. His photographs clearly indicate that this 'capsule' was in reality, Cytamoeba bacterifera. His 'oocysts' of H. catesbiana resemble figures 53 and 54 of this study, and photographs of K. clamatae are like my figures 51, 55, 56, 57 and 60. Ten per cent (7/70) of the R. catesbiana and ten per cent (4/40) of the R. clamitans examined by me were found to be infected with a large haemogregarine. Associated haematozoans were:

FROJ NLThffiER PARASITES 21 L. minima 34 " H. intestinalis, Pirhemocyton 66 If T. rotatorium, c. bacterifera 84 " 97 If " 121 Il "

Ins ufficient material from R. clamitans was available for detailed morphological study, and the following account is based on slides from R. catesbiana . Fully developed vermicules are large, their length often exceeding that of -27- a normal red blood cell (Figs. 41 and 55) .

.MEASUrtEi vJENT0 OF H. CATESBIA.NA :B'ROM R. CATESBIANA

LŒDIAN S.D. hi:'LXIlVl.UM MINI.MUM

Length 22.7 J.l 25.0 Il 5.45 Jl 30.5 Jl 10.0 Jl llidth 5.2 5.0 0.97 7.5 3.1

Most of the organisms observed were large forms . (Figs. 55, 56 and 57), al though a nu.mber of small ones

(Figs. 53 and 54) were seen in bone marrow smears. A few rounded parasites interpreted as developing schizonts were encountered. (Fig. 51). Morphological differences between large enàoerythro• èytic forms (Figs. 56 and 57) may be indicative of sexual forms. Sorne may be schizonts which, once lodged in the capillaries vdll undergo division. Smears from organs of animals dead for several hours contained free vermicules with altered nuclei which could be micro- and macro-gameto­ cytes or gametes . (Fig. 59). The nucleus of H. catesbiana is made up of discrete blacks or transverse bands of chromatin, and appears to lack a membrane. The cytoplasm i s granular and the organism is enclosed within a pale staining capsule . (Figs. 56, 56 and 57). -28-

During in vivo examination of freshly drawn blood, parasites were seen to leave the blood cell and move through the plasma, (Fig. 58), entering and leaving ether cells at will, often dragging behind them a tail of what appeared to be erythrocytic cytoplasm. This phenomenon does not take place in the peripheral circulation of living frogs, as free forms are never present in dried smears prepared from freshly drawn blood. Extreme alteration of the host cell is character­ istic. As the vermicule is often appreciably longer than the norml red blocxl cell, distortion following full maturation is not surprising. However, hypertrophy is exhibited by cells invaded by small forms. {Fig. 53}. The host is also hypertrophied and shows lateral or polar displacement. The extent of alteration of the size and shape of parasitized red cells is evident from the following figures. Simple multiplication of length by witlth gives an arbitrary factor for comparing overall size. The ratio of length to width gives an indication of dis­ tortion. -29-

l\.ŒAN MEDIAN S.D. MAXD~UM MINIMUM NON-PARA3ITIZED R.B.C. Length 22.5 Jl 23.0 p. 2.01 p. 2?.5 }l 18.? }l 'Nid th 15.1 15.0 1.23 1?.5 12.5 LW 350 360 90 450 2?0 L/W 1.5 1.5 0.15 1.8 1.3 PARA3ITIZED R.B.C. Length 28.5 28.5 2.93 34.5 22.5 Width 1?.8 1?.5 6.1 22.5 11.8

LN 515 510 103 810 300

L/'N 1.? 1.? 0.23 2.2 1.2

Chroma tin gramlle s are evenly dispersed thro ugho ut the host cell rendering it more deeply and coarsely stained and giving its membrane a beaded appearance. This is a conse­ quence of karyorrhexis, e.g. the nucleus of the cell dis­ integrates into formless granules which are then extruded. Figure 52 illustrates a small haemogregarine lodged in a cell altogether lacking the nucleus either following the completion of karyorrhexis or through extrusion. Six mature specimens from R. clamitans measured:

MEAN ~ŒDIAN S.D. MA.,TIMUM MINIMUM Length 25.5 Jl 25.0 Jl 2. 96 p. 29.0 p. 21.8 Jl Width 4 .0 5.0 1.28 6.2 4 . 4 -30-

The host cell in R. clamitans does not contain so much extruded chromatin, its cytoplasm is vacuolated and the nucleus is broken into four or five large blacks

(Fig. 60). No free vermicules were observed. These parasites are identified as H. catesbiana, des pite the facts that the original description of this species is inadequate and that detailed life history studies have yet to be made.

