Experimental Transmission of Sarcocystis from Icterid Birds to Sparrows and Canaries by Sporocysts from the Opossum

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Faculty Publications from the Harold W. Manter Laboratory of Parasitology Parasitology, Harold W. Manter Laboratory of

8-1978

Edith D. Box University of Texas Medical Branch

Donald W. Duszynski University of New Mexico, [email protected]

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Box, Edith D. and Duszynski, Donald W., "Experimental Transmission of Sarcocystis from Icterid Birds to Sparrows and Canaries by Sporocysts from the Opossum" (1978). Faculty Publications from the Harold W. Manter Laboratory of Parasitology. 128. https://digitalcommons.unl.edu/parasitologyfacpubs/128

This Article is brought to you for free and open access by the Parasitology, Harold W. Manter Laboratory of at DigitalCommons@University of Nebraska - Lincoln. It has been accepted for inclusion in Faculty Publications from the Harold W. Manter Laboratory of Parasitology by an authorized administrator of DigitalCommons@University of Nebraska - Lincoln. J. Parasitol., 64(4), 1978, pp. 682-688 0 American Society of Parasitologists 1978

EXPERIMENTALTRANSMISSION OF SARCOCYSTISFROM ICTERID BIRDSTO SPARROWSAND CANARIESBY SPOROCYSTSFROM THE OPOSSUM

Edith D. Box* and Donald W. Duszynskit

ABSTRACT: Cowbirds (Molothrus ater) and grackles (Cassidix mexicanus) infected with muscle cysts of Sarcocystis were fed to opposums (Didelphis virginiana) and fecal sporocysts from the latter were given to sparrows (Passer domesticus, Family Ploceidae), canaries (Serinus canarius, Family Fringilli- dae) and ducks (Anas platyrhynchos, Family Anatidae). Asexual parasites were found in the endothelium of sparrows and canaries but not in ducks. When birds were kept 10 weeks or more after infection, muscle cysts were found grossly and microscopically in the majority of sparrows, and in 1 canary, but not in ducks. Muscle zoites were found in digests of all sparrows and canaries but not in that of ducks. Metrocytes and forms dividing by endodyogeny also were found in the digest. Thus, avian Sarcocystis was transmitted experimentally from 2 genera of 1 family (Icteridae) to 2 different families of passerine intermediate hosts by sporocysts from the definitive host. This is the broadest intermediate host spectrum known for a species of Sarcocystis.

Transmission of Sarcocystis by obligatory the breeding season began so they were at least alternation of hosts for the sexual and asexual 7-8 months old when captured. They were caged in indoors and fed commercial starter has been with a number pairs mash. stages accomplished Experimental canaries (Serinus canarius) included of predator-prey combinations. The most in- 8 canaries reared coccidia-free in the laboratory tensively studied have been the cycles between the previous year and 3 canaries reared indoors the canine and feline predators and wild and at the senior author's home. Canaries were caged in to the of domestic meat animals (Levine, 1977). Trans- groups according origin sporocysts they were given. hatched (Anas mission of rabbit cats Newly ducklings Sarcocystis by (Fayer platyrhynchos) were purchased from Sears Roe- and Kradel, 1977; Crum and Prestwood, buck and infected 5 days after arrival. They were 1977) and murine Sarcocystis by cats (Ruiz kept in groups in brooders, separated according and Frenkel, 1976), owls (Cerna, 1976; Mun- to inoculum. As they grew, they were transferred to an inside and later to an outside until and snakes also pen pen day, 1977) (Rzepczyk, 1974) necropsied. has been described. We recently were able to transmit avian Sarcocystis from grackles and Inoculum cowbirds (the intermediate hosts) to opos- were collected from the feces of 2 sums, which served as definitive hosts (Dus- Sporocysts experimentally infected opossums (Didelphis vir- and zynski Box, 1978). Although previously giniana), one of which had been fed 7 Sarcocystis- Sarcocystis was thought to be rather host infected cowbirds (Molothrus ater) and the other, specific, parasitizing one genus of intermediate 2 infected grackles (Cassidix mexicanus) (for de- host this describes the tails, see Duszynski and Box, 1978). The sporo- (Levine, 1977), report in feces were in successful transmission of the muscle cysts refrigerated 2% (w/v) parasite aqueous K2Cr207,then concentrated by sugar flo- from Icteridae (cowbirds and grackles) to tation, washed, counted and administered to Ploceidae sparrows) and Fringillidae (ca- recipient birds by stomach tube. The age of the naries) by sporocysts from opossums. sporocysts was from 15 to 52 days when administered. Doses given sparrows and canaries varied from 10Qto 500 X 103 sporocysts except for birds MATERIALSAND METHODS killed 24 hr after infection which received 2 X 106 Experimental intermediate hosts sporocysts (Table I). Ducks were given 106 sporocysts. Adult sparrows (Passer domesticus) were captured in a Havahart trap on the University of Necropsy Texas Medical Branch campus in March before Birds were necropsied at various intervals were killed with Received for publication 16 December 1977. postinoculation (PI). They * Department of Microbiology, The University of chloroform, skinned, and muscles were inspected Texas Medical Branch, Galveston, Texas 77550. grossly under a magnifying lamp. A sample of t Department of Biology, The University of New thin abdominal muscle about 10 mm2 was pressed Mexico, Albuquerque, New Mexico 87131. between 2 slides and examined microscopically 682 BOX AND DUSZYNSKI-EXPERIMENTALTRANSMISSION OF AVIAN SARCOCYSTIS 683

