Characterisation of South African field Ehrlichia ruminantium using multilocus sequence typing.

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.

Heartwater: A situation report of the Southern district of Botswana from 2017 to 2019.

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.

The potential of Rhipicephalus microplus as a vector of Ehrlichia ruminantium in West Africa.

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.

Observations and perceptions of veterinarians and farmers on heartwater distribution, occurrence and associated factors in South Africa.

BACKGROUND: There is currently no scientific evidence regarding the current climatic or other epidemiological factors that could influence the occurrence of heartwater in South Africa. OBJECTIVES: The objective was to determine whether climatic changes or other epidemiological factors influence the occurence of heartwater in South Africa. METHOD: A survey was conducted to scrutinise these factors using both veterinarians and farmers working in known areas in which heartwater had previously been confirmed to establish the value of each of these factors. Based on the observations, meaningful tendencies were noted, and conclusions drawn. RESULTS: These include changes in the spatial distribution of heartwater in many areas, with serious expansion, in some instances, of up to 150 km. In total, 48% of veterinarians and 42% of farmers reported seeing increase in the number of farms affected by heartwater. Climate change as a causative factor indicated by observations of increased average temperatures, milder frosts, less rain and shorter rainy seasons was identified by the majority of farmers but not by as many veterinarians. Respondents in both groups considered vegetation change an important factor. Increasing number of wildlife, especially antelope, was seen as a major factor by most veterinarians and also by many farmers. Both groups identified the movement of livestock and wildlife as an increasingly important factor that should be of major concern for both industries because it leads to the avoidable spread of many diseases apart from heartwater. CONCLUSION: Movement controls should be reinstated and reinforced by vigorously enforced legislation. The role of genetically determined resistance or resilience to heartwater infection in ruminants should be investigated. Breeding better adapted animals could provide part of a sustainable approach to the disease.

Natural Ehrlichia ruminantium infection in two captive Arabian tahrs (Arabitragus jayakari) in Oman.

This study was investigated the cause of death of two captive adult Arabian tahrs (Arabitragus jayakari) died within 2-3 days after onset of fever and neurologic signs in a private farm in northern Batinah Region of Oman. Blood counting revealed leukocytosis attributed to neutrophilia and serum chemistry showed hypoproteinemia, increased creatine kinase and BUN. Upon autopsy, the animals exhibited mild ascites and hydrothorax, prominent hydropericardium, with large pale-yellow clear fluid coagulum, prominent epicardial petechiation, as well as severe pulmonary edema associated with frothy fluid in airways. Brain edema with congestion of meningeal and parenchymal vessels was prominent. Histopathology revealed severe congestion and edema of both lung and brain as well as cardiac myopathy. Ehrlichia ruminantium colonies (the causative organism of cowdriosis; OIE-listed disease) were demonstrated in the capillary endothelium of fresh brain squash and lung macrophages. This is the first report of natural E. ruminantium infection in Arabian tahr, the highly endangered species, based on typical clinical signs of acute cowdriosis and demonstration of E. ruminantium colonies in the brain capillary endothelial cells.

Molecular detection and characterization of Ehrlichia ruminantium from cattle in Mozambique.

Heartwater caused by Ehrlichia ruminantiumis a disease of domestic and wild ruminants and one of the most economically important tick-borne diseases in Africa. The present study aimed to investigate the occurrence and genetic diversity of E. ruminantium in blood samples from 210 cattle sampled in five districts of Maputo Province, Mozambique. DNA blood samples were initially submitted to PCR assays targeting E. ruminantium pCS20 gene fragments. Additionally, in order to assess the genetic diversity of E. ruminantium, the positive samples were submitted to a PCR assay targeting the E. ruminantium map1 gene. Finally, the amplicons were sequenced and phylogenetic position was inferred using the Maximum Likelihood method. PCR results revealed that the overall prevalence in Maputo Province was 15% of the animals sampled. E. ruminantium map1 sequences showed not to be conserved. In the phylogenetic analysis, E. ruminantium map1 genotypes were positioned into multiple-clades. This study provides information on the prevalence and genetic diversity of E. ruminantium in five localities of Maputo Province. The future immune control strategies against local E. ruminantium must be designed in the light of the genetic diversity of this parasite.

PCR detection of Ehrlichia ruminantium and Babesia bigemina in cattle from Kwara State, Nigeria: unexpected absence of infection.

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.

