Cysticercosis in Laboratory Rabbits

JAMES R. OWINY, BVM, PHD

There are no data on the current incidence of Taenia pisiformis in laboratory rabbits. Two cases of cysticercosis most likely due to T. pisiformis in laboratory rabbits (intermediate host) are presented. Both rabbits had no contact with dogs (final host); their caretakers did not work with dogs, and these caretakers changed into facility scrubs and wore gloves when working with the rabbits. Rabbit 1 may have been infected after being fed hay at our facility. In light of the life cycle of the parasite and the history of rabbit 2, it potentially could have been infected prior to arrival at our facility. There have been only three cases of tapeworm cysts in rabbits in our facility (average daily census, 250) during the last 10 years (incidence, Ͻ 1%). This report indicates that although cysticercosis is rare in laboratory rabbits, one should always be aware of such incidental findings. Although it may not produce overt illness in the rabbit, hepatic migration could adversely affect the outcome of some experimental procedures.

Two adult, female, Dutch Belted rabbits (Oryctolagus cuniculi) used in a toxicology study approved by the institutional animal use and care committee were submitted by an investigator for necropsy. Both rabbits were purchased from a commercial breeder (Myrtle’s Rabbitry, Thompson Station, Tenn.) and had been in our facility for 3 months. Vendor health records indi- cated freedom from Pasteurella multocida, Treponema cuniculi, CAR bacillus, Clostridium piliforme, Encephalitozoon cuniculi, Eimeria stiedae, Psoroptes cuniculi, Cheyletiella parasitovorax, Listrophorus gibbus, Passalurus ambiguus, and Taenia pisiformis. The rabbits were housed conventionally in banks of stainless-steel cages with drop pans containing autoclaved aspen chips (Northeastern Products, Warrensburg, N. Y.) in an AAALAC-accredited facility. Drop pans were changed twice a week, and cages were changed every 2 weeks. Temperature was maintained at 18 to 20ЊC, with approximately 40% relative humidity and 10-12 air changes per hour. A 14:10 h (light: dark) cycle was used. Rabbits were fed a breeder diet (Prolab breeder rabbit, 5P29, PMI Nutrition International, Inc., Brentwood, Mo.). Water was supplied in bottles to allow monitoring of daily water intake. Rabbit 1 was 20 days pregnant when it was presented for Figure 1. Cysticercus of Taenia pisiformis retrieved from the peritoneal necropsy. The rabbit had gone off feed 2 days earlier but report- cavity of a rabbit. Arrow (➞) indicates location of scolex (white spot). edly was drinking normally and was well hydrated. The rabbit Bar = 5 mm. was given grass hay from a local farm supply store to stimulate its appetite. On the morning of the third day after going off feed, rump) in length, were present in the uterus. The heart, lungs, and she became depressed, with a temperature of 37.1ЊC. A small kidneys were within normal limits. Several oval cysts (diameter, amount of reddish brown vaginal discharge was noticed. The approximately 1 cm) were found either attached to the omentum rabbit died shortly thereafter and was presented for necropsy. or floating free in the peritoneal cavity (Fig. 1). Each cyst con- Upon arrival at our facility 3 months earlier, she had developed tained clear fluid and a single white spot (diameter, approximately anorexia but recovered uneventfully after being fed hay. 1 to 3 mm). Selected tissues were fixed in 10% formalin (Fisher Rabbit 2 had a decreased appetite for several days and was Scientific, Fairlawn, N. J.), embedded in paraffin, sectioned at given hay and oral fluids plus nutritional supplement (Nutri- 5 ␮m, and stained with hematoxylin and eosin. Cal, Evsco Pharmaceuticals, Buena, N. J.). The rabbit delivered Microscopic examination of the mesentery revealed several fluid- a live pup at 29 days