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BACKGROUND: Members of the Anaplasmataceae family, such as the Anaplasma and Ehrlichia species, cause economic losses and public health risks. However, the exact economic impact has not been comprehensively assessed in Mozambique due to limited data available on its basic epidemiology. Therefore, we investigated the molecular occurrence and identity of Anaplasma and Ehrlichia spp. infecting beef cattle in Maputo province, Mozambique. METHODS: A total of 200 whole blood samples were collected from apparently healthy beef cattle. Whole blood DNA was extracted and tested for presence of Anaplasma spp. and Ehrlichia ruminantium DNA through amplification of the 16S rRNA and map1 genes. Positive samples to Anaplasma spp. were subject to PCR assay targeting the A. marginale-msp5 gene. Amplicons obtained were purified, sequenced and subject to phylogenetic analyses. RESULTS: Anaplasma spp., A. marginale and E. ruminantium were detected in 153 (76.5%), 142 (71%) and 19 (9.5%) of all the samples analyzed, respectively. On this same sample group, 19 (9.5%) were co-infected with A. marginale and E. ruminantium. The 16S rRNA sequences of Anaplasma spp. obtained were phylogenetically related to A. marginale, A. centrale and A. platys. Phylogenetic analysis revealed that A. marginale-msp5 nucleotide sequences were grouped with sequences from Asia, Africa and Latin America, whereas E. ruminantium-map1 DNA nucleotide sequences were positioned in multiple clusters. CONCLUSION: Cattle in Maputo Province are reservoirs for multiple Anaplasma species. A high positivity rate of infection by A. marginale was observed, as well as high genetic diversity of E. ruminantium. Furthermore, five new genotypes of E. ruminantium-map1 were identified.

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Heartwater, one of the major tick-borne diseases of some domestic and wild ruminants in Africa, is caused by Ehrlichia ruminantium. The genetic diversity of E. ruminantium isolates renders the available vaccine ineffective against certain virulent isolates. To better understand the E. ruminantium genotypes in South Africa, a total of 1004 Amblyomma hebraeum tick deoxyribonucleic acid (DNA) samples from cattle in three South African provinces were tested by pCS20 Sol1 real-time polymerase chain reaction (qPCR) and characterised by multilocus sequence typing (MLST) using five housekeeping genes. Out of 1004 samples tested, 222 (22%) were positive for E. ruminantium. The occurrence of E. ruminantium in Mpumalanga, KwaZulu-Natal and Limpopo provinces was 19%, 22% and 27%, respectively. The E. ruminantium positive samples were screened for housekeeping genes and sequenced. Phylogenetic analysis revealed three main lineages: clade 1 made up of worldwide isolates (eastern, southern Africa, and Caribbean isolates), clade 2 comprised only West African isolates and clade 3 consisted of Omatjenne, Kümm2 and Riverside. Some study sample sequences were not identical to any of the reference isolates. However, they could all be grouped into the worldwide clade. Genetic variation in the sequenced regions was observed in the form of single nucleotide polymorphisms (SNPs). Using MLST to characterise E. ruminantium field isolates allowed the South African genotypes to be clearly distinguished from the distinct West African isolates.Contribution: Characterisation of E. ruminantium field isolates is important for the control of heartwater and contributes to preliminary knowledge required for the development of a more practical vaccine against heartwater.

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Heartwater is a tick-borne haemoparasitic disease that can limit agro-business expansion in Botswana. It poses a threat to national food security due to low animal production as well as livestock morbidity and mortality. This report gives a snapshot view of heartwater in the Southern district of Botswana. Ixodid ticks parasitizing livestock in four Southern sub-districts of Botswana were collected and identified using morphological and molecular methods. A wide distribution of Amblyomma hebraeum in all four Southern sub-districts was revealed. The annual number of heartwater cases across the Southern district of Botswana was determined from veterinary clinical case reports and confirmed through Giemsa-stained brain smears. A concerning gradual annual increase in heartwater cases was shown in the Moshupa sub-district - a hardveld terrain with rock outcrops where the vector thrives. Goats were affected most (55%) by heartwater followed by sheep (37%) and then cattle (8%). Farmers were interviewed on the management of the heartwater burden within their respective sub-districts and they reported that their animals were affected by heartwater despite 17 out of the 27 farmers interviewed attempting to control vectors through acaricide use. The presented heartwater situation warrants further investigation of the prevalence of heartwater and the effectiveness of existing disease control interventions in the disease-endemic Southern district of Botswana.

