Unravelling the diversity of Anaplasma species circulating in selected African wildlife hosts by targeted 16S microbiome analysis.

Organisms in the genus Anaplasma are obligate intracellular alphaproteobacteria. Bovine anaplasmosis, predominantly caused by Anaplasma marginale, is the most prevalent tick-borne disease (TBD) of cattle worldwide. Other Anaplasma species are known to cause disease; these include A. ovis, A. platys in dogs, A. capra in goats and humans, and A. phagocytophilum in humans. The rapid advancement of next-generation sequencing technologies has led to the discovery of many novel sequences ascribed to the genus Anaplasma, with over 20 putative new species being proposed since the last formal organization of the genus. Most 16S rRNA gene surveys for Anaplasma were conducted on cattle and to a lesser extent on rodents, dogs, and ticks. Little is known about the occurrence, diversity, or impact of Anaplasma species circulating in wildlife species. Therefore, we conducted a 16S rRNA gene survey with the goal of identifying Anaplasma species in a variety of wildlife species in the Kruger National Park and neighbouring game reserves, using an unbiased 16S rRNA gene microbiome approach. An Anaplasma/Ehrlichia-group specific quantitative real-time PCR (qPCR) assay revealed the presence of Anaplasma and/or Ehrlichia species in 70.0% (21/30) of African buffalo, 86.7% (26/30) of impala, 36.7% (11/30) of greater kudu, 3.2% (1/31) of African wild dog, 40.6% (13/32) of Burchell's zebra, 43.3% (13/30) of warthog, 22.6% (7/31) of spotted hyena, 40.0% (12/30) of leopard, 17.6% (6/34) of lion, 16.7% (5/30) of African elephant and 8.6% (3/35) of white rhinoceros samples. Microbiome sequencing data from the qPCR positive samples revealed four 16S rRNA sequences identical to previously published Anaplasma sequences, as well as nine novel Anaplasma 16S genotypes. Our results reveal a greater diversity of putative Anaplasma species circulating in wildlife than currently classified within the genus. Our findings highlight a potential expansion of the Anaplasma host range and the need for more genetic information from other important genes or genome sequencing of putative novel species for correct classification and further assessment of their occurrence in wildlife, livestock and companion animals.

Distribution and Prevalence of Anaplasmataceae, Rickettsiaceae and Coxiellaceae in African Ticks: A Systematic Review and Meta-Analysis.

In Africa, ticks continue to be a major hindrance to the improvement of the livestock industry due to tick-borne pathogens that include Anaplasma, Ehrlichia, Rickettsia and Coxiella species. A systemic review and meta-analysis were conducted here and highlighted the distribution and prevalence of these tick-borne pathogens in African ticks. Relevant publications were searched in five electronic databases and selected using inclusion/exclusion criteria, resulting in 138 and 78 papers included in the qualitative and quantitative analysis, respectively. Most of the studies focused on Rickettsia africae (38 studies), followed by Ehrlichia ruminantium (27 studies), Coxiella burnetii (20 studies) and Anaplasma marginale (17 studies). A meta-analysis of proportions was performed using the random-effects model. The highest prevalence was obtained for Rickettsia spp. (18.39%; 95% CI: 14.23-22.85%), R. africae (13.47%; 95% CI: 2.76-28.69%), R. conorii (11.28%; 95% CI: 1.77-25.89%), A. marginale (12.75%; 95% CI: 4.06-24.35%), E. ruminantium (6.37%; 95% CI: 3.97-9.16%) and E. canis (4.3%; 95% CI: 0.04-12.66%). The prevalence of C. burnetii was low (0%; 95% CI: 0-0.25%), with higher prevalence for Coxiella spp. (27.02%; 95% CI: 10.83-46.03%) and Coxiella-like endosymbionts (70.47%; 95% CI: 27-99.82%). The effect of the tick genera, tick species, country and other variables were identified and highlighted the epidemiology of Rhipicephalus ticks in the heartwater; affinity of each Rickettsia species for different tick genera; dominant distribution of A. marginale, R. africae and Coxiella-like endosymbionts in ticks and a low distribution of C. burnetii in African hard ticks.

Temporal Dynamics of Anaplasma marginale Infections and the Composition of Anaplasma spp. in Calves in the Mnisi Communal Area, Mpumalanga, South Africa.

