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 Haemogregarina 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 Blood 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, Plasmodium-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 Red blood cell 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, Haemoproteus, Dactylosoma and Plasmodium. Two species of the last-named genus (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.B ER 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 amphibians 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 et al 1942 tl 1 0.44 " ~__;...;;..., " ,, 1anoraiea Fantbam et al 1942 1 0.44 ...;......;..__;.=:;.' " " 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.