Selection and characterisation of two attenuated vaccine lines of Eimeria tenella in Australia.

OBJECTIVE: To attenuate two strains of Eimeria tenella by selecting for precocious development and evaluate the strains in characterisation trials and by field evaluation, to choose one precocious line for incorporation into an Australian live coccidiosis vaccine for poultry. DESIGN: Two strains from non-commercial flocks were passaged through chickens while selecting for precocious development. Each strain was characterised for drug sensitivity, pathogenicity, protection against homologous and heterologous challenge, and oocyst output in replicated experiments in which the experimental unit was a cage of three birds. Oocyst output and/or body weight gain data collected over a 10 to 12 day period following final inoculation were measured. Feed conversion ratios were also calculated where possible. RESULTS: Fifteen passages resulted in prepatent periods reduced by 24 h for the Redlands strain (from 144 h to 120 h) and 23 h for the Darryl strain (from 139 h to 116 h). Characterisation trials demonstrated that each precocious line was significantly less pathogenic than its parent strain and each effectively induced immunity that protected chickens against challenge with both the parent strain and other virulent field strains. Both lines had oocyst outputs that, although significantly reduced relative to the parent strains, remained sufficiently high for commercial vaccine production, and both showed susceptibility to coccidiostats. CONCLUSION: Two attenuated lines have been produced that exhibit the appropriate characteristics for use in an Australian live coccidiosis vaccine.

Identification of antigenic differences that discriminate between cattle vaccinated with Anaplasma centrale and cattle naturally infected with Anaplasma marginale.

Monoclonal antibodies were raised against the vaccine strain of Anaplasma centrale used in Australia. A monoclonal antibody that reacted with an 80 kDa antigen was used to develop an A. centrale-specific fluorescent antibody test that will be useful for confirming species identity in patent infections. Another monoclonal antibody that reacted with a 116 kDa antigen was used to develop an A. centrale-specific competitive inhibition enzyme-linked immunosorbent assay (ELISA) for the serological identification of vaccinated cattle. The sensitivity of the ELISA was 100% in cattle experimentally infected with A. centrale, 97.1% in a vaccinated beef herd and 98.3% in a vaccinated dairy herd. The specificity of the ELISA was 98.6% in non-vaccinated cattle outside the Anaplasma marginale-endemic area, 97.9% in non-vaccinated cattle within the A. marginale-endemic area and 100% in cattle experimentally infected with A. marginale. The ELISA detected antibodies to A. centrale in cattle up to 9 years after vaccination with no apparent decrease in sensitivity. The assay has proved extremely valuable in Australia for investigating reported failures of multivalent live vaccines used to protect cattle against anaplasmosis and babesiosis, and should be similarly useful elsewhere in the world where these types of vaccines are used, e.g. Israel and South America.

Evaluation of a commercially available ELISA kit for detection of antibodies to Anaplasma marginale and Anaplasma centrale in cattle in Australia and Zimbabwe.

A newly available competitive inhibition ELISA kit for the serological diagnosis of anaplasmosis was evaluated in Australia and Zimbabwe. In Australia the performance of the test was compared with the card agglutination test (CAT). The assay was evaluated using negative sera collected from Anaplasma-free herds, positive sera from experimentally infected cattle and sera from Anaplasma marginale-endemic herds. The sensitivity and specificity of the ELISA in Australia were 100 % and 83,3 %, respectively, and the sensitivity and specificity of the CAT were both 100%. The agreement between the ELISA and CAT in the sera from endemic herds was 86,4 % (kappa = 0,718). The specificity of the ELISA in Zimbabwe was 100%. No meaningful estimate of sensitivity was possible in Zimbabwe because few known positive sera were available for testing, but all eight known positive sera that were available were clearly positive. We conclude that the ELISA is a useful alternative to the CAT for epidemiological studies. The ELISA kits have advantages over the CAT in that the ELISA is more robust and reagents are better standardized, but the kits are expensive.

Lysine requirements of growing emus.

1. The lysine requirement of growing emus between 23 and 65 d of age was determined according to growth response variables. 2. The optimal lysine requirement of emus was found to be 0.83 and 0.90 g/MJ ME for growth rate and gain:food ratio respectively. These findings are in accordance with the recommended value of 0.80 g/MJ ME, but is lower than the recommended value for ostriches (1.02 g/MJ ME) and higher than determined values for broilers (0.75 g/MJ ME) of the same age range.

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.

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.

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.

A survey of cattle producers in the Boophilus microplus endemic area of Queensland to determine attitudes to the control of and vaccination against tick fever.

A survey by mail was used to determine the views of beef producers in the Boophilus microplus endemic area of Queensland on the control of and vaccination against tick fever. Data from 448 questionnaires were analysed, representing 2.7% of beef producers in the survey area. Producers considered buffalo fly (Haematobia irritans exigua) infestation as the most important problem whereas tick fever ranked sixth overall. Private veterinarians were regarded as the most important source of information on vaccines for cattle followed by a weekly rural newspaper. From the survey we estimate that about 33% of producers used the tick fever vaccine produced by the Tick Fever Research Centre of Queensland Department of Primary Industries but there were significant (P < 0.05) variations between regions and herds. Large herds (> or = 400 head) in south-east Queensland were the most likely to be vaccinated against tick fever. Of the producers who did not use the vaccine, over 70% replied that there was no need to vaccinate because of the low risk of the disease in their herds. In 52% of unvaccinated herds the treatment of animals with acaricide was considered the most important means of tick fever control and 61% of these herds comprised Bos indicus x Bos taurus or Bos indicus cattle.

The effect of age on resistance of cattle to Babesia bovis.

Susceptible Hereford cattle of different ages were inoculated with 2 X 10(8) Babesia bovis organisms. Experiment I consisted of cows aged 6 to 7 years, steers aged 17 to 18 months and calves aged 5 to 6 months, while experiment 2 consisted of cows aged 6.5 to 7.5 years, steers aged 23 to 24 months and yearlings aged 11 to 12 months. Daily measurements of temperature, parasitaemia and packed cell volume were made in order to determine susceptibility of the different ages. Twenty-four of the 36 animals in experiment I, which included all 12 cows, required treatment. One cow died as a result of an enlarged ruptured spleen, and 2 steers and 1 calf died with classical babesiosis symptoms. No treatement was given to experiment II animals, and 5 of the 12 cows died, but the steers and yearlings underwent relatively mild reactions. Statistical analysis confirmed the high susceptibility to B. bovis of the aged cows in both experiments, and the innate resistance of 5 to 6 month old calves in experiment I. The reaction of the 18-month-old steers in experiment I was significantly greater than that of the calves, but significantly less severe than that of the aged cows. Two-year-old steers and yearlings in experiment 2 underwent similar mild reactions, suggesting that innate immunity may persist for longer periods when compared to aged cows. Age groups showing reduced susceptibility were found to reach peak parasitaemia, temperature and anaemia before the more susceptible age groups. Heterologous challenge of the remaining experiment 1 and experiment 2 animals at 6 and 8 months respectively after primary inoculation, revealed all animals of all ages had a solid resistance to B. bovis.