Productivity and health effects of anaplasmosis and babesiosis on Bos indicus cattle and their crosses, and the effects of differing intensity of tick control in Australia.

Tick fever is an important disease of cattle where Rhipicephalus (Boophilus) microplus acts as a vector for the three causal organisms Babesia bovis, Babesia bigemina and Anaplasma marginale. Bos indicus cattle and their crosses are more resistant to the clinical effects of infection with B. bovis and B. bigemina than are Bos taurus cattle. Resistance is not complete, however, and herds of B. indicus-cross cattle are still at risk of babesiosis in environments where exposure to B. bovis is light in most years but occasionally high. The susceptibility of B. indicus cattle and their crosses to infection with A. marginale is similar to that of B. taurus cattle. In herds of B. indicus cattle and their crosses the infection rate of Babesia spp. and A. marginale is lowered because fewer ticks are likely to attach per day due to reduced numbers of ticks in the field (long-term effect on population, arising from high host resistance) and because a smaller proportion of ticks that do develop to feed on infected cattle will in turn be infected (due to lower parasitaemia). As a consequence, herds of B. indicus cattle are less likely than herds of B. taurus cattle to have high levels of population immunity to babesiosis or anaplasmosis. The effects of acaricide application on the probability of clinical disease due to anaplasmosis and babesiosis are unpredictable and dependent on the prevalence of infection in ticks and in cattle at the time of application. Attempting to manipulate population immunity through the toleration of specific threshold numbers of ticks with the aim of controlling tick fever is not reliable and the justification for acaricide application should be for the control of ticks rather than for tick fever. Vaccination of B. indicus cattle and their crosses is advisable in all areas where ticks exist, although vaccination against B. bigemina is probably not essential in pure B. indicus animals.

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.

Vaccination against bovine babesiosis.

Bovine babesiosis is an important disease caused by Babesia bovis, B. bigemina, and B. divergens. Solid immunity develops after infection and this feature has been exploited with the use of live attenuated organisms as immunogens. Attributes of live vaccines include a durable immunity to heterologous challenge after one vaccination. To overcome disadvantages relating to poor quality control (risk of contamination and adverse reactions), production procedures have been modified to meet the requirements of codes of good manufacturing practice. This includes development of methods to allow production of cryopreserved vaccine and limit antigenic drift. Killed vaccines have also been used on a limited basis and consist of antigens extracted from cultured material or blood of infected calves, and given with adjuvant. The degree and duration of immunity against heterologous challenge is not well documented. Attempts are being made to develop subunit vaccines but the progress has been slow. A better understanding of the mechanisms involved in the expression of protective immunity against Babesia spp will aid in the identification of protective antigens.

A msp1alpha polymerase chain reaction assay for specific detection and differentiation of Anaplasma marginale isolates.

Anaplasma marginale is the causative agent of bovine anaplasmosis, a disease which can be protected by vaccination with the less pathogenic Anaplasma species, A. centrale. Currently, there is no polymerase chain reaction (PCR) assay available which differentiates between different species of Anaplasma or which can differentiate isolates of A. marginale within outbreaks and between different countries. A molecular test specific for A. marginale would be ideal for the identification of Anaplasma species in wild ruminants, as possible reservoirs of anaplasmosis, and to differentiate between A. marginale from A. centrale. A PCR assay was designed to amplify the major surface protein 1alpha gene of the rickettsial bovine pathogen, A. marginale both as an inter- and intra-specific test. The test did not amplify A. centrale or A. ovis, and discriminated A. marginale by amplifying repeat regions within the msp1alpha gene which vary in number between many isolates. The nested A. marginale amplicons varied in size from 630 to 1190bp representing one to eight internal repeats. All 22 Australian isolates tested amplified a 630bp product (one repeat) in contrast to all 19 non-Australian isolates tested. Eight sequences from Australian isolates from different geographical regions confirmed the conserved nature of the Australian A. marginale msp1alpha genes. The Australian 'repeat unit' MSP1a deduced amino acid sequence has been designated as Australian type 1. The msp1alpha PCR method developed here enabled the amplification and comparison of A. marginale isolates originating from North and South America, Africa, Israel and Australia. The method is sensitive and specific for A. marginale. Although additional msp1alpha products were amplified from at least two Australian isolates, the results suggest limited introduction of A. marginale into Australia.

