Detection of Eimeria acervulina using the polymerase chain reaction.

A polymerase chain reaction (PCR)-based assay was developed for the detection of Eimeria acervulina. Primers were designed to amplify a fragment of the EASZ240/160 sporozoite antigen gene. The PCR assay detected as few as 10 E. acervulina oocysts in a mixed population containing a total of 10(6) oocysts. No nonspecific reaction was observed with any other species of avian Eimeria known to occur in Australia. PCR products from genomic DNA were 237 bp larger than predicted from previously reported cDNA sequences. Sequencing of the product revealed the presence of a probable intron. This work demonstrates the potential of PCR-based assays for identification and detection of avian Eimeria. Potential uses include identification of minor species present in mixed infections and quality control in the production of live vaccines.

Isolation and pathogenicity of Australian strains of Eimeria praecox and Eimeria mitis.

OBJECTIVE: To determine the presence of E praecox and E mitis in Australia, to isolate representative strains of these species from chickens and determine their pathogenicity. DESIGN: Morphological, physiological and cross protection studies were undertaken to confirm the identity of Australian isolates of E praecox and E mitis. PROCEDURE: Oocysts were isolated from a backyard flock at Jimboomba, southeastern Queensland and numbers of E praecox and E mitis enriched by passage in chickens immune to five other species of poultry Eimeria. Oocysts of mean conformation and size of the two species were purified by single oocyst passage. Two isolates that closely matched recorded parameters for E praecox and E mitis were selected and designated JP and JM respectively. The cross protection between the isolates and E acervulina was determined by infection and challenge experiments. The virulence of the two isolates was determined by comparing weight gains of groups of birds inoculated with JP isolate or JM isolate with untreated groups. RESULTS: Isolates JP and JM most closely matched recorded parameters of E praecox and E mitis respectively. Groups of chickens, previously infected with JP and JM isolates, showed no significant protection against infection with E acervulina. In a separate trial, groups of susceptible chickens inoculated with 10(5) oocysts of JP and JM isolates showed significantly reduced weight gains compared with untreated controls. CONCLUSION: Isolates JP and JM are E praecox and E mitis respectively, confirming the presence of these species in Australia. These isolates were found capable of causing significant reductions in weight gains in susceptible chickens.

Review of Australian species of Theileria, with special reference to Theileria buffeli of cattle.

This review describes 4 species of parasites in Australia generally regarded as Theileria. The physiological and epidemiological characteristics of the species that occurs in cattle are discussed. Confusion still surrounds the relationship of this species with benign species of Theileria in other countries. Arguments are presented to support the proposal that the bovine species in Australia is correctly designated Theileria buffeli and that this name should apply to all the benign species of Theileria of cattle in Eurasia. The other 3 species considered in the review are proposed species from marsupials. Of these, only Theileria tachyglossi can confidently be included in the genus Theileria, because schizonts have yet to be detected in the other 2 species.

The distribution and prevalence of Theileria buffeli in cattle in Queensland.

The distribution and prevalence of Thelleria buffeli in Queensland cattle were investigated using serum samples and blood films collected primarily for brucellosis surveillance and tick fever diagnosis. Serums from 8654 cattle from 357 farms throughout Queensland were examined by an indirect fluorescent antibody test for antibody to T buffeli. In addition, 347 peripheral blood films collected from 147 farms in south-eastern Queensland were examined for piroplasms of T buffeli. The overall herd and animal prevalences for T buffeli were 75% and 41%, respectively. There was significant variation among regions in both herd and animal prevalences (P less than 0.001). Herd and animal prevalences were highest in the north and east decreasing westward. The results indicate that T buffeli is more widespread in Queensland than previously thought.

Some effects of tick infestations on juvenile northern brown bandicoot (Isoodon macrourus).

The effect of tick infestations on body weight and various blood parameters was monitored in juvenile northern brown bandicoots (Isoodon macrourus) after release into tick-infested or tick-free enclosures. Three species of ticks were observed in the enclosures, Haemaphysalis humerosa, Ixodes tasmani and Ixodes holocyclus. Bandicoots released into tick-infested enclosures showed a reduced growth rate (1.8 versus 2.5 g/day increase in body weight), a reduced haematocrit value (27.4 versus 40.0%) and an increased number of white blood cells when compared with bandicoots released into tick-free enclosures. These results suggest that tick infestations may influence the health of juvenile I. macrourus.