Cytamoeba bacterifera Labb~ (Plate VII and VIII, Figs. 64 to 76) Kruse, the first to record c. bacterifera was convinced it was not 3 living organism (15). Since then several workers have examined it and reported their opinions as to its nature. One regarded it as a protozoan similar to what is now known as Plasmodiwa, ethers doubted its protozoan nature altogether (3,15) while still ethers thought it to be a protozoan parasitized by ba ccill iform bacteria. Laveran (24) isola ted the bacil liform bodies, which he designated Bacillus krusie. 'dorkers in Europe, Africa (11, 44,45) and North Ame rica have found t his enigmat ic parasite in the red blood cel ls of a variety of amphibie, including the two species of bullfrogs examined by me (15). This mos t unusual organism or organisms occurred in erythrocytes of 8.6 per cent (6;'70) of the R. catesbiana -31- and 5.0 per cent (2/40) of the R. clamitans examined during the present project. Fresh and Giemsa stained preparations were studied. Observation of living specimens reveals an amoeboid body in the cytoplasm of a red blood cell. Movement takes the form of rapid pulsation, and considerable granulation is evident by phase contrast. Pseudopods are extended in one part of the body and then another (Figs. 70 to 72). Prolonged observation and recoràing of the position of the parasite failed to reveal any progressional movement within the host cell. 3mall parasites (Figs. 64 to 66, 70 to 72) continue this ceaseless activity for many hours in carefully sealed wet preparations. After about one hour pseudopods are no longer formed, but the contents of the body exhibit rippling movements ~rlthin the confines of an elastic bounding membrane (Fig. 70). One particularly large Cytamoeba (Fig. 73) showed increasingly violent activity, in the course of which its pseudopods be came smoothly rounded. After about an ho ur the Cytamoeba had rounded up although its pulsations continued

(Fig . 74). The host cell was n~~ hypertrophied and its nucleus and cytoplasm appeared paler. ~uite suddenly, myriads of tiny rods in rapid movement became apparent within the disintegr ating erythrocyte. No longer confined -32- by a limiting membrane, they extended themselves in all directions from the main mass (Fig. '75), from which none, however, broke loose. The mass having first burst partly out of the host cell, now withdrew once more behind the cell membrane, the space between this and the nucleus becoming fully occupied within ten minutes. The wall of the host cell, its membrane contacted by the pulsating mass within, appe~red as a large amoeboid body (Fig. '76). Observations bad now gone on for four hours, and cou1d not be continued beyond this stage. The staining properties of parasitized ce11s always remained unchanged . (Figs. 64 to 68). Nuclear displace­ ment was common, but the erythrocyte itself -was seldom grossly enlarged.

MEAN MEDIAN S.D. MAXIMUM :MINIMUM C. BACTERIFERA Length 9.4 p. 10.0 p. 0.97 p. 15.0 p. 5.0 fl Width '7.4 ?.5 1.01 14.0 3.0 PARASITIZED ERYTHROCYTES

Length 22.5 23.0 2.6'7 29.0 19.0 Width 14.8 14.4 2.9 22.5 10.6

L iN 370 340 90 580 250 L/W 1.7 1.6 0.34 2.2 1.0 -33-

MEAN MEDIAN S.D. MAXIMUM MINIMUM NON-PARASITIZED ERYTHROCYTES

Length 22.5 Il 23.0 p. 2.01 p. 2'7.5 p. 1'7.'7 p

~Nid th 15.1 15.3 1.23 1'7.5 12.5 LW 350 360 90 450 2'70 L/W 1.5 1.5 0.23 2.2 1.2

Prolonged and careful searching of stained smears revealed a number of extracellular parasites. These clumps of pink stained bacilliform bodies (Fig. 69) were often found near the nucleus of a disintegrated red blood cell. Measurements of 50 rods were made.

MEAN :WiliDIAN MAXIMUM l\JITNIMUM Length 2.3 p. 2.0 p. 4.0 Jl 1.0 p.

The width was about 0.2 P.· Individuel rods were occasion­ ally found near a mass of others but were never scattered throughout the plasma. Stained intracellular parasites are surrounded by what seems to be a thick capsule (Figs. 65 and 66). Stebbins (39) mistakenly believed them to be schizonts of H. catesbiana. Close examination leads me to believe that this peripheral zone cons ists of closely packed rods (Figs . 64 , 65 and 66). There i s no evidence of s uch a capsule i n living specimens . Sta ined, the central zone is -34- structureless, occasionally containing a few pink rods. Nuclei and blue staining cytoplasm are never seen, nor are any other structures which could be interpreted as protozoan organelles. Figures 64 to 69 represent the organism as seen in Giemsa-stained slides. Figures 70 to 76 are comparable bodies as viewed alive by phase contrast. Two parasitized R. catesbiana (85-A and 26-A} were kept under observation for several months. Daily and weekly smears were examined and blocd counts recorded. On August 4, 1958 blood from a small frog exhibiting Pirhemocyton but not Cytamoeba (as far as could be ascer­ tained from repeated searching of smears) was injected into the peritoneal cavity of 85-A. Within 12 hours several large cytamoebae were noticed in a wet smear. The frog was sacrificed 16 days later, its heart blood being used to inoculate another ani.rral. V/ithin ten days the second frog wa s positive for c. bacterifera. Attempts to transmit the infection to B. americanus and X. laevis were unsuccessful. Blood counts were made, based on the number of parasites and various leucocytes per 1,000 erythrocytes. Daily smears were examined from 85-A for 20 days, weekly smears from a frog with a naturel infection for a period -35- of four months. Changes in the differentiai count were of low magnitude, and showed no significant correlation with the level of cytamoebae in blood. They could as easily be explRined as due to seasonal factors, if not, indeed, to intestinal helminths. More extensive and controlled data must be collected bef ore an adequate account of changes in the blood pic ture can be presented. It is tentatively considered thAt the org­ anism is àt most onl:y slightly pathogenic, but that when the condition of the host deteriorates through ether causes cytamoebae increase in the peripheral blood. Sections of heart, brain, liver, spleen and were examined but no ether stages or sites of Cytamoeba were found. Several theories as to the nature of Cytamoeba have been proposed, the authors concerned differing as to whether the para si te is a prot.ozoan alone, bac illi alone, or a protozoan pa rasitized by bacilli, Laveran (24 ) contended that the organism is not referable to the Protozoa. Regner (15) differed with him, favouring the view that Cytamoeba i s a protozoan parasiti zed by baci l liform bodies. Brumpt (3) was convinced that it is a degenerate form of a no the r para si te, most:· likely Lank:esterella or Dactylo soma. Though the Cytamo eba pulsates in an amoeboid fashion, -36- it does not change position in the red blood cell. Tiny rods are rapidly forced against a confining membrane and sometimes extend beyond it. ~Vhen the membrane disinte­ grates and rods fill the cytoplasmic area of the dead erythrocytes (Figs. ?5 and ?6), the same amoeboid movement is evident as the rods press against and stretch the outer membrane of the host cell. Regner did not see free bacilliform bodies, though he did not deny their existence. If cytamoebae were in fact protozoans parasitized by bacilli, one would expect to find sorne of them only lightly parasitized. However, rods are invariably plentiful. It is concluded that cytamoebae are clumps of mutually adhesive bacilliform organisms. The confining membrane, which is present in intra-erythrocytic forms only, is probably of host cell origin. These have some characteristics of Rickettsiae (34) and it is suggested that c. bacterifera may prove referable to this group.