TABLE I. Size and origin of inoculum given experimental birds.

Sporocyst origin No. sporocysts Cassidix Molothrus Recipient in inoculum bird X 103 No. birds Mortality* No. birds Mortality* Total

Sparrow 1 2 - 2 100 3 3 1D 6 500 3 2D 3 1D 6 2,000 1 1K 1 1K 2

Canary 1 1 - 1 200 3 1KM 3 2KM 6 2,000 1 1K 1 1K 2 0 -- 2

Duck 1,000 5 2K 5 2K 10

* Mortality by 16 days PI; D = died; KM = moribund when killed; K = killed, not sick. for cysts. The head, feet, wing tips, and viscera sporocysts of grackle origin. The first inocu- were removed from sparrows and canaries and the lum was given about 24 hr and the second remainder of the carcass and Mc- digested (Box about 2 hr before In both birds, Guinness, 1978). A 50 g sample of combined breast necropsy. in and thigh muscle was taken from each duck for many extracellular sporozoites were found digestion. The material to be digested was ground the small intestine and a few were in mono- for 30 sec at high speed in a commercial Waring nuclear phagocytes (Fig. 1). They were most Blendor with solution w/v enough pepsin (0.75% numerous about 6 cm from the duodenal loop. w/v NaCl and v/v HC1 in pepsin, 0.75% 1% Of the internal the were water) to cover it. Pepsin solution was added to organs sampled, lungs the blend to equal 10 X the weight of the ma- the most heavily parasitized. Here, the para- terial to be digested. Digestion was for 1 hr at sites were commonly inside mononuclear 37 C while with a stirrer. The agitating magnetic 2). An occasional sporozoite was then strained double of phagocytes (Fig. digest through layers was found in liver and smears. for 10 min at 700 g, and a spleen Sporo- gauze, centrifuged = drop of sediment was examined for zoites at 400 X zoites were 2.35 X 5.65 (2-3.5 X 5-7, n using phase optics on a Zeiss photomicroscope. 10); one end was pointed, the other rounded and often stained red with Giemsa's. These Slides parasites inside mononuclear phagocytes re- Impression smears were made of liver, spleen, sembled stages of Isospora serini (Box, 1977) lung, brain, and duodenal loop of sparrows and but the nucleus was more compact and cyto- canaries. They were wet-fixed in Bouin's and were less abundant than in stained in Giemsa's in buffered water plasmic granules 4% (pH I. serini. 7.2) overnight. Zoites from muscle cyst digests were stained by smearing the sediment on a slide, A similar experiment with sparrows was dif- air-drying, fixing with absolute methanol and ficult to interpret because they were naturally staining with 2% aqueous Giemsa's (pH 7.2) for infected with Isospora. Stages of the latter in 45 min. For tissues were fixed in buff- sections, mononuclear were difficult to dis- ered formalin, cut 4-5 um thick and stained with phagocytes hematoxylinand eosin (HE). tinguish from intracellular sporozoites of Sar- Measurements were by ocular micrometer at cocystis but some extracellular sporozoites 1,000 X of Giemsa-stained parasites and at 100 X could be identified in intestinal smears by their of in muscle between slides. All cysts compressed similarity to those seen in canaries. measurements are in ,um unless otherwise speci- fied. One of the ducklings given 106 sporocysts of cowbird origin and one given 106 sporocysts RESULTS of grackle origin were killed 20 hr PI. A single sporozoite was seen after examination of Early asexual stages smears taken from five equally spaced seg- For information on early phases of infection, ments of the small intestine from the duck- e.g. excystation, two canaries were each given lings. two doses of 106 sporocysts; one was given The majority of the birds were scheduled to sporocysts of cowbird origin and the other be held for 10-20 weeks at which time it was 684 THE JOURNAL OF PARASITOLOGY,VOL. 64, NO. 4, AUGUST 1978