Concurrent infection with Theileria buffeli caused depression of parasitaemia in Babesia bovis and Anaplasma centrale infections in splenectomised calves but not in B bigemina infections.

In five experiments undertaken in splenectomised calves it was found that Theileria buffeli infections depressed the parasitaemias of superimposed Babesia bovis and Anaplasma centrale infections, but not B bigemina infections. The course of A centrale infections did not appear to be affected by a concurrent Eperythrozoon teganodes infection.

Study of virulence and vector transmission of Babesia bovis by use of cloned parasite lines.

Cloned lines of Babesia bovis were prepared from the avirulent vaccine strain, Ka, by an in vivo limiting dilution procedure. The virulence of these clones for adult Bos taurus cattle varied from completely avirulent to highly virulent. This suggests that the parent strain, Ka, is composed of a mixture of subpopulations of varied virulence. Passage of the avirulent clone K-19-47 in intact (nonsplenectomized) cattle resulted in its full reversion to virulence. This suggests that two mechanisms are operating to enable virulence to be a readily modified characteristic in this parasite: differential gene expression and phenotypic selection of subpopulations. A series of experiments demonstrated that all clones were non-tick transmissible. This lack of vector transmission was a stable characteristic and could not be altered by the passage of K-19-47 in intact cattle, despite the fact that passage in intact animals caused this clone to revert to the fully virulent phenotype. A mechanism is suggested for the virulence and vector transmission variations observed in B. bovis.

Babesia bovis: evidence for selection of subpopulations during attenuation.

DNA probes were used to detect variation in subpopulations of virulent and serially passaged Babesia bovis. Two distinct patterns were evident after hybridization to genomic DNA; the first was a basic profile typical of virulent B. bovis and the second, a more variable array, was characteristic of B. bovis after various stages of attenuation. Tick transmission of avirulent B. bovis causes reversion to the virulent genomic pattern, suggesting that selective enrichment of a small residual subpopulation caused reversion to a virulent profile of subpopulations. Certain genomic fragments, predominant in either virulent or avirulent parasite forms, are putative "markers" or actual elements responsible for these biological characteristics.

Elimination of Theileria buffeli infections from cattle by concurrent treatment with primaquine phosphate and halofuginone lactate.

Fifty splenectomised calves naturally infected with Theileria buffeli were treated with primaquine phosphate (ICI, UK) and halofuginone lactate (Hoechst, Australia) either separately or in combination. Infections in treated calves were monitored for up to 26 weeks by examining Giemsa stained peripheral blood films for piroplasms and by an immunofluorescent antibody test. When used alone neither of the drugs eliminated infection. The most successful results were obtained when two treatments of halofuginone lactate, at a rate of 1 mg kg-1 body weight and six treatments of primaquine phosphate, at a rate of 2 mg kg-1 body weight, were administered concurrently. No theilerial relapses were observed in 14 of 16 calves so treated, and no antibody to T. buffeli was detected in these calves beyond the twelfth week after treatment. The results have application in the preparation of Theileria-free calves for use in the production of living vaccines against babesiosis and anaplasmosis.

Elimination of Theileria buffeli infections from cattle by concurrent treatment with buparvaquone and primaquine phosphate.

Attempts (some successful) were made to eliminate Theileria buffeli infections from 23 naturally or experimentally infected, splenectomised calves. The anti-theilerial hydroxynaphthoquinone derivative buparvaquone was used either alone or in combination with primaquine phosphate. After treatment the calves were monitored for infection for up to 26 weeks. Blood films were examined for piroplasms and serum antibody levels were measured using an immunofluorescence technique. Buparvaquone alone failed to eliminate infections. Infections were eliminated from 11 of 16 calves treated twice with buparvaquone and three or six times with primaquine phosphate. Theilerial parasites were not subsequently seen in these 11 calves nor were antibodies detected beyond the eighth week after treatment.

Transmission of Theileria buffeli to cattle by Haemaphysalis bancrofti fed on artificially infected mice.