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BACKGROUND: The majority of the African population lives in rural areas where they heavily depend on crop and livestock production for their livelihoods. Given their socio-economic importance, we initiated a standardized multi-country (Benin, Burkina Faso, Ghana, Nigeria, Ethiopia Tanzania and Uganda) surveillance study to assess the current status of important tick-borne haemoparasites (TBHPs) of cattle. METHODS: We assessed pathogen prevalences (Anaplasma marginale, Anaplasma centrale, Babesia bigemina, Babesia bovis, Ehrlichia ruminantium, and Theileria parva) in the blood of 6447 animals spread over fourteen districts (two districts per country). In addition, we screened for intrinsic (sex, weight, body condition) and extrinsic (husbandry, tick exposure) risk factors as predictors of infections with TBHPs. RESULTS: There was a large macro-geographic variation observed in A. marginale, B. bigemina, B. bovis and E. ruminantium prevalences. Most correlated with the co-occurrence of their specific sets of vector-competent ticks. Highest numbers of infected cattle were found in Ghana and Benin, and lowest in Burkina Faso. While T. parva was seldomly found (Uganda only: 3.0%), A. marginale was found in each country with a prevalence of at least 40%. Babesia bovis infected individuals had lower body condition scores. Age (as estimated via body weight) was higher in A. marginale infected cattle, but was negatively correlated with B. bigemina and E. ruminantium prevalences. Ehrlichia ruminantium infection was more often found in males, and A. marginale more often in transhumance farming. High levels of co-infection, especially the combination A. marginale × B. bigemina, were observed in all countries, except for Uganda and Burkina Faso. Babesia bigemina was more or less often observed than expected by chance, when cattle were also co-infected with E. ruminantium or A. marginale, respectively. CONCLUSIONS: Tick-borne pathogens of cattle are ubiquitous in African's smallholder cattle production systems. Our standardized study will help a wide range of stakeholders to provide recommendations for TBHP surveillance and prevention in cattle, especially for B. bovis which heavily impacts production and continues its spread over the African continent via the invasive Rhipicephalus microplus tick.

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Heartwater, or cowdriosis, is a virulent tick-borne rickettsial disease of ruminants caused by Ehrlichia ruminantium, biologically transmitted by Amblyomma species (A. variegatum in West Africa). In West Africa, this bacterium was recently reported to naturally infect the invasive cattle tick, Rhipicephalus microplus (Rm) through trans-ovarian transmission from replete adult females to offspring. A 'sheep-tick-sheep' cycle was set up to determine whether feeding the progeny of these ticks on naïve sheep could lead to infection, and to compare clinical outcomes resulting from this transmission with those observed following infection by the natural A. variegatum (Av) vector. Using local strains of ticks (KIMINI-Rm and KIMINI-Av) and of E. ruminantium (BK242), we recorded, using the PCR technique, the presence of bacterial DNA in ticks (larvae for Av and females for Rm) engorged on sheep inoculated by BK242-infected blood. The bacterial DNA was also detected in the next stages of the lifecycle of R. microplus (eggs and larvae), and in sheep infested either by those R. microplus larvae or by A. variegatum nymphs moulted from larvae engorged on blood-inoculated sheep. Bacterial infection in these sheep was demonstrated by detecting antibodies to E. ruminantium using the MAP1-B ELISA and by isolation of the bacterium on cell culture from blood. The sequences of PCS20 gene detected in ticks and sheep were identical to that of the BK242 strain. Our results confirm that R. microplus can acquire and transmit E. ruminantium to the next stage. However, this transmission resulted in a mild subclinical disease whereas severe clinical disease was observed in sheep infested by A. variegatum infected nymphs, suggesting differences in the tick/bacteria relationship. Future studies will focus on replicating these findings with ticks of different isolates and life stages to determine if R. microplus is playing a role in the epidemiology of heartwater in West Africa. Additionally, studies will investigate whether sheep that are seropositive due to infestation by E. ruminantium-infected R. microplus are subsequently protected against heartwater. Such data will add to our understanding of the possible impact of R. microplus in areas where it has become recently established.

