Sarcocystis leporum in cottontail rabbits and its transmission to carnivores.

Muscle from Sarcocystis-infected cottontail rabbits (Sylvilagus floridanus) was fed to coccidia-free cats (Felis domestica) and dogs (Canis familiaris). Only cats became infected and shed sporocysts in their feces. The prepatent period ranged from 10 to 25 days and the patent period from 3 to 46 days. Sporocysts were fully sporulated when shed. They contained 4 sporozoites and a coarse granular residuum and averaged 9.4 by 13.6 micron (N=55). Doses of 200-75,000 sporocysts were orally administered to 5 domestic rabbits (Oryctolagus cuniculus). Domestic rabbits did not become infected, suggesting a strict host specificity for the intermediate host S. floridanus.     

Prevalence of sarcocystis species sporocysts in wild-caught opossums (Didelphis virginiana)

Sarcocystis sporocysts were found in intestinal scrapings from 24 (54.5%) of 44 opossums (Didelphis virginiana). The number of sporocysts varied from a few (< 10,000) to 245 million. Sporocysts from 23 of 24 opossums were fed to captive budgerigars (Melopsittacus undulatas), and the inocula from 21 opossums were infective, indicating the presence of Sarcocystis falcatula. Sporocysts from 24 opossums were fed to gamma-interferon-knockout (KO) or nude mice; inocula from 14 opossums were infective to mice. Sarcocystis neurona was detected in tissues of KO mice by specific staining with anti-S. neurona antibodies, and the parasite was cultured in vitro from the brains of KO mice fed sporocysts from 8 opossums. Sarcocystis speeri was identified by specific staining with anti-S. speeri antibodies in tissues of KO mice fed inocula from 8 opossums; 3 opossums had mixed S. neurona and S. speeri infections. Thus, the prevalences of sporocysts of different species of Sarcocystis in opossums were: S. falcatula 21 of 44 (47.7%), S. neurona 8 of 44 (18.1%), and S. speeri 8 of 44 (18.1%) opossums. Sarcocystis neurona alone was found in 1 opossum, and S. speeri alone was found in 1 opossum. Mixed Sarcocystis infections were present in 21 opossums.     

Isolation of Sarcocystis speeri Dubey and Lindsay, 1999 parasite from the South American opossum (Didelphis albiventris) from Argentina

Sarcocystis sporocysts from the intestines of 2 opossums (Didelphis albiventris) from Argentina were fed to gamma-interferon knockout (KO) and nude mice. Protozoal schizonts were seen in brain, liver, spleen, and adrenal glands of mice examined 33-64 days after feeding sporocysts. Sarcocysts were seen in skeletal muscles of KO mice 34-71 days after feeding sporocysts. Schizonts and sarcocysts were structurally similar to Sarcocystis speeri Dubey and Lindsay, 1999 seen in mice fed sporocysts from the North American opossum Didelphis virginiana from the United States.     

Experimental transmission of Sarcocystis from icterid birds to sparrows and canaries by sporocysts from the opossum.

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 Fringillidae) 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.     

Experimental infection of dogs with Sarcocystis from wapiti.

Ten domestic dogs became infected with Sarcocystis when fed simple portions of heart, esophagus and diaphragm from a two-year-old female wapiti (Cervus canadensis). The prepatent period was 14 days in all exposed dogs; the patent period ranged from 8 to 20 days. Neither the 10 control dogs, nor two dogs fed sporocysts collected from the infected dogs passed sporocysts within the study period. Sporocysts averaged 16.5 by 11.1 micron in size.     

The striped skunk (Mephitis mephitis) is an intermediate host for Sarcocystis neurona

Striped skunks, initially negative for antibodies to Sarcocystis neurona, formed sarcocysts in skeletal muscles after inoculation with S. neurona sporocysts collected from a naturally infected Virginia opossum (Didelphis virginiana). Skunks developed antibodies to S. neurona by immunoblot and muscles containing sarcocysts were fed to laboratory-reared opossums which then shed sporulated Sarcocystis sporocysts in their faeces. Mean dimensions for sporocysts were 11.0 x 7.5 microm and each contained four sporozoites and a residuum. Sarcocysts from skunks and sporocysts from opossums fed infected skunk muscle were identified as S. neurona using PCR and DNA sequence analysis. A 2-month-old, S. neurona-naive pony foal was orally inoculated with 5 x 10(5) sporocysts. Commercial immunoblot for antibodies to S. neurona performed using CSF collected from the inoculated pony was low positive at 4 weeks p.i., positive at 6 weeks p.i., and strong positive at 8 weeks p.i. Gamma-interferon gene knockout mice inoculated with skunk/opossum derived sporocysts developed serum antibodies to S. neurona and clinical neurologic disease. Merozoites of S. neurona present in the lung, cerebrum, and cerebellum of mice were detected by immunohistochemistry using polyclonal antibodies to S. neurona. Based on the results of this study, the striped skunk is an intermediate host of S. neurona.     