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.

PCR methods for the discrimination of Babesia bovis isolates.

Three different polymerase chain reaction assays for the typing of isolates of Babesia bovis have been developed and compared with a hybridisation based method. Primers were designed within conserved regions flanking the variable length tandem repeats of the Bv80 and BvVA1 genes. For the long array of repeats in BvVA1, up to 7.5 kb, a modified long template PCR method was developed. The assays were compared using ten independent isolates of Babesia bovis. Using the BvVA1 and Bv80 PCR assays, 13 and 10 genotypes could be discriminated, respectively, with some isolates containing several genotypes. Combining the two PCR assays, 17 genotypes were identified within the ten Babesia bovis isolates. Whilst simpler and requiring less DNA, the BvVA1 PCR analysis exhibited significant bias towards some genotypes of the BvVA1 repeats. Further discrimination of BvVA1 PCR products was achieved using AccI digests producing population specific ladders. Genomic DNA fingerprints were also generated by PCR of DNA using an arbitrary primer (randomly amplified polymorphic DNA, RAPD) revealing polymorphic genotypes that were isolate specific. No amplification of host DNA resulted from any of the three PCR procedures. Babesia bigemina DNA was not amplified by the Bv80 or BvVA1 primers. Applications demonstrating changes in composition of populations of Babesia bovis parasites during attenuation and prolonged culture maintenance are described.

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.

A serological survey of bovine babesiosis in northern and eastern Zimbabwe.

The geographical distribution of Babesia bovis and Babesia bigemina antibodies in communal herds in northern and eastern Zimbabwe was determined using the ELISA technique. The animals in different herds in the study region had different levels of natural exposure to B. bovis (mean 32%, range 0-79%) and B. bigemina (mean 52%, range 5-92%) infections. The majority of herds (90%) were endemically unstable for B. bigemina and 62% were unstable for B. bovis. Natural region 5 and Manicaland province had the highest seroprevalence of B. bovis infection, while natural region 5 and Masvingo province had the highest seroprevalence of B. bigemina infection.

Suspected botulism in three intensively managed Australian cattle herds.

Serious outbreaks of a paralytic disease in cattle occurring in the spring and summer of 1988 were investigated on three farms in south eastern Queensland, Australia. On one farm 237 (31 per cent) of 770 cattle died, on the second 109 (40 per cent) of 271 cattle died and on the third 30 (8 per cent) of 380 cows died. Botulism was suspected on the basis of the clinical signs, the lack of significant pathology, a failure to incriminate other agents and a positive feeding trial in one sheep. Laboratory tests for the presence of botulinum toxin failed to confirm this diagnosis, and further feeding trials using ingredients of two rations were also negative.

Immunity following use of Australian tick fever vaccine: a review of the evidence.

OBJECTIVE: To review the evidence available on the degree and duration of immunity provided by Australian tick fever vaccines against Babesia bovis, B. bigemina and Anaplasma marginale infections in Australia and overseas. BACKGROUND: Vaccines containing attenuated strains of B bovis and B bigemina as well as A. centrale grown in splenectomised calves have been used in Australia since 1964 to immunise cattle against tick fever. About 800,000 doses of vaccine are supplied annually and much of the evidence for protection is field evidence rather than conventional immunological measures or pen trials. CONCLUSIONS: Immunity to Babesia bovis and B. bigemina--A single inoculation generally provides sound, long-lasting protection both in Australia and overseas. No evidence was found of a loss of immunity with time. Vaccine failures to B. bovis do occur, but are uncommon and evidently caused by a number of factors, including immune responsiveness of the vaccinated animals, and immunogenicity of the vaccine strain. Immunity to Anaplasma marginale--The vaccine containing A. centrale provides partial, variable protection against A. marginale. Protection against challenge in Australia is adequate in most cases to prevent disease and use of the vaccine in this country appears to be justified. Protection against antigenically diverse, highly virulent stocks of A. marginale in other countries is, at times, clearly inadequate and better vaccines are required in situations where the challenge is severe.