of gestation, but the adult was found dead filled cysts surrounded by fibrous connective tissue with abundant the next morning. heterophils and mononuclear inflammatory cells. The cysts had profiles of immature cestode larvae containing an invaginated Pathologic findings scolex armed with hooks and suckers but no ova (Fig. 2). Each larva had a thick cuticle, which covered a segmented At necropsy, rabbit 1 was in fairly good body condition. Approxi- parencymatous body containing numerous calcenous corpuscles mately 50 ml of straw-colored fluid was present in the peritoneal (Fig. 3)—features consistent with a cestode larva (1). The liver cavity. The liver was firm and contained scattered yellowish, had multifocal areas of fibrosis in portal triads (Fig. 4). These foci shrunken areas (diameter, 2 to 5 cm) interspersed with promi- contained numerous mononuclear inflammatory cells and, in some nent white streaks (width, 2 to 4 mm). There was very little ingesta cases, macrophages with brown pigment. There were few tracts in the intestine. Eight pups, each approximately 4 cm (crown-to- containing mononuclear inflammatory cells, with minimal fibrosis and loss of hepatocytes adjacent to these well-delineated fibrotic Laboratory Animal Resources, Painter Center, Colorado State University, Fort Collins, Colo- foci. The liver lesions were attributed to migration of cestode lar- rado 80523-2007 Address correspondence to: James R. Owiny, Johns Hopkins University School of Medi- vae. There was no necrosis or fatty change in the liver. None of the cine, 459 Ross Research Building, 720 Rutland Avenue, Baltimore, Maryland 21205 liver lesions were thought severe enough to cause death. The pla-

Volume 40, No. 2 / March 2001 CONTEMPORARY TOPICS © 2001 by the American Association for Laboratory Animal Science 45 Analysis of the peritoneal fluid revealed a hazy, yellow-colored transudate with 2.3 g/dl protein and 100 cells/␮l. The cytocentrifuge preparation contained 2% heterophils, 78% lymphocytes, and 20% large mononuclear cells. Lymphocytes were predominantly small, with only a few intermediate-sized ones. A few lymphoid cells were plasmacytoid in appearance, and the large mononuclear cells were vacuolated. No microorganisms were present, and aerobic culture of the fluid yielded no growth. The underlying cause of the fluid effusion was not apparent. Rabbit 2 was in very good body condition. She had a bloody vaginal discharge and a smearing of blood on the front paws and muzzle. The liver was pale, soft, and friable, with a prominent reticular pattern. The stomach contained a large hairball. Most of the gastrointestinal tract was empty except for a few fecal pellets. The bladder also was empty. The uterus contained seven full-term pups. The lungs were dark and oozed blood from the cut surface. Aerobic culture of the lungs yielded no growth. Figure 2. Section through a cysticercus cyst showing scolex (➞) and cyst Five ovoid cysts (diameter, approximately 1 cm) containing a wall (➤). Bar = 1 mm. prominent white spot were attached to the abdominal fat. One cyst was floating free in the abdominal cavity. Cysts were filled with a clear fluid and resembled those found in rabbit 1. Histologically, the cysts from rabbit 2 resembled those from rabbit 1. There was marked midzonal fatty change in the liver, moderate congestion and multifocal edema of the lungs, marked lymphoid atrophy of the spleen accompanied by mildly increased heterophils, and multifocal subacute inflammation of the kidney. The uterus and placenta had large areas of necrosis and marked congestion without inflammation. Diagnoses of mesenteric cestodiasis in a probable intermediate host, marked fatty liver, pulmonary congestion, and splenic atrophy with mild acute splenitis were made. The cause of death most likely was preg- nancy toxemia, as suggested by presence of a fatty liver.

