Prevalence, transmission, and pathogenicity of Sarcocystis gigantea of sheep

Between March and May 1983, tongues and esophagi of 355 adult ewes from Colorado and Idaho were examined for grossly visible sarcocysts. Sarcocysts of Sarcocystis gigantea were found in 35 sheep. Cats fed sarcocysts from these naturally infected sheep shed sporocysts in their feces. Two adult ewes and 12 lambs inoculated with 1,000 to 1,000,000 sporocysts were euthanatized at postinoculation days (PID) 146, 230, 265, 391, 721, and 882, and their tissues were fed to Sarcocystis-free cats. All inoculated sheep remained clinically normal except for mild pyrexia between PID 12 and 18. Sarcocysts first became grossly visible at PID 391 and sarcocysts from sheep first became infectious for cats at PID 230.     

The in vitro excystation of Sarcocystis gigantea sporocysts

Studies on the in vitro excystation of Sarcocystis gigantea sporocysts revealed that pretreatment before exposure to trypsin and bile was an essential prerequisite. However, in contrast to Sarcocystis tenella and Sarcocystis capracanis, incubation in cysteine hydrochloride under CO2 was largely unsuccessful for excysting Sarcocystis gigantea: of the pretreatments tested, only exposure to sodium hypochlorite proved effective. Excystation from sodium hypochlorite-pretreated S. gigantea sporocysts took place in trypsin and bile between temperatures of 30 and 43 degrees C and occurred rapidly at 39 degrees C. While the presence of bile or bile salts was essential for this process, that of trypsin was not, although more sporocysts excysted in its presence than in its absence. Excystation occurred in the presence of all bile types tested but not when Tween 80 was substituted for bile. The highest levels of excystation were recorded when cattle or sheep bile or sodium taurocholate were used and the lowest when chicken or pig bile were employed. Neither the concentration of sheep bile above 2.5%, nor hydrogen ion concentration (pH range 5.0-10.0) appeared to have any marked effect on the level of excystation obtained.     

Antibody development and cellular immune responses in sheep immunized and challenged with Sarcocystis tenella sporocysts

Four specific-pathogen-free (SPF) sheep were experimentally infected with 10(3) or 10(4) Sarcocystis tenella (syn. S. ovicanis) sporocysts and another two sheep served as uninfected controls. All sheep were challenged 49 days later by infection with 2.5 X 10(5) sporocysts and their humoral and cellular responses to infection and challenge were assessed weekly by enzyme immunoassays and lymphocyte transformation assays. The control sheep died from acute sarcocystosis 29-30 days after challenge, whereas the immunized sheep survived and were protected against acute disease. Specific IgM and IgG antibodies were detected in the immunized sheep from 28 days after infection onwards. Lymphocytes collected before and after challenge did not exhibit any significant differences in their responses to stimulation with S. tenella cystozoite or sporozoite antigens. Furthermore, lymphocytes collected before challenge did not differ from the controls in their responses to stimulation with the mitogens lipopolysaccharide or phytohaemagglutinin. However, lymphocytes collected after challenge did exhibit increased blastogenic responses to stimulation with both mitogens from 21-28 days after challenge onwards. The infected sheep were necropsied 46 days after challenge, and histological and ultrastructural studies revealed numerous infiltrates of lymphocytes, histiocytes and plasma cells in the skeletal muscles, sometimes in association with degenerating parasitic cysts and macrophage myophagia. Parasites were not completely eliminated nor prevented from further establishment, therefore the protective immunity was not sterile but rather a state of premunition.     

The in vivo excystation of Sarcocystis gigantea and S. tenella sporocysts

In vivo studies on the excystation of Sarcocystis gigantea and S. tenella sporocysts indicated that this process was, as in vitro, a diphasic one involving both pretreatment and treatment phases. The studies also tended to support in vitro observations that the requirements for the excystation of these two species are quite different. The results suggested that for neither species was the pretreatment stimulus likely to be provided by conditions in the rumen alone. However, exposure to abomasal conditions only induced moderate levels of excystation in both when they were subsequently treated with trypsin and bile. For S. gigantea, 0.25 to 4 h abomasal exposure was most effective; for S. tenella, 24 hours. The stimuli necessary to complete the excystation process could, apparently, be provided by 1 h placement in the duodenum for S. gigantea but not for S. tenella.     

IS SARCOCYSTIS TENELLA TWO SPECIES?

When mutton containing microscopic sarcocysts of Sarcocystis spp was fed to dogs and cats, only dogs excreted sporocysts in their faeces. Conversely, mutton containing macroscopic sarcocysts produced infection in cats but not dogs. Sheep were experimentally infected with sporocysts from the faeces of dogs and cats either separately or together, and reciprocal feeding trials with their meat carried out with dogs and cats. The results of the experiments strongly suggest that Sarcocystis tenella of sheep may be 2 distinct species, one with the cat as definitive host, and the other parasitising the dog.     