Bovine anaplasmosis, caused by Anaplasma marginale, is one of the most important tick-borne diseases of cattle. Anaplasma marginale is known to be present in the Mnisi community, Mpumalanga Province, with frequent cases of anaplasmosis reported. This study investigated the infection dynamics in calves (n = 10) in two habitats in the study area over 12 months. A duplex real-time PCR assay targeting the msp1ß gene of A. marginale and the groEL gene of A. centrale confirmed the presence of A. marginale in five calves in a peri-urban area from the first month, but in only two calves at the wildlife-livestock interface and only after six months. These results were confirmed by 16S rRNA microbiome analysis. Over 50 A. marginale msp1α genotypes were detected in the calves along with five novel Msp1a repeats. Calves in the peri-urban area were more likely to be infected with A. marginale than calves in the wildlife-livestock interface. Cattle management, acaricide treatment, and cattle density could explain differences in infection prevalence in the two areas. Our results revealed that most calves were superinfected by distinct A. marginale strains within the study period, indicating continuous challenge with multiple strains that should lead to robust immunity in the calves and endemic stability in the area.

Genetic diversity in Babesia bovis from southern Africa and estimation of B. bovis infection levels in cattle using an optimised quantitative PCR assay.

Babesia bovis is a causal agent of bovine babesiosis, a disease which leads to mortality and morbidity and impacts the cattle industry worldwide. We amplified, cloned and sequenced the B. bovis merozoite surface antigen-2b (msa-2b) gene (∼940 bp) and the near full-length 18S rRNA gene (∼1600 bp) from cattle samples from South Africa and Mozambique to determine sequence variation between B. bovis parasites in the region. A TaqMan quantitative real-time PCR (qPCR) assay (18S rRNA gene) was optimised for the detection of B. bovis and estimation of parasitaemia in field samples from cattle from southern Africa. Phylogenetic analysis grouped the Msa-2b sequences in six clades and these were 59.7 to 99.6% identical to reference sequences. Sequence variation amongst B. bovis 18S rRNA sequences was found at 2 to 36 positions, and the sequences were 97 to 99% identical to published sequences. Mismatches between the B. bovis 18S rRNA sequences and a previously published qPCR forward primer (BoF) were observed; therefore, we developed a new forward primer (BoF2), and optimised the qPCR assay. Six 10-fold dilution series of B. bovis infected erythrocytes (2 × 108 to 2 × 103 infected red blood cells [iRBC]/ml) were analysed in triplicate in each of six separate qPCR runs, to determine the efficiency of the assay. The qPCR assay amplified the B. bovis 18S rRNA gene with 92.0 to 94.9% efficiency. The detection limit of the qPCR assay was approximately 6 iRBCs/µl. The performance of the optimised assay to diagnose B. bovis in field samples was assessed by testing DNA from 222 field samples of cattle from South Africa and Mozambique using three methods: the optimised qPCR assay, the reverse line blot (RLB) hybridisation assay, and the previously published qPCR assay. The detection rate of B. bovis using the optimised qPCR assay (31.1%, 69/222) was significantly higher (p<0.001) than both that using RLB (20.7%, 46/222) and the previously published qPCR assay (5.4%; 12/222). The B. bovis parasitaemia in samples from infected cattle ranged from 6 iRBCs/µl to 101,852 iRBCs/µl of blood. Our study revealed marked sequence variation between B. bovis parasites from southern Africa. The optimised qPCR assay will be useful in epidemiological studies and clinical diagnosis of B. bovis in southern Africa, and can be used to determine parasitaemia and potential carrier status in cattle populations, which is essential in the control of babesiosis.

Anaplasma phagocytophilum and Other Anaplasma spp. in Various Hosts in the Mnisi Community, Mpumalanga Province, South Africa.