Detection of Ehrlichia ruminantium infection in cattle in Cameroon.

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.

Occurrence of tick-borne haemoparasites in cattle in the Mungwi District, Northern Province, Zambia.

Little is known about the occurrence of haemoparasites in cattle in communal grazing areas of Mungwi District of Northern Province, Zambia. Clinical signs and post mortem lesions are pathognomonic of mixed tick-borne infections especially babesiosis, anaplasmosis and East Coast fever. The main objective of this study was to screen selected communal herds of cattle for tick-borne haemoparasites, and identify the tick vectors associated with the high cattle mortalities due to suspected tick-borne diseases in the local breeds of cattle grazing along the banks of the Chambeshi River in Mungwi District, Northern Province, Zambia. A total of 299 cattle blood samples were collected from July to September 2010 from Kapamba (n = 50), Chifulo (n = 102), Chisanga (n = 38), Kowa (n = 95) and Mungwi central (n = 14) in the Mungwi District. A total of 5288 ticks were also collected from the sampled cattle from April to July 2011. DNA was extracted from the cattle blood and the hypervariable region of the parasite small subunit rRNA gene was amplified and subjected to the reverse line blot (RLB) hybridization assay. The results of the RLB assay revealed the presence of tick-borne haemoparasites in 259 (86.6%) cattle blood samples occurring either as single (11.0%) or mixed (75.6%) infections. The most prevalent species present were the benign Theileria mutans (54.5%) and T. velifera (51.5%). Anaplasma marginale (25.7%), Babesia bovis (7.7%) and B. bigemina (3.3%) DNA were also detected in the samples. Only one sample (from Kapamba) tested positive for the presence of T. parva. This was an unexpected finding; also because the tick vector, Rhipicephalus appendiculatus, was identified on animals from Kowa (14.0%), Chisanga (8.5%), Chifulo (6.0%) and Kapamba (1.4%). One sample (from Kapamba) tested positive for the presence of Ehrlichia ruminantium even though Amblyomma variegatum ticks were identified from 52.9% of the sampled animals from all study areas. There was significant positive association between T. mutans and T. velifera (p < 0.001) infections, and between A. marginale and B. bovis (p = 0.005). The presence of R. microplus tick vectors on cattle was significantly associated with B. bovis (odds ratio, OR = 28.4, p < 0.001) and A. marginale (OR = 42.0, p < 0.001) infections, while A. variegatum presence was significantly associated with T. mutans (OR = 213.0, p < 0.001) and T. velifera (OR = 459.0, p < 0.001) infections. Rhipicephalus decoloratus was significantly associated with B. bigemina (OR = 21.6, p = 0.004) and A. marginale (OR = 28.5, p < 0.001). Multivariable analysis showed a significant association between location and tick-borne pathogen status for A. marginale (p < 0.001), T. mutans (p = 0.004), T. velifera (p = 0.003) and T. taurotragi (p = 0.005). The results of our study suggest that the cause of cattle mortalities in Mungwi during the winter outbreaks is mainly due to A. marginale, B. bovis and B. bigemina infections. This was confirmed by the clinical manifestation of the disease in the affected cattle and the tick species identified on the animals. The relatively low prevalence of T. parva, B. bigemina, B. bovis and E. ruminantium could indicate the existence of endemic instability with a pool of susceptible cattle and the occurrence of disease outbreaks.

Prevalence, risk factors, and genetic diversity of veterinary important tick-borne pathogens in cattle from Rhipicephalus microplus-invaded and non-invaded areas of Benin.

