Comparison of a competitive inhibition ELISA and the card agglutination test for detection of antibodies to Anaplasma marginale and Anaplasma centrale in cattle.

OBJECTIVE: To compare a recently developed recombinant MSP-5 competitive inhibition ELISA with a card agglutination test for detection of antibodies to Anaplasma marginale and Anaplasma centrale in Australian cattle. MATERIALS AND METHODS: The ELISA was compared with the card agglutination test using 208 sera from cattle in Anaplasma-free herds, 86 sera from cattle experimentally infected with A marginale or A centrale and 757 sera from cattle in areas endemic for A marginale. RESULTS: The specificity of the ELISA, based on testing 208 sera from cattle in Anaplasma-free areas, was 99.5%, and the sensitivities for detection of antibodies to A marginale and A centrale in sera from the experimentally infected cattle were 98.0% and 100%, respectively. For the same sets of sera, the specificity of the card agglutination test was 98.6% and the sensitivities for detection of antibodies to A marginale and A centrale were 98.0% and 100%, respectively. For the 757 sera collected from cattle in areas endemic for A marginale, the agreement between the ELISA and the card agglutination test depended on the positive threshold selected for the ELISA. The maximum achievable agreement was 91.5% (kappa = 0.73; 95% confidence interval 0.66, 0.79). CONCLUSION: We conclude that the competitive inhibition ELISA is a useful alternative to the card agglutination test for detection of A marginale or A centrale infection in cattle. The assay should be particularly useful for epidemiological applications such as prevalence studies and control programs.

Observations on the use of Anaplasma centrale for immunization of cattle against anaplasmosis in Zimbabwe.

A total of 93 Bos taurus cattle was used in pen trials to compare vaccine stocks of Anaplasma centrale from South Africa and Australia (which stock came from South Africa in 1934) in protecting against three virulent field isolates from clinical Anaplasma marginale infections. In addition, field observations were made on the use of a vaccine, prepared from the Australian stock, in over 9553 cattle of mixed age and breeds on 16 co-operator farms and at one communal dip. The results of the pen trials indicated that the two vaccine stocks were comparable and that neither provided adequate protection against two of the three isolates of A. marginale. The field observations indicated that the vaccine was highly infective and produced mild reactions in most recipient cattle, and that users were generally satisfied with the vaccine. These somewhat conflicting results are discussed in the context of observations in Australia and future vaccination against anaplasmosis in Zimbabwe.

Development of an enzyme-linked immunosorbent assay for detection of antibodies to Babesia bigemina in cattle.

Monoclonal antibodies, directed against a 58-kDa Babesia bigemina merozoite antigen that reacted strongly with immune sera from experimentally and naturally infected cattle in Western blots, were used to develop a competitive-inhibition enzyme-linked immunosorbent assay (ELISA). As based on the testing of 70 antibody-positive sera from experimentally infected cattle and 166 antibody-negative sera collected in non-endemic areas of Australia, the sensitivity and specificity of the ELISA were 95.7% and 97.0%, respectively. In sequential sera collected from six calves during the course of experimental B. bigemina infections the ELISA detected seroconversion at about 10 days post-inoculation. The specificity of the ELISA was not affected by the presence of antibodies to B. bovis, Anaplasma marginale or Theileria buffeli. In 42 sera from cattle experimentally infected with B. bovis but negative for B. bigemina the specificity of the ELISA was 95.2%. The use of a competitive-inhibition ELISA format detecting only antibody directed against a single epitope on the 58-kDa antigen appears to have overcome many of the specificity problems that have plagued serological tests for B. bigemina in the past. The test should be useful for epidemiology studies, particularly in areas where B. bovis and B. bigemina have overlapping distributions.

Evaluation of an ELISA for detection of antibodies to Babesia bovis in cattle in Australia and Zimbabwe.

An enzyme-linked immunosorbent assay (ELISA) for antibodies to Babesia bovis was evaluated in comparison with the indirect fluorescent antibody test (IFAT) in Australia and Zimbabwe. Positive and negative threshold values for the ELISA were set using sera from cattle of known infection status. Sensitivity and specificity estimates for the ELISA based on 158 positive sera from cattle experimentally infected with Australian isolates of B. bovis and 318 negative sera collected from B. bovis-free herds in Australia were 100% and 99.4%, respectively. The specificity of the assay in Africa, based on 328 sera from B. bovis-free herds in Kenya and South Africa, was 99.7%. The ELISA was compared with the IFAT using sequential sera from 16 calves experiencing primary B. bovis infections, and a total of 777 field sera collected from B. bovis-endemic herds in Australia and Zimbabwe. In primary infections, the ELISA and IFAT detected antibodies at or about the same time. With sera from endemic herds, the performance of the ELISA was at least comparable with that of the IFAT. Two hundred and fourteen of 221 sera that were negative by IFAT, were negative by ELISA, and 428 of 439 sera that were clearly positive by IFAT were positive by ELISA. Of 117 sera that gave equivocal (suspect or weak positive) results in the IFAT, 20 were positive by ELISA, 7 were suspect and 90 were negative. We conclude that the ELISA will be useful for epidemiological studies on B. bovis in Australia and Zimbabwe, and probably elsewhere.