DNA samples from 74 patients with non-malarial acute febrile illness (AFI), 282 rodents, 100 cattle, 56 dogs and 160 Rhipicephalus sanguineus ticks were screened for the presence of Anaplasma phagocytophilum DNA using a quantitative PCR (qPCR) assay targeting the msp2 gene. The test detected both A. phagocytophilum and Anaplasma sp. SA/ZAM dog DNA. Microbiome sequencing confirmed the presence of low levels of A. phagocytophilum DNA in the blood of rodents, dogs and cattle, while high levels of A. platys and Anaplasma sp. SA/ZAM dog were detected in dogs. Directed sequencing of the 16S rRNA and gltA genes in selected samples revealed the presence of A. phagocytophilum DNA in humans, dogs and rodents and highlighted its importance as a possible contributing cause of AFI in South Africa. A number of recently described Anaplasma species and A. platys were also detected in the study. Phylogenetic analyses grouped Anaplasma sp. SA/ZAM dog into a distinct clade, with sufficient divergence from other Anaplasma species to warrant classification as a separate species. Until appropriate type-material can be deposited and the species is formally described, we will refer to this novel organism as Anaplasma sp. SA dog.

Anaplasma marginale outer membrane protein vaccine candidates are conserved in North American and South African strains.

Bovine anaplasmosis is a globally economically important tick-borne disease caused by the obligate intraerythrocytic rickettsia, Anaplasma marginale. A live Anaplasma centrale blood-based vaccine is available, but it does not protect against all A. marginale field strains and may also transmit other blood-borne pathogens. Five potential outer membrane protein (OMP) vaccine candidates have been well-characterised in A. marginale strains from the USA, however, their levels of conservation in other countries must be ascertained in order to inform their use in a vaccine with regional or global efficacy. This study assessed the amino acid variation in vaccine candidate OMPs in South African strains of A. marginale, and also compared the immunogenic properties between South African and US strains. OMP genes Am779, Am854, omp7, omp8 and omp9 were amplified and sequenced from a set of genetically diverse South African samples with different msp1α-genotypes. OMPs Am854 and Am779 were highly conserved, with 99-100 % amino acid identity, while Omp7, Omp8 and Omp9 had 79-100 % identity with US strains. As has been shown previously, Omp7-9 possess conserved N- and C- termini, a central variable region, and a highly conserved CD4 T-cell epitope, FLLVDDA(I/V)V, in the N-terminal region. Western blot analysis of recombinant OMPs indicates strong antigenic conservation between South African and US strains of A. marginale, suggesting that they are good candidates for use in a novel global vaccine cocktail, although further work on the best formulation and delivery methods will be necessary.

Improved detection of Babesia bigemina from various geographical areas in Africa using quantitative PCR and reverse line blot hybridisation.

Babesia bigemina is one of the aetiological agents of bovine babesiosis, which causes economic losses through mortality, loss of production and control costs. Effective means of detecting and quantifying B. bigemina in cattle populations is therefore important to inform control approaches. In order to examine the parasite genetic diversity in African countries, B. bigemina 18S rRNA genes from cattle from South Africa, Uganda and Angola were sequenced. The 25 distinct B. bigemina 18S rRNA gene sequences obtained in this study showed 99 to 100% identity with previously published sequences of strains from African and other continents. The sequences of the previously published B. bigemina 18S rRNA gene-specific quantitative PCR (qPCR) primers and probe, developed based on American and Asian strains, were conserved in the African B. bigemina sequences. The qPCR assay was evaluated using 10-fold and 2-fold serial dilutions of B. bigemina-infected erythrocytes to determine the efficiency and analytical sensitivity. The qPCR assay had an efficiency of 98.14 ± 1.71%, and the limit of detection was approximately 1.5 infected red blood cells (iRBCs) per microlitre (µl) of blood. The detection rate of B. bigemina from duplicates of field-collected blood samples from cattle from South Africa, Mozambique and Angola was 37% (30/81), 12% (6/49) and 50% (38/76), respectively. Reverse line blot hybridisation (RLB) results obtained from the same samples in previous studies, using a previously published B. bigemina-specific probe, detected the parasite DNA in only 1.5% (3/206) of the samples. A new B. bigemina-specific RLB oligonucleotide probe was designed in the hypervariable V4 region of the 18S rRNA gene. Screening of field blood samples from cattle showed that the new probe was specific, and its frequency of detection of B. bigemina was three times higher than the previously published probe. The qPCR assay and the newly developed B. bigemina-specific RLB probe provide good tools for epidemiological studies, which are essential in the control of bovine babesiosis.

The Pathology of Pathogenic Theileriosis in African Wild Artiodactyls.