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Ehrlichia ruminantium (E. ruminantium) is the causative agent of heartwater disease and it is mainly transmitted to livestock by Amblyomma hebraeum (A. hebraeum) tick in South Africa. This study investigated the occurrence of E. ruminantium and its genetic diversity in ticks within Ngaka Modiri Molema district of North West Province in South Africa. Genomic DNA was extracted from whole A. hebraeum ticks totaling 876 and resulted in a total of 292 pooled DNA samples. Firstly, conventional PCR was used to detect Ehrlichia spp. targeting the dsbA gene, followed by nested PCR targeting the Map1 gene performed on DNA pool samples that were positive from the first PCR. One hundred and six tick DNA pool samples were positive by dsbA gene PCR for the presence of Ehrlichia spp. with minimum infection rate (MIR) of 121, while 13/106 were positive by Map1 PCR with MIR of 15. Different E. ruminantium Map1 genotypes (NWUe1, NWUe2, NWUe3, NWUe5, and NWUe6) were detected from tick samples and were closely related to more than 13 gene sequences of E. ruminantium from the NCBI GenBank database. These findings suggest that there is a significant diversity of E. ruminantium infecting ticks in the study area.

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Heartwater, Ehrlichia ruminantium infection in cattle, sheep, goats, and some wild ruminants, is an economically important disease in Africa characterized by high mortality rates in susceptible populations. In South Africa, the current commercial heartwater vaccine is an infection and treatment type of immunization using virulent live E. ruminantium organisms generated from blood of infected sheep with subsequent treatment of the animals with antibiotics at specific times during the course of infection. This vaccine has several inherent problems preventing its wide use as the vaccine must be administered intravenously and it does not protect against all the South African field isolates. A vaccine based on inactivation of Zimbabwean E. ruminantium Mbizi strain organisms produced in endothelial cell cultures can be a sustainable option because it will not require antibiotic treatment and will be safe as there is no potential for reversion to virulence. Previous data generated in laboratory trials and under natural field setting provides support for this vaccine approach. Four inactivated vaccine formulations using the E. ruminantium Mbizi strain were tested for their efficacy in Merino sheep compared to an unvaccinated control group (11 sheep per group). Two vaccines were prepared by beta-propiolactone (BPL) inactivation, and two were inactivated with binary ethylenimine (BEI) while purification was done with both percoll and polyethylene glycol (PEG). The four vaccine preparations were formulated with Montanide ISA 50V2 adjuvant and administered twice subcutaneously (2 ml per dose) at an interval of 4 weeks. All groups were challenged with a virulent homologous cell-cultured E. ruminantium inoculated via the intra-venous route on day 56. The primary variable of efficacy was measured by the percentage survival rate or mortality between the Controls and Vaccine Groups. Three vaccine formulations (BEI/Percoll (Group 3), BEI/PEG (Group 4), BPL/Percoll, (Group 1) had a significantly higher percent of animal surviving challenge compared to the unvaccinated control (p-values 0.001, 0.035, 0.030, respectively). The highest number of survivors was obtained in Group 3 BEI/Percoll; 10/11 (91%). Groups 4 (BEI/PEG) and Group 1 (BPL/Percoll) produced similar percentage of survivals of 64%. In contrast, the lowest survival rate of 50% was observed in Group 2 (BPL/PEG) which was numerically different but not significantly different from the unvaccinated control which had an 18% survival rate (2/11). The inactivated vaccine using BEI or BPL as inactivating agents blended with ISA 50 adjuvant induced protective immunity against challenge. The BEI/Percoll (Group 3) vaccination regimen was most efficacious against a lethal heartwater challenge as it significantly protected sheep against mortality which is the most important aspect of heartwater infections. Future work should be directed towards improvement of this vaccine formulation especially from the down-stream processing point of view as the percoll method is not scalable for commercialization purposes.