Babesiosis, theileriosis, anaplasmosis, and heartwater are tick-borne diseases (TBD) that threaten livestock production in sub-Saharan Africa including Benin. This country has been faced with an invasion of Rhipicephalus microplus, a major vector for babesiosis, theileriosis, and anaplasmosis over the last decade. Yet, data on TBD and the impact of the invasive ticks are lacking, making risk level evaluation and disease control arduous. In this study, epidemiological features of Babesia bovis, B. bigemina, Theileria spp., Anaplasma marginale and Ehrlichia ruminantium infections in Benin cattle were investigated in R. microplus-invaded and non-invaded areas. Detection of pathogens was based on species-specific PCR assays and resulting data were used to identify risk factors. Genetic diversity and phylogenies were then evaluated using several markers. Out of 207 samples examined, 170 (82.1%), 109 (52.7%), 42 (20.3%) 24 (11.6%) and 1 (0.5%) were positive for T. mutans, A. marginale, B. bigemina, B. bovis and E. ruminantium, respectively. Animal gender (for B. bovis), exposure to R. microplus (for B. bigemina and A. marginale), animal age (for B. bigemina and A. marginale) and cattle breed and/or antiprotozoal treatment (for T. mutants) significantly modulated pathogen occurrence. In addition, R. microplus exposure was significantly related to co-infection patterns and cases of clinical theileriosis and/or anaplasmosis were recorded among cattle highly exposed to the tick. In the genetic characterization, Theileria spp. and E. ruminantium sequences were conserved. Babesia spp. and A. marginale, however, showed high sequence polymorphisms that indicate the presence of several strains and may be linked to R. microplus invasion. Taken together, these results ascertain the endemicity of tick-borne infections in Benin and suggest that the characteristics of Babesia spp. and A. marginale infections in R. microplus-invaded and non-invaded areas are different.

Identification and genetic characterization of Piroplasmida and Anaplasmataceae agents in feeding Amblyomma variegatum ticks from Benin.

In many African countries including Benin, the reluctance of some livestock owners to blood collection from their cattle makes epidemiological surveys cumbersome and prevents regular monitoring of tick-borne diseases. In the present study, Amblyomma variegatum ticks were used to find out more about bovine tick-borne pathogens. DNA extracts from 910 adult ticks collected off cattle in North East Benin were examined for Babesia bigemina, B. bovis, Theileria taurotragi, T. annulata, T. orientalis, T. parva, T. mutans, Anaplasma marginale and Ehrlichia ruminantium using pathogen-specific PCR assays and sequence analyses. Altogether, 21.6% of the ticks carried at least one pathogen. A. marginale (142/910) was the most frequent pathogen, followed by E. ruminantium (57/910), B. bovis (10/910), T. mutans (3/910) and B. bigemina (1/910). Theileria taurotragi, T. annulata, T. orientalis, T. parva were not detected in the samples. Babesia bigemina, B. bovis and T. mutans were present in only one location whereas A. marginale and E. ruminantium were found in ticks from 7/8 locations surveyed. Coinfections occurred in 7.1% of all positive ticks. The analyses of partial sequences of B. bovis spherical body protein 4, B. bigemina rhoptry-associated protein-1a, T. mutans 18S rRNA and A. marginale major surface protein 5 showed high sequence conservation and homologies between Benin isolates and those from other African countries. However, E. ruminantium pCS20 partial sequences were different from published West African isolates and presented similar genetic variation with South and East African isolates. These results provide information on the pathogens circulating in North East Benin and suggest that Am. variegatum, one of the most abundant ticks in Africa, may play a role in the transmission of A. marginale.

Efficient high-throughput molecular method to detect Ehrlichia ruminantium in ticks.

BACKGROUND: Ehrlichia ruminantium is the causal agent of heartwater, a fatal tropical disease affecting ruminants with important economic impacts. This bacterium is transmitted by Amblyomma ticks and is present in sub-Saharan Africa, islands in the Indian Ocean and the Caribbean, where it represents a threat to the American mainland. METHODS: An automated DNA extraction method was adapted for Amblyomma ticks and a new qPCR targeting the pCS20 region was developed to improve E. ruminantium screening capacity and diagnosis. The first step in the preparation of tick samples, before extraction, was not automated but was considerably improved by using a Tissue Lyser. The new pCS20 Sol1 qPCR and a previously published pCS20 Cow qPCR were evaluated with the OIE standard pCS20 nested PCR. RESULTS: pCS20 Sol1 qPCR was found to be more specific than the nested PCR, with a 5-fold increase in sensitivity (3 copies/reaction vs 15 copies/reaction), was less prone to contamination and less time-consuming. As pCS20 Sol1 qPCR did not detect Rickettsia, Anasplasma and Babesia species or closely related species such as Panola Mountain Ehrlichia, E. chaffeensis and E. canis, its specificity was also better than Cow qPCR. In parallel, a tick 16S qPCR was developed for the quality control of DNA extraction that confirmed the good reproducibility of the automated extraction. The whole method, including the automated DNA extraction and pCS20 Sol1 qPCR, was shown to be sensitive, specific and highly reproducible with the same limit of detection as the combined manual DNA extraction and nested PCR, i.e. 6 copies/reaction. Finally, 96 samples can be tested in one day compared to the four days required for manual DNA extraction and nested PCR. CONCLUSIONS: The adaptation of an automated DNA extraction using a DNA/RNA viral extraction kit for tick samples and the development of a new qPCR increased the accuracy of E. ruminantium epidemiological studies, as well as the diagnostic capabilities and turn-over time for surveillance of heartwater. This new method paves the way for large-scale screening of other bacteria and viruses in ticks as well as genetic characterization of ticks and tick-pathogen coevolution studies.

