Molecular characterization of the Israeli B. bigemina vaccine strain and field isolates.

The present study demonstrated the genetic character of the Israeli Babesia bigemina vaccine strain and field isolates, based on rap-1a and rap-1c gene sequences. The RAP-1a of blood-derived Israeli B. bigemina field isolates shared 100% amino acid sequence identity. However, comparison of RAP-1c from various Israeli B. bigemina field isolates revealed that the total sequence identity among the field isolates ranged from 98.2 to 100%. High identity was observed when RAP-1a sequences from the Israeli vaccine strain and field isolates were compared with RAP-1a from Egypt, Syria, Mexico and South Africa, while, the Israeli RAP-1c sequences showed the highest identity to the Mexican isolate JG-29 and to the PR isolate from Puerto-Rico. Based on sequence variations between the rap-1a of the vaccine strain and that of the field isolate, and between the rap-1c of the vaccine strain and that of the field isolates, nPCR-RFLP procedures were developed that enable, for the first time differentiation between the Israeli B. bigemina vaccine strain and field-infection isolates. These assays could serve as fast and sensitive methods for detection and differentiation between Israeli B. bigemina vaccine strains and field isolates, as well as for epidemiological investigations.

Differentiation between Israeli B. bovis vaccine strain and field isolates.

The present study demonstrated for the first time the ability to distinguish between the Israeli Babesia bovis vaccine strain and field isolates. The existence of an additional EcoRI restriction site in the rhoptry-associated protein-1 (rap-1) gene, which is unique to the Israeli vaccine strain, and the abolition of one of the HaeIII restriction sites in the rap-1 gene of the vaccine strain enabled distinction between the Israeli B. bovis vaccine strain and field isolates, and this was the basis for polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) development. ClustalW sequence alignment of RAP-1-deduced amino acids of the Israeli B. bovis strains and of field isolates showed that the total sequence identity among the RAP-1 amino acid sequences ranged from 97.5% to 100%. However, comparison between amino acids of RAP-1 of the Israeli vaccine strain and of field isolates, on the one hand, and B. bovis strains from Argentina, Mexico, Brazil, and USA, on the other hand, revealed 90% identity. The PCR-RFLP assay offered the great advantage of being able to distinguish between vaccine and field isolates in mixtures and provide new insight into the molecular epidemiology of B. bovis infections in Israel.

Genetic polymorphism of Babesia bovis merozoite surface antigens-2 (MSA-2) isolates from bovine blood and Rhipicephalus annulatus ticks in Israel.

This study demonstrated the genetic diversity among MSA-2c, MSA-2a1 and MSA-2b proteins of Babesia bovis isolates obtained from bovine blood and Rhipicephalus annulatus tick samples. The least identities that were observed among the deduced amino acid sequences of MSA-2c, MSA-2a1 and MSA-2b were 55, 63, and 71%, respectively. During the study four B. bovis calves, aged about 1 month, were found to be infected with virulent field strains and developed babesiosis. Probably, the calves had received insufficient antibodies, or the antibodies raised against the vaccine strain did not cross-protect against virulent field isolates. The complete msa-2 locus from the Israeli B. bovis vaccine strain and two field isolates were characterized. Similarly to the Australian strains and isolates, the msa-2 loci of the examined Israeli strain and isolates had only two msa-2 genes - msa-2c and msa-2a/b - located between msa-2c and orfB. Several of the examined samples, contained different MSA-2 genotypes concurrently. No obvious geographical relationships among isolates from various regions of Israel were established. Moreover, in the phylogenetic analyses, the Israeli deduced MSA-2 amino acid sequences of the three examined genes were clustered together with sequences derived from other countries, proving that the msa-2 gene sequences of B. bovis shared the same genetic characters worldwide. The present study clearly showed that the MSA-2 proteins of B. bovis isolates from Israel were genetically distinct from the vaccine strains. Thus, further research will be needed in order to understand the genetic diversity mechanisms of B. bovis, and the immunological responses of the infected animals.