.. L BOX AND DUSZYNSKI-EXPERIMENTALTRANSMISSION OF AVIAN SARCOCYSTIS 685 anticipated that muscle stages would be pres- TABLE II. Muscle parasites in passerines given ent if development time was similar to that of Sarcocystis sporocysts collected from opossums previously fed either cowbirds or grackles. other Sarcocystis species. However, three of six canaries given 200 X 103 sporocysts be- Muscles at necropsy came moribund and were killed on days 7-12 Positive/total no. birds PI. One of the canaries had been inoculated Opossum sporocyst Recipient Days after Abdom- with sporocysts of grackle origin and two had origin bird infection Gross inal Digest been inoculated with sporocysts of cowbird Grackle Sparrow 84-136 4/6 5/6 6/6 origin (Table I). Breathing was labored, the Canary 95-142 0/3 0/3* 3/3 Duckling 58-122 0/3 0/3 0/3 lungs were grey and consolidated and the spleens were enlarged. In sections, lung cap- Cowbird Sparrow 105-127 4/4 4/4 4/4 illaries were found outlined the schizonts Canary 120 0/1 0/1 1/1 by Duckling 58-75 0/3 0/3 0/3 (Fig. 3) and extracellular merozoites and schiz- Controlt Canary 0 0/2 0/2 0/2 onts were found in lung smears (Fig. 4). * Merozoites in smears were 1.9 X 5.5 (1.5-2 Muscle cysts found in leg muscle of 1 bird. x n = mero- t Six sparrows died prior to 3 mo after infection; all were 4-7, 11). Occasionally, single grossly negative for muscle cysts. zoites were in mononuclear phagocytes in the lung (Fig. 5). Four of 12 100-500 X 103 sparrows given Muscle stages sporocysts died on days 11-16 PI. Two sparrows had received sporocysts originating from Sparrows dying before 10 weeks PI were grackles and the other two had received cow- examined grossly for muscle cysts; all were bird origin sporocysts (Table I). They were negative. Surviving canaries and sparrows were not observed to be sick, but a certain propor- necropsied from 12 to 20 weeks PI and all tion of these wild birds often die of stress, were found by the digestion method to be coccidiosis, etc. after being confined in cages. infected (Table II). Most of the sparrows had However, asexual stages, similar to those found heavy infections; cysts were visible on gross in canaries, were seen in smears and sections. inspection and numerous cysts were seen in Most schizonts and merozoites were found in each microscopic field of the abdominal mus- the lungs; an occasional extracellular mero- culature preparations (Fig. 8). Canaries, on zoite was found in spleen and liver. In addi- the other hand, would have been evaluated as tion, several schizonts were found in endo- negative without the use of the digestion thelial cells in the brain smear from a sparrow technique. No cysts could be seen grossly and which died 11 days PI (Fig. 6), and two none was found in the abdominal muscle. schizonts with apparently different-sized mero- After finding canaries positive by digestion, an zoites were seen in the lung of a sparrow extra effort was made to find muscle cysts and necropsied 14 days PI (Fig. 7). Sizes of mero- one cyst was discovered microscopically by zoites in smears were 2 X 4-8 (mean = 2 x 6, teasing apart leg muscles of a canary necrop- n = 14). No parasites were found in smears of sied on day 119 (Fig. 9). Sections of leg lungs, liver, and spleen of two ducks killed 2 muscle from this same bird contained two weeks PI. additional cysts. No muscle parasites were

