Prevalence of Ticks Infesting Dairy Cattle and the Pathogens They Harbour in Smallholder Farms in Peri-Urban Areas of Nairobi, Kenya

This study aimed at determining the tick species infesting dairy cattle in Nairobi, Kenya, and the pathogens they harbour. While ticks are well-known vectors of major bacterial pathogens of both veterinary importance and public health importance, few studies have investigated the range of the tick species and the associated pathogens, especially present in unique dairy production systems, which compromise animal welfare, such as those in peri-urban areas. A cross-sectional study was undertaken involving 314 randomly selected dairy cattle in 109 smallholder farms. Each animal was examined for attached ticks followed by morphological tick identification at the species level. Genomic DNA was extracted from each of the ticks, and 16S rDNA gene was amplified for pathogen identification. Sequencing of the amplicons and subsequent BLASTn analysis, multiple sequence alignment, and phylogenetic reconstruction were performed to confirm the species of the pathogens. Sixty-six (21.0%) of the cattle examined had ticks. A total of 94 adult ticks were found on the cattle, and of these, 63 (67.0%), 18 (19.1%), and 13 (13.8%) were in the genera Rhipicephalus, Amblyomma, and Hyalomma, respectively. Twelve tick species in Rhipicephalus genus and two in Amblyomma and Hyalomma genera were identified. Although Rh. decoloratus was the most prevalent tick (24.5% (23/94)), the emerging Rh. microplus (6.4% (6/94)) was also identified. The DNA of Rickettsia was detected in the ticks, with Rickettsia conorii in H. rufipes and A. variegatum, and Rickettsia aeschlimannii in Rh. microplus and H. rufipes, while Ehrlichia ruminantium and E. canis were in A. variegatum. In conclusion, the study reported a wide range of tick species present in the study area including Rhipicephalus microplus, which is an emerging tick species in parts of Kenya. The ticks harboured DNA of Rickettsia and Ehrlichia, highlighting possible animal and human health concerns. Hence, effective tick control strategies remain paramount to prevent potential diseases associated with the harboured pathogens.

Molecular prevalence and risk factors associated with tick-borne pathogens in cattle in western Kenya

Background Tick-borne pathogens (TBPs) are of global importance, especially in sub-Saharan Africa where they represent a major constraint to livestock production. Their association with human disease is also increasingly recognized, signalling their zoonotic importance. It is therefore crucial to investigate TBPs prevalence in livestock populations and the factors associated with their presence. We set out to identify TBPs present in cattle and to determine associated risk factors in western Kenya, where smallholder livestock production is important for subsistence and market-driven income. Results Tick-borne pathogen infections in blood samples collected from cattle at livestock markets and slaughterhouses between May 2017 and January 2019 were identified by high-resolution melting analysis and sequencing of PCR products of genus-specific primers. Of the 422 cattle sampled, 30.1% (127/422) were infected with at least one TBP, while 8.8% (37/422) had dual infections. Anaplasma spp. (19.7%) were the most prevalent, followed by Theileria (12.3%), Ehrlichia (6.6%), and Babesia (0.2%) spp. Sequence analysis of the TBPs revealed them to be Anaplasma platys-like organisms (13.5%), Theileria velifera (7.4%), Anaplasma marginale (4.9%), Theileria mutans (3.1%), Theileria parva (1.6%), and Babesia bigemina (0.2%). Ehrlichia ruminantium, Rickettsia spp., and arboviruses were not detected. Exotic breeds of cattle were more likely to be infected with A. marginale compared to local breeds (OR: 7.99, 95% CI: 3.04–22.02, p < 0.001). Presence of ticks was a significant predictor for Anaplasma spp. (OR: 2.18, 95% CI: 1.32–3.69, p = 0.003) and Ehrlichia spp. (OR: 2.79, 95% CI: 1.22–7.23, p = 0.022) infection. Cattle sampled at slaughterhouses were more likely to be positive for Anaplasma spp. (OR: 1.64, 95% CI: 1.01–2.70, p = 0.048) and A. marginale (OR: 3.84, 95% CI: 1.43–12.21, p = 0.012), compared to those sampled at livestock markets. Conclusion This study reports TBP prevalence and associated risk factors in western Kenya, factors which are key to informing surveillance and control measures.