Redescription of the sarcocysts of Sarcocystis rileyi (Apicomplexa: Sarcocystidae)

The intermediate hosts for Sarcocystis rileyi (Stiles 1893) Minchin 1913 are ducks (Anas spp.), and the striped skunk (Mephitis mephitis) is its definitive host. The structure of sarcocysts from an experimentally infected shoveler duck (Anas cylpeata) fed sporocysts from an experimentally-infected M. mephitis was studied and compared with type specimens from a naturally infected duck. The experimentally infected duck was killed 154 d after feeding sporocysts. By light microscopy the sarcocyst wall was 3-5 microm thick with indistinct villar protrusions. Ultrastructurally, the sarcocyst wall was a type-23 cyst wall with anastomosing villar protrusions that were up to 7.5 microm long. The villar projections contained filamentous structures. The bradyzoites were 12-14 microm long. Structurally, the sarcocyst from the naturally infected and experimentally infected ducks appeared similar.     

Lack of Sarcocystis neurona antibody response in Virginia opossums (Didelphis virginiana) fed Sarcocystis neurona-infected muscle tissue

Serum was collected from laboratory-reared Virginia opossums (Didelphis virginiana) to determine whether experimentally infected opossums shedding Sarcocystis neurona sporocysts develop serum antibodies to S. neurona merozoite antigens. Three opossums were fed muscles from nine-banded armadillos (Dasypus novemcinctus), and 5 were fed muscles from striped skunks (Mephitis mephitis). Serum was also collected from 26 automobile-killed opossums to determine whether antibodies to S. neurona were present in these opossums. Serum was analyzed using the S. neurona direct agglutination test (SAT). The SAT was modified for use with a filter paper collection system. Antibodies to S. neurona were not detected in any of the serum samples from opossums, indicating that infection in the opossum is localized in the small intestine. Antibodies to S. neurona were detected in filter-paper-processed serum samples from 2 armadillos naturally infected with S. neurona.     

Sarcocystis falcatula of opossums: transmission by cockroaches with fatal pulmonary disease in psittacine birds.

Old World psittacines experienced an acute fatal illness in outdoor breeding collections in South Florida. Toxoplasma-like organisms were found histologically in pulmonary capillaries and elsewhere. Because the organisms underwent schizogony and could not be transmitted to mice, we looked for a cause other than Toxoplasma gondii. An opossum was trapped on the premises of 1 facility and was found to be shedding sporocysts similar to Sarcocystis falcatula in its feces. Cockroaches were prevalent and suspected as transport hosts. Cockroaches that had ingested opossum feces and subsequently were fed to cockatoos induced an identical fatal illness. Obstruction of pulmonary capillaries by developing schizonts and pulmonary edema were the most important pathologic findings. The epidemic was stopped by biological insect control employing flightless chickens to reduce cockroach populations and by an electric fence restricting access of opossums to these outdoor psittacine breeding facilities.     

Are Sarcocystis neurona and Sarcocystis falcatula synonymous? A horse infection challenge

Equine protozoal myeloencephalitis (EPM) is a debilitating neurologic disease of the horse. The causative agent. Sarcocystis neurona, has been suggested to be synonymous with Sarcocystis falcatula, implying a role for birds as intermediate hosts. To test this hypothesis, opossums (Didelphis virginiana) were fed muscles containing S. falcatula sarcocysts from naturally infected brown-headed cowbirds (Molothrus ater). Ten horses were tested extensively to ensure no previous exposure to S. neurona and were quarantined for 14 days, and then 5 of the horses were each administered 10(6) S. falcatula sporocysts collected from laboratory opossums. Over a 12-wk period, 4 challenged horses remained clinically normal and all tests for S. neurona antibody and DNA in serum and cerebrospinal fluid were negative. Rechallenge of the 4 seronegative horses had identical results. Although 1 horse developed EPM, presence of S. neurona antibody prior to challenge strongly indicated that infection occurred before sporocyst administration. Viability of sporocysts was confirmed by observing excystation in equine bile in vitro and by successful infection of naive brown-headed cowbirds. These data suggest that S. falcatula and S. neurona are not synonymous. One defining distinction is the apparent inability of S. falcatula to infect horses, in contrast to S. neurona, which was named when cultured from equine spinal cord.     