Discussion Both animals were diagnosed as having mesenteric cestodiasis consistent with the larval stage (cysticercus) of T. pisiformis, in light of the appearance and location of the cysts in the rabbits. Figure 3. Scolex and part of the cyst wall (➤) containing mixed inflam- The cysts appear as transparent spheres about 10 mm in diam- matory cells. A thick segmented cuticle (➞), hooks (➤), and eter that contain a characteristic scolex, which is seen as a white parenchymatous body (asterisk) are evident. Bar = 100 ␮m. spot. In addition, white raised 1- to 3-mm foci or 2- to 3-mm linear lesions as well as fibrin deposits may be seen on the hepatic surface (2-4) and are usually attributed to parasite migration. The foci or linear lesions are solitary and coalescing granulomas with caseous cores containing monocytes, giant cells, and eosinophils surrounded by a layer of mixed inflammatory cells and fibroblasts. Some granulomas may contain parasites. In chronic cases, the lesions undergo fibrosis and mineraliza- tion (2,3). The liver lesions we saw were mild but still consistent with larval migratory tracks. T. serialis (Mulitceps serialis) forms a coenurus in the inter- muscular connective tissue, commonly in the flank and occasionally in the abdomen or brain. The coenurus is a fluid- filled bladderworm (diameter, 4 to 5 cm) with many scolices, each of which is on a separate stalk that invaginates into a common bladder. Echinococcus granulosus forms a similar coenurus (hydatid cyst) and can occur in the abdominal cavity of the rabbit. Both of these organisms are easy to differentiate from a cysticercus. E. cuniculi can cause granulomatous and fibrosing hepatitis; however, it does not produce cysts, nor does it histo- Figure 4. Section of a liver showing fibrosis (➤), loss of hepatocytes, and logically resemble a cysticercus. mixed cellular infiltrate. Bar = 100 ␮m. Serologic and skin tests to detect rabbit cysticercosis have been developed (5,6) but are not commercially available. Because of centa appeared normal, and although there was some autolysis, the difficulty of ante-mortem diagnosis, treatment usually is not there was no evidence of necrosis or inflammation. There were no attempted; however, mebendazole at 1 g/ kg of feed (approxi- lesions in the kidneys. A diagnosis of mesenteric cestodiasis in a mately 50 mg/kg body weight daily) for 14 days has been reported probable intermediate host was made. to kill T. pisiformis cysticerci (7).

46 CONTEMPORARY TOPICS © 2001 by the American Association for Laboratory Animal Science Volume 40, No. 2 / March 2001 T. pisiformis usually does not produce overt disease in rabbits, ing with rabbits. All the pelleted feed and bedding was sup- although massive infections can result in extensive liver dam- plied by a reputable commercial vendor. Hay was obtained from age, abdominal distension, lethargy, weight loss, and death (8, a local farm supplier. We can not rule out contamination of 9). Bowman et al. (10) reported that 5 of 10 Flemish Giant rab- the hay prior to delivery to our facility. Rabbit 1 was fed hay 3 bits infected with 500 oncospheres died 17 to 22 days months before and just prior to death. It is possible that this postinfection. Necropsies were not performed on the rabbits, so animal could have been infected from the first hay feeding if it it is difficult to determine the cause of death. In contrast, none contained cestode eggs. Rabbit 2 was fed hay in our facility only of 4 (6) or 60 (7) New Zealand White rabbits died or developed during last 2 days of its life. Because it takes 15 to 30 days for severe lesions after administration of 2000 oncospheres. These the larva to reach the surface of the liver and pass into the peri- findings may reflect potential breed differences in the response toneal cavity (3, 18), this rabbit was not infected by eating to infection or differences in the virulence of the parasite. In contaminated hay at our facility. the Dutch Belted rabbits we examined, the liver lesions were A review of the medical records of our