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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.

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Ticks and tick-borne diseases (TBDs) significantly affect cattle production and the livelihoods of communities in pastoralist areas. Data on protozoan and rickettsial pathogens in ticks infesting cattle in Uganda is scanty; while it is an indicator of the likelihood of disease transmission and occurrence. A cross-sectional study was conducted amongst cattle in the Karamoja Region, northeastern Uganda, from July through September 2017, to determine the tick species diversity, identify protozoan and rickettsial pathogens in the ticks, and characterise pathogenic species by sequence and phylogenetic analyses. About 50 % of the ticks detected from each predilection site on each animal were collected from 100 purposively-selected cattle from 20 randomly-selected herds. Twelve tick species belonging to the genera Amblyomma, Rhipicephalus and Hyalomma were identified, the most abundant being Amblyomma lepidum (93.9 %), followed by Amblyomma variegatum (2.0 %) and Rhipicephalus evertsi evertsi (1.0 %). Tick species that have not been reported in recent studies amongst cattle in Uganda were found, namely Rhipicephalus pravus, Rhipicephalus praetextatus and Rhipicephalus turanicus. The ticks were grouped into 40 pools, by species and location, and the reverse line blot (RLB) hybridisation assay was used to detect pathogens from the ticks. The most frequently detected tick-borne parasites were Theileria mutans, Theileria velifera and Theileria parva, each observed in 25 % (10/40) of the tick pools. Tick-borne pathogens, namely Babesia rossi, Babesia microti and Theileria sp. (sable) that are not common to, or not known to infect, cattle were identified from ticks. The gene encoding Ehrlichia ruminantium pCS20 region, the Ehrlichia and Anaplasma 16S rRNA gene, and T. parva p67 sporozoite antigen gene were amplified, cloned and sequenced. Seven novel E. ruminantium pCS20 variants were identified, and these grouped into two separate clusters with sequences from other parts of Africa and Asia. The T. parva p67 sequences were of the allele type 1, and parasites possessing this allele type are commonly associated with East Coast fever in eastern Africa. Analysis of the Ehrlichia and Anaplasma 16S rRNA gene sequences showed that they were closely related to Rickettsia africae and to a new Ehrlichia species variant recently found in China. Our R. africae 16S rRNA sequences grouped with R. africae isolates from Nigeria, Egypt and Benin. The information on tick species diversity and pathogens in the various tick species provides an indicator of potential transmission amongst cattle populations, and to humans, and can be useful to estimate disease risk and in control strategies.

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Heartwater is a non-contagious tick-borne disease of domestic and wild ruminants. Data regarding the complex processes involved during pathogen-vector-host interaction during Ehrlichia ruminantium infection is lacking and could be improved with knowledge associated with gene expression changes in both the pathogen and the host. Thus, in the current study, we aimed to identify E. ruminantium genes that are up-regulated when the pathogen enters the host and before the disease is established. Identification of such genes/proteins may aid in future vaccine development strategies against heartwater. RNA-sequencing was used to identify E. ruminantium genes that were exclusively expressed at the tick bite site in sheep skin biopsies (SB) and in adult tick salivary glands (SG). RNA was extracted from pooled samples of the SB or SG collected at different time points during tick attachment and prior to disease manifestation. Ribosomal RNA (rRNA) was removed and the samples were sequenced. Several E. ruminantium genes were highly expressed in all the samples while others were exclusively expressed in each. It was concluded that E. ruminantium genes that were exclusively expressed in the SB or both SB and SG when compared to the transcriptome datasets from bovine elementary bodies (BovEBs) from cell culture may be considered as early antigenic targets of host immunity. In silico immunogenic epitope prediction analysis and preliminary characterization of selected genes in vitro using ELIspot assay showed that they could possibly be ideal targets for future vaccine development against heartwater, however, further epitope characterization is still required.