Plasmodium-like artefacts: (Plate VII, Figs. 61 to 63) Plasmodium was not believed to parasitize amphibians until Fantham et al. (13) reported two light infections from R. catesbiana and B. americanus from the Montreal area. -3?-

The former comprised tiny oval bodies 3 hy 2.2 p, rings 6.6 11 in diameter, schizonts ?.2 by 6.2 p. and macro­ and micro-gametocytes. Some of the trophozoites contained one or more pigmented granules. Division stages forming 4, 6 and 8 merozoites were described. Extrema hypertrophy of the host celland its nucleus was characteristic. Inta-erythrocytic schizogony of P. bufonis resulted in the division of the nucleus into 8 merozoites. Pigment occupied one pole of the organism. Two forms of endo­ erythrocytic gametocytes were found, oval or slightly reniform macro~gametocytes and vermiform micro-gametocytes 18.6 to 20.3 r by 4.1 to 5.3 r' bent upon themselves in the host cell. Heart blood smears from one R. clamitans examined in the course of this study exhibited bodies which at first sight could be mistaken for pigmented haematozoans (Fig~. 61, 62 and ô3). The frog, caught in May, died in an emaciated condition 18 days later. Peripheral blood contained H. catesbiana and L. minima. The film was contaminated by bacteria and debris. Some of the Plasmodium-like bodies appeared to be endo-erythrocytic (61 and 63) but many were obviously exoerythrocytic (Fig. 62). The quantity of pigmented granules associated with them varied considerably, and large clumps of free -38- pigment granules were seen. These artifacts were not regularly distributed throughout the film, their presence in which is considered due to accidental contamination during the smearing process.

Pirhemocyton chattoni n. sp. (Plates IV and V, Figs. 11 to 40) Two types of parasites have been as E" igned to the genus Pirhemocyton, which Chatton and Blanc (6,7) described from the blood of a gecko. Life history stages of the genotype, P. tarentolae, incl ude anaplasmoid bodier; and for .rrs exhibiting albuminoid globules, irregular vacuoles and vesicular nuclei. Jobhston (17) recorded a similar para­ site from the South African snakes Echis carenatus and Causus rhombeatus. Doubting its protozoan nature, he did not name it. The South African frog , Rana galamensis, was found by Dutton, Todd and Tobey (11) to harbour an unidenti­ fied parasite of similar morphology but associated with one or more crystals. A similar parasite of Bufo regularis was described by França (38) as a new genus and species,Toddia bufonis. The validity of this genus was upheld by Scorza and Boyer (38) who recorded a second species, î oddia carbonelli, from Bufo marinus in Venezuela. It is evident from their illustra tions and description that these authors were dealing with a n organism very similar to P . tarentolae . -39-

In 1935 Brumpt and Lavier described Pirhemocyton lacertae from the blood of a , Lacerta viridis (5). The only stage seen was an amoeboid one having several chromatin masses. There was no globular refractile body, no vacuolization, and no granular, vesicular nuclei. Garnham's P. granosa (14) from tbe African skin.k: Agama colonarum much resembles P. lacertae. Neither of these parasites resemble Pirhemocyton as originally characterized by Cha tt on and Blanc. Eleven per cent (8/70) of the R. catesbiana harboured the parasite described hereunder. Two of the hosts were very large but the remainder small and presumably just emerging from their first winter's hibernation as frogs. They were collected at stations 5 and 6 (Plate I). Four of the hasts harboured other haematozoans.

FROG NUlrBER PARASITES

34 L. minima, H. catesbiana 71 " 64 Tt 107 c. bacterifera

The organism is characterized by an early anaplasmoid form found in erythroblasts or erythrocytes. This chromatin body develops into a larger Aegyptianella-like one. It is -40- accompanied, though not always, by an irregŒlar body which appears as a vacŒole on dried smears but as a pyriform amoeboid body in vivo. With maturation a large retractile body develops in the host cell cytoplasm. Multiplication of smal1 vesicular nuclei occurs in the red b1ood ce1ls. There is hypertrophy of non-infected cells and hypertrophy fo1lowed by hypotrophy of infected ce11s. Haematopoietic centers are affected. The spleen of infected anima1s is a1ways enlarged. Tbe àisease may be acute and fatal or chronic and not fatal.