Recombination Is a Major Driving Force of Genetic Diversity in the Anaplasmataceae Ehrlichia ruminantium.

The disease, Heartwater, caused by the Anaplasmataceae E. ruminantium, represents a major problem for tropical livestock and wild ruminants. Up to now, no effective vaccine has been available due to a limited cross protection of vaccinal strains on field strains and a high genetic diversity of Ehrlichia ruminantium within geographical locations. To address this issue, we inferred the genetic diversity and population structure of 194 E. ruminantium isolates circulating worldwide using Multilocus Sequence Typing based on lipA, lipB, secY, sodB, and sucA genes. Phylogenetic trees and networks were generated using BEAST and SplitsTree, respectively, and recombination between the different genetic groups was tested using the PHI test for recombination. Our study reveals the repeated occurrence of recombination between E. ruminantium strains, suggesting that it may occur frequently in the genome and has likely played an important role in the maintenance of genetic diversity and the evolution of E. ruminantium. Despite the unclear phylogeny and phylogeography, E. ruminantium isolates are clustered into two main groups: Group 1 (West Africa) and a Group 2 (worldwide) which is represented by West, East, and Southern Africa, Indian Ocean, and Caribbean strains. Some sequence types are common between West Africa and Caribbean and between Southern Africa and Indian Ocean strains. These common sequence types highlight two main introduction events due to the movement of cattle: from West Africa to Caribbean and from Southern Africa to the Indian Ocean islands. Due to the long branch lengths between Group 1 and Group 2, and the propensity for recombination between these groups, it seems that the West African clusters of Subgroup 2 arrived there more recently than the original divergence of the two groups, possibly with the original waves of domesticated ruminants that spread across the African continent several thousand years ago.

Seroprevalence of Ehrlichia ruminantium antibodies and its associated risk factors in indigenous goats of South Africa.

The present study investigated the seroprevalence of antibodies to Ehrlichia ruminantium and the associated risk factors in goats from five different farming provinces of South Africa. Sera collected from 686 goats of the commercial meat type (n=179), mohair type (n=9), non-descript indigenous goats from Eastern Cape (n=56), KwaZulu-Natal (n=209), Limpopo (n=111), North West (n=61) and Northern Cape (n=11) provinces and a feral Tankwa goat (n=50) were tested for the presence of immunoglobulin G (IgG) antibodies to antigens of E. ruminantium using the indirect fluorescent-antibody test (IFAT). Fifty two percent of these goats had ticks. The overall seroprevalence of antibodies to E. ruminantium was 64.87% (445/686) with the highest seroprevalence reported for Limpopo (95.50%) and lowest for Northern Cape (20.29%). Highest seroprevalence for antibodies to E. ruminantium was observed in goats from endemic regions (76.09%), and from smallholder production systems (89.54%). High seroprevalence was also observed in non-descript indigenous goats (85.04%), adult goat (69.62%), in does (67.46%) and goats infested with ticks (85.79%). The logistic model showed a gradient of increasing risk for commercial meat type Savanna (OR=3.681; CI=1.335-10.149) and non-descript indigenous (OR=3.466; CI=1.57-7.645) compared to Boer goats and for goats from the smallholder production system (OR=2.582; CI=1.182-5.639) and those with ticks (OR=3.587; CI=2.105-6.112). Results from this study showed that E. ruminantium infections were prevalent but were widely and unevenly distributed throughout South Africa. Findings from the study facilitate identification and mapping of risk areas for heartwater and its endeminicity in South Africa and should be taken into consideration for future disease control strategies and local goat improvement programs.

Development of a Quantitative PCR Assay for Differentiating the Agent of Heartwater Disease, Ehrlichia ruminantium, from the Panola Mountain Ehrlichia.