Genotypic diversity in field isolates of Babesia bovis from cattle with babesiosis after vaccination.

OBJECTIVE: To determine whether particular genotypes of Babesia bovis were common to field isolates obtained from cattle properties in Queensland where the B bovis vaccine had apparently failed. DESIGN: A comparative study of polymerase chain reaction genotypes in different populations of B bovis. PROCEDURE: Two polymerase chain reaction assays were applied to analyse DNA extracts of B bovis vaccine (K, T and Dixie strains) and 27 field isolates from 24 properties where disease outbreaks had occurred despite the use of the vaccine. To evaluate the stability of the genotypes identified, 11 of the field isolates were inoculated into experimental cattle that had either been previously vaccinated with T strain or not vaccinated. RESULTS: No particular genotype of B bovis was responsible for the problems observed in previously vaccinated herds. None of the isolates had genotypes identical to the vaccine strains used. No geographic trends among the genotypes were observed. Isolates that originated from the same property also had different genotypes. Blood passage of the 11 field isolates in either previously vaccinated or nonvaccinated cattle did not alter the original genotype. CONCLUSION: No particular genotypes identified by the Bv80 and BvVA1 polymerase chain reaction assays could be associated with vaccine failures.

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.

Development of effective living vaccines against bovine babesiosis--the longest field trial?

Between 1959 and 1996, research was performed to change a vaccine against babesiosis in Australia and to improve it as actual or threatened untoward field responses became apparent. The most significant change occurred in 1964 with the traditionally used carriers of Babesia being replaced as vaccine donors by acutely infected splenectomised calves. This ensured the infectivity of the vaccine and was fortuitously associated with a reduction in the virulence of Babesia bovis in vaccine. Since then, more than 27 million doses of highly infective vaccine have been supplied from the laboratory at Wacol near Brisbane. This vaccine reduced serious losses from babesiosis in vaccinated cattle in Australia to very low levels and has now gained acceptance worldwide. Research to ensure the continuing effectiveness of the vaccine has proved to be essential.

Current state and future trends in the diagnosis of babesiosis.

An overview is given of the currently available methods to diagnose babesiosis in livestock. Microscopic techniques are still the only appropriate techniques to diagnose acute disease. Thin or thick blood films stained with Giemsa's stain are sufficient. The sensitivity ranges from 10(-5) to 10(-6), i.e. one parasite per 10(5)-10(6) erythrocytes can be detected. Thick films stained with acridine orange (sensitivity approximately 10(-7)) and the Quantitative Buffy Coat (QBC) analysis tube system (sensitivity approximately 10(-7)-10(-8)) are applicable for diagnosis in the laboratory. DNA probes are very specific tools to identify haemoparasites in organs post mortem and in ticks. For the identification of carrier animals the sensitivity (approximately 10(-5)-10(-6)) is generally not sufficient. For the latter the polymerase chain reaction (PCR) technique is a very powerful tool (sensitivity approximately 10(-9)). Many different serodiagnostic tests have been described; however, the immunofluorescence antibody test is the most widely used, while the enzyme-linked immunosorbent assay (ELISA) is the test system which holds the greatest promise for the future. Thus far, improvements to the ELISA have been limited as the quality of antigen preparations made from infected blood is generally poor with a few exceptions (Babesia bovis, Babesia caballi). Potentially, most of the problems associated with crude antigens can be overcome by the production of recombinant antigens. Several ELISAs based on highly defined recombinant antigens have been described and show promise. None of these tests has been validated to the extent that it could be applied globally. Future research requirements as well as the need for coordination of the research effort and collaboration between institutions involved in the diagnosis of babesiosis are discussed.

Investigations of breakdowns in protection provided by living Babesia bovis vaccine.

Field investigations of protection afforded by live Babesia bovis vaccine in Australia revealed that a ninefold increase in vaccine failures occurred in the period from 1985 to 1990. Laboratory trials using 189 experimental cattle were conducted to evaluate the protection afforded by the Babesia bovis strain used in the commercial vaccine during this time. Four isolates from clinical cases of babesiosis in vaccinated cattle were assessed. The results showed that the strain used in the vaccine during the 5 year period was poorly protective against three isolates while a recently isolated and prepared vaccine strain was strongly protective. Circumstantial evidence is provided that indicates the vaccine failures were due to change in the field populations of Babesia bovis, rather than change in the strain used in the vaccine. Implications of the results for the future of Babesia bovis vaccines are discussed.