FIGURES 1-7. Precystic stages of avian Sarcocystis. X 2,250. Figures 1, 2, 4, 5, 6, Giemsa-stained, Figs. 3, 7, HE-stained. 1. Sporozoites in canary intestine smear, 24 hr postinoculation (PI). 2. Sporozoite in mononuclear phagocyte of canary lung smear, 24 hr PI. 3. Schizont in canary lung section, 11 days PI. 4. Schizont in canary lung smear, 11 days PI. 5. Merozoite in mononuclear phagocyte, canary lung smear, 11 days PI. 6. Sparrow brain smear showing schizont in capillary, 11 days PI. 7. Schizonts with two sizes of merozoites in sparrow lung section, 14 days PI. FIGURES 8-12. Cystic stages of avian Sarcocystis. 8. Abdominal wall of sparrow, 110 days PI. X 53. 9. Canary leg dissection, 119 days PI. X 211. 10. Metrocyte and zoites in endodyogeny from digest of sparrow, 84 days PI. HE X 2,250. 11. Cyst wall showing villi, canary, 119 days PI. X 2,250. 12. Cyst wall showing villi, cowbird, natural infection. HE X 2,250. 686 THEJOURNAL OF PARASITOLOGY,VOL. 64, NO. 4, AUGUST1978

TABLE III. Comparative size of Sarcocystis in muscle of sparrowsand canaries infected with sporocysts from opossums.

Cowbird origin Grackle origin Mean size (range) /um Mean size (range) /um Muscle cyst: Sparrow* 102 X 716 (50-210 X 330-1,350) n = 40 84 X 1,037 (50-120 X 750-1,680) n = 24 Canary none found 50 X 500, n = 1 Zoites: Sparrow 2.1 X 6.4 (2-3 X 5-7) n = 40 2.1 X 6.5 (1.5-3 X 5.5-8) n = 60 Canary 2.2 X 6.3 (1.5-3 X 5-7.5) n = 10 2.1 X 6.6 (1.5-3 X 5-8) n = 30 * Age of infection = 105-127 days for sparrows infected with sporocysts of cowbird origin and 84-136 days for sparrows infected with sporocysts of grackle origin. found in six ducks given sporocysts of the include cattle to sheep and sheep to cattle via same origin and necropsied 8-17 weeks PI or dogs and cats (Gestrich et al., 1975; Rickard in two control uninfected canaries. and Munday, 1976), mule deer to cattle and No difference was observed between the in- sheep via dogs and coyotes (Hudkins and fections initiated by sporocysts of cowbird or Kistner, 1977), mouse to rat, guinea pig and grackle origin. Abdominal muscle cysts were hamster via the cat (Ruiz and Frenkel, 1976), measured and the sizes are recorded in Table mouse to rat via the owl (Munday, 1977) and III. Although the muscle cysts originating cottontail to domestic rabbit via the cat from sporocysts of cowbird origin were some- (Fayer and Kradel, 1977). A broader inter- what wider and shorter on the average than mediate host range is suggested by reports of those resulting from the grackle origin sporo- muscle cysts in humans because it seems un- cysts, the size ranges overlap. Size of zoites likely that humans serve as food for predators from Giemsa-staineddigest was almost identi- often enough to perpetuate the cycle. cal from either source of infecting sporocysts Our experiments clearly show that sporo- and from either the canary or the sparrowserv- cysts orginatingfrom one family of avian hosts ing as intermediate host (Table III). Zoite (Icteridae) excyst in the gut, undergo asexual size also was comparable to those recorded schizogony in lung endothelium and eventually from the donor intermediate hosts (Box and develop into muscle cysts in experimentalhosts Duszynski, 1977; Duszynski and Box, 1978). of two different avian families (Fringillidae Metrocytes and forms undergoing endodyo- and Ploceidae) within the Order Passeri- geny were found in digest smears with zoites formes. However, although sporocysts origi- (Fig. 10). Cyst walls were similar in donor nating from icterid intermediatehosts excysted and recipient intermediate hosts. In HE in the intestines of ducks (Order Anseri- stained sections, they were from 1-2 [umthick formes), the parasite did not develop to the with short villi (Fig. 11). Cysts from donor muscle stage in these hosts. birds usually were larger, presumablybecause The evidence that canaries were infected these cysts were older, and had thinner walls, experimentally with Sarcocystis originating stretched by the multiplying zoites (Fig. 12). from icterid birds appears to be valid. The Compartmentsformed by septa were present canaries had been reared indoors with no in the cysts in both donor and recipient hosts. predator contact. Furthermore, successive stages of developing Sarcocystiswere found in DISCUSSION the experimentallyinfected birds, first in the Although a few attempts have been made to lung endothelium and later in characteristic transmit Sarcocystisfrom one species of inter- muscle cysts. Although early stages of Sarco- mediate host to another by sporocysts from a cystis resemble asexual stages of I. serini, the definitive host, none has been reported to in- latter species does not form schizonts in endo- fect more than a single genus. Examples of thelium as seen in sections of Sarcocystis- some unsuccessful cross-transmissionattempts infected birds. The possibility that coccidia BOXAND DUSZYNSKI-EXPERIMENTALTRANSMISSION OF AVIANSARCOCYSTIS 687