The published literature on schizont-"transforming," or pathogenic theileriosis, in African wild artiodactyls is dated and based on limited information. Here the authors review the taxonomy, diagnosis, epidemiology, hematology, pathology, and aspects of control in various species. Molecular studies based on 18S and 16S rRNA gene sequences have shown that African wild artiodactyls are commonly infected with diverse Theileria spp., as well as nontheilerial hemoprotozoa and rickettsia-like bacteria, and coinfections with pathogenic and nonpathogenic Theileria species are often recorded. Although theileriosis is still confusingly referred to as cytauxzoonosis in many species, the validity of a separate Cytauxzoon genus in artiodactyls is debated. The epidemiology of theileriosis is complex; the likelihood of fatal disease depends on the interplay of parasite, vertebrate host, tick vector, and environmental factors. Roan calves (Hippotragus equinus) and stressed animals of all host species are more susceptible to fatal theileriosis. Even though regenerative anemia is common, peripheral blood piroplasm parasitemia does not correlate with disease severity. Other than anemia, common macroscopic lesions include icterus, hemorrhages (mucosal, serosal, and tissue), fluid effusions into body cavities, lung edema, and variably sized raised cream-colored foci of leukocyte infiltration in multiple organs. Histopathologic findings include vasocentric hyperproliferation and lysis of atypical leukocytes with associated intracellular schizonts, parenchymal necrosis, hemorrhage, thromboembolism, and edema. Immunophenotyping is required to establish the identity of the schizont-transformed leukocytes in wild ungulates. Throughout the review, we propose avenues for future research by comparing existing knowledge on selected aspects of theileriosis in domestic livestock with that in African wild artiodactyls.

Evidence confirming the phylogenetic position of Anaplasma centrale (ex Theiler 1911) Ristic and Kreier 1984.

In 1911, Sir Arnold Theiler isolated and described a parasite that was very similar to Anaplasma marginale but which was more centrally located within the erythrocytes of the host cells, and was much less pathogenic than A. marginale. He named the parasite A. marginale variety centrale. The name Anaplasma centrale, referring to the same organism, was published in Validation List No. 15 in 1984, but the publication was based on an erroneous assumption that Theiler had indicated that it was a separate species. Many authors have subsequently accepted this organism as a separate species, but evidence to indicate that it is a distinct species has never been presented. The near full-length 16S rRNA gene sequence, and the deduced amino acid sequences for groEL and msp4 from several isolates of A. marginale and A. centrale from around South Africa were compared with those of the A. marginale type strain, St Maries, and the A. centrale Israel strain and other reference sequences. Phylogenetic analyses of these sequences demonstrated that A. centrale consistently forms a separate clade from A. marginale, supported by high bootstrap values (≥90 %), revealing that there is divergence between these two organisms. In addition, we discuss distinctive characteristics which have been published recently, such as differences in Msp1a/Msp1aS gene structure, as well as genome architecture that provide further evidence to suggest that A. centrale is, in fact, a separate species. Our results, therefore, provide evidence to support the existing nomenclature, and confirm that A. centrale (ex Theiler 1911) Ristic and Kreier 1984 is, indeed, a distinct species.

Selection and evaluation of housekeeping genes as endogenous controls for quantification of mRNA transcripts in Theileria parva using quantitative real-time polymerase chain reaction (qPCR).

The reliability of any quantitative real-time polymerase chain reaction (qPCR) experiment can be seriously compromised by variations between samples as well as between PCR runs. This usually result from errors in sample quantification, especially with samples that are obtained from different individuals and tissues and have been collected at various time intervals. Errors also arise from differences in qPCR efficiency between assays performed simultaneously to target multiple genes on the same plate. Consequently, the derived quantitative data for the target genes become distorted. To avoid this grievous error, an endogenous control, with relatively constant transcription levels in the target individual or tissue, is included in the qPCR assay to normalize target gene expression levels in the analysis. Several housekeeping genes (HKGs) have been used as endogenous controls in quantification studies of mRNA transcripts; however, there is no record in the literature of the evaluation of these genes for the tick-borne protozoan parasite, Theileria parva. Importantly, the expression of these genes should be invariable between different T. parva stocks, ideally under different experimental conditions, to gain extensive application in gene expression studies of this parasite. Thus, the expression of several widely used HKGs was evaluated in this study, including the genes encoding ß-actin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 28S rRNA, cytochrome b and fructose-2.6-biphosphate aldolase (F6P) proteins. The qPCR analysis revealed that the expression of genes encoding cytochrome b, F6P and GAPDH varied considerably between the two T. parva stocks investigated, the cattle-derived T. parva Muguga and the buffalo-derived T. parva 7014. 28S rRNA and ß-actin gene expression was the most stable; thus, these genes were considered suitable candidates to be used as endogenous control genes for mRNA quantification studies in T. parva.