MEAN MEDIAN S • D. W~XIMUM MINIMUM

CHROMA TIN BODY ( Diiù/.ŒTER)

Anaplasma-like 1.5 p 1.4 ~ 0.86 ~ 1.85 ~ 1.25 f Aegyptianella-1ike 3.5 3.3 0.25 3.75 3.2

REFRACTILE BODY Length 9.9 8.8 2.46 12.5 3.8 .ïNidth 7.3 8.1 2.3 10.6 3.8 MULTIPLE NUCLEI 1.7 1.6 0. 54 2.8 0.8

The coŒrse of the infection was not followed from inception throŒgh convalescence in any single case, early and late stages being seen in different individuals. -41-

CHROMATIN BCDY (anaplasmoid and Aegyptianella-like) Anaplasmoid forms may be seen unassociat ed with ether structQres (Fig. 12) or together with a very small vacQole. (Figs. 13 and 14). They are easily distingQishable by phase contrast, and over 50 per cent of the erythrocytes may contain them. Their chromatin is homogeneoQS. MeasQre­ ments of 50 individQals per day for ten days indicated an increase of aboQt 0.6 ~ in diameter as the parasite matQred and other structures developed.

Aegyptianella-like forms \~re largest in the erythro­ blasts, ·the ir development clearly accompanied by the appearance and growth of the retractile body. With ma tura­ tien they continQe to stain deeply, but especially so centrally and peripherally (Figs. 17, 26 and 29) • VIi th rare exceptions (Fig. 29) no more than one such body is present in each host cell. In figures 3i to 40 various forms of the chromatin body can be seen as they appear in life. Many were seen free in the plasma (Fig. 19) at a time when matQre forms were foQlld in the erythroblasts and just prior to the rr.assive erythrocytic invasion which preceded the frog's death. The central nuclear body is clearly seen but the outer granular membrane appears to have disinte­ grated. -42-

MULTIPLE :t-.IUCLEI By phase contrast one often sees several nuclei such as those found in figures 35, 36, 39 and 40. These are not readily apparent on dried films. However, they are sometimes evident in favourable smears as very pale circular or amoeboid structures with a darker central body and granular chromatin on the nuclear membrane (Figs. 16, 17 and 30). These multiple nuclei are only found in association with the mature chromatin and retractile bodies. They may lie at the edge of the latter (Fig. 16) or be closely associated with the host cell nucleus (Fig. 36). Some are se en unassociat ed in the host cell cytoplasm. At times, when fresh ma terial is under examination, the large chromatin bodies became elongated and their granular periphery separated into lobes (Fig. 40). This could indicate the formation of multiple nuclei by mitosis or budding.

REFRACTILE BODIES Large retractile bodies develop in association with the larger Aegyptianella-like nuclei (Figs. 14, 16 to 18 and 22 to 30 and Text Figure 2}. More than one may be found per host cell (Figs. 25, 28 and 29). In some indivi­ duels they stain a distinct, though pale blue, while in ethers the body, containing very fine pink granules, assumes -43-

b. a..

Text Figure 2. Pirhemocyton chattoni n. sp. {phase contrast x 2550) a. Mature retractile body within an erythroblast or hypotrophied erythrocyte. b. Free refractile bod~es. almost exactly the same stain as does the host cell cyto- plasm. The pink bodies contrast clearly with the host cell cytoplasm of basophilie erythroblasts (Figs. 23 to 30). Free forms are seen as rather more basophilie than mature erythrocytes (Fig. 20).

VACUOLES A distinctive type of vacuole formation is associated with this haemotozoan. Phase contrast examination of fresh material reveals bodies of comparable size and shape as -44- amoeboid shadows, sorne of them pyriform (Figs. 33 to 35 and 37). Very often they are in close association with the host ce11 nucleus. Vacuolation of red blood ce1ls, unassociated with P. chattoni is depicted in Fig. 31. Stained preparations reveal an irregular clear body edged by condensed cytoplasm of host cell origin (Fig~. 13, 15 and 17). They are on1y occasiona1ly united with the chromatin or refractile body. No structure can be seen in them by phase contrast or bright field microscopy. The shape and size are by no means uniform. The 1ast indica­ tion of the disease in #124 was the presence of these vacuoles in red blood cells, and in the last stages of the disease in #123 they were not seen at a11. In one indivi­ duel they accompanied multiplicative stages (Fig. 17), in another they were seen onlywith the anap1asma-like bodies.