Larval and nymphal Haemaphysalis bancrofti became infected with Theileria buffeli following the intraperitoneal inoculation of infected bovine blood into CBA mice on which they were feeding. Subsequent instars of fed ticks were released on calves and transmitted theileriosis at each of 10 attempts. Suspensions made from ticks moulted in vitro and then incubated for 4 days at 37 degrees C were also infective for calves inoculated subcutaneously. The findings provide a convenient method for infecting ticks with field isolates of Theileria.

Growth of Babesia bovis parasites in stationary and suspension cultures and their use in experimental vaccination of cattle.

A combination of stationary culture and suspension culture was used to produce litre quantities of Babesia bovis parasites suitable for use as live vaccine. The Australian vaccine strain of B bovis, Ka, was maintained continuously in microaerophilus stationary phase (MASP) cultures, and for a short period in batch and flow-through spinner flask cultures. Although continuous culturing was not achieved in spinner flasks, the production of litre quantities of heavily parasitised erythrocytes was achieved more simply than by using MASP cultures. Ka strain parasites were maintained continuously in MASP culture for 174 days without altering their virulence or immunogenicity when compared to calf-derived parasites. Cultured parasites also survived storage at 4 degrees C for six days in basal medium, adding to their potential usefulness as a live vaccine in field situations.

Effect of different methods of maintenance on the pathogenicity and infectivity of Babesia bigemina for the vector Boophilus microplus.

Observations were made on the effects of five different methods of laboratory maintenance on the infectivity and virulence of Babesia bigemina for the tick Boophilus microplus. The original isolate was highly infective and virulent, causing premature death of engorged female ticks and reduced egg production. Maintenance of the strain by syringe passage in unsplenectomised calves at six to 10 week intervals reduced both its infectivity and virulence for ticks. When slow passages were preceded by a series of rapid passages in splenectomised calves, the changes to the strain were less pronounced. The other three procedures, rapid syringe passage in splenectomised calves and tick passage in either splenectomised or intact calves, had no statistically significant effect on the characteristics measured.

Observations on the development of tick-transmitted Theileria buffeli (syn T orientalis?) in cattle.

The development and morphology of Australian Theileria buffeli in cattle were studied after infection had been experimentally transmitted by the tick, Haemophysalis humerosa. Macroschizonts of T buffeli were demonstrated in Giemsa's stained lymph node preparations for between six and 20 days following tick infestation. The presence of schizonts was confirmed by immunofluorescence with sera from known infected animals. Microschizonts were seen infrequently. The schizonts and piroplasms of T buffeli are morphologically similar to those of T orientalis.

Babesia bovis: molecular and biological characteristics of cloned parasite lines.

An in vivo limiting dilution technique was used to produce several Babesia bovis cloned lines with which to study the basis of virulence and immunogenicity in this parasite. DNA hybridization using a cloned DNA fragment from the BabR locus demonstrated that the cloned lines were a more restricted genetic population than the parent strain. Biosynthetic labeling and immunoprecipitation studies indicated that the cloned lines differed from each other and from the parentals in the expression of a small number of polypeptides and antigens. Animal trials with three of the lines demonstrated that the parental line contains both virulent and avirulent parasite populations, at least three of which are not tick transmissible, and that while the lines do provide significant protection against heterologous challenge, they may not give as effective protection as the parental line. These experiments demonstrated the existence of subpopulations with distinctive molecular and biological properties, providing evidence that the attenuation process is based on the selection of preexisting parasite subpopulations combined with the ability of these parasites to vary genetically.

Effect of different methods of maintenance on the development and morphology of Babesia bigemina in the gut of Boophilus microplus.

The development and morphology of Babesia bigemina in the gut of Boophilus microplus ticks were studied during laboratory maintenance of the babesia by four methods: tick transmission in unsplenectomised (intact) calves; tick transmission in splenectomised calves; syringe passage at intervals of five days or less in splenectomised calves; and syringe passage at intervals of six weeks or more in intact calves. The first method had no apparent effect on the development of B bigemina in ticks compared to that of the original isolate. The other three methods had obvious effects, the most pronounced being increased numbers of babesial forms with processes, particularly during early stages of development. The findings emphasise the importance of maintaining laboratory strains of parasites by natural means in life cycle studies.