Panola Mountain Ehrlichia (PME) is an emerging Ehrlichia sp. reported in ten US states. Based on the sequence homology of all known genes, PME is closely related to Ehrlichia ruminantium (ER), the causative agent of heartwater. Heartwater is an economically important tick-borne disease of cattle, sheep and goats responsible for stock losses in sub-Saharan Africa. Unfortunately, ER was imported to the Caribbean islands in the 19th century, and the presence of this foreign animal disease in the Caribbean poses a threat to the US mainland. If introduced, a heartwater outbreak would cause massive losses of naïve livestock. The serologic assay of choice to diagnose heartwater is cross-reactive with Ehrlichia spp., including PME, as we demonstrate here, which would confound disease surveillance in the event of a heartwater outbreak. The purpose of this study was to develop a diagnostic assay capable of rapidly distinguishing between these pathogens. Using synthetic MAP-1B peptides for ER and PME, we tested the cross-reactivity of this assay using sera from infected livestock. The MAP-1B ELISA cannot distinguish between animals infected with PME and ER. Therefore, a dual-plex Taqman™ qPCR assay targeting the groEL gene of PME and ER was developed and validated. Primers were designed that are conserved among all known strains of ER, allowing for the amplification of strains from the Caribbean and Africa. The assay is highly sensitive (10 copies of DNA) and specific. This assay distinguishes between infection with PME and ER and will be a valuable tool in the event of heartwater outbreak on the US mainland, or for epidemiological studies involving either disease-causing organism.

Heartwater--Ehrlichia ruminantium infection.

Summary Heartwater is a notifiable disease that is listed by the World Organisation for Animal Health. It is caused by Ehrlichia ruminantium, an obligately intracellular Gram-negative bacterium in the order Rickettsiales and the family Anaplasmataceae. The disease is borne byticks in the genus Amblyomma and causes heartwater, or cowdriosis, in wild and domestic ruminants, primarily in Africa, but also in parts of the Caribbean. The disease was recognised in South Africa in the 19th Century and determined to be tick borne in 1900, while the organism was identified in 1925 and first cultured in vitro in 1985. This latter achievement boosted research into the disease at a time when biology was moving into the molecular genetic age. Over the last 20 years, there have been significant improvements in our understanding of E. ruminantium, yielding major advances in diagnosis, epidemiology, genetic characterisation, phylogeny, immunology, and vaccine development. The organism is genetically highly variable; this has important implications for future control measures, and is making it difficult to develop an effective vaccine for protection against tick challenge. Research is continuing into three different types of vaccine, inactivated, attenuated, and recombinant, and the current state of development of each is discussed.

An assessment of zoonotic and production limiting pathogens in rusa deer (Cervus timorensis rusa) from Mauritius.

A population of approximately 70,000 rusa deer (Cervus timorensis russa) represents the most important mammal species reared for food on the island of Mauritius, being the main source of red meat for the local population. However, very limited information is available on the circulation of pathogens affecting the productivity and health of this species. To produce baseline data on the circulation of infectious pathogens in rusa deer under production, a serological survey and/or direct pathogen detection for six selected infectious diseases was undertaken in 2007 in a sample of 53% of the herds reared in semi-free-ranging conditions in hunting estates. Seropositive results were recorded for Johne's disease with an indirect ELISA test (1.7%, n = 351), heartwater with an immunofluorescence antibody test (IFAT) (95.5%, n = 178) and leptospirosis with a Microscopic Agglutination Test (MAT) (25.9%, n = 363). Significant associations were found between seroprevalence to some of the leptospiral serogroups detected (Tarassovi, Pomona, Sejroe and Mini) and age of the animals, animal density or location of the estates (being more prevalent in hotter and more humid areas). In addition, Mycobacterium bovis and M. avium subspecies paratuberculosis were confirmed in two deer carcasses by culture and PCR, respectively. No antibodies against Brucella spp. nor Rift Valley Fever virus were detected with the use of respective indirect ELISA's. The results obtained suggest that the population of rusa deer from Mauritius is exposed to a wide range of pathogens which may affect their productivity. In addition, the results highlight the potential public health risks incurred by deer industry workers and consumers. This survey fills an important gap in knowledge regarding the health of tropical deer meat in Mauritius and justifies the need to implement more regular surveys of selected pathogens in the deer population.

Opportunities in diagnostic and vaccine approaches to mitigate potential heartwater spreading and impact on the American mainland.