ALTERATION OF BLOOD CELLS The size and shape of parasitized and non-parasitized erythrocytes are altered as intra-erythrocytic development proceeds. Plates XIII and XV illustrate the change in size of the surface area (L W). Fifty cells of each type were measured for each reading. The simple mean is recorded. Plate XIII records these measurements made from a frog infected by èn intra peritoneal injection of para­ sitized blood. No significant change in healthy cells was -45- noted for the first 14 days after injection and the first few days of the appearance of parasites in erythroblasts. However, the situation altered when erythrocytes became infected. From the 17th day onward healthy and infected erythrocytes became hypertrophied, the latter to a lesser extent. The infected erythroblasts showed early hyper­ trophy but later hypotrophy. Plate XV illustrating recovery in another larger frog, shows increasing hypo­ trophy of infected cells. A similar examination for cell distortion (L/N) revealed increased irregularity of infected cells as the parasites matured within them. Cells containing multiple nuclei were especially altered (Figs. 16, 17 and 30). The hypertrophy of healthy cells is comparable to pernicious anaemia in mammals. Maturation of the parasite in erythroblasts is illustrated in figures 2.l to 31. The nucleus becomes increasingly pycnotic and may be extruded (Fig. 18). Many of these cells are destroyed and disintegrating nuclei are numerous in the plasma. Surviving erythro­ blasts mature and in these one may find multiplying parasites (Fig. 30), Figures 35, 36 and 37 show infected erythroblasts as seen in vivo by phase contrast. -46-

Other authors (6,17,37) have described similar distortion of blood cells. Scorza and Boyer (37) attribute five phases to the disease caused by Toddia

cattonelli. The early stage is characterized by ?fiaplasmoid bodies, low parasitaemia, and little change in the blood picture. Then come phases of higher parasitaemia with the formation of one or more octagonal crystals, hyper­ trophy and karyolysis. In late phases of acute infection and in splenectomized animals the Bufos became prostrate and died, some of them exhibiting no uninvaded red cells whatsoever.

BLOOD COUNTS The disease is accompanied by a rise in the propor­ tion of granulocytes (Plate XII). Eosinophils are more numerous than neutrophils. Sorne dark staining granula- cytes are probably basophils. These disappear with the disappearance of parasites in convalescing animals. The

~ . cells are very much like bodies wh.ich Ivan~c (16} described from R. esculenta under the name of Erythro- cytonucleophaga ranae. The high eosinophilia persisted in #124 long after the parasite and may have been related to helminths. The change in #67-A (Plate XII) can be attributed directly to Pirhemocyton. Small lymphocytes were not effected. Large lymphocytes, many of them -47- haemoplasts, were seen. Changes in the relative numbers of red cells (erythroblasts, immature and mature erythrocytes, infected and non-infected) were recorded. The data of Plates XI, XIV and XVI were obtained by examining 1,000 such cells for each day recorded. The readings are on the basis of numbers of each type per 1,000 of all types. Plate :cr illustrates the increase of parasitized blood cells in frog ;j;67-A. The first indication of change was the appearance of a relatively large number of uninvaded erythroblasts nine days after injection. Four days later all of the erythroblasts in the peripheral blood were infected. The relative proportion of erythro­ blasts decreased as the infection advanced in the mature erythrocytes. In these the increase vms dramatic. One the day of death over 85 per cent of the cells were invaded. The frog was in a very emaciated condition just prior to death. Plates XIV and À~I show similar counts from a series of slides taken from convalescing animals. Plate XIV clearly indicates regeneration of blood to an extent relative to destruction by the parasite. Plate XVI illustrates almost complete regeneration of blood following infection. -48-

TR.:\.NffiUSSION EXPERIMENTS Heart blood of a naturally infected R. catesbiana was injected into the peritoneal cavity of another, which had consistantly yielded negative blood films. Within 12 hours anaplasmoid bodies appeared in the erythrocytes. Their nwnbers never increased beyond 5.5/1,000 per blood cells. Four days later the parasites had disappeared. On the 16th day this frog was sacrificed and its heart blood was used to inoculate a second animal (#67-A). Plates X, XI and XII illustrate the changes which occurred. This frog died 23 days later in an acute state of parasi­ taemia. Other transfer attempts were not successful. Blood of convalescent frogs was injected into three other R. catesbiana, three R. clamitans and five B. americanus. In sorne there was an inorease of erythroblasts. Possibly the R. catesbiana (the only species found with natural infections) were immune from earlier infections, or the blood used was no longer infective. Inoculation of five X. laevis did not result in a normal development of the parasite, but 30 days after inoculation three of them passed whole blood or pigments into the water, giving it a yellow colour. This persisted for 24 hours. Irregular vacuoles as already described (pg. 44) were seen in these frogs, and the percentages of eosinophils and erythroblasts -49- increased, but in ten weeks of observation there was no ether manifestation of the parasite.

DISCU3SION An anaplasmoid chromatin body is characteristic of beth Pirhemocyton and Toddia, and is recognized as the post-invasive form. This develops into an Aegyptianella­ like body which Scorza and Boyer (39), observing its slow amoeboid motility, interpreted as a trophozoite. My phase contrast stcrlies lead me to believe that the pseu­ dopods are actually new nuclei being formed by budding or multipolar division (Fig. 40). Their structure and staining properties are commenserate with those of a protozoan nucleus. Chatton and Blanc (7) saw no forms which could be interpreted as schizonts, but Scorza and Boyer described the formation of schizonts with 4 to 17 anaplasmoid merozoites (38). The method of erythrocytic invasion remains undeter­ mined. Two forms of the parasite were seen free in the plasma (Figs. 19 and 20), but it is not known whether either of these is capable of re-entering a red blood cell. Tiny amoeboid bodies (Fig. 33) were discerned moving along the outside of red blood cells(phase contrast). Those in figure 34 were observed for sorne time as they moved within -50- the host cell. These bodies recall the irregular vacuoles described earlier (compare figures 15 and 34). Pyriform and irregular vacuoles were described by Chatton and Blanc, who suggested that these areas were the paths through which an amoeboid body had moved. They could also be due to pathological changes in the host cell. The nature of the refractile body is not known. Cha tton and Blanc were convinced that it is not parasit ic but the results of host cell reaction. Its stain1ng properties make it appear to be homogeneous with the cytoplasm of a mature erythrocyte. However in P. chattoni the bodies were clearly seen in all erythroblasts infected and in many erythrocytes they stained a distinct pale blue. Dividing forms of P. chattoni nuclei were usually closely associated with this body (Figs. 16, 30, 39 and 40}, which may be the cytoplasm of a mature trophozoite, containing reserve chromatin materiel, or metabolites resulting from the assimilation of material from the host cell nucleus. In its free form (Fig. 20) it could be the final stage of development in the host. As in F. tarentolae and Toddia cattonelli, the para si te is of ten associat ed with the host cell nucleus. -51-

