Class-specific antibody responses in cattle following experimental challenge with sporocysts or merozoites of Sarcocystis cruzi.

An ELISA using antigen produced from merozoites of Sarcocystis cruzi was developed to monitor specific IgM and IgG antibody, following challenge of cattle with either merozoites or sporocysts of S. cruzi. This assay was compared with an ELISA using antigen produced from the cystozoite stage of the parasite. Both ELISAs were able to detect significant increases in levels of circulating IgM and IgG antibodies against Sarcocystis in all challenged cows; however, the magnitude of the titres was greater in the ELISA which used the antigen derived from the merozoites. This immunoassay also detected increases in the levels of IgG earlier than did the assay using antigen derived from cystozoites of S. cruzi. Since this rise coincided with the presence of clinical signs, and was persistent for several weeks, the IgG-ELISA using antigen derived from merozoites appears to be suitable for the diagnosis of acute sarcocystiosis in cattle. Furthermore, since significant increases in the levels of circulating IgM and IgG antibodies against Sarcocystis were detected in the cows infected with merozoites of S. cruzi, it is evident that merozoites of S. cruzi cultured in vitro maintain their capability to replicate in the natural intermediate host.

Sensitivities and specificities of two ELISA tests for detecting infection with Sarcocystis in cattle of Western Australia.

The accuracies of two ELISAs, one using antigen from merozoites of S. cruzi grown in vitro and the other using antigen from cystozoites of S. cruzi, for detecting infection of cattle with Sarcocystis, were evaluated by testing the sera of 303 cattle from 36 Western Australian herds. The results were compared with those obtained by digestion of oesophageal samples collected from the same animals. A similar proportion of infected animals were detected by the three methods. The sensitivity of the assays for detecting infected cattle was comparable (98 and 95% for the assay using antigen from merozoites and cystozoites, respectively), however the specificity (97%) of the assay which used antigen derived from merozoites was significantly higher (P < 0.001) than that (84%) which used antigen from cystozoites. When herds which had at least five animals sampled were considered, the same infected and non-infected herds were detected by the ELISA employing antigen from merozoites and the digestion methods (sensitivity and specificity of 100%). The sensitivity and specificity of the assay using cystozoite antigen were 100 and 67%, respectively. The kappa values for agreement beyond chance between the two ELISAs were calculated as 78% for the animal-based data and 72.5% for the herd-based data. We conclude that because of the high sensitivity and specificity of the ELISA using antigen derived from merozoites, this assay would be a useful and reliable tool for general sero-epidemiological studies into infection with Sarcocystis.

Excystation rates and infectivity of sporocysts of Sarcocystis cruzi exposed to different treatments and storages.

The effect of some chemical and anatomical factors on the excystability and infectivity in cell culture of sporocysts of Sarcocystis cruzi was investigated. A significantly (P < 0.001) higher excystation rate (ER) occurred when sodium bicarbonate was added to the excysting fluid at concentrations between 0.075 and 0.3 M. Sporocysts collected from different dogs had different ER. In contrast, although not uniformly distributed along the intestinal lumen, the sporocysts collected from the different tracts of the intestine showed similar ER. The period of storage did not affect the excystability of the sporocysts; however, it influenced the pattern of growth of the sporozoites in cell culture. Sporozoites excysted from sporocysts stored for approximately 2 years grew slower and produced significantly fewer merozoites compared to those excysted from sporocysts stored for 5-7 months.

Viability of the sporocysts of Sarcocystis cruzi after exposure to different temperatures and relative humidities.

The effect of temperature and relative humidity (RH) on the survival of sporocysts of S. cruzi were investigated in vitro. Under all experimental conditions (temperature of 4 degrees C, 37 degrees C, or room temperature; RH of 18%, 75%, or 100%) some sporozoites retained their viability to excyst for at least 90 days. The best conditions for survival were 4 degrees C at 100% RH (more than 240 days) and 37 degrees C at 18% RH (more than 180 days). Sporocysts maintained at room temperature at all humidities had the lowest level of survival. It is concluded that sporocysts of S. cruzi are able to survive in most environments for several months and that the fluctuation of the daily ambient temperature is likely to influence the viability of the sporocysts.

