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A quantitative PCR, based on the gene encoding Babesia ovis Surface Protein D (BoSPD) was developed and applied to investigate the presence of Babesia ovis (B. ovis) in its principal vector, the tick Rhipicephalus bursa (R. bursa), and in the ovine host. Quantification of B. ovis in experimentally-infected lambs showed a sharp increase in parasitemia 10-11 days in blood-inoculated and adult tick-infested lambs, and 24 days in a larvae-infested lamb. A gradual decrease of parasitemia was observed in the following months, with parasites detectable 6-12 months post-infection. Examination of the parasite load in adult R. bursa during the post-molting period using the quantitative PCR assay revealed a low parasite load during days 2-7 post-molting, followed by a sharp increase, until day 11, which corresponded to the completion of the pre-feeding period. The assay was then used to detect B. ovis in naturally-infected sheep and ticks. Examination of samples from 8 sheep and 2 goats from infected flocks detected B. ovis in both goats and in 7 out of the 8 sheep. Additionally, B. ovis was detected in 9 tick pools (5 ticks in each pool) and two individual ticks removed from sheep in infected flocks.

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.

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.

Trichinellosis is a worldwide disease caused by nematode worms of the genus Trichinella, frequently diagnosed in Israel. However, the identity of the Israeli isolates have not been studied. Here we describe the molecular characterization of 58 isolates collected from jackals (Canis aureus), wild boar (Sus scrofa), foxes (Vulpes vulpes) and a wolf (Canis lupus) in central and northern Israel. Isolates were analyzed using the multiplex PCR analysis encompassing expansion segment V (ESV) and internal sequence 1 (ITS-1) markers, which identified 52 of the 58 samples. Out of the six unidentified samples, four were successfully identified using extended PCR assays for ESV and ITS-1, developed in this study. Our analysis identified 44 isolates as T. britovi, 8 as T. spiralis, four mixed infections, and two isolates were not identified. Clonal analysis of the ITS-1 sequences from six isolates confirmed the initial identification of four mixed infections. These results show that the prevalent species in Israel are T. britovi and T. spiralis, with nearly 7% (4 of 58) incidence of mixed infection.

The gene encoding Babesia ovis surface protein D (BoSPD) was cloned from B. ovis cDNA library. This gene encodes a polypeptide chain of 155 amino acids, including a predicted 22 amino acid signal peptide. Sequence analysis of the BoSPD suggested that it is a surface protein with no known domains. BLAST analysis followed by multiple alignments showed four orthologs from other Apicomplexan species and suggested that BoSPD is specific for B. ovis. BoSPD-based PCR was then developed to specifically detect B. ovis in experimentally-infected sheep and Rhipicephalus bursa ticks, as well as in field samples. The PCR enabled detection of B. ovis at a calculated parasitemia of 0.0016% and was shown to be specific for B. ovis. Moreover, the BoSPD PCR allowed detection of prolonged subclinical infection in experimentally-infected lambs and in dissected organs of experimentally-infected ticks. Finally, the PCR was used to detect parasitemia in blood samples from naturally-infected sheep and in R. bursa ticks collected from sheep in an infected flock. These results suggest that the BoSPD gene sequence can be used as a specific and sensitive marker, allowing detection of subclinical parasitemia in sheep and in ticks. Based on its predicted properties, BoSPD may be considered as a candidate for anti-B. ovis vaccine development or a target for anti-B.ovis treatment.

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.