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Heartwater is an economically important tick-borne disease of ruminants in Africa. The current commercial vaccine uses live Ehrlichia ruminantium from blood of infected sheep, requires antibiotic treatment during infection, needs to be administered intravenously and does not protect against all South African isolates. An attenuated tissue culture vaccine not requiring antibiotic treatment and effective against different field strains in small groups of goats and sheep was reported previously. The objective of the present study was to test safety and efficacy of this vaccine administered by intramuscular (i.m.) inoculation in larger groups of sheep, Angora goats and cattle. Animals were vaccinated via intravenous (i.v.) and i.m. routes and received E. ruminantium homologous challenge by feeding of infected ticks or by i.v. inoculation of infected blood. For vaccine titration in sheep and goats, the optimum safe and efficacious dose was determined using 2 ml equivalent of 102-105 culture-derived live elementary bodies (EBs). Similarly, the vaccine was titrated in cattle using 5 ml containing 105-107 EBs. Seventy percent of i.v. vaccinated and 9.7% of i.m. vaccinated Angora goats receiving 105 EBs, developed severe reactions to vaccination and were treated. These treated animals and the remaining 90.3% of i.m.- vaccinated goats showed 100% protection against i.v. or tick challenge. Sheep and Angora goats vaccinated i.m. with 104 EBs had no vaccination reactions and were fully protected against i.v. or tick challenge. Similarly, vaccinated cattle (dose 106 EBs) did not react to vaccine inoculation and were fully protected against i.v. or tick homologous challenge. Control non-vaccinated animals reacted severely to challenge and required oxytetracycline treatment. This successfully demonstrated that Angora goats, sheep and cattle can be safely vaccinated with the attenuated E. ruminantium Welgevonden vaccine via the i.m. route, with no clinical reactions to vaccination and 100% protection against virulent i.v. and homologous tick challenge.

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Three isolates of Ehrlichia ruminantium (Kümm 2, Omatjenne and Riverside), the causative agent of heartwater in domestic ruminants, were isolated in Ixodes scapularis (IDE8) tick cell cultures using the leukocyte fraction of infected sheep blood. All stocks were successfully propagated in IDE8 cells, whereas initiation attempts using endothelial cell cultures were unsuccessful. Therefore, the new technique should be included in any attempt to isolate field strains of E. ruminantium to enhance the probability of getting E. ruminantium isolates which might not be initiated in endothelial cells. Draft genome sequences of all three isolates were generated and compared with published genomes. The data confirmed previous phylogenetic studies that these three isolates are genetically very close to each other, but distinct from previously characterised E. ruminantium isolates. Genome comparisons indicated that the gene content and genomic synteny were highly conserved, with the exception of the membrane protein families. These findings expand our understanding of the genetic diversity of E. ruminantium and confirm the distinct phenotypic and genetic characteristics shared by these three isolates.

