Expression of Anaplasma marginale ankyrin repeat-containing proteins during infection of the mammalian host and tick vector.

Transmission of tick-borne pathogens requires transition between distinct host environments with infection and replication in host-specific cell types. Anaplasma marginale illustrates this transition: in the mammalian host, the bacterium infects and replicates in mature (nonnucleated) erythrocytes, while in the tick vector, replication occurs in nucleated epithelial cells. We hypothesized that proteins containing ankyrin motifs would be expressed by A. marginale only in tick cells and would traffic to the infected host cell nucleus. A. marginale encodes three proteins containing ankyrin motifs, an AnkA orthologue (the AM705 protein), AnkB (the AM926 protein), and AnkC (the AM638 protein). All three A. marginale Anks were confirmed to be expressed during intracellular infection: AnkA is expressed at significantly higher levels in erythrocytes, AnkB is expressed equally by both infected erythrocytes and tick cells, and AnkC is expressed exclusively in tick cells. There was no evidence of any of the Ank proteins trafficking to the nucleus. Thus, the hypothesis that ankyrin-containing motifs were predictive of cell type expression and nuclear localization was rejected. In contrast, AnkA orthologues in the closely related A. phagocytophilum and Ehrlichia chaffeensis have been shown to localize to the host cell nucleus. This difference, together with the lack of a nuclear localization signal in any of the AnkA orthologues, suggests that trafficking may be mediated by a separate transporter rather than by endogenous signals. Selection for divergence in Ank function among Anaplasma and Ehrlichia spp. is supported by both locus and allelic analyses of genes encoding orthologous proteins and their ankyrin motif compositions.

Identification of Anaplasma marginale proteins specifically upregulated during colonization of the tick vector.

The transition between infection of the mammalian host and colonization of an arthropod vector is required for the ongoing transmission of a broad array of pathogens, from viruses to protozoa. Understanding how this transition is mediated provides opportunities to disrupt transmission through either chemotherapy or immunization. We used an unbiased proteomic screen to identify Anaplasma marginale proteins specifically upregulated in the tick compared to the mammalian host. Comparative mass spectrometric analysis of proteins separated by two-dimensional gel electrophoresis of uninfected and infected ISE6 cells and infected mammalian cells identified 15 proteins exclusively expressed or upregulated in tick cells. All 15 had originally been annotated as hypothetical proteins. We confirmed quantitative upregulation and expression in situ within the midgut epithelial and salivary gland acinar cells of vector ticks during successful transmission. The results support the hypothesis that A. marginale gene expression is regulated by the specific host environment and, in a broader context, that the core genome evolved in the arthropod vector with differential regulation, allowing adaptation to mammalian hosts. Furthermore, the confirmation of the in situ expression of candidates identified in ISE6 cell lines indicates that this approach may be widely applicable to bacteria in the genera Anaplasma and Ehrlichia, removing a major technical impediment to the identification of new targets for vaccine and chemotherapeutic blocking of transmission.

Association of pathogen strain-specific gene transcription and transmission efficiency phenotype of Anaplasma marginale.

Efficient transmission of pathogens by an arthropod vector is influenced by the ability of the pathogen to replicate and develop infectiousness within the arthropod host. While the basic life cycle of development within and transmission from the arthropod vector are known for many bacterial and protozoan pathogens, the determinants of transmission efficiency are largely unknown and represent a significant gap in our knowledge. The St. Maries strain of Anaplasma marginale is a high-transmission-efficiency strain that replicates to a high titer in the tick salivary gland and can be transmitted by <10 ticks. In contrast, A. marginale subsp. centrale (Israel vaccine strain) has an identical life cycle but replicates to a significantly lower level in the salivary gland, with transmission requiring >30-fold more ticks. We hypothesized that strain-specific genes expressed in the tick salivary gland at the time of transmission are linked to the differences in the transmission efficiency phenotype. Using both annotation-dependent and -independent analyses of the complete genome sequences, we identified 58 strain-specific genes. These genes most likely represent divergence from common ancestral genes in one or both strains based on analysis of synteny and lack of statistical support for acquisition as islands by lateral gene transfer. Twenty of the St. Maries strain-specific genes and 16 of the strain-specific genes in the Israel strain were transcribed in the tick salivary gland at the time of transmission. Although associated with the transmission phenotype, the expression levels of strain-specific genes were equal to or less than the expression levels in infected erythrocytes in the mammalian host, suggesting that function is not limited to salivary gland colonization.

Seroprevalence of Selected Tick Borne Pathogens and Diversity and Abundance of Ixodid Ticks (Acari: Ixodidae) at the Wildlife-Livestock Interface in Northern Botswana.