Detection and Characterisation of Anaplasma marginale and A. centrale in South Africa.

Bovine anaplasmosis is endemic in South Africa and it has a negative economic impact on cattle farming. An improved understanding of Anaplasma marginale and Anaplasma marginale variety centrale (A. centrale) transmission, together with improved tools for pathogen detection and characterisation, are required to inform best management practices. Direct detection methods currently in use for A. marginale and A. centrale in South Africa are light microscopic examination of tissue and organ smears, conventional, nested, and quantitative real-time polymerase chain reaction (qPCR) assays, and a reverse line blot hybridisation assay. Of these, qPCR is the most sensitive for detection of A. marginale and A. centrale in South Africa. Serological assays also feature in routine diagnostics, but cross-reactions prevent accurate species identification. Recently, genetic characterisation has confirmed that A. marginale and A. centrale are separate species. Diversity studies targeting Msp1a repeats for A. marginale and Msp1aS repeats for A. centrale have revealed high genetic variation and point to correspondingly high levels of variation in A. marginale outer membrane proteins (OMPs), which have been shown to be potential vaccine candidates in North American studies. Information on these OMPs is lacking for South African A. marginale strains and should be considered in future recombinant vaccine development studies, ultimately informing the development of regional or global vaccines.

Molecular detection and phylogenetic analysis of Anaplasma marginale and Anaplasma centrale amongst transhumant cattle in north-eastern Uganda.

There is little molecular data from Anaplasma marginale and Anaplasma centrale isolates from cattle in Uganda. Between November 2013 and January 2014, blood was collected from 240 cattle in 20 randomly-selected herds in two districts of the Karamoja Region in north-eastern Uganda. A duplex quantitative real-time polymerase chain reaction (qPCR) assay was used to detect and determine the prevalence of A. marginale (targeting the msp1ß gene) and A. centrale (targeting the groEL gene). The qPCR assay revealed that most cattle (82.9%; 95% confidence interval [CI] 78.2-87.7%) were positive for A. marginale DNA, while fewer cattle (12.1%; 95% CI 7.9-16.2%) were positive for A. centrale DNA. A mixed effects logistic regression model showed that the age of cattle was significantly associated with A. centrale infection, while the prevalence of A. marginale varied significantly according to locality. The near full-length 16S ribosomal RNA (16S rRNA) gene and the heat shock protein gene, groEL, for both Anaplasma species were amplified from a selection of samples. The amplicons were cloned and the resulting recombinants sequenced. We found three novel A. marginale 16S rRNA variants, seven A. marginale groEL gene sequence variants and two A. centrale groEL gene sequence variants. Phylogenetic trees were inferred from sequence alignments of the 16S rRNA gene and GroEL amino acid sequences determined here and published sequences using maximum likelihood, Bayesian inference and parsimony methods Phylogenetic analyses classified the 16S rRNA gene and GroEL amino acid sequences into one clade for A. marginale and a separate clade for A. centrale. This study reveals a high prevalence and sequence variability of A. marginale and A. centrale, and is the first report on the phylogenetic characterisation of A. marginale and A. centrale from cattle in Uganda using molecular markers. Sequence variation can be attributed to mobile pastoralism, communal grazing and grazing with wildlife. These data support future epidemiological investigations for bovine anaplasmosis in Uganda.

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.

Co-infections with multiple genotypes of Anaplasma marginale in cattle indicate pathogen diversity.