Detection of calves persistently infected with bovine pestivirus in a sample of dairy calves in south-eastern Queensland.

OBJECTIVE: To determine the proportion and incidence of calves persistently infected with bovine pestivirus in calves (n = 1521) supplied to the Tick Fever Research Centre and to assess the test regime to detect calves persistently infected with bovine pestivirus. DESIGN: Calves, 1 to 6 weeks old, selected for use in the production of the tick fever vaccine were collected from 21 properties in 56 separate groups between October 1990 and December 1996. Each group was examined for the presence of calves persistently infected with bovine pestivirus. PROCEDURE: All calves were routinely tested for antibody to bovine pestivirus and bovine pestivirus antigen using a serum neutralisation test and an antigen-capture ELISA, respectively. Pooled lymphocyte samples from calves were also monitored for bovine pestivirus by inoculation of sheep. Whole herd testing was carried out in eight herds, using a serum neutralisation test as a screen test followed by an antigen-capture ELISA of cattle with a serum neutralisation test titre of less than 32. RESULTS: Fourteen of the 1521 calves tested (0.9%), were detected as persistently infected and the incidence ranged from 0.0 to 3.0% per year over 6 years. Persistently infected calves were found in 13 of the 59 groups and originated from 7 of the 21 herds used. In whole herd testing on the properties of origin, cattle persistently infected with bovine pestivirus were detected in four of the eight herds tested. CONCLUSIONS: The proportion of calves persistently infected with bovine pestivirus is similar to that in other countries and indicates that bovine pestivirus could be a significant cause of economic loss in Australian cattle herds. In detecting calves persistently infected with bovine pestivirus, the combination of sheep inoculation, paired antigen-capture ELISA and serum neutralisation tests appeared to be highly sensitive and specific.

Development of an enzyme linked immunosorbent assay (ELISA) for the detection of bovine serum antibody to bovine viral diarrhoea virus.

An enzyme linked immunosorbent assay (ELISA) was developed to detect antibody to bovine viral diarrhoea virus (BVDV) in bovine serum. The ELISA results were compared with those of the serum neutralisation test (SNT) using serums from 6 experimentally infected calves bled at intervals from 0 to 154 days postinfection and 886 field samples. The optical density (OD) produced by a single dilution of test serum was compared with a standard curve and the result expressed in ELISA units. Despite wide variation between absolute ELISA and SNT results, an agreement of 97% was obtained when reciprocal SNT titres greater than or equal to 8 and ELISA units greater than or equal to 10 were taken as indicative of a specific reaction. The ELISA was shown to be an efficient method of measuring antibody in bovine serum samples and would assist in any large scale screening of cattle herds for BVDV antibody.

Effect of breed of cattle on transmission rate and innate resistance to infection with Babesia bovis and B bigemina transmitted by Boophilus microplus.

OBJECTIVE: To assess the effect of breed of cattle on the transmission rates of and innate resistance to Babesia bovis and B bigemina parasites transmitted by Boophilus microplus ticks. DESIGN: Groups of 56 purebred B indicus and 52 B indicus cross B taurus (50%, F1 generation) steers were placed in a paddock seeded with and also naturally infested with B microplus which were the progeny of females ticks fed on B taurus cattle specifically infected with a virulent isolate of B bovis. The cattle were placed in the infested paddock 50 days after seeding had started. PROCEDURE: Cattle were inspected from horseback daily for 50 days. Clinically ill cattle were brought to yards and assessed by monitoring fever, depression of packed-cell volume, parasitaemia and severity of clinical signs. Any animals that met preset criteria were treated for babesiosis. Blood samples were collected from all cattle on day 28, 35 and 42 after exposure and antibodies to Babesia spp and packed cell volume measured. RESULTS: All steers, except for one crossbred, seroconverted to B bovis and B bigemina by day 35 and 75% of the crossbred steers showed a maximum depression in packed cell volume of more than 15% due to infection with Babesia spp compared with only 36% of the B indicus group. Ten of the 52 crossbreds and 1 of the 56 B indicus steers showed severe clinical signs. Two of the crossbreds required treatment of which one died 2 weeks after initial treatment. CONCLUSIONS: Pure-bred B indicus cattle have a high degree of resistance to babesiosis, but crossbred cattle are sufficiently susceptible to warrant the use of preventive measures such as vaccination. Transmission rates of B bovis and B bigemina to B indicus and crossbred cattle previously unexposed to B microplus were the same.