Studies on pathogenesis, tissue infection and congenital transmission in cows experimentally infected with Sarcocystis cruzi by various routes.

A group of nine cows, naturally infected with Sarcocystis, were challenged with Sarcocystis cruzi: three intrarumenally with sporocysts, two intrarumenally with water (controls), two intravenously with merozoites grown in vitro and two intravenously with saline solution (controls). The animals intrarumenally challenged with sporocysts developed acute sarcocystiosis and produced stillborn calves, whereas those intravenously challenged with merozoites suffered from subclinical sarcocystiosis with premature births. No parasites were found in calves from cows challenged with sporocysts; however, a meront of Sarcocystis was found within a macrophage in the cerebrospinal fluid of a calf from a cow intravenously inoculated with merozoites of S. cruzi. This is the first time that merozoites of S. cruzi grown in vitro have been demonstrated to retain the ability to infect their natural intermediate host and complete their life cycle.

Evaluation of a serological test system for the diagnosis of Sarcocystis cruzi infection in cattle using S. cruzi merozoite antigen.

Fifty serum samples were examined by enzyme-linked immunosorbent assay (ELISA) for antibodies directed against crude antigens isolated from cystozoites and merozoites of Sarcocystis cruzi and Sarcocystis tenella/Sarcocystis arieticanis and merozoites of Toxoplasma gondii. Of these 50 samples, 25 were from cattle originating from an area where the prevalence of S. cruzi was found to be very low (9%) and in which no cystozoites were detected, and 25 were from cattle which were found by a digestion method to be heavily infected with S. cruzi. A very high correlation was observed between the parasitological data and the results obtained from the serological assays which used antigens from either cystozoites or merozoites of S. cruzi. The assay using the antigen derived from merozoites provided the best result for discriminating infected and non-infected animals. There was some cross-reactivity between the antigen derived from cystozoites of heterologous species of Sarcocystis and S. cruzi antibodies, and some cross-reactivity between antigen of T. gondii and antibodies to S. cruzi. The reproducibility of the assays was found to be high and similar results were observed when the sera were tested on two separate occasions. The unpurified S. cruzi merozoite antigen produced in vitro is relatively accurate in discriminating positive and negative animals and may be used for diagnosis in economically important hosts such as cattle and sheep.

The ecology of Haemonchus contortus in a winter rainfall region in Australia: the development of eggs to infective larvae.

The seasonal pattern of development of Haemonchus contortus eggs to infective larvae was investigated on the south coast of Western Australia. Sheep faeces containing H. contortus eggs were deposited on pasture plots at intervals over a 3-year period, and pasture grass and faeces were sampled periodically for infective larvae (L3). The maximum recoveries occurred in late autumn and in late spring, when adequate moisture coincided with warm temperatures. Larval development was low and sporadic over the hot and dry summer period, and depressed during winter, although most egg depositions in winter yielded L3 at some time. The proportion of L3 recovered was related to temperature and moisture parameters, and the major constraint appeared to be the availability of moisture at pasture level. The best indicator of the suitability of conditions for the development of H. contortus was the proportion of green pasture material present, based on a visual assessment.

The ecology of Haemonchus contortus in a winter rainfall climate in Australia: the survival of infective larvae on pasture.

