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.

The effect of a live Neospora caninum tachyzoite vaccine in naturally infected pregnant dairy cows.

Neosporosis, caused by the intracellular protozoan Neospora caninum, is a major cause of abortion and reproductive failure in cattle worldwide. The principal route of transmission of neosporosis is via in utero infection of the offspring. There is no effective prophylactic treatment or vaccine available against bovine neosporosis. A N. caninum NcIs491 isolate was examined for its ability to immunize and reduce abortions in naturally infected dairy cows under field conditions. N. caninum-seropositive pregnant dams were inoculated with 10(8) live tachyzoites during mid-term pregnancy. A total of 520 N. caninum seropositive dams were included in this study, of these, 146 were immunized and 374 cows served as a non-vaccinated control group. A significantly lower incidence of abortion was observed in vaccinated compared to non-vaccinated cows, 16 and 26% respectively (P=0.01), with a vaccine efficacy of 39%. However, the number of seropositive offspring remained similar in both groups. Overall, this field trial suggests that vaccination with live N. caninum tachyzoites should be considered as an effective measure to reduce abortions caused by neosporosis in naturally infected cows.

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.

Comparative analysis of mitochondrial markers from four species of Rhipicephalus (Acari: Ixodidae).

Mitochondrial sequences of four mitochondrial markers: 12S rRNA, 16S rRNA, cytochrome c oxidase subunit I (COX1) and cytochrome b (CytB) from four Rhipicephalus species were analyzed to establish genetic relationships and enable molecular identification. Field-collected samples from the species Rhipicephalus annulatus, Rhipicephalus bursa, Rhipicephalus sanguineus and Rhipicephalus turanicus were amplified by PCR and compared with GenBank™ annotated sequences. PCR products were obtained using primers that were designed to amplify orthologous sequences from different tick species and genera. The average intra-species sequence identity was 98.5-99.5%, while the average inter-species identity was 86.5-89.6%, reflecting a ≈ 10% decrease in the identity, when different species are compared. The "closest" two species, in terms of sequence identity, were R. sanguineus and R. turanicus, while the "least close" ones were R. annulatus and R. sanguineus. Molecular identification of each species was accomplished by a combined restriction analysis of 12S, COX1 and CytB markers, obviating the need for field sample sequencing. The restriction mapping data suggest that by using several markers, each with a unique digestion pattern, the identity of a given sample could be determined at the species level. It is anticipated that with the accumulation of more information on additional species and markers, molecular identification will become a standard approach for tick classification, complementing morphological taxonomy.

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.