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Heartwater is a tick-borne disease caused by the intracellular rickettsial parasite Ehrlichia ruminantium and transmitted by Amblyomma hebraeum ticks. Heartwater is problematic in endemic areas because it causes high mortality in ruminants and leads to economic losses that threaten productivity and food security. This may indicate that there is augmented genetic diversity in the field, which may result in isolates that are more virulent than the Ball3 and Welgevonden isolates. The genetic diversity of E. ruminantium was investigated in this study, focussing on the pCS20 gene region and four polymorphic open reading frames (ORFs) identified by subtractive hybridisation. The 16S ribosomal ribonucleic acid gene confirmed E. ruminantium in brain, blood and tick genomic deoxyribonucleic acid samples (n = 3792) collected from 122 farms that were randomly selected from seven provinces of South Africa where heartwater is endemic. The conserved E. ruminantium pCS20 quantitative polymerase chain reaction (qPCR) assay was used to scan all collected field samples. A total of 433 samples tested positive with the qPCR using the pCS20 gene region, of which 167 were sequenced. The known stocks and field samples were analysed, and phylogenetic trees were generated from consensus sequences. A total of 25 new clades were identified; of these, nine isolates from infected blood could be propagated in cell cultures. These clades were not geographically confined to a certain area but were distributed amongst heartwater-endemic areas in South Africa. Thus, the knowledge of strain diversity of E. ruminantium is essential for control of heartwater and provides a basis for further vaccine development.

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BACKGROUND: In sub-Saharan Africa, Amblyomma ticks are vectors of heartwater disease in domestic ruminants, caused by the rickettsial pathogen Ehrlichia ruminantium. Immature tick stages often bite humans, whereby they act as vectors of tick-bite fever caused by Rickettsia africae. Moreover, Amblyomma ticks cause damage to livestock due to their feeding behaviour. In South Africa, we studied the abundance of Amblyomma hebraeum ticks on goats of emerging farmers in Mpumalanga Province. A selected number of A. hebraeum nymphs and adult ticks was tested for co-infection with E. ruminantium and R. africae. METHODS: A total of 630 indigenous goats, belonging to farmers in the Mnisi Community area, were examined for ticks in 2013 and 2014. All ticks were identified, and a selected number was tested by PCR with reverse line blot hybridisation. RESULTS: In total, 13,132 ticks were collected from goats distributed over 17 different households. Amblyomma hebraeum was the predominant species, followed by R. microplus. Rhipicephalus appendiculatus, R. simus and R. zambeziensis were also identified. Amblyomma hebraeum was present throughout the year, with peak activity of adults in summer (November) and nymphs in winter (July). The ratio between adults and nymphs ranged from 1:2.7 in summer to 1:55.1 in winter. The mean prevalence of infection for E. ruminantium by PCR/RLB in adult ticks was 17.4% (31/178), whereas 15.7% (28/178) were infected with R. africae. In pooled nymphs, 28.4% were infected with E. ruminantium and 38.8% carried R. africae infection. Co-infections of E. ruminantium and R. africae in adult and pooled nymphal ticks were 3.9% (7/178) and 10% (14.9), respectively. Lameness of goats due to predilection of ticks for the interdigital space of their feet was observed in 89% of the households. CONCLUSIONS: Goats act as important alternative hosts for cattle ticks, which underscored the necessity to include goats in control programs. It is suggested to use acaricide-impregnated leg-bands as a sustainable method to kill ticks and prevent lameness in goats. The challenge of goats by considerable numbers of E. ruminantium-infected ticks is a major obstacle for upgrading the indigenous goat breeds. Humans may be at risk to contract tick-bite fever in this area.