T. cattonelli, though certainly not the same species, is very similar, especially in regard to the structure of the vesicular nucleus, and the pathological changes of the host cells. This disease resembles a babesiosis, and is accompanied by jauntice. The octagonal crystal may be analogous to the refractile body of Pirhemocyton. These facts point to sorne relationship between

Toddia, Pirhemocyton and of ma rr~.m.als. The parasites found in R. catesbiana cannot be placed in the genus Toddia, which is characterized by the consistant appearance of crystals. The parasite found by Johnston in African snakes is considered referable to Pirhemocyton sensu stricto. It is submitted that P. lacertae and P. granosa require new categories. The parasite discussed in this study is included in the genus Pirheruocyton as originally described by Chatton and Blanc. Due to the morphological differences detailed above and their occurrence in another species of Amphibie, they are considered as a new species and given the name, Pirhemocyton chattoni n. sp •• -52-

MITOSIS DJ THE CIRCULA.TDTG BLOOD OF R. CATESBIANA RECOVERING

FROM IJ\IFECTION CHITH PIRHAEMOCYTON CHATTONI N. SP ••

Due to the stimulation of haematopoietic centers after destruction of a large number of erythrocytes, the blood of frogs convalescing from P. chattoni gives an excellent opportunity to observe various stages in the development of erythrocytic and leucocytic cells. Extensive studies of haemotopoiesis in amphibia have been undertaken (1,2,18,19). In tadpoles the centers of blood cell formation are largely the kidney and spleen. In the adult the spleen is the chief organ, though the general circulation, heart, or bone marrow may also con­ tributs. The monophyletic theory of blood cell origin, (18) holds that all blood cells originate from identical "polypotential" haemoblasts derived from the mesenchyma. Differentiation is due to extrinsic factors. The erythro­ cytes and thrombocytes develop intravascularly, the leucocytes, extravascularly and the lymphocytes both intra­ and extravascularly. Early forms are not normally found in peripheral blood of adults, though they may be seen in larvae. Red bone marrow develops in the femur of the adult in the spring , giving rise to an increase of red blood cells after hibernation. -53-

The flooding of haemoblasts into the circulation of frogs harbouring P. chattoni recalls the blood picture of pernicious anaemia in mammals (43). A series of myelo­ blasts and erythroblasts could be traced from very immature forms to the fully developed ones usually found in the circulating blood. Large hypertrophied lymphocytes, monocytes and small lymphocytes were also seen. Very small rounded forms with deep staining polychromatic granules were probably early thrombocytes. The material also offered a unique opportunity to illustrate the stages of mitosis in erythroblasts (Figs. 78 to 92). Haemoblasts about to divide become rounded, and much resemble small lymphocytes. Their cytoplasm (Giemsa) is deeply basophilie and finely granular. The nucleus is irregularly rounded, occupies the greater part of the cell (Fig . 78 ), a nd differs from tha t of a small lymphocyte (Fig. 77) in having its chromatin condensed into discrete blocks. When observed in vivo by phase contrast the nucleus of a haemoblast in late prophase was seen whirling and tumbling into the cell, as finger-like chromos omes appeared. (Text Fig. 3) Figures 79 and 80 show early prophase as seen in stained films. Figure 81 illustrates a spireme, formed in late prophase. Centr ioles and -54-

Text Figure 3. Late prophase in erythroblast in R. catesbiana recovering from P. chattoni. (phase contrast x 2550}. astral rays could not be seen at any time. Metaphase is depicted in ~igures 82 to 84. The separate chromosomes can again be distinguished as polar anaphase commences (Figs. 85 to 87). Telophase and final constriotion are seen in figures 88 to 92. The daughter cells have super­ ficial pseudopodia and stain a delicate blue. They soon become rounded and their cytoplasm more homogeneous as the cell membrane forms. -55-