BACKGROUND: Only a few studies have examined the presence of Anaplasma marginale and Anaplasma centrale in South Africa, and no studies have comprehensively examined these species across the whole country. To undertake this country-wide study we adapted a duplex quantitative real-time PCR (qPCR) assay for use in South Africa but found that one of the genes on which the assay was based was variable. Therefore, we sequenced a variety of field samples and tested the assay on the variants detected. We used the assay to screen 517 cattle samples sourced from all nine provinces of South Africa, and subsequently examined A. marginale positive samples for msp1α genotype to gauge strain diversity. RESULTS: Although the A. marginale msp1ß gene is variable, the qPCR functions at an acceptable efficiency. The A. centrale groEL gene was not variable within the qPCR assay region. Of the cattle samples screened using the assay, 57% and 17% were found to be positive for A. marginale and A. centrale, respectively. Approximately 15% of the cattle were co-infected. Msp1α genotyping revealed 36 novel repeat sequences. Together with data from previous studies, we analysed the Msp1a repeats from South Africa where a total of 99 repeats have been described that can be attributed to 190 msp1α genotypes. While 22% of these repeats are also found in other countries, only two South African genotypes are also found in other countries; otherwise, the genotypes are unique to South Africa. CONCLUSIONS: Anaplasma marginale was prevalent in the Western Cape, KwaZulu-Natal and Mpumalanga and absent in the Northern Cape. Anaplasma centrale was prevalent in the Western Cape and KwaZulu-Natal and absent in the Northern Cape and Eastern Cape. None of the cattle in the study were known to be vaccinated with A. centrale, so finding positive cattle indicates that this organism appears to be naturally circulating in cattle. A diverse population of A. marginale strains are found in South Africa, with some msp1α genotypes widely distributed across the country, and others appearing only once in one province. This diversity should be taken into account in future vaccine development studies.

Comparison of three nucleic acid-based tests for detecting Anaplasma marginale and Anaplasma centrale in cattle.

Several nucleic acid-based assays have been developed for detecting Anaplasma marginale and Anaplasma centrale in vectors and hosts, making the choice of method to use in endemic areas difficult. We evaluated the ability of the reverse line blot (RLB) hybridisation assay, two nested polymerase chain reaction (nPCR) assays and a duplex real-time quantitative polymerase chain reaction (qPCR) assay to detect A. marginale and A. centrale infections in cattle (n = 66) in South Africa. The lowest detection limits for A. marginale plasmid DNA were 2500 copies by the RLB assay, 250 copies by the nPCR and qPCR assays and 2500, 250 and 25 copies of A. centrale plasmid DNA by the RLB, nPCR and qPCR assays respectively. The qPCR assay detected more A. marginale- and A. centrale-positive samples than the other assays, either as single or mixed infections. Although the results of the qPCR and nPCR tests were in agreement for the majority (38) of A. marginale-positive samples, 13 samples tested negative for A. marginale using nPCR but positive using qPCR. To explain this discrepancy, the target sequence region of the nPCR assay was evaluated by cloning and sequencing the msp1ß gene from selected field samples. The results indicated sequence variation in the internal forward primer (AM100) area amongst the South African A. marginale msp1ß sequences, resulting in false negatives. We propose the use of the duplex qPCR assay in future studies as it is more sensitive and offers the benefits of quantification and multiplex detection of both Anaplasma spp.

Characterization of Anaplasma marginale subsp. centrale Strains by Use of msp1aS Genotyping Reveals a Wildlife Reservoir.

Bovine anaplasmosis caused by the intraerythrocytic rickettsial pathogen Anaplasma marginale is endemic in South Africa. Anaplasma marginale subspecies centrale also infects cattle; however, it causes a milder form of anaplasmosis and is used as a live vaccine against A. marginale There has been less interest in the epidemiology of A. marginale subsp. centrale, and, as a result, there are few reports detecting natural infections of this organism. When detected in cattle, it is often assumed that it is due to vaccination, and in most cases, it is reported as coinfection with A. marginale without characterization of the strain. A total of 380 blood samples from wild ruminant species and cattle collected from biobanks, national parks, and other regions of South Africa were used in duplex real-time PCR assays to simultaneously detect A. marginale and A. marginale subsp. centrale. PCR results indicated high occurrence of A. marginale subsp. centrale infections, ranging from 25 to 100% in national parks. Samples positive for A. marginale subsp. centrale were further characterized using the msp1aS gene, a homolog of msp1α of A. marginale, which contains repeats at the 5' ends that are useful for genotyping strains. A total of 47 Msp1aS repeats were identified, which corresponded to 32 A. marginale subsp. centrale genotypes detected in cattle, buffalo, and wildebeest. RepeatAnalyzer was used to examine strain diversity. Our results demonstrate a diversity of A. marginale subsp. centrale strains from cattle and wildlife hosts from South Africa and indicate the utility of msp1aS as a genotypic marker for A. marginale subsp. centrale strain diversity.