other than Sarcocystiswere in the digests was may show that S. falcatula (Stiles 1893) from considered but ruled out for several reasons: Pheucticus ludovicianus (rose-breasted gros- (1) stages of Isosporawere not found in touch beak) is the same species as that in our ex- smears of tissues likely to contain them such periments and thus has priority. Zoites of as duodenal loop mucosa and lung; (2) eight S. falcatula are described as 2 X 5-6; this of the nine infected canaries had been reared bird is a fringillid, migratoryin Texas and thus coccidia-free in the laboratory and had been could be exposed to infections common to the repeatedly negative for fecal oocysts; (3) no cowbirds and grackles used to infect opossums zoites were found in digests of two canaries in our experiments. not given sporocysts; and (4) fully formed Canaries were less susceptible than spar- cysts were found in the leg muscles of one rows to muscle infection with Sarcocystis. experimentallyinfected canary. They also seemed more susceptible to pathol- It is possible that some of the sparrowswere ogy resulting from early asexual schizogony infected before capture, but the incidence of than the sparrows. Possibly a host reaction to Sarcocystis in sparrows in this area is ex- early schizogony destroyed many of the forms tremely low. In a 1967 survey (unpublished which would have developed into muscle cysts data by EDB) only one of 120 adult sparrows in canaries. One wonders if species of Sarco- trapped on the Medical Branch campus was cystis may evolve in abnormal hosts by light found infected. In addition, none of the six infections such as this which gradually adapt sparrows that died before 12 weeks PI had to a given predator-preyrelationship when it visible muscle cysts, whereas eight of the ten is sustained over a period of time. This would experimentally infected birds necropsied 12 help explain the apparently numerous species weeks or more PI had visible muscle cysts and of Sarcocystiswhich seem to be quite specific all had zoites in muscle digest. These findings for a given intermediatehost. support our contention that such muscle cysts The digestion technique proved to be very resulted from our experimental inoculations useful in finding light infections. Although with sporocysts produced in opossums that ate cysts could be found in one canary by exten- cowbirdsor grackles. sive examination (microdissection and histo- Our transmissionstudies suggest that Sarco- logic sections), other canaries subjected to the cystis of cowbirds and grackles and probably same type of examinationwere negative except Sarcocystis found in many passerines are the by digestion. Staining of the digest concen- same species. In our earlier paper (Duszynski trate with Giemsa's provides a permanent and Box, 1978) we used the species name S. record of stages in the muscle cysts. Contrary dubonei for the parasite from icterid birds of to our expectations, cyst stages apparently do the genera Molothrus, Cassidix, and Quiscalus not have to be infectious to be resistant to on the basis of a description by Vogelsang in digestion since metrocytes were found in these 1929 of a Sarcocystisfrom another member of preparations. the cowbird genus, M. bonarensis. However, the size of zoites of S. corderoi from Passer LITERATURECITED domesticus, given in the same paper fits the Box, E. D. 1977. Life cycles of two Isospora size of zoites in our experimental infections species in the canary,Serinus canarius Lin- better. Zoites of S. corderoi were 3-6 x 1-2 naeus. J Protozool24: 57-67. in size with 8-9 x 3-4 of S. dubonei , AND D. W. DUSZYNSKI. 1977. Survey compared for in the brownheaded from M. bonarensis. is assumed that Sarcocystis cowbird (It Vogel- (Molothrusater): A comparisonof macro- sang misplaced the decimal point in describing scopic, microscopicand digestiontechniques. the zoites as 0.03-0.06 X 0.01-0.02 mm for J Wildl Dis 13: 356-358. S. corderoi and as 0.08-0.09 X 0.03-0.04 mm , AND T. B. MCGUINNESS. 1978. Sarco- in beef fromretail for S. dubonei.) The size range of S. corderoi cystis outletsdemonstrated by digestiontechnique. J Parasitol64: 161- zoites fits that of zoites from both natural and 162. experimentalhosts in our transmissionstudies; CERNA, 2. 1976. Relationship of oocysts of furthermore, sparrows were susceptible to "Isosporabuteonis" from the barn owl (Tyto to muscle of sporocysts from an opossum which had eaten alba) cysts sarcosporidiansfrom the house mouse (Mus musculus). Fol Para- a cowbird. Future transmission experiments sitol 22: 285. 688 THEJOURNAL OF PARASITOLOGY,VOL. 64, NO. 4, AUGUST1978