Effect of breed of cattle on innate resistance to infection with Anaplasma marginale transmitted by Boophilus microplus.

OBJECTIVE: To assess the innate resistance of and transmission in naive Bos taurus cross Bos indicus and purebred Bos indicus cattle when placed in a paddock with cattle infected with Anaplasma marginale and carrying Boophilus microplus ticks. DESIGN: A group of 49 purebred B indicus, and 48 B indicus cross B taurus (50%, F1 generation) 24-month-old steers were kept in the same paddock with cattle artificially infected with a virulent isolate of A marginale and Boophilus microplus. The cattle were seronegative for A marginale at the start of the trial but had previously been exposed to Babesia bovis and B bigemina. PROCEDURE: Cattle were inspected twice weekly for 118 days. Whole blood, blood smears and serum samples were collected from the cattle on day 37 after exposure and then at regular intervals to day 83 after exposure to measure packed-cell volumes, parasitaemias and antibody titres to A marginale. Any animals that met preset criteria were treated for anaplasmosis. On day 83 all cattle were treated with an acaricide and cattle infected with A marginale were removed from the rest of the group. RESULTS: A marginale was detected in blood smears from 14 crossbred and 9 B indicus steers between days 56 and 72 after exposure. Five and two of the infected crossbred and B indicus steers required treatment, respectively. One of the Bos indicus cattle died as a result of the A marginale infection despite treatment. Antibodies to A marginale were detected in the 23 infected cattle. The mean packed-cell volume depression was 40 and 37% in the affected crossbred and Bos indicus groups, respectively. There was no significant difference detected in susceptibility between these two groups. CONCLUSIONS: Innate resistance of purebred B indicus and crossbred cattle was not significantly different. The results confirm that purebred B indicus and crossbred cattle are sufficiently susceptible to warrant the use of vaccination against Anaplasma infections.

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.

Studies on failure of T strain live Babesia bovis vaccine.

Field investigations of the protection afforded by the Australian live Babesia bovis vaccine used in the early 1990s (T strain) revealed inadequate vaccine-induced protection in certain herds. Vaccination/challenge trials using 207 experimental cattle were conducted to evaluate the protection afforded by T strain B bovis against field isolates from these herds. The trials investigated whether isolates that could 'break-through' T strain immunity were present in the field, the ability or inability of specific cattle to develop protective immunity after vaccination with T strain and the effect of attenuation and maintenance procedures on the immunogenicity of T strain. The results showed that B bovis parasites present early in the process of attenuation of T strain were more protective than those remaining late in the process. They also showed that cattle from properties experiencing vaccine failures were less protected by T strain vaccination than Bos taurus cattle randomly selected from the general population if vaccinated with highly attenuated T strain. A hypothesis is offered to explain these findings.

Effect of breed of cattle on innate resistance to infection with Babesia bovis, B bigemina and Anaplasma marginale.

OBJECTIVE: To assess the innate resistance of naive Bos taurus, Bos taurus cross Bos indicus and Bos indicus cattle to virulent Babesia bovis, B bigemina and Anaplasma marginale parasites. DESIGN: Groups of 10, pure B indicus, 1/2 B indicus cross, 1/4 B indicus cross and pure B taurus steers were infected with virulent B bovis, B bigemina and A marginale parasites [corrected]. PROCEDURE: Sequential infections were carried out by intravenous inoculation of infected blood containing 1 x 10(8) parasites of B bovis, followed by B bigemina and then A marginale. To assess resistance, measurements were made of parasitaemia, rectal temperature, packed cell volume and the number within a group requiring chemotherapy to control infection. There was a recovery period between each infection. RESULTS: Infection with B bovis showed that pure B indicus steers were significantly more resistant to B bovis infection than the other groups, with none of this group requiring treatment. There was no significant difference between 1/2 B indicus cross and 1/4 B indicus cross with 30% and 20%, respectively, of steers in these groups requiring treatment [corrected]. The pure B taurus steers were significantly more affected then those in the other three groups with 80% requiring treatment. Infections of B bigemina produced a mild response in comparison to that of B bovis and none of the steers required treatment. However, the pure B taurus group was significantly more affected than the other three groups for all other measurements. After the A marginale infection, B indicus steers were moderately affected with 50% requiring treatment, whereas 70% of the 1/2 B indicus group, 80% of the 1/4 B indicus cross group and 100% of the pure B taurus group required treatment [corrected]. CONCLUSIONS: All breeds of cattle, ranging from pure B indicus to pure B taurus may be at risk of severe disease if exposed to virulent A marginale. The results confirm that pure B indicus cattle are relatively resistant to B bovis, but there could be a significant risk of severe mortalities if cross-bred herds are exposed to virulent infection.