rabbit colony during mild and of chronic fibrosing nature, suggesting a chronic, low the last 10 years produced one other case of tapeworm cysts, parasite burden. which were identified as larval stages of T. serialis. This incident We could not determine the reason for the accumulation of occurred 5 years prior to the writing of this manuscript, in a 6- fluid in the peritoneal cavity in rabbit 1. LaPage (9) reported month-old New Zealand White rabbit obtained from a that abdominal distension, presumably due to accumulation of nontraditional vendor. This rabbit was fed a diet consisting of fluid in the peritoneal cavity, can occur during massive infec- 70% alfalfa in a field prior to arrival in our facility, and this tion with T. pisiformis in the rabbit. Flatt and Moses (3) found 3 practice was thought to be the source of contamination. Inter- to 10 ml of clear fluid in the peritoneal cavity of some of their estingly, this rabbit also had peritoneal effusion (35 cc) and infected rabbits. This amount is far less than the 50 ml we col- hydrothorax (10 cc). Considering that we have had three cases lected. Because the parasite burden was low, the rabbit may have of cysticercosis (including the subjects of this paper) during had underlying cardiac or chronic hepatic disease that was not the last 10 years, this frequency would translate to a less than diagnosed. We did not see any gross or histopathologic cardiac, 1% incidence in our facility, which has an average daily census hepatic, or kidney lesions in this rabbit to explain the effusion. of about 250 rabbits. We do not have clinical chemistry results and can not determine This report indicates that although the prevalence of cysticer- whether the animal was hypoproteinemic. cosis is low in laboratory rabbits, this disease occasionally can T. pisiformis has a worldwide distribution (4, 9, 11-16). Adults occur. Furthermore, liver lesions secondary to parasite migra- occur in the small intestine of carnivores (definitive hosts). The tion can confound studies of liver function, toxicology, or worms grow, mature, and pass gravid proglottids and embryo- pharmacology and others. To limit the possibility of exposure to nated eggs (hexacanth embryos or oncospheres) into the feces viable cestode eggs, unprocessed feeds should be screened and by 7 weeks postinfection (10). Oncospheres are dark brown, rejected or treated. round to ellipsoidal, and measure 34 to 41 mm by 29 to 35 mm (9). Intermediate hosts are infected after ingesting feed or bed- Acknowledgments ding that is contaminated by feces of the definitive host. Dogs I thank Ms. Catherine Hyden for taking care of the rabbits and pro- often harbor these tapeworms and provide potential sources of viding all the medical and feeding history. I thank Ms. Elisa French for infection for rabbits (15) by fecal contamination of rabbit feed, her technical assistance. particularly grass (17). In a suitable intermediate host, the oncospheres hatch in the small intestine, and the embryos mi- grate to the liver, reach the surface of the liver after about 15 References days, and pass into the peritoneal cavity (3, 18). The larval stages 1. Chitwood, M., and J. R. Lichtenfels. 1972. Identification of para- (Cysticercus pisiformis) are found in the liver and peritoneal cavity sitic metazoa in tissue sections. Exp. Parasitol. 32:407-519. 2. Flatt, R. E., and W. W. Campbell. 1974. Cysticercosis in rabbits: of lagomorphs and rodents worldwide (4, 9, 11-15). Transmis- incidences and lesions of the naturally occurring disease in young sion to the definitive host is by ingestion of the viscera of an domestic rabbits. Lab. Anim. Sci. 24:914-918. intermediate host. 3. Flatt, R. E., and R. W. Moses. 1975. Lesions of experimental cys- Among wild rabbits and hares, the incidence of cysticercosis ticercosis in domestic rabbits. Lab. Anim. Sci. 25:162-167. due to T. pisiformis has been reported to be 3 to 93% (4, 12, 13, 4. Lepitzki, D. A. W., A. Woolf, and B. M. Bunn. 1992. Parasites of 16), depending on age, intermediate and definitive host inter- cottontail rabbits of southern Illinois. J. Parasitol. 