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Endemic stability is a widely used term in the epidemiology of ticks and tick-borne diseases. It is generally accepted to refer to a state of a host-tick-pathogen interaction in which there is a high level of challenge of calves by infected ticks, absence of clinical disease in calves despite infection, and a high level of immunity in adult cattle with consequent low incidence of clinical disease. Although endemic stability is a valid epidemiological concept, the modelling studies that underpinned subsequent studies on the epidemiology of tick-borne diseases were specific to a single host-tick-pathogen system, and values derived from these models should not be applied in other regions or host-tick-pathogen systems., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

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.

The merozoite surface antigen 2 (MSA-2) proteins of Babesia bovis are members of the variable merozoite surface antigen (VMSA) family that have been implicated in erythrocyte invasion and are important targets for antibody-mediated blocking of invasion. Extensive sequence variation in another VMSA member, MSA-1, has been shown in all vaccine breakthrough isolates. To test the hypothesis that the msa-2 genes of vaccine breakthrough isolates would also encode a diverse set of proteins, the complete msa-2 locus was characterized from 12 Australian B. bovis strains and isolates, including two vaccine strains and eight vaccine breakthrough isolates, and compared to the loci in previously and newly characterized American strains. In contrast to American strains, the msa-2 loci of all Australian strains and isolates examined contain, in addition to msa-2c, only a solitary gene (designated msa-2a/b) closely related to American strain msa-2a and msa-2b. Nevertheless, the proteins encoded by these genes are quite diverse both between and within geographic regions and harbor evidence of genetic exchange among other VMSA family members, including msa-1. Moreover, all but one of the Australian breakthrough isolate MSA-2a/b proteins is markedly different from the vaccine strain from which immune escape occurred, consistent with their role in strain-specific protective immunity. The densest distribution of polymorphisms occurs in a hypervariable region (HVR) within the carboxy third of the molecule that is highly proline rich. Variation in length and content of the HVR is primarily attributable to differences in the order and number of degenerate nucleotide repeats encoding three motifs of unknown function.

The Babesia bovis merozoite surface antigen 1 (MSA-1) is an immunodominant membrane glycoprotein that is the target of invasion-blocking antibodies. While antigenic variation has been demonstrated in MSA-1 among strains from distinct geographical areas, the extent of sequence variation within a region where it is endemic and the effect of variation on immunologic cross-reactivity have not been assessed. In this study, sequencing of MSA-1 from two Australian B. bovis vaccine strains and 14 breakthrough isolates from vaccinated animals demonstrated low sequence identity in the extracellular region of the molecule, ranging from 19.8 to 46.7% between the T vaccine strain and eight T vaccine breakthrough isolates, and from 18.7 to 99% between the K vaccine strain and six K vaccine breakthrough isolates. Although MSA-1 amino acid sequence varied substantially among strains, overall predicted regions of hydrophilicity and hydrophobicity in the extracellular domain were conserved in all strains examined, suggesting a conserved functional role for MSA-1 despite sequence polymorphism. Importantly, the antigenic variation created by sequence differences resulted in a lack of immunologic cross-reactivity among outbreak strains using sera from animals infected with the B. bovis vaccine strains. Additionally, sera from cattle hyperinfected with the Mexico strain of B. bovis and shown to be clinically immune did not cross-react with MSA-1 from any other isolate tested. The results indicate that isolates of B. bovis capable of evading vaccine-induced immunity contain an msa-1 gene that is significantly different from the msa-1 of the vaccine strain, and that the difference can result in a complete lack of cross-reactivity between MSA-1 from vaccine and breakthrough strains in immunized animals.

Sensitive assays are required to detect bovine retroviruses in donor cattle used for the in vivo preparation of Australian tick fever vaccines. 5' Taq nuclease assays using 3' minor groove binder DNA probes (TaqMan)MGB) were developed and compared to conventional PCR assays for the sensitive detection of bovine syncytial virus (BSV) and bovine immunodeficiency virus (BIV). Seven beef and dairy herds were screened to evaluate these tests. Comparative sensitivities of PCR tests were determined by testing log(10) dilutions of plasmids with inserts containing corresponding provirus sequences. Published PCR assays targeting BIV env sequences did not adequately amplify Australian BIV sequences. Pol sequences from Australian strains of BIV and BSV were used to design TaqMan MGB assays, which improved sensitivity 10-fold (BIV) and 100-fold (BSV), respectively, over conventional PCR tests. This is the first report of Australian sequences of BIV and BSV and the first 5' Taq nuclease assays described to detect these viruses. These methods could be applied to future studies requiring sensitive detection of these two bovine retroviruses.

Sensitive assays are required to detect proviral bovine leukemia virus (BLV) in donor cattle used for the in vivo preparation of Australian tick fever vaccines. 5' Taq nuclease assays using 3' minor groove binder DNA probes (TaqManMGB) were developed and compared to conventional PCR assays for sensitive detection of Australian BLV. Seven beef and dairy herds were screened using DNA prepared by a variety of protocols to evaluate these tests. Comparative sensitivities of PCR tests were determined by testing log(10) dilutions of plasmids with inserted BLV sequences. Animals were also screened by the BLV standard agar-gel immunodiffusion test (AGID) and commercial enzyme linked immunosorbent assays (ELISA) for antibodies, and an ELISA for detecting viral antigens expressed (VAE) in lymphocyte cultures. The TaqMan MGB assay based on the pol region was the most sensitive and specific for the detection of BLV. This is the first report of a sensitive BLV 5' Taq nuclease assay.

A 14-year-old cow (Dawn) born and kept in a Boophilus microplus-free region gave birth to a calf, which showed the presence of an Anaplasma marginale infection after splenectomy. The calf's grand dam was from a B. microplus infected area and we assume the infection originated via the transplacental route over two generations. An isolate, prepared from the calf, had similar or lower pathogenicity as Anaplasma centrale, and previously exposed steers were resistant to challenge by four A. marginale field isolates. Two attempts to transmit the isolate using B. microplus were unsuccessful. Our results indicate that Dawn A. marginale may be a useful vaccine in Australia and warrants larger scale validation of its safety and potency locally as well as of the protection it affords against African and New World isolates.

Demand for live trivalent tick fever vaccine containing Babesia bovis, Babesia bigemina and Anaplasma centrale produced by the Department of Primary Industries, Queensland, has increased from less than 10,000 doses in 1988 to 500,000 doses in 2001. This paper describes a series of trials aimed at overcoming certain constraints to obtain B. bigemina parasitised erythrocytes (PEs) on a large enough scale from infected splenectomised calves to meet the demand. Passage through a series of splenectomised calves failed to increase the yield per calf but we showed that the dose rate of infected cells could be reduced from the long-time standard of 1x10(7) to 2.5x10(6) without affecting immunogenicity and still leaving a safety margin of at least 50-fold for infectivity. This change quadrupled the potential yield of doses per calf and allowed the DPI to meet the increased demand for B bigemina in vaccine. Due to the high cost and limited availability of suitable, health tested donors, calves previously infected with B. bovis or A. centrale were used to provide B. bigemina organisms but the practice resulted in red cell agglutination in some batches of prepared vaccine. A trial is described where B. bigemina-infected red cells were washed by centrifugation to remove agglutinating antibodies. Washing had no effect on parasite viability and this method is now in routine use in the production of trivalent vaccine.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.