These observations were made from blood of large frogs, at least four years old. As mitosis \~S occurring in the circulating blood, it was possible to follow daily changes without destroying the specimen. Detailed studies of haematopoiesis and mitosis could be undertaken very readily on such convenient and easily available material. -56-

sm~nv-LARY Two hundred and twenty-eight frogs and toads from the Montreal area of Q.uebec were exanp.ned during these studies. The six species collected were, Rana catesbiana, R. clamitans, R. sylvatica, Hyla versicolor and Bufo americanus. The incidence of haematozoans is much higber than earlier investigations implied. Six genera and species of blood parasites were iden­ tified, Trypanosoma rotatorium, Hexamita intestinalis, Lankesterella minima, Haemogregariœ catesbiana, Cytarnoeba bacterifera and Pirhemocyton chattoni n. sp. These are described and illustrated in detail, as are some Plasmodium­ like artifacts, in view of the fact that two species of this genus, the first and only ones recorded from amphibiens, were described during an earlier survey in the Montreal area. Hexamita xenopi Fantham, 1923 is rejected as a synonym of H. intestinalis Dujardin, 1841. The controversial c. bacterifera consists of a clump of mut œ lly adhesive rads, hel d w:i thin a vacuole which is considered to be of host cell origin. It is suggested tbat the affinities of this organism lie with the Rickettsiae rather than the Protozoa. Observation of the blood picture lead to the tentative conclusion that it is at most only slightly pathogenic. -5?-

Pirhemocyton Chattoni n. sp. was found in 11.4 per cent of the R. catesbiana. It is a babesioid organism, exhibiting a vesicular nucleus, irregular vacuoles, large refractile bodies, and multiple nuclei, and producing a disease which may be acute and fatal or chronic and not fatal. Comparisons with Toddia suggest that these two genera are closely related. Mitosis in the peripheral blood of R. catesbiana recovering from infection with P. chattoni n. sp. is discussed and illustra ted. -58-

PLATE I.

Maps indicating collection areas. The circle in the top map is enlarged for the lower one and numbered circles designate specifie location for:- 1. R. pipiens and R. sylvatica; 3. B. americanus; 2, 4 , 5 and 6. R. catesbiana and R. clamitans. MILES 20 PLATEr·

15

10

5

0

1 LE PERROT

0c=:===--~====:, 1 1 M 1 LE 4- 2 -59-

PL.\TE II.

Diagram of an aquarium arranged to trap night flying insects. Small Amphibia remained within the container and fed nightly. PLATE Il

Wl N DOW

Wl DE

AQUARIUM -60-

PLATE III.

Al1 drawings made with the aid of a camera 1ucida.

Figs. 1-6. Trypanosoma rotatori~n (Mayer, 1843) from b1ood of R. catesbiana, R. c1amitans and R. pipiens. Dried amears stained with Giemsa, (x 1600). 2, 5 and 6. F1age11ated forms (Group 1). 1 and 3. Non-f1age11ated forms with an undulating membrane (Group 2). 4. Rounded form from heart b1ood (Group 3).

Figs. ?-10. F1age11ates from the b1ood and rectum of R. catesbiana and X. 1aevis (x 1600). 7. Intestinal flagellate on contaminated blood smear (Giemsa). 8. Hexamita intes­

tina1is Dujardin, lA~J in blood of R. catesbiana (Giemsa). 9 and 10.

H. intestinali~ from rectum of R. catesbiana and X. laevis (fixed in Schaudinn's fluid, stained with Shortt's Haematoxy1in). . PLATE Ill

2 -61-

PLA'rE IV.

Fig. 11. Normal erythrocyte of R. catesbiana.

Figs. 12-30. Pirhemocyton chattoni n. sp. from blood of H. catesbiana.l2-17. Parasitized erythrocytes. 18. Hypotrophied erythroblast. 19 and 20. Free forms. 21-29. Development in erythro­

blasts. 30. Multiple nuclei in i~ture erythrocyte. (Giemsa, x 1600). - PLATE IV

11 13 14

17 18 19

26 27 30 -62-

FLA.TE V.

Fig. 31 Non-parasi tized erythrocyte of R. catesbiana containing vacuoles. (Phase-contrast, x 2550).

Figs. 31-40. Pirhemocyton Chattoni n. sp. as seen by phase-contrast microscopy. (living material, x 2550). 32 and 33. In an hypertrophied

and an hypotrophied erythrocyte. ~4. Amoeboid bodies which correspond in size and shape to the irregular vacuoles seen on dried smears. 35-40. Aegyptianella-like forms, some host cells containing multiple nuclei. 40. Formation of multiple nuclei by division or budding of the Aegyptianella­ like chroma tin body. PLATE V

36

IOP.

38 39 40 -63-

PLATE VI.

Fig. 41. Normal erythrocyte of R. catesbiana.

Figs. 42-50. Lankesterella minima (Chaussat, 1850 ). 42-46. Forms associated with a capsule. 47-50. Forms not associated with a capsule.

Figs. 51-57. Haemogregarina catesbiana, Stebbins, 1903, in erythrocytes. 51-54. Forms seen in organ smears from freshly killed specimens. 55-57. Forms from the peripheral blood. (Giemas, x 1600). PLATE VI

42 43

·· ~ ·· · · 9 50 45

' . ·.. . (. ~ :-4 ~-t_l~:.:. ~~~1-~~.~ . . •:· '),'ft''%;.~ Il.-~;; ~-· G . _,

:;#<' lî/

52

56 57 -64-

PLATE VII.

Figs. 58-60. .t~r.... catesbiana. 58. Free form in freshly drawn b1ood. 59. Free forms in 1iver smear from R. catesbiana dead severa1 hours. 60. H. catesbiana as seen in R. clamitans. (Giemsa, x 1600).

Figs. 61-63. Plasmodium-1ike artifacts from contaminated heart b1ood smear. (R. catesbiana). (Giemsa, x 1600).