At sites near Albany, on the south coast of Western Australia, sheep faecal pellets containing either eggs or infective larvae (L3) of Haemonchus contortus were deposited onto natural pasture plots at intervals over 3 years. Faecal and pasture samples were collected periodically and processed to detect L3. On annual pasture plots, L3 were recovered for from 10 to over 20 weeks from depositions made between autumn and early spring, at which time temperatures were mild or low, rainfall was frequent and the pasture was visibly green. During the hot and dry summer, when the pasture was completely dry, L3 often failed to develop or were recovered on only a single occasion, and the mean larval survival period on positive plots was less than 5 weeks. In contrast, L3 deposited on green perennial pasture plots in summer were recovered for up to 4 months in faecal pellets and on pasture. The longest periods of larval survival were associated with the lowest temperature and highest rainfall recordings, and with the greatest quantity of green plant material in the pasture. It is suggested that the poor survival of L3 during the dry summer period could be used in a strategic treatment programme to interrupt the transmission cycle of H. contortus in winter rainfall climates. However, areas of green pasture which persist in summer may provide a sufficiently moist microclimate to permit the survival of H. contortus L3 despite otherwise unfavourable environmental conditions.

The epidemiology of Sarcocystis spp. in cattle of Western Australia.

Oesophagus samples from 714 cattle from Western Australia were examined by artificial digestion to detect the presence of Sarcocystis spp. The overall prevalence of infection was 52%. The prevalence of infection increased with age and was highest in the entire males (92%). The prevalence was lower in cattle which originated from arid and semiarid regions (9 and 31% respectively) than those from tropical (87%) and temperate (60%) regions. Possible reasons for these differences are discussed and it is concluded that environmental and management factors as well as host age and sex influence the prevalence of infection with Sarcocystis spp. in cattle.

Plasma thromboxane B2 levels in horses experimentally infected with Strongylus vulgaris.

Plasma thromboxane B2 (TXB2) the stable inactive metabolite of thromboxane A2 (TXA2), was measured daily by specific radioimmunoassay in three groups of animals before and after experimental infection with Strongylus vulgaris. Infection of four 'parasite naive' foals produced a typical acute syndrome with intermittent but statistically insignificant rises in TXB2 levels. Interpretation of results was complicated by the presence of a non-septic peritonitis associated with implantation of the foals with electrodes for recording myoelectrical activity. In two foals of similar age, with some natural exposure to S. vulgaris, there was little or no clinical response to infection and increases in TXB2 were absent. Baseline levels were also much lower, indicating that the peritonitis may have affected the results obtained in the first group of foals. Severe mesenteric arteritis was confirmed at necropsy in all six foals. A third group of yearling horses, all with natural exposure to the parasite, were generally resistant to infection. One animal developed arteritis with clinical signs of diarrhoea and mild colic, and also showed intermittent increases in TXB2. The mean plasma TXB2 level after infection was significantly higher than in the control period, although absolute levels were lower than those recorded in the 'parasite naive' foals. Other animals in this group had low TXB2 levels and minimal arteritis was found at necropsy. These results indicate that although infection appears to have an effect on plasma TXB2, the changes are inconsistent and not reliable indicators of the presence of verminous arteritis. The results also confirm the difficulty in establishing infection and the variability of the response in animals with previous exposure.

Climate and the distribution of intestinal Trichostrongylus spp. of sheep.

The relative prevalences of Trichostrongylus vitrinus, T. colubriformis and T. rugatus in sheep in south-west Western Australia were examined for evidence of associations with climatic conditions in different localities. The region has a mediterranean-type climate with hot, dry summers and cool, wet winters. The intestinal Trichostrongylus spp. populations in sheep sampled comprised 50% T. vitrinus, 38% T. colubriformis and 12% T. rugatus. The prevalence of T. vitrinus was negatively correlated with mean autumn, winter and spring temperatures of a locality. The prevalence of T. colubriformis was positively correlated with mean autumn, winter and spring temperatures. The prevalence of T. rugatus was not correlated with temperature of any season, but was negatively correlated with mean annual rainfall and length of growing season of a locality. There were suggestions of association of amount of rainfall of a locality and prevalence of T. rugatus was not correlated with temperature of any season, but was negatively correlated with mean annual rainfall and length of growing season of a locality. There were suggestions of association of amount of rainfall of a locality and prevalence of T. colubriformis and of differences in seasonal prevalence of T. vitrinus, but the significance of these was obscured by confounding of some climatic factors. The differences in prevalence of the three Trichostrongylus spp. at different locations was attributed to differences in preferred ambient conditions for development and survival of the free-living stages.