Characterization of monoclonal antibodies against ovine Sarcocystis spp. antigens by immunoblotting and immuno-electron microscopy

Six monoclonal antibodies were raised in mice against purified cytozoite extracts of Sarcocystis gigantea and S. tenella from sheep. Each monoclonal antibody was evaluated for specificity by enzyme immunoassay, immunoblotting and immuno-electron microscopy using homologous and heterologous antigenic preparations. All six monoclonal antibodies exhibited good species-specificity when reacted against crude soluble cystozoite antigens in enzyme immunoassays. However, only two monoclonal antibodies (IgM and IgG2a) exhibited reactivity in Western blots against specific protein bands. Both reacted against S. gigantea antigens of 100,000, 43,000 and 39,000 molecular weight. Neither monoclonal antibody reacted against the heterologous species S. tenella. Ultrastructural studies performed with colloidal-gold conjugated antisera revealed that both monoclonal antibodies reacted against antigens located around micronemes and amylopectin granules in S. gigantea cystozoites. Another monoclonal antibody (IgGI) reacted only against microneme determinants in S. tenella cystozoites. In contrast, polyclonal sheep and rabbit immune sera cross-reacted against a wide range of cystozoite antigens.     

Demonstration of schizogonous stages of Sarcocystis gigantea in experimentally infected sheep

Sarcocystis-free lambs were orally dosed with 1 X 10(6) sporocysts of Sarcocystis gigantea. Schizonts were found in endothelial cells of capillaries and arterioles of the brain, lung and kidney of lambs 7 and 14 days post-inoculation (d.p.i.). Between 21 and 35 d.p.i. there was extensive multi-focal encephalitis; however no organisms were detected in association with these lesions.     

Sarcocystis infection and myocardial pathological changes in cattle from south-eastern Norway

The incidence of myocardial sarcocystis infection and of myocardial pathological changes was recorded in samples of 79 healthy cattle obtained from an abattoir. The incidence rate of thin-walled cysts of S. cruzi was 81.0 %, while mixed infection with thick-walled cysts of S. hominis and/or S. hirsuta was found in 5.0 %. Focal interstitial myocarditis was found in 31.6 % of the samples. The sarcocystis infection and the interstitial mononuclear cell infiltrates were positively associated (P < 0.05). Intimai proliferations of musculo-elastic or fibro-elastic tissues in the intramural coronary arteries were found in 75.0 % of the cattle older than 3% years of age, and in 45.7 % of the cattle less than 3½ years old. No association of the arterial lesions and the sarcocystis infection was demonstrated.     

Coxiella burnetii shedding and environmental contamination at lambing in two highly naturally-infected dairy sheep flocks after vaccination

Abortion due to Coxiella burnetii was confirmed in the 2007/08 season in two naturally-infected dairy sheep flocks. Proportion of C. burnetii shedders and bacterial loads in vaginal mucus were high among aborted or lambed ewes, as was within-flock seroprevalence. Before the next reproductive season (2008/09) 75% of ewes and 50% of replacement lambs were vaccinated (Coxevac, CEVA Santé Animale) keeping the remaining as untreated controls. Compared with the previous year results when abortion outbreak started, a great reduction in the percentage of abortions, in the number of shedders and in the bacterial burden excreted by the ewes was found in both flocks. However, seroconversion in non-vaccinated yearlings from both flocks and the presence of C. burnetii DNA in bioaerosols taken at sheep premises at lambing indicated that infection was still active. No differences were observed between vaccinated and control groups in terms of proportion of C. burnetii shedders. These results suggest that optimal results of vaccination in heavily infected flocks may not be obtained in a short-term period.     

Sarcocystis infections in Georgia swine

Tissues from 168 mature sows obtained at slaughter in northern and southern Georgia were examined for infection with Sarcocystis spp. Digestion techniques revealed zoites in 28 (16.5%) sows. Infected meat was fed to laboratory-reared dogs, cats, raccoons (Procyon lotor), and opossums (Didelphis marsupialis). Dogs and raccoons shed sporocysts (8.3 mum X 11.2 mum) 12 to 14 days after infection. Cats and opossums were refractory to infection. Sarcocystis-free pigs were infected with 50,000 sporocysts of swine-dog origin. Tissues from laboratory-infected swine were fed to dogs and raccoons. Both species shed sporocysts 14 days later. This is the 1st time in which a definitive host has been demonstrated for species of Sarcocystis occurring in North American swine. The raccoon constitutes a new definitive host for S suicanis Erber 1977. In contrast to previously reported low prevalences of Sarcocystis infections in swine, the relatively high prevalence reported here indicates that S suicanis may be of importance to swine producers in Georgia.     