Figs. 64-69. Cytamoeba bacterifera Labbé, 1894, in erythrocytes of R. catesbiana. 64-68. Endoerythrocytic forms. 69. C1wmp of ba­ cilliforrn rods free in plasma. {Giemsa, x 1600). pL A T E VIl

58

61 63

64 65 66

68 69 -65-

PLATE VIII.

Figs. 70-76. C. bacterifera as seen by phase-contrast microscopy. (drawn from living material, x 2550). 70-72. Forms usually seen in

erythrocytes . 73 -76. Ba c~lli form rods {B. krusei) as they became free of the restraining me mbrane and filled the cy topl asmic a rea of a dying erythrocyte. PLATE V Ill

70

14

76 PLATE IX.

Fig. 7?. Normal small lymphocyte of R. catesbiana.

Figs. ?8-92. ~itosis of erythroblasts in the peripheral blood of an adult R. catesbiana convalescing from an infection of P. chattoni. ?8-80. Early prophase. 81. Spireme. 82-8?. Polar anaphase. 88-92. Telophase and final constriction. (Giemsa, x 1600). PLATE IX

71

81 82 83 84

85 86 87 88

89 90 91 92

~~ ~ r , ·. ·.. . . 1 ' '~··.···' .rJ··".: .,.: )N• ) '·'..,,..··... ,.·' -67-

PLATE X.

Key to Histograms. KEY TO HISTOGRAMS

NORMAL ERYTHROCYTES mrmmmum , r­ PARASI Tl ZED ERYTHROCYTES ~mmmm~ l> -f IMMATURE ERYTHROCYTES uma m x

NORMAL ERYTHROBLASTS 1//llllllllll/1/lllllllll/1/lllllillllllllll//lllll/11/lll/li

~·:: ~:-::.-.--.·.•:;---c. ·:·.·...::::~·.-.~··.·. 1. .: ...... • .! .• .• • ••••••••• ·. PARASITIZED ERYTHROBLASTS • , • 1 ., 0 • 1 -68-

PLATE :xi.

Red blood cell count from R. catesbiana (#67-A} inoculated with P. chattoni. Erythroblasts were non-parasitized when they suddenly appeared on the nin th day, a ft er which t ime all of them contained parasites. The frog died on the 23rd day. (Data based on actual count of 1,000 cells for each reading). PLATE Xl

100

(/) _J _J LLJ 800 (.) 0 0 0 _J m 0 600 LLJ 0:: 0 0 Q 0:: ~ 400 a.. 0:: LLJ CQ ~ :::> z 200

0 9 13 15 16 17 NU M 8ER OF DA YS A FT ER 1N 0 C ULAT 10 N -69-

PLATE XII.

Count of granulocytes from R. catesbiana (/f67-A) inoculated with P. chattoni. Parasites were first seen in the blood on the 15th day. (Data based on moving average of count per 1,000 red blood cells). PLATE X Il

Cl) ...J ...J L&J 0 0 0 28 0 ...J m 24 Q l&J 0::: 20 0 0 0 16

2

8 NEUTROPHILS

4

0 1 3 5 7 9 15 17 19 21 23 NUMBER 0 F DAYS AFTER INOCULATION -70-

PLA.TE XIII.

Changes in the surface areas (LN) of red blood cells of R. catesbiana (#67-A) inoculated with P. chattoni. Normal erythrocytes becoms more hypertrophied than parasitized ones and erythroblasts become hypotrophied. {Data based on mean average of 50 cells measured for each reading) • PLATE Xlii

z 0 -t­

0:: ~ t­ LL ­

0 0 0 0 0 0 0 0 ~ M N - SQUARE MICRONS OF SURFACE AREA -?1-

PLATE 2ŒV.

Red blood cell count from R. catesbiana ( ;/124) recoverirg from a natural infection of P. chattoni. Erythroblasts appeared in just sufficient quantity to replace the parasitized erythrocytes. (Data based on actual count of 1,000 red blood cells for each reading) • 1000 . IIIIIIIHIUI~~ z c ~ 800 (Tl ::0 ""0 "l) r (Tl ::0 )>

0- -1 0 600 l'Tl 0 ::0 (Tl 0 x CD - b 400 < 0 0 0 ITI r- r- (/) 200

0 20 1 10 15 25 -0 1 10 15 25 30 21 DATE JULY AUGUST SEPTEMBER OCTOBER -72-

PLATE X.V.

Surface area (LW') of red blood cells from R. catesbiana (#124). Parasitized erythrocytes show marked hypo­ trophy. (Data based on mean average of 50 cells measured for each reading). Cf) 0 c l> :::0 400 l'Tl ~ 0- :::0 0 z "'1J CJ) r 0 300 )> "TT --i CJ) c l'Tl :::0 "TT x l> (") < l'Tl l> 200 ::0 l'Tl l>

100 21 25 27 29 1 15 21 30 2 6 9 11 17 DATE JULY AUGUST SEPTEMBER -?3-

PU~.TE XVI •

Red blood cell count from large R. catesbiana (#123) recovering from a naturel infection of P. chattoni. All of the mature erythrocytes were destroyed and replaced by erythrob1asts in the periphera1 blood. (Data based on actual count of 1,000 red b1ood cells for each reading). PLATE XVI

1000

Cl) ..J ..J 800 IJJ 0 0 0 0 ..J m 0 600 LIJ a:: 0 0 0- a:: 400 L&J a.. 0:: LU m ::E ::l z 200

0 3 2 DATE J U L Y -74-

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