CRUM, J. M., AND A. K. PRESTWOOD. 1977. LEVINE, N. D. 1977. Nomenclature of Sarco- Transmission of Sarcocystis leporum from a cystis in the ox and sheep and of fecal coc- cottontail rabbit to domestic cats. J Wildl cidiosis of the dog and cat. J Parasitol 63: Dis 13: 174-175. 36-51. DUSZYNSKI, D. W., AND E. D. Box. 1978. The MUNDAY, B. L. 1977. A species of Sarcocystis opossum (Didelphis virginiana) as a host for using owls as definitive hosts. J Wildl Dis Sarcocystis debonei from cowbirds (Molo- 13: 205-207. thrus ater) and grackles (Cassidix mexicanus, RICKARD,M. D., AND B. L. MUNDAY. 1976. Host Quiscalus quiscula). J Parasitol 64: 326-329. specificity of Sarcocystis spp. in sheep and FAYER, D., AND D. KRADEL. 1977. Sarcocystis cattle. Aust Vet J 52: 48. leporum in cottontail rabbits and its trans- Rurz, A., AND J. K. FRENKEL. 1976. Recognition mission to carnivores. J Wildl Dis 13: 170- of cyclic transmissionof Sarcocystis muris by 173. cats. J Infect Dis 133: 409-418. GESTRICH,R., A. O. HEYDORN, AND N. BAYSU. RZEPCZYK, C. M. 1974. Evidence of a rat-snake 1975. Beitrige zum Lebenszyklus der Sarko- life cycle for Sarcocystis. Int J Parasitol 4: sporidien. VI. Untersuchungen zur Artendif- 447-449. ferenzierung bei Sarcocystis fusiformis und STILES, C. W. 1893. On the presence of Sarco- Sarcocystis tenella. Berl Miinch Tierartzl sporidia in birds. U.S.D.A. B.A.I. Bull. No. Wochenschr 88: 191-197. 3, p. 79-88. HUDKINS,G., AND T. P. KISTNER. 1977. Sarco- VOGELSANG,E. G. 1929. Beitrage zur Kenntnis cystis hemionilatrantis (sp. n.) life cycle in der Parasitenfauna Uruguays. Sarkosporidien mule deer and coyotes. J Wildl Dis 13: bei Vigeln. Z Bakt Parasitenkd I Orig 113: 80-84. 206-208.

ANNOUNCEMENT ...

i. Parasitol., 64(4), 1978, p. 688 ? American Society of Parasitologists 1978

Ernest Bueding Honored

On 12 April 1978, Dr. Ernest Bueding received the first Theodore Weicker Memorial Award in Pharmacology and Experimental Therapeutics at the annual meeting of the American Society for Pharmacology and Experimental Therapeutics in Atlantic City, New Jersey. The honor, $10,000 and a certificate, is given to an active investigator who has made sustained dis- tinguished contributions to pharmacology.