Molecular Survey of Anaplasmataceae Agents and Coxiellaceae in Non-Hematophagous Bats and Associated Ectoparasites from Brazil

The Anaplasmataceae family (order Rickettsiales) encompasses obligately intracellular bacteria of the genera Anaplasma, Ehrlichia, and Neorickettsia. Together with Coxiella burnetii (Coxiellaceae family, order Legionellales), these bacteria represent important causative agents of diseases in humans and animals. The scarcity of studies that investigated the occurrence of these agents in bats and their associated ectoparasites, emphasizes the need to achieve a better understanding of the role of these animals in the maintenance of such bacteria. Herein, 418 samples (133 blood, 135 spleen, and 150 ectoparasites) are collected from 135 non-hematophagous bats belonging to 12 species in a periurban area of Campo Grande city, Mato Grosso do Sul state, midwestern Brazil. In the results, 1.65% (7/418), 12.04% (50/418), and 13.63% (57/418) of samples are positive in PCR assays for Anaplasma spp. (16S rRNA gene), Ehrlichia spp. (dsb gene), and Neorickettsia spp. (16S rRNA gene), respectively. Anaplasma spp. and Neorickettsia spp. are detected in one (5.26%) Ornithodoros hasei tick larva. Ehrlichia spp. is detected in 14% of bat flies (represented by Megistopoda aranea, Trichobius costalimai, and Strebla hertigi), 6% of tick larvae (O. hasei), 12% of Spinturnicidae mites (represented by Periglischrus sp., P. torrealbai, and P. acutisternus), and 38% of Macronyssidae mites (Steatonyssuss sp.). The obtained sequences are observed to be similar to Anaplasma phagocytophilum (97.42–97.6% identified), Ehrlichia minasensis (96.73–100% identified), Neorickettsia risticii (96.7–100% identified), and Neorickettsia findlayensis (95.07–100% identified) by BLASTn analyses, and closely related to Ehrlichia ruminantium by phylogenetic analyses based on the gltA gene. No bat samples (blood/spleen) are positive in the qPCR assay for C. burnetii based on the IS1111 gene. The present work shows, for the first time, the occurrence of Anaplasmataceae in bats and associated ectoparasites (ticks, mites, and bat flies) from Brazil.

The Influence of Eroding Djallonke Genotype on the Incidence of Diseases and Mortalities in Sahalian X Djallonke Cross-bred Sheep

Despite cross breeding of Djallonke and Sahelian sheep due to the higher growth rate and final body weight of the Sahelian breeds over the years, little scientific evidence exists on the ability of the crosses to withstand diseases compared to their Djallonke parents. A study involving 700 Djallonke and two crosses of the Djallonke and Sahelian breeds (50% Sahel × 50% Djallonke, 75% Sahel × 25% Djallonke) was conducted, and documented incidence of diseases, parasites and mortalities in these animals. Enteritis (82%), Ehrlichia ruminantium (6.2%) and orf (6.2%) were more prevalent in 25% Djallonke than both 100% and 50% Djallonkes. Similarly, pneumonia was more prevalent in 50% Djallonkes, individuals of all age groups and females than all other genotypes, adults and males, respectively. Enteritis prevalence was also higher in lambs and males than animals of all age groups and females, respectively. Coccidiosis was more prevalent in individuals of all age groups and males, than lambs and females, respectively. More abscess and trauma cases were found in males than females. Significantly higher levels of tapeworms and flea infestations were seen in lambs than weaners and adults. Conversely, more mange mite and tick infestations were seen in adults than weaners and lambs. Higher levels of mortalities were recorded in all other genotypes, lambs, females and in haemoncosis cases than in 100% Djallonke, all other age groups, males and taeniasis cases, respectively. The Sahel x Djalloke crosses are more susceptible to infectious diseases and record higher mortality rates than the pure Djallonke genotypes.

Comparative Genomics of Clinical Isolates of the Emerging Tick-Borne Pathogen Neoehrlichia mikurensis