Isolation in immunodeficient mice of Sarcocystis neurona from opossum (Didelphis virginiana) faeces, and its differentiation from Sarcocystis falcatula

Sarcocystis neurona was isolated in nude mice and gamma-interferon knockout mice fed sporocysts from faeces of naturally infected opossums (Didelphis virginiana). Mice fed sporocysts became lethargic and developed encephalitis. Protozoa were first found in the brain starting 21 days post-inoculation. Sarcocystis neurona was recovered in cell culture from the homogenate of liver, spleen and brain of a nude mouse 11 days after feeding sporocysts. The protozoa in mouse brain and in cell culture multiplied by schizogony and mature schizonts often had a residual body. Sarcocystis falcatula, which has an avian-opossum cycle, was not infective to nude or knockout mice. Protozoa were not found in tissues of nude mice or knockout mice after subcutaneous injection with culture-derived S. falcatula merozoites and sporocysts from the faeces of opossums presumed to contain only S. falcatula. Results demonstrate that S. neurona is distinct from S. falcatula, and that opossums are hosts for both species.     

Genetically different isolates of Trypanosoma cruzi elicit different infection dynamics in raccoons (Procyon lotor) and Virginia opossums (Didelphis virginiana)

Trypanosoma cruzi is a genetically and biologically diverse species. In the current study we determined T. cruzi infection dynamics in two common North American reservoirs, Virginia opossums (Didelphis virginiana) and raccoons (Procyon lotor). Based on previous molecular and culture data from naturally-exposed animals, we hypothesised that raccoons would have a longer patent period than opossums, and raccoons would be competent reservoirs for both genotypes T. cruzi I (TcI) and TcIIa, while opossums would only serve as hosts for TcI. Individuals (n = 2 or 3) of each species were inoculated with 1 × 10⁶ culture-derived T. cruzi trypomastigotes of TcIIa (North American (NA) – raccoon), TcI (NA – opossum), TcIIb (South American – human), or both TcI and TcIIa. Parasitemias in opossums gradually increased and declined rapidly, whereas parasitemias peaked sooner in raccoons and they maintained relatively high parasitemia for 5 weeks. Raccoons became infected with all three T. cruzi strains, while opossums only became infected with TcI and TcIIb. Although opossums were susceptible to TcIIb, infection dynamics were dramatically different compared with TcI. Opossums inoculated with TcIIb seroconverted, but parasitemia duration was short and only detectable by PCR. In addition, raccoons seroconverted sooner (3–7 days post inoculation) than opossums (10 days post inoculation). These data suggest that infection dynamics of various T. cruzi strains can differ considerably in different wildlife hosts.     

Evaluation of Cruzia americana, Turgida turgida, and Didelphostrongylus hayesi infection in the Virginia opossum (Didelphis virginiana) and risk factors along the California coast

Three nematodes, Turgida turgida, Cruzia americana, and Didelphostrongylus hayesi, have been documented to cause morbidity and mortality in the Virginia opossum (Didelphis virginiana). The present study was designed to determine the frequency of infection of these nematodes in opossums at 2 study sites in California and to determine if there are risk factors associated with shedding of eggs or larvae in the feces. Turgida turgida and C. americana adults were found in 84.4% (stomach; n = 45) and 62.5% (intestinal wash and feces; n = 16) of sampled opossums. Eggs were present in opossum feces (n = 105) less frequently (40% T. turgida and 35.2% C. americana). Didelphostrongylus hayesi larvae were found in 79.0% of opossum feces examined (n = 105). Adult age and wet season (December through April) were significant predictive factors for the presence of T. turgida eggs, whereas the dry season (May through November) was significantly associated with the presence of C. americana eggs in feces. Adult opossums were more likely to have eggs and larvae from all 3 nematodes in the feces.     

Sarcocystis spp. in white-tailed deer. I. Definitive and intermediate host spectrum with a description of Sarcocystis odocoileocanis n. sp

Sporocysts containing four sporozoites and measuring (avg.) 15.2 micrometers X 10.7 micrometers (N = 195) were shed in the feces of dogs (Canis familiaris) 8 to 16 days (avg. 11.6 days) after the first feeding of venison infected with Sarcocystis sp. Sporocysts containing four sporozoites and measuring (avg.) 11.5 micrometers X 8.1 micrometers (N = 35) were shed by a cat (Felis catus) 14 days after ingesting Sarcocystis-infected venison. Statistical (pooled t-test) comparison of the mean measurements of the sporocysts passed by the dog and cat demonstrated a significant difference (P less than .01). The raccoon (Procyon lotor) and opossum (Didelphis virginiana) could not be infected with Sarcocystis from white-tailed deer (Odocoileus virginianus). The name, Sarcocystis odocoileocanis, is proposed for the species transmitted from white-tailed deer to dogs. Sarcocystis odocoileocanis is differentiated from S. hemionilatrantis Hudkins and Kistner, 1977 of mule deer (Odocoileus hemionus), S. ovicanis Heydorn, Gestrich, Mehlhorn and Rommel, 1975 of sheep (Ovis aries) and S. cruzi Hasselmann, 1926 (=S. bovicanis Heydorn, Gestrich, Mehlhorn and Rommel, 1975) of cattle (Bos taurus) because S. odocoileocanis has (1) low infectivity for calves and sheep and (2) apparent insignificant pathogenicity for its intermediate host.     