Prevalence of Babesia bovis and Anaplasma marginale at selected localities in Sri Lanka.

Sera were collected from a minimum of 20 cattle aged nine to 36 months at each of 14 localities in five climatic zones of Sri Lanka. Sera were tested for antibodies to Babesia bovis and Anaplasma by an indirect fluorescent antibody test and a card agglutination test, respectively. Antibodies to B. bovis and Anaplasma were detected in all samples tested from each of 14 and 12 localities respectively. In general, prevalences were consistently high among localities below 1,200 m and lower and more variable in the hill country. Management practice rather than climate was considered to be the main factor influencing prevalences. Management is discussed in relation to the risk of disease outbreak.

Infectivity of cryopreserved Babesia bovis, Babesia bigemina and Anaplasma centrale for cattle after thawing, dilution and incubation at 30 degrees C.

Blood containing either Babesia bovis, Babesia bigemina or Anaplasma centrale was mixed with an equal volume of 3 M glycerol in phosphate-buffered saline with or without glucose and then stored in liquid nitrogen for 2-30 days. After being thawed, the parasitized blood was subjected to various procedures, including dilution up to 1000-fold followed by incubation at 30 or 4 degrees C for 8 h, before infectivity of the parasites was tested in a total of 70 cattle. The results showed that the blood cryopreserved with glycerol remained highly infective after thawing, despite dilution and incubation for 8 h at 30 degrees C. The results have practical application in the use of frozen, live vaccines against bovine babesiosis and anaplasmosis.

Comparison between Sri Lankan and Australian strains of Babesia bovis in the vaccination of imported cattle in Sri Lanka.

A Sri Lankan strain of Babesia bovis (designated A strain) was isolated from larval ticks and prepared for use as vaccine by syringe-passage in 20 splenectomised calves followed by irradiation. The A strain and a vaccine strain of Babesia bovis (designated K strain) brought in frozen form from Australia were used to vaccinate 37 susceptible bulls imported from southern Australia. Rectal temperatures, packed cell volumes, parasitaemias and overt clinical signs were monitored for three weeks following vaccination. The results indicated that the A strain was slightly more virulent than the K strain but suitable for the vaccination of well-supervised cattle.

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.

Molecular vaccines against parasites.

Prophylactic vaccines can be expected to be one of the major practical outputs of parasitology research. Various groups within Australia have pursued the vaccine objective for several years, with particular emphasis on blood-stage falciparum malaria in man, intestinal helminths of sheep and cattle, cutaneous myiasis (blowfly strike) in sheep, cysticercosis in sheep and cattle, bovine babesiosis, and cattle ticks. Other vaccine programmes are concerned with giardiasis, filariasis, toxoplasmosis, fascioliasis, coccidiosis in poultry, cutaneous leishmaniasis and schistosomiasis japonica. For many years, the only available vaccine against a parasite in Australia has been the attenuated Babesia bovis vaccine produced by the Tick Fever Research Centre of the Queensland Department of Primary Industries. Strategies for achieving molecular vaccines are generally similar within the various research groups. They involve analysis of the immunology and immunochemistry of a model or in-vitro system; development of functional monoclonal antibodies; analysis of antibody specificities in clinically and/or functionally defined polyclonal sera; screening of cDNA or genomic expression libraries; peptide synthesis; identification of an appropriate vaccination schedule involving adjuvants or new recombinant DNA-based antigen delivery systems. Outlined below are five of the major vaccine programmes.

Effect of imidocarb dipropionate prophylaxis on the infectivity and immunogenicity of a Babesia bovis vaccine in cattle.

Imidocarb dipropionate (IDP), a potent prophylactic drug against Babesia bovis infections in cattle, was tested for its effect on the infectivity and immunogenicity of a live B. bovis vaccine marketed in Australia. At the recommended prophylactic dose rate of 3 mg/kg, IDP suppressed infectivity of the vaccine for at least 6 weeks. The vaccine infected all cattle inoculated either 8 weeks after treatment with 3 mg/kg, or 4 weeks after treatment with the recommended therapeutic dose of 1.2 mg/kg. These infections were, however, partially suppressed and the level of immunity to a subsequent heterologous virulent challenge was reduced. Cattle that failed to become infected after vaccination were fully susceptible to the challenge. It is concluded that where B. bovis vaccination is contemplated, prophylactic use of IDP should be avoided.

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.