Radioimmunoassay of thromboxane B2 in horse plasma.

A radioimmunoassay for thromboxane B2 (TXB2) in unextracted horse plasma was evaluated. Sensitivity of the assay was 14.0 (SD 5.6) pg ml-1 of plasma. Interassay and intra-assay variation were 21.3 per cent and 4.3 per cent, respectively. The percentage of tracer bound in unextracted plasma in the absence of TXB2 was often higher than that in buffer. Therefore standard curves were obtained using standards diluted in plasma from horses treated with aspirin or in charcoal treated TXB2-free plasma. Standard curves determined in plasma and buffer were parallel. This assay was used to determine the half-life of exogenous TXB2 in horses. The mean value of 20.7 minutes (n = 3) is similar to that determined in other species. Storage of plasma samples at -20 degrees C for four months was found to alter the TXB2 levels in an unpredictable manner. Mean recovery was 102.4 per cent (SD 44.1). Effect of variability in sample collection and handling was assessed, but no consistent source of artifactual generation of TXB2 was found.

Prevalence and epidemiology of the major gastrointestinal parasites of horses in Perth, Western Australia.

A survey was conducted on the prevalence of the major gastrointestinal parasites in 140 horses necropsied in Perth, Western Australia, during 1979 to 1982. Adult Strongylus vulgaris were found in 22.5 per cent of horses and verminous arteritis in 62.9 per cent. The peak worm prevalence was in November to January (summer). S edentatus had a similar prevalence and seasonality but S equinus was not found in this survey. Draschia megastoma and Habronema muscae were found in 66.2 per cent and 35.3 per cent of horses respectively. Infection is probably acquired in summer when 8 per cent of the Musca domestica in the vicinity of the stables carried third stage spiruroid larvae. Gasterophilus intestinalis and G nasalis occurred in 36.4 per cent and 22.1 per cent of the horses respectively and 52.1 per cent of horses were infected with one or both species. The peak prevalence of G intestinalis larvae occurred in December with a trough in February-April; the peak prevalence of G nasalis was in May with a trough in November-December. Parascaris equorum was found in 9.9 per cent of the horses and in 21.3 per cent of those less than three years old. Anoplocephala perfoliata was found in 4.9 per cent of the horses and most of these were in older horses.

Integrated control of Strongylus vulgaris infection in horses using ivermectin.

An attempt was made to control or eliminate Strongylus vulgaris from a closed group of three horses at pasture near Perth, Western Australia, by dosing with ivermectin on four occasions during the time of year when it was believed that environmental conditions would eliminate all the non-parasitic stages of that species. At necropsy, five months after the last dose of anthelmintic and after continually grazing the same pastures, no S vulgaris or arterial lesions were found in those horses and S edentatus, Draschia megastoma and Habronema species were also almost completely eliminated.

Prevalence and survival of Toxocara canis eggs in the urban environment of Perth, Australia.

Using a technique of proven efficiency no Toxocara canis eggs were recovered from a total of 266 sand samples collected from "dog beaches" and parks in the Perth urban area. This observation emphasises that the major risk to humans is from an environment in which puppies are found. Other observations, made at a heavily contaminated site, showed that the eggs of T. canis disappeared from sandy soil over about 6 months during a period in which it would have been expected that conditions were favourable for egg survival. Other data are also presented indicating that the prevalence of T. canis in adult dogs in Perth is quite low. However, T. canis eggs were recovered in large numbers from the majority of soil samples collected from Perth dog-breeding establishments.