Genetic diversity of Babesia bovis in virulent and attenuated strains.

The aim of this study was to compare the genetic diversity of the single copy Bv80 gene sequences of Babesia bovis in populations of attenuated and virulent parasites. PCR/ RT-PCR followed by cloning and sequence analyses of 4 attenuated and 4 virulent strains were performed. Multiple fragments in the range of 420 to 744 bp were amplified by PCR or RT-PCR. Cloning of the PCR fragments and sequence analyses revealed the presence of mixed subpopulations in either virulent or attenuated parasites with a total of 19 variants with 12 different sequences that differed in number and type of tandem repeats. High levels of intra- and inter-strain diversity of the Bv80 gene, with the presence of mixed populations of parasites were found in both the virulent field isolates and the attenuated vaccine strains. In addition, during the attenuation process, sequence analyses showed changes in the pattern of the parasite subpopulations. Despite high polymorphism found by sequence analyses, the patterns observed and the number of repeats, order, or motifs found could not discriminate between virulent field isolates and attenuated vaccine strains of the parasite.

Concomitant infection of cattle with the vaccine strain Anaplasma marginale ss centrale and field strains of A. marginale.

Bovine anaplasmosis, caused by Anaplasma marginale, the intraerythrocytic rickettsia, is controlled by vaccination with live Anaplasma marginale ss centrale (A. centrale), a subspecies of relatively low pathogenicity. We have experimentally demonstrated that an animal primarily infected with A. marginale, or with the related vaccine subspecies A. centrale can be infected with the heterologous subspecies, and carries both bacteria. The co-infection was detected in experimentally cross-infected calves for up to 3 months after the last inoculation with the heterologous subspecies. The occurrence of characteristic cyclic rickettsemia of A. centrale and A. marginale was observed by examination of Giemsa-stained blood smears, or by the presence of specific rickettsial DNA confirmed in PCR assays based on specific msp1a and msp4 for A. marginale, and on specifically designed msp3 and msp4 primers for A. centrale. Sequence analysis of msp4-specific fragments for each subspecies revealed the presence of dual infection in both calves on days 30 and 60 after cross-inoculation with the heterologous Anaplasma subspecies. The experimental cross-infection of calves clearly demonstrated that the concept of "infection exclusion" does not apply to Anaplasma infection in cattle; as there was no infection exclusion of A. marginale in A. centrale-infected cattle, and vice versa. The present results confirmed our previous findings that cattle grazing in an anaplasmosis-endemic field were subject to concomitant infection with both the vaccine A. centrale and the field A. marginale strains.

Molecular and serological detection of A. centrale- and A. marginale-infected cattle grazing within an endemic area.

A reverse line blot hybridization (RLB) one-stage nested PCR (nPCR) for Anaplasma centrale and a nested PCR for Anaplasma marginale were used to detect infected cattle grazing within an endemic region in Israel. A novel set of PCR primers and oligonucleotide probes based on a 16S ribosomal RNA gene was designed for RLB detection of both Anaplasma species, and the performance of the molecular assays compared. The immunofluorescent antibody test (IFA) was used to detect antibodies to both Anaplasma species, whereas, a highly sensitive and specific competitive enzyme-linked immunosorbent assay (cELISA) was used to detect antibodies in A. centrale-vaccinated cattle. The RLB and the nested PCR procedures showed bacteremia with sensitivity of 50 infected erythrocytes per milliliter. Up to 93% of the A. centrale vaccinates carried specific antibodies that were detected by cELISA, and up to 71% of the vaccinated cattle were found to be naturally infected with A. marginale according to the PCR and the RLB assays. Nevertheless, no severe outbreaks of A. marginale infection occurred among vaccinated herds in this endemic region. It appears that both, molecular tools and serology are useful for evaluation of the vaccine efficacy. In the light of wide natural field infection with A. marginale, strong recommendations to continue the A. centrale vaccination program regime will continue until a new generation of non-blood-based vaccine will be developed.