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 enzymelinked immunosorbent assay (cELISA) was used to detect antibodies in A. centralevaccinated 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-bloodbased vaccine will be developed. Article Outline

A system biology approach was used to gain insight into tick biology and interactions between vector and pathogen. Rhipicephalus annulatus is one of the main vectors of Babesia bigemina which has a massive impact on animal health. It is vital to obtain more information about this relationship, to better understand tick and pathogen biology, pathogen transmission dynamics, and new potential control approaches. In ticks, salivary glands (SGs) play a key role during pathogen infection and transmission. RNA sequencing obtained from uninfected and B. bigemina infected SGs obtained from fed female ticks resulted in 6823 and 6475 unigenes, respectively. From these, 360 unigenes were found to be differentially expressed ( p < 0.05). Reversed phase liquid chromatography–mass spectrometry identified a total of 3679 tick proteins. Among them 406 were differently represented in response to Babesia infection. The omics data obtained suggested that Babesia infection lead to a reduction in the levels of mRNA and proteins ( n = 237 transcripts, n = 212 proteins) when compared to uninfected controls. Integrated transcriptomics and proteomics datasets suggested a key role for stress response and apoptosis pathways in response to infection. Thus, six genes coding for GP80, death-associated protein kinase (DAPK-1), bax inhibitor-1 related (BI-1), heat shock protein (HSP), heat shock transcription factor (PHSTF), and queuine trna-ribosyltransferase (QtRibosyl) were selected and RNA interference (RNAi) performed. Gene silencing was obtained for all genes except phstf. Knockdown of gp80 , dapk-1 , and bi-1 led to a significant increase in Babesia infection levels while hsp and QtRibosyl knockdown resulted in a non-significant decrease of infection levels when compared to the respective controls. Gene knockdown did not affect tick survival, but engorged female weight and egg production were affected in the gp80 , dapk-1 , and QtRibosyl -silenced groups in comparison to controls. These results advanced our understanding of tick– Babesia molecular interactions, and suggested new tick antigens as putative targets for vaccination to control tick infestations and pathogen infection/transmission.

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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.

Infection of cattle with

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