Heartwater, caused by the Rickettsiales Ehrlichia ruminantium (ER), is a tropical tick-borne disease of wild and domestic ruminants, transmitted by Amblyomma ticks. It causes significant economic losses due to high mortality and the high cost of antibiotic treatment of affected animals, limiting herd productivity. It is present in sub-Saharan Africa, islands in the Indian Ocean and two Caribbean islands (Guadeloupe and Antigua) from where it threatens the American mainland due to risk of the spread of infected A. variegatum by migratory birds or by uncontrolled movement of animals. If an accidental introduction of a tick-free ER carrier animal occurs, autochthonous A. maculatum has proven to be a good experimental vector for heartwater. Modeling A. variegatum population dynamics has been developed, but further work is needed to predict favourable habitats and allow targeted surveillance. We overview here the advances in diagnostics, vaccines and epidemiology of heartwater and analyze the research gaps and needs to mitigate potential ER introduction and spread on the American mainland. Effective serologic ELISA tests allow prevalence studies, and several PCR-based diagnostic tests are currently available to detect ER in sick animals. However, the development of rapid assays, including multi-pathogen tests, would enhance the efficacy and cost-effectiveness of heartwater diagnosis. Several experimental vaccines (inactivated, attenuated and recombinant) are under development. Attenuated and inactivated vaccines are effective against homologous strains but their efficacy in the field is decreased due to broad antigenic diversity of ER. New molecular typing assays are now being used to study the genetic structure of ER populations worldwide, but the linking of genotyping to cross-protection is still not straightforward. Currently an inactivated vaccine would be the most appropriate vaccine for the American mainland due to its safety, the availability of a fully controlled bioprocess allowing ER mass production and the possibility to design "regional cocktail vaccines". This would require the selection and isolation of Caribbean ER strains supported by data of molecular epidemiology studies in this region. Development of an universal recombinant vaccine requires increased knowledge of ERbiology, including virulence mechanisms. Comparison of virulent and attenuated strains using"omic approaches" is on-going and will be crucial to understand these mechanisms and to develop improved vaccines.

Prevalence of Ehrlichia ruminantium in adult Amblyomma variegatum collected from cattle in Cameroon.

Ehrlichia ruminantium, the etiologic agent of the economically important disease heartwater, is an obligate intracellular bacterium transmitted by ticks of the genus Amblyomma, particularly A. hebraeum and A. variegatum. Although serologic and microscopic evidence of the presence of heartwater have been reported in ruminants in Cameroon, knowledge of E. ruminantium infection in the tick vector, A. variegatum, is lacking. In order to determine the infectivity of A. variegatum ticks by E. ruminantium, we analysed 500 un-engorged A. variegatum ticks collected by hand-picking from predilection sites from 182 cattle [115 ticks from 82 cattle at Société de Développement et d'Exploitation des Productions Animales (SODEPA) Dumbo ranch (SDR) and 385 ticks from 100 cattle at the Upper Farms ranch (UFR)] by amplification of the open reading frame (ORF) 2 of the pCS20 region of E. ruminantium. PCR amplification of the 279 bp fragment of the pCS20 region detected E. ruminantium DNA in 142 (28.4 %) of the 500 ticks with a higher infection rate (47/115; 40.9 %) observed in ticks from SDR and 24.7 % (95/385) of ticks collected from cattle at UFR. Twenty five randomly selected PCR products were sequenced and results indicated that some of the isolates shared homology with one another and to sequences of E. ruminantium in the GenBank. This report represents the first molecular evidence of E. ruminantium infection in A. variegatum ticks in Cameroon and suggests possible exposure of cattle to this pathogen in our environment.

Survey of ixodid ticks and two tick-borne pathogens in African buffaloes, Syncerus caffer, from the Caprivi Strip, Namibia.

A capture operation to ascertain health status in free-ranging buffaloes from six different areas in the Caprivi Strip in the northeast corner of Namibia was conducted in October 2009. Basic information on the ticks and tick-borne pathogens normally found in wildlife from this area are scarce. The objective of this study was to assess the host status of African buffaloes, Syncerus caffer, for ixodid ticks and two selected tick-borne pathogens in the Caprivi Strip, a key area bordering Angola, Zambia, Botswana, and Zimbabwe. Four different tick species have been identified among the 233 collected specimens, and, of 95 tested buffaloes, 54 (57%) were positive for Theileria parva, whereas only 3 (3%) showed evidence of being infected with Ehrlichia ruminantium.