Tick-borne ‘Neoehrlichia (N.) mikurensis’ is the cause of neoehrlichiosis, an infectious vasculitis of humans. This strict intracellular pathogen is a member of the family Anaplasmataceae and has been unculturable until recently. The only available genetic data on this new pathogen are six partially sequenced housekeeping genes. The aim of this study was to advance the knowledge regarding ‘N. mikurensis’ genomic relatedness with other Anaplasmataceae members, intra-species genotypic variability and potential virulence factors explaining its tropism for vascular endothelium. Here, we present the de novo whole-genome sequences of three ‘N. mikurensis’ strains derived from Swedish patients diagnosed with neoehrlichiosis. The genomes were obtained by extraction of DNA from patient plasma, library preparation using 10x Chromium technology, and sequencing by Illumina Hiseq-4500. ‘N. mikurensis’ was found to have the next smallest genome of the Anaplasmataceae family (1.1 Mbp with 27% GC contents) consisting of 845 protein-coding genes, every third of which with unknown function. Comparative genomic analyses revealed that ‘N. mikurensis’ was more closely related to Ehrlichia chaffeensis than to Ehrlichia ruminantium, the opposite of what 16SrRNA sequence-based phylogenetic analyses determined. The genetic variability of the three whole-genome-sequenced ‘N. mikurensis’ strains was extremely low, between 0.14 and 0.22‰, a variation that was associated with geographic origin. No protein-coding genes exclusively shared by N. mikurensis and E. ruminantium were identified to explain their common tropism for vascular endothelium.

Ticks and Tick-Borne Pathogens Associated with Dromedary Camels (Camelus dromedarius) in Northern Kenya

Ticks and tick-borne pathogens (TBPs) are major constraints to camel health and production, yet epidemiological data on their diversity and impact on dromedary camels remain limited. We surveyed the diversity of ticks and TBPs associated with camels and co-grazing sheep at 12 sites in Marsabit County, northern Kenya. We screened blood and ticks (858 pools) from 296 camels and 77 sheep for bacterial and protozoan TBPs by high-resolution melting analysis and sequencing of PCR products. Hyalomma (75.7%), Amblyomma (17.6%) and Rhipicephalus (6.7%) spp. ticks were morphologically identified and confirmed by molecular analyses. We detected TBP DNA in 80.1% of blood samples from 296 healthy camels. “Candidatus Anaplasma camelii”, “Candidatus Ehrlichia regneryi” and Coxiella burnetii were detected in both camels and associated ticks, and Ehrlichia chaffeensis, Rickettsia africae, Rickettsia aeschlimannii and Coxiella endosymbionts were detected in camel ticks. We also detected Ehrlichia ruminantium, which is responsible for heartwater disease in ruminants, in Amblyomma ticks infesting camels and sheep and in sheep blood, indicating its endemicity in Marsabit. Our findings also suggest that camels and/or the ticks infesting them are disease reservoirs of zoonotic Q fever (C. burnetii), ehrlichiosis (E. chaffeensis) and rickettsiosis (R. africae), which pose public health threats to pastoralist communities.

Ehrlichia ruminantium uses its transmembrane protein Ape to adhere to host bovine aortic endothelial cells

Ehrlichia ruminantium is an obligate intracellular bacterium, transmitted by ticks of the genus Amblyomma and responsible for heartwater, a disease of domestic and wild ruminants. High genetic diversity of E. ruminantium strains hampers the development of an effective vaccine against all strains present in the field. In order to develop strategies for the control of heartwater through both vaccine and alternative therapeutic approaches, it is important to first gain a better understanding of the early interaction of E. ruminantium and its host cell. Particularly, the mechanisms associated with bacterial adhesion remain to elucidate. Herein, we studied the role of E. ruminantium membrane protein ERGA_CDS_01230 (UniProt Q5FFA9), a probable iron transporter, in the adhesion process to host bovine aortic endothelial cells (BAEC). The recombinant version of the protein ERGA_CDS_01230, successfully produced in the Leishmania tarentolae system, is O-glycosylated. Following in vitro culture of E. ruminantium in BAEC, the expression of CDS ERGA_CDS_01230 peaks at the extracellular infectious elementary body stages. This result suggest the likely involvement of ERGA_CDS_01230, named hereafter Ape for Adhesion protein of Ehrlichia, in the early interaction of E. ruminantium with its host cells. We showed using flow cytometry and scanning electron microscopy that beads coated with recombinant ERGA_CDS_01230 (rApe) adheres to BAEC. In addition, we also abserved that rApe interacts with proteins of the cell lysate, membrane and organelle fractions. Additionally, enzymatic treatment degrading dermatan and chondroitin sulfates on the surface of BAEC is associated with a 50% reduction in the number of bacteria in the host cell after a development cycle, indicating that glycosaminoglycans seem to play a role in the adhesion of E. ruminantium to the host cell. Finally, Ape induces a humoral response in vaccinated animals. Globally, our work identifying the role of Ape in E. ruminantium adhesion to host cells makes it a gold vaccine candidate and represents a first step toward the understanding of the mechanisms of cell invasion by E. ruminantium.