Ticks and tick borne diseases (TBDs) undermine livestock production with considerable economic losses to livestock producers in endemic areas worldwide. Despite the impact of ticks and TBDs in livestock production, there is a paucity of information on ticks and diseases they transmit in Botswana. To address this gap, a cross-sectional study was conducted to determine (i) the seroprevalence of selected tick borne (TB) pathogens and (ii) the diversity and abundance of ixodid ticks among 301 cattle foraging around two protected areas in northern Botswana, differing by the presence or absence of a physical barrier (fence) separating wildlife and livestock. Competitive inhibition enzyme linked immuno-sorbent assay (cELISA) was used to test for Anaplasma spp. infection and Indirect Fluorescence Antibody Test (IFAT) was used to test for Theileria parva, Babesia bovis, and B. bigemina. Ticks were identified morphologically at either genus or species level. Seroprevalence of cattle was found to be 90% for Anaplasma spp., followed by 38.6% for Babesia spp. and 2.4% for T. parva. Except for Babesia spp., comparisons of the seroprevalence of the selected haemoparasites between the two wildlife-livestock interface areas were not significantly different. The overall prevalence of ticks was found to be 73.4% with Amblyomma variegatum being the most abundant (53.1%) followed by Rhipicephalus evertsi evertsi (31.7%) and R. (B.) decoloratus (7.7%). Except for Babesia spp., comparisons of the seroprevalence of the selected haemoparasites between the two study areas were not significantly different while comparisons of the burden of tick infestation between the study sites revealed significant difference for A. variegatum and R. evertsi evertsi with both tick infestations higher where there is no barrier. Our work provided baseline data on TBD pathogens and tick infestation in cattle populations exposed to different levels of contact with adjacent buffalo populations. The presence of a veterinary fence did not significantly influence the seroprevalence of the selected TBD pathogens (except for Babesia spp.) but seemed to reduce tick burdens in cattle. Findings from this study can be used for guiding future epidemiological study designs to improve our understanding of ticks and TBDs dynamics in northern Botswana.

First-time detection of bovine viral diarrhoea virus, BVDV-1, in cattle in Botswana.

Infectious diseases are serious constraints for improving livestock productivity. Bovine viral diarrhoea virus (BVDV) is a virus causing grave economic losses throughout the cattle producing world. Infection is often not apparent, but the virus can also cause respiratory signs, diarrhoea, reproductive problems and immunosuppression. Risk factors for disease transmission include, but are not limited to, herd size, animal trade and grazing on communal pastures. Several prevalence studies have been conducted in southern Africa, but in Botswana the occurrence is largely unknown. In this study, blood samples were obtained from 100 goats from three villages around the capital city, Gaborone. Also, 364 blood samples from cattle around Gaborone, collected as part of another study, were analysed. The detected antibody prevalence was 0% in goats and 53.6% in cattle when using a competitive enzyme-linked immunoassay. Three animals from two different herds were positive for viral nucleic acids on polymerase chain reaction. The two herds with viraemic animals had significantly higher antibody prevalence compared to the other herds. Also, two of the detected viruses were sequenced and found to be most similar to BVDV-1a. To the authors' knowledge, this is the first time that sequencing has been performed on BVDV isolated in Botswana.

Anaplasma infection prevalence in beef and dairy cattle in the south east region of Botswana.

Infection of cattle by the tick-borne intra-erythrocytic bacteria of the genus Anaplasma occurs worldwide. Nevertheless, prevalence rates in specific regions are still required to inform cattle farming management decisions. A study was carried out to determine Anaplasma infection prevalence in beef and dairy cattle in the south east region of Botswana. Two methods were used: competitive inhibition enzyme-linked immune-sorbent assay (cELISA) and conventional polymerase chain reaction (PCR). A total of 429 cattle consisting 207 beef and 222 dairy animals were sampled and tested. The prevalence was 91% and 31% by cELISA and PCR, respectively. A Z test revealed a statistical difference between the point prevalence as determined by cELISA compared to PCR (p=0). There was no statistical difference between the point prevalence of Anaplasma infection as determined by cELISA (p=0.45) between beef and dairy cattle. But there was a significant difference (p=0.001) between the animals by PCR with the prevalence in beef cattle nearly double that in dairy cattle. Individual herd prevalence ranged from 79% to 100% by cELISA, and 0 to 100% by PCR. Though not statistically significant sero-prevalence in both beef and dairy animals tended to be higher in urban/peri-urban areas compared to rural areas. The cELISA mean percentage inhibition (PI) for all cattle was found to be 58.6 (95% CI: 56.8-60.4). There was no statistically significant difference between the mean PI of sera from beef cattle (56.4 (95% CI: 54.1-58.7)) as compared to dairy cattle (60.7 (95%CI: 58.0-63.3)). However, there was a tendency towards statistical significance with beef animals having a lower PI value than dairy animals. Anaplasma infection was endemic in cattle in the south east region of Botswana with similar infection in beef and dairy animals. Further research should be done to identify the strains prevalent in the cattle herds.