Coyote as a final host for Sarcocystis species of goats, sheep, cattle, elk, bison, and moose in Montana

Tissues (1 kg) from sheep, goats, cattle, moose, bison, or elk naturally infected with Sarcocystis species were fed to one to four Sarcocystis-free coyotes and the number of sporocysts in feces and intestines were counted. All 12 coyotes fed naturally infected tissues shed Sarcocystis in feces, with a prepatent period of 9 to 15 days. The four coyotes fed infected beef had 15, 25, 113, and 201 million sporocysts in their feces and intestines. The coyotes fed elk, moose, or bison had 2.5, 15, and 2.5 million sporocysts in their intestines, respectively. Sporocysts in feces of coyotes fed musculature of cattle, sheep, goats, and elk were structurally similar to those described previously from the feces of dogs. This is evidently the first report of the completion of life cycle of Sarcocystis species in moose and bison. Cross-transmission experiments indicated that one species of goat Sarcocystis completes its life cycle in both dog and coyote and that the ovine Sarcocystis is not transmissible to goats.     

Diclazuril preventive therapy of gamma interferon knockout mice fed Sarcocystis neurona sporocysts

Gamma interferon knockout (KO) mice (n=74) were fed a lethal dose of approximately 1000 sporocysts of the SN15-OP isolate of Sarcocystis neurona. Groups of mice were given pelleted rodent feed containing 50ppm of diclazuril at different times before and after feeding sporocysts. All mice were examined at necropsy and their tissues were examined immunohistochemically for S. neurona infection. Twenty mice were fed sporocysts and given diclazuril starting 5 days before feeding sporocysts and continuing 30-39 days post-infection (p.i.). One mouse died of causes unrelated to S. neurona with no demonstrable parasites; the remaining 19 mice remained clinically normal and S. neurona organisms were not found in their tissues. Sarcocystis neurona organisms were not demonstrable by bioassay of the brains of these 19 mice in uninfected KO mice. Sarcocystis neurona organisms were not found in tissues of five mice treated with diclazuril, starting 7 days after feeding sporocysts and continuing up to 39 days p.i. Therapy was less efficient when diclazuril was given 10 days p.i. Sarcocystis neurona organisms were found in two of 19 mice treated with diclazuril starting 10 days after feeding sporocysts, in two of five mice starting therapy 12 days p.i., and in 10 of 10 mice when treatment was delayed until 15 days p.i. All 15 mice fed S. neurona, but not given diclazuril, developed neural sarcocystosis and were euthanized 22-30 days after feeding sporocysts. Six mice not fed S. neurona, but given diclazuril for 44 days, remained clinically normal. Results indicate that diclazuril can kill the early stages of S. neurona.     

Prevalence of Sarcocystis neurona sporocysts in opossums (Didelphis virginiana) from rural Mississippi

Sarcocystis species sporocysts were found in intestinal scrapings from 24 of 72 opossums (Didelphis virginiana) from rural Mississippi. The number of sporocysts in each opossum varied from a few ( < 100000) to 187 million. Sporocysts from 24 opossums were bioassayed for Sarcocystis neurona infections by feeding to gamma-interferon knockout (KO) mice. S. neurona was detected in the brains of KO mice fed sporocysts from 19 opossums by immunohistochemical staining with anti-S. neurona specific polyclonal rabbit serum, and by in vitro culture from the brains of KO mice fed sporocysts. The isolates of S. neurona from opossums were designated SN16-OP to SN34-OP. Merozoites from 17 of 19 isolates tested at the 25/396 locus were identical to previously described S. neurona isolates from horses. The high prevalence of S. neurona sparocysts in D. virginiana suggests that this opossum constitutes an ample reservoir of infection in the southern United States.     

The effect of Sarcocystis tenella on wool growth in sheep

A dose of 1 × 10⁴Sarcocystis tenella sporocysts produced significant depression of wool growth in lambs dosed at 1 month of age. No significant effect on wool growth was produced when lambs were dosed at 5 months of age.     

Clinical, haematological and plasma biochemical changes in specified-pathogen-free (sporozoa) lambs experimentally infected with low numbers of Sarcocystis tenella sporocysts

Two groups of lambs raised free of sporozoan infection were inoculated with Sarcocystis tenella sporocysts and compared with controls. Lambs from Group 1 were inoculated with 5000 sporocysts and those in Group 2 were given 20,000. Transient increases in rectal temperatures occurred between 23 and 39 days post-inoculation (dpi), although the lambs appeared normal and retained their appetites. Packed cell volumes (PCV) of lambs given 20,000 sporocysts decreased dramatically from 28 to 38 dpi after which they slowly returned to near pre-inoculation levels by 99 dpi. The anaemia was normocytic/normochromic. White cell counts (WCC) rose in infected lambs from 49 dpi, reflecting principally an increase in lymphocyte numbers. Plasma albumin of Group 2 decreased at 28 dpi and remained depressed until the experiment was terminated at 99 dpi. Plasma globulin of infected groups increased from 31 (Group 2) and 35 dpi (Group 1). Plasma alkaline phosphatase (ALP) of Group 2 decreased from 28 dpi and remained depressed to 99 dpi. Lactate dehydrogenase (LDH) of Group 2 was elevated at 24 and 28 dpi and from 42 to 78 dpi, while aspartate aminotransferase (AST) of the same group was elevated from 45 to 66 dpi. Creatine kinase (CK) of Group 2 was elevated from 52 to 71 dpi.     