78:1080-1083. actions, and environmental factors (4, 11, 13-15). In domestic 5. Rickard, M. D., and P. M. Outteridge. 1974. Antibody and cell- rabbits raised for meat production, Flatt and Campbell (2) found mediated immunity in rabbits infected with larval stages of Taenia pisiformis. Zeitschrift fur Parasitenkunde 44:187-201. that 7% of more than 17,000 livers examined carried the para- 6. Craig, P.S. 1984. Circulating antigens, antibodies and immune com- site. A study in 1967 found tapeworm cysts in rabbits from 5 of plexes in experimental Taenia piriformis infections of rabbits. 10 commercial rabbit suppliers (19). Unfortunately, the tape- Parasitology 89:121-131. worm cysts, which were present in 14 of the 100 rabbits evaluated, 7. Heath, D. D., M. J. Christie, and R. A. Chevis. 1975. The lethal were not speciated. There are no data on the current incidence effect of mebendazole on secondary Echinococus granulosus, cysticerci of T. pisiformis in laboratory rabbits. of Taenia pisiformis and tetrathyridea of Mesocestoides corti. Parasitol- Cysticercosis should not be a problem in the laboratory set- ogy 70:273-285. ting in which current husbandry practices such as thorough 8. Bundesen, P. G., and P. A. Janssens. 1971. Biochemical tracing of parasitic infections. III. Taenia pisiformis in rabbits—a quantitative and frequent sanitation, protective clothing, proper feed and study. Int. J. Parasitol. 1:15-20. bedding storage, and separation of species make it extremely 9. Lapage, G. 1968. Class Cestoda (tapeworms), p. 370-443. In G. difficult for the tapeworm to complete its life cycle. These pro- Lapage, Veterinary parasitology, 2nd ed. Charles C. Thomas, Spring- cedures eliminate contact between dogs or dog feces and field, Ill. rabbits, rabbit feed, and bedding. It is interesting to speculate 10. Bowman, D. D., D. S. Lin, R. C. Johnson, et al. 1991. Effects of upon the possible sources of infection for rabbits 1 and 2. Rab- nitroscanate on adult Taenia pisiformis in dogs with experimentally bits used in this study were not housed in the same facility as induced infections. Am. J. Vet. Res. 52:1542-1544. were dogs or cats. The caretakers did not work with dogs, and these people wore personal protective equipment when work-

Volume 40, No. 2 / March 2001 CONTEMPORARY TOPICS © 2001 by the American Association for Laboratory Animal Science 47 11. Andrews, C. L., and W. R. Davidson. 1980. Endoparasites of se- 16. Keith, L. B., J. R. Cary, T. M. Yuill, et al. 1985. Prevalence of helm- lected populations of cottontail rabbits (Sylvilagus floridanus) in the inths in a cyclic snowshoe hare population. J. Wildlife Dis. southeastern United States. J. Wildlife Dis. 16:395-401. 21:233-253. 12. Boag, B. 1985. The incidence of helminth parasites from the wild 17. Shanks, P. L. 1962. Common diseases in rabbits, p. 49-53. In G. rabbits, Oryctolagus cuniculus (L.), in Eastern Scotland. J. Helminthol. Porter and W. Lane-Petter (ed.), Notes for breeders of common 59:61-69. laboratory animals. Academic Press, New York. 13. Boggs, J. F., S. T. McMurry., D. M. Jr. Leslie, et al. 1990. Influence 18. Shield, J. M., D. D. Heath, and J. D. Smyth. 1973. Light microscope of habitat modification on the intestinal helminth community ecol- studies of the early development of Taenia pisiformis cysticerci. Int. ogy of cottontail rabbit populations. J. Wildlife Dis. 26:157-169. J. Parasitol. 3:471-480. 14. Lawson, J. R., and M. A. Gemmel. 1990. Transmission of taeniid 19. Poole, C. M., W. G. Keenan, D. V. Tolle, et al. 1967. Disease status tapeworm eggs via blowflies to intermediate hosts. Parasitology of commercially produced rabbits, p. 219-220. In Biological and 100:143-146. medical research division annual report 1967. ANL-7409. Argonne 15. Rashed, R. M., P. J. Whitfield, and J. W. Lewis. 1991. The epidemi- National Laboratory, Argonne, Illinois E.18ANL-7409XAB. ology of Taenia pisiformis infections in domestic dogs in Cairo. J. Egypt. Soc. Parasitol. 21:597-610.