Experimental transmission of Sarcocystis speeri Dubey and Lindsay, 1999 from the South American opossum (Didelphis albiventris) to the North American opossum (Didelphis virginiana)

Sarcocystis speeri Dubey and Lindsay, 1999 from the South American opossum Didelphis albiventris was successfully transmitted to the North American opossum Didelphis virginiana. Sporocysts from a naturally infected D. albiventris from Argentina were fed to 2 gamma-interferon knockout (KO) mice. The mice were killed 64 and 71 days after sporocyst feeding (DAF). Muscles containing sarcocysts from the KO mouse killed 71 DAF were fed to a captive D. virginiana; this opossum shed sporocysts 11 days after ingesting sarcocysts. Sporocysts from D. virginiana were fed to 9 KO mice and 4 budgerigars (Melopsittacus undulatus). Schizonts, sarcocysts, or both of S. speeri were found in tissues of all 7 KO mice killed 29-85 DAF; 2 mice died 39 and 48 DAF were not necropsied. Sarcocystis stages were not found in tissues of the 4 budgerigars fed S. speeri sporocysts and killed 35 DAE These results indicate that S. speeri is distinct from Sarcocystis falcatula and Sarcocystis neurona, and that S. speeri is present in both D. albiventris and D. virginiana.     

Eimeria indianensis sp. n. and an Isospora sp. from the Opossum Didelphis virginiana (Kerr)*

Two of 15 road-killed opossums examined for coccidia were found to be infected with a hitherto undescribed species of Eimeria, herein named Eimeria indianensis . The oocysts were spherical (63%) or slightly subspherical (37%) with a double-layered wall. The outer layer was ～1.5 μm thick, yellowish, striated, and appeared rough and pitted on the surface. A micropyle was absent. The spherical oocysts were 16.3 (13–18) μm in diameter; the subspherical ones, 17.6 (15–18) × 16.4 (14–17) μm. The sporocysts measured 9.1 (8–10) × 6.2 (6–7) μm and contained a granular residuum. The sporozoites were elongate, measuring 13.4 (13–15) × 1.8 (1.6-2.0) μm; no refractile globules were seen. The prepatent period was 10 days and the patent period ranged from 9–15 days. A few oocysts of an Isospora sp. were present in one opossum. It was not possible to confirm whether they were specifically of the opossum or of spurious origin.     

Habitat selection for resting sites by the water opossum (Chironectes minimus) in the Brazilian Atlantic Forest

We evaluated the selection of resting sites occupied by the water opossum Chironectes minimus, between 2004 and 2010, in streams of the Brazilian Atlantic Forest. Fourteen radio-tracked adult (males and females) opossums used natural cavities as resting sites. Opossums selected narrow river stretches and selected their resting sites mainly according to characteristics favoring protection against adverse weather conditions. Likewise, opossums avoided disturbed habitats and established their resting sites in well-preserved riparian forest sites, selecting river sections containing a high density of trees and a high proportion of forest cover between the river banks and 50 m from the river. Besides increasing our knowledge on this species habitat selection, such findings further highlight this species’ sensitivity to human disturbance.     

Prevalence of agglutinating antibodies to Sarcocystis neurona in skunks (Mephitis Mephitis), raccoons (Procyon lotor), and opossums (Didelphis Virginiana) from Connecticut

Equine protozoal myeloencephalitis is the most important protozoan disease of horses in North America and is usually caused by Sarcocystis neurona. Natural cases of encephalitis caused by S. neurona have been reported in skunks (Mephitis mephitis) and raccoons (Procyon lotor). Opossums (Didelphis spp.) are the only known definitive host. Sera from 24 striped skunks, 12 raccoons, and 7 opossums (D. virginiana) from Connecticut were examined for agglutinating antibodies to S. neurona using the S. neurona agglutination test (SAT) employing formalin-fixed merozoites as antigen. The SAT was validated for skunk sera using pre- and postinfection serum samples from 2 experimentally infected skunks. Of the 24 (46%) skunks 11 were positive, and all 12 raccoons were positive for S. neurona antibodies. None of the 7 opossums was positive for antibodies to S. neurona. These results suggest that exposure to sporocysts of S. neurona by intermediate hosts is high in Connecticut. The absence of antibodies in opossums collected from the same areas is most likely because of the absence of systemic infection in the definitive host.