The prevalence and identity of Sarcocystis in beef cattle in New Zealand

Muscle tissue from the oesophagus and diaphragm of 500 beef cattle slaughtered in New Zealand was examined for Sarcocystis infection by microscopic examination of cysts isolated from muscle samples. All cattle were infected with Sarcocystis; based on light microscopy of cysts, 98% had thin-walled Sarcocystis cruzi cysts and 79.8% had thick-walled (Sarcocystis hirsuta/Sarcocystis hominis) cysts. Cysts were also collected for electron microscopy and transmission experiments. Thick-walled cysts could not be distinguished as S. hirsuta or S. hominis by light or electron microscopy. Thick-walled cysts were fed to three cats and one human volunteer; one cat shed sporocysts but not the human volunteer. Electron microscopy of the cysts revealed many features that have not been described previously.     

Listeriosis in sheep. Listeria monocytogenes excretion and immunological state in healthy sheep.

Listeriosis in sheep. Listeria monocytogenes excretion and immunological state in healthy sheep. Acta vet. scand. 1979, 20, 168–179. — The excretion of Listeria monocytogenes (Lm) in the faeces and milk, and humoral and cell mediated immunity against Lm, were examined in a sheep flock where no cases of listeriosis had occurred during the last 3 years. The investigation was carried out during the indoor season. During the first part of the season 2 of the 10 pregnant, 8 months old lambs excreted Lm in the faeces, but none of the 106 ewes, 2–10 years old. At lambing the organism was isolated from the faeces of 6 of the 10 1 year old lambs and from 64% of the ewes, and from the milk of 1 of the lambs and 41% of the ewes. Nearly all the isolates (98.5%) belonged to serotype 1.Antibody titres against Lm were found in sera and whey by an indirect haemagglutination method. The titres were higher for the ewes than for the hoggs and seemed to be influenced by the number of foetuses the animals carried.Cell mediated immunity was determined by a skin test where delayed hypersensitivity against an antigen prepared from Lm, was measured. Animals fed grass silage had a stronger reaction than animals fed hay, and a stronger reaction was found in animals with ≥ 3 foetuses than in the remainder.The investigation indicates that even in a healthy sheep flock all the animals may be exposed to Lm, and the majority may be latent carriers and excrete this organism in the faeces and milk during periods of stress.     

Premature parturition in ewes inoculated with Sarcocystis ovicanis

A dose of 6 X 10(4) Sarcocystis ovicanis sporocysts produced premature parturition in ewes and eventually proved fatal. The main pathological findings were myositis, myocarditis and encephalitis. Dose rates over the range of 2.5 X 10(3) to 6 X 10(4) depressed the haematocrit levels of pregnant ewes during the period 5-9 weeks after inoculation. Previous infection with S. gigantia did not protect from subsequent challenge with S. ovicanis.     

Colostral transmission of maedi visna virus: sites of viral entry in lambs born from experimentally infected ewes

Maedi visna virus (MVV) vertical transmission in sheep via infected colostrums is a very important route of infection in lambs. To verify colostral transmission and to study early viral entry in lambs, colostrum samples, and small intestine and mesenteric lymph nodes of lambs born from experimentally infected ewes were examined by histopathology, immunohistochemistry (IHC) and in situ hybridisation (ISH) studies. In particular, newborn lambs were naturally fed maternal colostrum and humanely killed at 10, 24, 48, 72, 96 h and 7 and 10 days after birth; two caesarian-derived lambs served as uninfected controls. No lesions suggestive of MVV infection were found, but marked immunoreactions for MVV capsid antigen (CA, p28) were detected in lambs fed maternal colostrum and in macrophages cultured from colostrum. IHC results in lambs suggest an initial viral absorption by intestinal epithelial cells at the tip of the villi, passage to mononuclear cells in the lamina propria and involvement of ileum Peyers' patches and mesenteric lymph nodes, with different staining patterns depending on infection times. ISH on intestinal sections of the 72 h lamb revealed the presence of proviral DNA in epithelial cells at the tip of the villi, suggesting a role for these cells in early MVV replication. The results contribute to knowledge about the pathogenesis of ovine lentivirus infection suggesting that the small intestine and mesenteric nodes are the sites of entry and propagation of MVV in lambs fed colostrums from infected ewes.