Molecular investigation of tick-borne haemoparasite infections among transhumant zebu cattle in Karamoja Region, Uganda.

Tick-borne diseases (TBDs) are a major constraint to cattle production in pastoral areas in Africa. Although information on tick-borne infections is important to prioritise control approaches, it is limited for transhumant zebu cattle in Karamoja, Uganda. We conducted a study to determine the occurrence and level of tick-borne infections among cattle in Karamoja Region. A total of 240 cattle were selected for blood collection using systematic sampling in 20 randomly-selected herds in two districts. The hypervariable V4 region of the 18S rRNA gene for Theileria/Babesia and the V1 region of the 16S rRNA gene for Ehrlichia/Anaplasma were amplified and hybridised to genus- and species-specific oligonucleotide probes on a reverse line blot (RLB) membrane. A duplex quantitative real-time polymerase chain reaction (qPCR) assay based on msp1ß and groEL genes was used for the detection of Anaplasma marginale and A. centrale, while monoplex qPCR assays were used for the detection of Ehrlichia ruminantium (226bp fragment of the pCS20 region) and Theileria parva (18S rRNA gene). The RLB hybridisation assay demonstrated the presence of tick-borne haemoparasites in all but one sample (99.6%), mostly as mixed infections (97.5%). The most frequently detected species were Theileria mutans (88.3%, 95% confidence interval: 84.6-91.7%), A. marginale (73.8%: 68.3-78.8%), Theileria velifera (71.3%: 65.8-76.7%) and Anaplasma sp. Omatjenne (63.3%: 57.5-68.8%). Other virulent pathogens, namely Babesia bigemina (5.0%) and T. parva (2.9%), were also detected with RLB, but not E. ruminantium. The proportions of qPCR positive samples were 82.9% (A. marginale), 12.1% (A. centrale), 3.3% (T. parva), and 1.7% (E. ruminantium). The full-length 18S rRNA genes from 6 out of 47 samples that were positive on RLB for the Babesia genus-specific probe and not for any of the Babesia species-specific probes were amplified, cloned and sequenced. The sequences were used to construct phylogenetic trees. Variations (5 to 9 nucleotides) in the 18S rRNA gene sequences of B. bigemina were identified, when compared with B. bigemina sequences from other parts of the world. Three nucleotide differences in the B. bigemina probe region may explain the failure of the RLB hybridisation assay to detect B. bigemina in some samples. T. mutans and B. bigemina sequences grouped in separate clades from previously published sequences. In conclusion, this study demonstrated high and widespread occurrence, and sequence variation of tick-borne haemoparasites among cattle in the pastoral area of Karamoja, which is useful for diagnosis and control of TBDs.

Co-infections determine patterns of mortality in a population exposed to parasite infection.

Many individual hosts are infected with multiple parasite species, and this may increase or decrease the pathogenicity of the infections. This phenomenon is termed heterologous reactivity and is potentially an important determinant of both patterns of morbidity and mortality and of the impact of disease control measures at the population level. Using infections with Theileria parva (a tick-borne protozoan, related to Plasmodium) in indigenous African cattle [where it causes East Coast fever (ECF)] as a model system, we obtain the first quantitative estimate of the effects of heterologous reactivity for any parasitic disease. In individual calves, concurrent co-infection with less pathogenic species of Theileria resulted in an 89% reduction in mortality associated with T. parva infection. Across our study population, this corresponds to a net reduction in mortality due to ECF of greater than 40%. Using a mathematical model, we demonstrate that this degree of heterologous protection provides a unifying explanation for apparently disparate epidemiological patterns: variable disease-induced mortality rates, age-mortality profiles, weak correlations between the incidence of infection and disease (known as endemic stability), and poor efficacy of interventions that reduce exposure to multiple parasite species. These findings can be generalized to many other infectious diseases, including human malaria, and illustrate how co-infections can play a key role in determining population-level patterns of morbidity and mortality due to parasite infections.