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BACKGROUND AND AIM: Ehrlichiosis caused by Ehrlichia ruminantium is a tick-borne disease of great economic importance in cattle production worldwide. Despite its economic impact, limited knowledge is available on its epidemiology in Africa, including Kenya. Suspected cases of E. ruminantium infections have been reported in the recent past to the University of Nairobi's Veterinary Hospital, prompting the need to investigate their possible re-emergence. Therefore, this study was aimed at determining the prevalence of E. ruminantium among smallholder dairy cattle in Nairobi City County and to assess potential risk factors. This knowledge may guide the development of appropriate control strategies of ehrlichiosis, subsequently reducing associated losses. MATERIALS AND METHODS: A total of 107 smallholder dairy farms from Nairobi City County were recruited for the study. Blood samples were collected from 314 apparently healthy dairy cattle, and Giemsa-stained blood smears were screened under the microscope for Ehrlichia species. A commercial antigen enzyme-linked immunosorbent assay (ELISA) kit was then used to confirm the presence of the infections in serum samples. A pre-tested questionnaire was used to collect data on management practices that may be potential risk factors. A univariate and mixed-effects logistic regression was then used to determine significant risk factors. RESULTS: On microscopy, 79.3% (249/314) of the sampled animals had Ehrlichia-like inclusion bodies in white blood cells, though only 18.6% (95% confidence interval [CI] 14.2-23.0) of these were confirmed to be E. ruminantium on ELISA. A farm-level prevalence of 35.5% (95% CI 27.0-45.3) was reported. Female-headed households (p=0.013), farms in Langata region (p=0.027), cleaning of cowsheds fortnightly (p=0.019), and roofing of cowshed (p=0.022) were factors significantly associated with E. ruminantium infections. CONCLUSION: There is a relatively high prevalence of E. ruminantium infections in apparently healthy cattle in smallholder dairy farms in this area, warranting control measures. It is critical to improve animal welfare-related factors, such as cowshed cleaning and roofing, as well as the strategic location of farms, especially, since reservoirs may reduce infection levels in the farms, in relation to wildlife. However, since Ehrlichia-like inclusion bodies other than those of E. ruminantium were observed in this study, there is a need to investigate further these factors and the possibility of other Ehrlichia species infecting cattle in the study area.

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Unraveling which proteins and post-translational modifications (PTMs) affect bacterial pathogenesis and physiology in diverse environments is a tough challenge. Herein, we used mass spectrometry-based assays to study protein phosphorylation and glycosylation in Ehrlichia ruminantium Gardel virulent (ERGvir) and attenuated (ERGatt) variants and, how they can modulate Ehrlichia biological processes. The characterization of the S/T/Y phosphoproteome revealed that both strains share the same set of phosphoproteins (n = 58), 36% being overexpressed in ERGvir. The percentage of tyrosine phosphorylation is high (23%) and 66% of the identified peptides are multi-phosphorylated. Glycoproteomics revealed a high percentage of glycoproteins (67% in ERGvir) with a subset of glycoproteins being specific to ERGvir (n = 64/371) and ERGatt (n = 36/343). These glycoproteins are involved in key biological processes such as protein, amino-acid and purine biosynthesis, translation, virulence, DNA repair, and replication. Label-free quantitative analysis revealed over-expression in 31 proteins in ERGvir and 8 in ERGatt. While further PNGase digestion confidently localized 2 and 5 N-glycoproteins in ERGvir and ERGatt, respectively, western blotting suggests that many glycoproteins are O-GlcNAcylated. Twenty-three proteins were detected in both the phospho- and glycoproteome, for the two variants. This work represents the first comprehensive assessment of PTMs on Ehrlichia biology, rising interesting questions regarding ER-host interactions. Phosphoproteome characterization demonstrates an increased versatility of ER phosphoproteins to participate in different mechanisms. The high number of glycoproteins and the lack of glycosyltransferases-coding genes highlight ER dependence on the host and/or vector cellular machinery for its own protein glycosylation. Moreover, these glycoproteins could be crucial to interact and respond to changes in ER environment. PTMs crosstalk between of O-GlcNAcylation and phosphorylation could be used as a major cellular signaling mechanism in ER. As little is known about the Ehrlichia proteins/proteome and its signaling biology, the results presented herein provide a useful resource for further hypothesis-driven exploration of Ehrlichia protein regulation by phosphorylation and glycosylation events. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium with the data set identifier PXD012589.