Ticks and tick-borne pathogens associated with dromedary camels (Camelus dromedarius) in northern Kenya

Ticks and tick-borne pathogens (TBPs) are major constraints to camel health and production, yet epidemiological data on their diversity and impact on dromedary camels are limited. We sur-veyed the diversity of ticks and TBPs associated with camels and co-grazing sheep at 12 sites in Marsabit County, northern Kenya. We screened blood and ticks (858 pools) collected from 296 camels and 77 sheep for bacterial and protozoan TBPs by high-resolution melting analysis and sequencing of PCR products. Hyalomma (75.7%), Amblyomma (17.6%) and Rhipicephalus (6.7%) spp. ticks were morphologically identified and confirmed by molecular analyses. We detected TBP DNA in 80.1% of blood samples from 296 healthy camels. “Candidatus Anaplasma camelii”, “Candidatus Ehrlichia regneryi” and Coxiella burnetii were detected in both camels and associ-ated ticks, and Ehrlichia chaffeensis, Rickettsia africae, Rickettsia aeschlimannii and Coxiella endo-symbionts were detected in camel ticks. We also detected Ehrlichia ruminantium, responsible for heartwater disease in ruminants, in Amblyomma ticks infesting camels and sheep and in sheep blood, indicating its endemicity in Marsabit. Our findings also suggest that camels and/or the ticks infesting them are reservoirs of zoonotic Q fever (C. burnetii), ehrlichiosis (E. chaffeensis), and rickettsiosis (R. africae), which pose a public health threat to pastoralist communities.

Revisiting Ehrlichia ruminantium Replication Cycle Using Proteomics: The Host and the Bacterium Perspectives

The Rickettsiales Ehrlichia ruminantium, the causal agent of the fatal tick-borne disease Heartwater, induces severe damage to the vascular endothelium in ruminants. Nevertheless, E. ruminantium-induced pathobiology remains largely unknown. Our work paves the way for understanding this phenomenon by using quantitative proteomic analyses (2D-DIGE-MS/MS, 1DE-nanoLC-MS/MS and biotin-nanoUPLC-MS/MS) of host bovine aorta endothelial cells (BAE) during the in vitro bacterium intracellular replication cycle. We detect 265 bacterial proteins (including virulence factors), at all time-points of the E. ruminantium replication cycle, highlighting a dynamic bacterium–host interaction. We show that E. ruminantium infection modulates the expression of 433 host proteins: 98 being over-expressed, 161 under-expressed, 140 detected only in infected BAE cells and 34 exclusively detected in non-infected cells. Cystoscape integrated data analysis shows that these proteins lead to major changes in host cell immune responses, host cell metabolism and vesicle trafficking, with a clear involvement of inflammation-related proteins in this process. Our findings led to the first model of E. ruminantium infection in host cells in vitro, and we highlight potential biomarkers of E. ruminantium infection in endothelial cells (such as ROCK1, TMEM16K, Albumin and PTPN1), which may be important to further combat Heartwater, namely by developing non-antibiotic-based strategies.

Molecular Characterization of Ticks and Tick-Borne Pathogens in Cattle from Khartoum State and East Darfur State, Sudan

Ticks transmit many pathogens with public health and veterinary importance. Despite the wide distribution of tick-borne pathogens in Sudan, the information on the tick–pathogen relationship needs to be updated, particularly using modern molecular techniques. This cross-sectional study, conducted between September and November 2019, used morphology, PCR, and sequencing to confirm the identity of adult cattle ticks (male and female; n = 536) from Khartoum State (n = 417) and East Darfur State (n = 119). Moreover, the presence of Theileria annulata, Babesia bigemina, B. bovis, Anaplasma marginale, and Ehrlichia ruminantium was detected and confirmed in each tick using species-specific PCR or nested PCR and sequencing. The most economically important tick genera, Rhipicephalus, Hyalomma, and Amblyomma, were prevalent in the study area, and 13 different tick species were identified. The most prevalent tick species were Rhipicephalusevertsi evertsi (34.3%) and Hyalomma anatolicum (57.3%) in Khartoum State, and Rhipicephalus annulatus (27%), Rhipicephalus decoloratus (25%), and Hyalomma rufipes (29%) in East Darfur State. We detected all five pathogens in both states. To the best of our knowledge, this is the first study to report the presence of E. ruminantium, its vector Amblyomma variegatum, and B. bovis in Khartoum State. Further, this is the first report on most tick and pathogen species identified in East Darfur State. Our findings indicate the migration of some tick and pathogen species beyond their distribution areas in the country, and this consideration is necessary to develop future control strategies.