The epidemiology of tick-borne haemoparasites as determined by the reverse line blot hybridization assay in an intensively studied cohort of calves in western Kenya.

The development of sensitive surveillance technologies using PCR-based detection of microbial DNA, such as the reverse line blot assay, can facilitate the gathering of epidemiological information on tick-borne diseases, which continue to hamper the productivity of livestock in many parts of Africa and elsewhere. We have employed a reverse line blot assay to detect the prevalence of tick-borne parasites in an intensively studied cohort of indigenous calves in western Kenya. The calves were recruited close to birth and monitored for the presence of infectious disease for up to 51 weeks. The final visit samples from 453 calves which survived for the study period were analyzed by RLB. The results indicated high prevalences of Theileria mutans (71.6%), T. velifera (62.8%), Anaplasma sp. Omatjenne (42.7%), A. bovis (39.9%), Theileria sp. (sable) (32.7%), T. parva (12.9%) and T. taurotragi (8.5%), with minor occurrences of eight other haemoparasites. The unexpectedly low prevalence of the pathogenic species Ehrlichia ruminantium was confirmed by a species-specific PCR targeting the pCS20 gene region. Coinfection analyses of the seven most prevalent haemoparasites indicated that they were present as coinfections in over 90% of the cases. The analyses revealed significant associations between several of the Theileria parasites, in particular T. velifera with Theileria sp. sable and T. mutans, and T. parva with T. taurotragi. There was very little coinfection of the two most common Anaplasma species, although they were commonly detected as coinfections with the Theileria parasites. The comparison of reverse line blot and serological results for four haemoparasites (T. parva, T. mutans, A. marginale and B. bigemina) indicated that, except for the mostly benign T. mutans, indigenous cattle seem capable of clearing infections of the three other, pathogenic parasites to below detectable levels. Although the study site was located across four agroecological zones, there was little restriction of the parasites to particular zones.

Tick-borne haemoparasites in African buffalo (Syncerus caffer) from two wildlife areas in Northern Botswana.

BACKGROUND: The African buffalo (Syncerus caffer) is a host for many pathogens known to cause economically important diseases and is often considered an important reservoir for livestock diseases. Theileriosis, heartwater, babesiosis and anaplasmosis are considered the most important tick-borne diseases of livestock in sub-Saharan Africa, resulting in extensive economic losses to livestock farmers in endemic areas. Information on the distribution of tick-borne diseases and ticks is scarce in Northern Botswana. Nevertheless, this data is necessary for targeting surveillance and control measures in livestock production at national level. METHODS: In order to address this gap, we analyzed 120 blood samples from buffalo herds for the presence of common tick-borne haemoparasites causing disease in livestock, collected in two of the main wildlife areas of Northern Botswana: the Chobe National Park (CNP, n=64) and the Okavango Delta (OD, n=56). RESULTS: Analysis of the reverse line blot (RLB) hybridization assay results revealed the presence of Theileria, Babesia, Anaplasma and Ehrlichia species, either as single or mixed infections. Among the Theileria spp. present, T. parva (60%) and T. mutans (37%) were the most prevalent. Other species of interest were Anaplasma marginale subsp. centrale (30%), A. marginale (20%), Babesia occultans (23%) and Ehrlichia ruminantium (6%). The indirect fluorescent antibody test (IFAT) indicated 74% of samples to be positive for the presence of T. parva antibodies. Quantitative real-time PCR (qPCR) detected the highest level of animals infected with T. parva (81% of the samples). The level of agreement between the tests for detection of T. parva positive animals was higher between qPCR and IFAT (kappa=0.56), than between qPCR and RLB (kappa=0.26) or the latter and IFAT (kappa=0.15). CONCLUSIONS: This is the first report of tick-borne haemoparasites in African buffalo from northern Botswana, where animals from the CNP showed higher levels of infection than those from OD. Considering the absence of fences separating wildlife and livestock in the CNP and the higher levels of some parasite species in buffalo from that area, surveillance of tick-borne diseases in livestock at the interface in the CNP should be prioritized.