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Ticks and tick-borne diseases (TBDs) continue to pose an insidious and ever-present threat to livestock and livelihoods across the globe. Two of the most significant TBDs of cattle in Africa are heartwater and babesioisis, caused by Ehrlichia ruminantium and Babesia bigemina respectively. Both pathogens are endemic in Nigeria. However, to date, little data has been published regarding the number of cattle infected. In this study, blood samples were collected from cattle of the Kwara State, north-central Nigeria. Probe-based quantitative PCR (qPCR) and semi-nested PCR were used to investigate the presence of both pathogens, respectively. Our study found all samples (n = 157) to be surprisingly negative for both B. bigemina and E. ruminantium. These results contribute new information on the current burden of these two pathogens in Kwara State and may be helpful in informing more effective targeting of control strategies in Nigeria.

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Heartwater is an economically important disease of ruminants caused by the tick-borne bacterium Ehrlichia ruminantium. The disease is present throughout sub-Saharan Africa as well as on several islands in the Caribbean, where it poses a risk of spreading onto the American mainland. The dominant immune response of infected animals is directed against the variable outer membrane proteins of E. ruminantium encoded by a polymorphic multigene family. Here, we examined the full-length sequence of the major antigenic protein 1 (map1) family genes in multiple E. ruminantium isolates from different African countries and the Caribbean, collected at different time points to infer the possible role of recombination breakpoint and natural selection. A high level of recombination was found particularly in map1 and map1-2. Evidence of strong negative purifying selection in map1 and balancing selection to maintain genetic variation across these samples from geographically distinct countries suggests host-pathogen co-evolution. This co-evolution between the host and pathogen results in balancing selection by maintaining genetic diversity that could be explained by the demographic history of long-term pathogen pressure. This signifies the adaptive role and the molecular evolutionary forces underpinning E. ruminantium map1 multigene family antigenicity.

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Theileriosis and ehrlichiosis are two important tick-borne diseases affecting cattle farming in China. However, limited information is available regarding prevalence and molecular characterization of Theileria annulata and Ehrlichia ruminantium in cattle in Xinjiang Uygur Autonomous Region (XUAR), northwestern China. In this study, a total of 176 blood samples of cattle from three rural areas of XUAR were collected in June 2017 and were tested by nested-PCR. A total of 34 (19.3%) samples were found to be infected with one or two pathogens. The overall prevalence rates of T. annulata and E. ruminantium were 18.2% and 1.7%, respectively. Phylogenetic analyses revealed that the E. ruminantium isolates from XUAR were located in the same clade but diverged from the isolates from African countries using pCS20 gene while T. annulata isolates from XUAR revealed differences in the genotypes using Tams1 sequences. To our knowledge, this is the first report of E. ruminantium infection in cattle in China. It also provides the first genetic characterization of T. annulata in cattle in XUAR. The current findings are important for understanding the distribution of agents of theileriosis and ehrlichiosis and in designing measures for the prevention and control of tick-borne diseases in cattle, other animals, and humans.

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OBJECTIVES: Ehrlichia ruminantium infection (heartwater) is a major constraint that impacts negatively on the cattle industry development in sub-Saharan Africa and so far, little is known of the presence of heartwater in cattle in Cameroon. This study sought to investigate the prevalence of E. ruminantium infection in cattle in Cameroon and to determine the predictors of infection. RESULTS: A species-specific semi-nested pCS20 polymerase chain reaction was used to screen the buffy coats from 182 cattle (comprising 82 cattle that received intensive tick control regimen and 100 cattle on strategic tick control) from two study sites in Cameroon for E. ruminantium DNA in a cross-sectional study. E. ruminantium infection was confirmed in 12 (6.6%) of the 182 cattle comprising 11 that received intensive tick control and one on strategic tick control. Of the 12 cattle detected, 11 were apparently healthy and one was clinically diagnosed of heartwater. All DNA sequences of pCS20 amplicons were identical to each other (a representative sequence deposited in GenBank under accession number JQ039939). These findings which have veterinary and epidemiological significance, suggest the need for further investigation to determine the extent and role of heartwater in cattle in Cameroon.

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