Serological evidence of exposure to tick fever organisms in young cattle on Queensland dairy farms.

OBJECTIVES: To compare the features of farms on which the exposure of young cattle to tick fever organisms is sufficient to ensure that immunity is high and the risk of clinical disease is low (endemic stability) with those of farms on which exposure is insufficient to induce widespread immunity (hence without endemic stability); to examine the relationships between the management of ticks and tick fever, and endemic stability to Babesia bovis, B. bigemina and Anaplasma marginale. DESIGN: Cross-sectional study of 874 cattle between the ages of 6 and 15 months on 64 dairy farms, from three centres in south-eastern Queensland (Mutdapilly, Dayboro and Kenilworth) and one centre in far-north Queensland (Malanda). PROCEDURE: Blood samples collected from between 5 and 20 calves from each farm were submitted for serological assay to determine exposure to B. bovis, B. bigemina and A. marginale. A questionnaire about the farm characteristics and the management of ticks and tick fever was completed with each farmer. RESULTS: On 73% of farms, confirmed clinical cases of tick fever were recalled by the farmer, indicating that tick fever was a threat on most farms. The majority of herds in the study (54 of 64) did not have sufficient numbers of seropositive animals aged between 6 and 15 months to have a low risk of tick fever. Region had an effect on the likelihood of endemic stability for all tick fever organisms. Cattle near Malanda in Far-north Queensland were more likely to be seropositive to B. bovis and B. bigemina. The method, strategy and intensity of tick control were not related to the likelihood of endemic stability when the effect of region was considered. The decision to leave a few ticks on cattle in an effort to induce endemic stability did increase the likelihood of endemic stability to A. marginale. However, in practical terms, it was ineffective, because only 26% of these farms had endemic stability against all three organisms. CONCLUSIONS: Given the low proportion of farms that have endemic stability to the tick fever organisms and the high likelihood of clinical disease, vaccination is recommended to protect dairy cattle from tick fever throughout the tick infested area of Queensland. However, further work is required to determine the economic value of vaccination, taking into account the costs of vaccination, of outbreaks and the protective value of vaccination.

Application of PCR assays to determine the genotype of Babesia bovis parasites isolated from cattle with clinical babesiosis soon after vaccination against tick fever.

OBJECTIVE: To demonstrate the value of PCR assays to determine the genotypes of Babesia bovis in cattle with clinical signs of babesiosis within 3 weeks after vaccination against tick fever. DESIGN: Samples from 5 cases of babesiosis in cattle soon after vaccination against tick fever were analysed in two PCR assays. PROCEDURE: Parasite DNA was purified from blood taken from cattle with signs of babesiosis within 3 weeks of vaccination against tick fever. DNA was also prepared from the tissues of animals that died of babesiosis. Two PCR assays that amplify repeat sequences of DNA within the B bovis genes, Bv80 and BvVA1, were used to differentiate the genotypes of field isolates and vaccine strains of B bovis. RESULTS: One of the five cases of babesiosis was found to be caused by a vaccine strain, but PCR analyses showed that the predominant isolate in the other four cases was not the vaccine strain. CONCLUSIONS: PCR assays on the DNA of B bovis obtained from the blood or tissues of cattle clinically affected with tick fever within 3 weeks after vaccination are useful to distinguish between vaccine strains and field isolates as the source of infection.

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.