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1.  Development and evaluation of PCR assay for detection of low levels of Cowdria ruminantium infection in Amblyomma ticks not detected by DNA probe. 
Journal of Clinical Microbiology  1995;33(1):166-172.
The sensitivities of a PCR assay and a DNA probe assay were compared for the detection of Cowdria ruminantium in Amblyomma ticks that were fed on C. ruminantium-infected, clinically reacting, and recovered carrier animals. The PCR assay and DNA probe detected infection in 86.0 and 37.0%, respectively, of 100 ticks fed on a febrile animal. In 75 ticks fed on carrier animals, PCR and the DNA probe detected infection in 28.0 and 1.33% of ticks, respectively. This demonstrates that the DNA probe has poor sensitivity for the detection of low levels of infection in ticks and that PCR is necessary for this purpose. The PCR assay had a detection limit of between 1 and 10 C. ruminantium organisms and did not amplify DNA from Ehrlichia canis, which is phylogenetically closely related to C. ruminantium, Theileria parva, or uninfected Amblyomma hebraeum or A. variegatum. PCR detected infection in A. hebraeum and A. variegatum adult ticks infected with one of six geographically different C. ruminantium strains. Amplification was also possible from desiccated ticks and ticks fixed in 70% ethanol, 10% buffered formalin, or 2% glutaraldehyde. The PCR assay supersedes the DNA probe and older detection methods for the detection of C. ruminantium in ticks, particularly those fed on carrier animals, and is suitable for both prospective and retrospective studies which require accurate detection of C. ruminantium in individual ticks. Application of the PCR assay should significantly improve the understanding of heartwater epidemiology, particularly through the determination of field tick infection rates.
PMCID: PMC227901  PMID: 7699036
2.  Detection of Cowdria ruminantium by means of a DNA probe, pCS20 in infected bont ticks, Amblyomma hebraeum, the major vector of heartwater in southern Africa. 
Epidemiology and Infection  1993;110(1):95-104.
A DNA probe, pCS20, previously described for use in detection of Cowdria ruminantium infections in Amblyomma variegatum (the principal vector of heartwater) hybridized with C. ruminantium DNA in organs of laboratory-infected A. hebraeum adult ticks (the major southern African vector of heartwater). The probe hybridized with C. ruminantium DNA in 46/49 midguts from male ticks and 26/29 from females, thus indicating infection. Corresponding salivary glands were less heavily infected, but infections were more numerous in glands from males. Infection in ticks was confirmed by transmission of the disease to susceptible goats. The probe did not hybridize with DNA from uninfected ticks or with DNA from a spotted fever group rickettsia commonly associated with A. hebraeum in Zimbabwe. The C. ruminantium specific pCS20 DNA probe can be applied to determine accurately the infection rates in the two major vectors of heartwater and the risk of exposure of ruminants in endemic areas.
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PMCID: PMC2271963  PMID: 8432329
3.  Detection of the Agent of Heartwater, Cowdria ruminantium, in Amblyomma Ticks by PCR: Validation and Application of the Assay to Field Ticks 
Journal of Clinical Microbiology  2000;38(4):1539-1544.
We have previously reported that the pCS20 PCR detection assay for Cowdria ruminantium, the causative agent of heartwater disease of ruminants, is more sensitive than xenodiagnosis and the pCS20 DNA probe for the detection of infection in the vector Amblyomma ticks. Here, we further assessed the reliability of the PCR assay and applied it to field ticks. The assay detected DNA of 37 isolates of C. ruminantium originating from sites throughout the distribution of heartwater and had a specificity of 98% when infected ticks were processed concurrently with uninfected ticks. The assay did not detect DNA of Ehrlichia chaffeensis, which is closely related to C. ruminantium. PCR sensitivity varied with tick infection intensity and was high (97 to 88%) with ticks bearing 107 to 104 organisms but dropped to 61 and 28%, respectively, with ticks bearing 103 and 102 organisms. The assay also detected C. ruminantium in collections of Amblyomma hebraeum and Amblyomma variegatum field ticks from 17 heartwater-endemic sites in four southern African countries. Attempts at tick transmission of infection to small ruminants failed with four of these collections. The pCS20 PCR assay is presently the most characterized and reliable test for C. ruminantium in ticks and thus is highly useful for field and laboratory epidemiological investigations of heartwater.
PMCID: PMC86485  PMID: 10747140
4.  A cloned DNA probe identifies Cowdria ruminantium in Amblyomma variegatum ticks. 
Journal of Clinical Microbiology  1991;29(11):2571-2577.
Heartwater, caused by Cowdria ruminantium and transmitted by ticks of the genus Amblyomma, is a constraint to ruminant animal production in sub-Saharan Africa. This rickettsial disease could spread from endemically infected areas of sub-Saharan Africa and certain Caribbean islands to other countries, including the United States, in which Amblyomma ticks exist. To detect C. ruminantium in tick vectors and animals, we made DNA probes from C. ruminantium DNA isolated from endothelial cell cultures. Two clones were evaluated; pCS20 from Crystal Springs (Zimbabwe) strain DNA had a 1,306-bp insert, and pCR9 from Kiswani (Kenya) strain DNA had a 754-bp insert. Both DNA probes detected 1 ng of Crystal Springs DNA; however, the pCS20 probe had a 10-fold-greater ability to discriminate between C. ruminantium DNA and DNA from other organisms. Also, the pCS20 probe did not hybridize to 400 ng (highest amount tested) of DNA from bovine cells, 3 protozoa, 3 rickettsiae, and 12 bacteria. In all experiments, C. ruminantium DNA was detected in midguts from 99 of 160 Amblyomma variegatum nymphs infected as larvae and in midguts from 38 of 80 adult ticks infected as nymphs but not in midguts from control nymphs and adults. The presence of C. ruminantium in nymphs and adults was confirmed by transmission of heartwater to goats. The DNA sequences of both probes were determined; synthetic oligonucleotides from pCS20 are recommended as DNA probes for C. ruminantium.
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PMCID: PMC270375  PMID: 1774264
5.  A cloned DNA probe for Cowdria ruminantium hybridizes with eight heartwater strains and detects infected sheep. 
Journal of Clinical Microbiology  1992;30(4):981-986.
The DNA probe pCS20, which was cloned from the DNA of the Crystal Springs heartwater strain from Zimbabwe, cross-reacted with DNAs of heartwater strains from all endemic areas, including four heartwater strains from Zimbabwe, two strains from South Africa, one strain from Nigeria, and the Gardel strain from the Caribbean island of Guadeloupe. By nucleic acid hybridization, the pCS20 DNA probe detected Cowdria ruminantium DNA in all DNA preparations made from plasma samples from infected sheep before and during the febrile reaction. Synthetic oligonucleotides were prepared for amplification of specific C. ruminantium DNA sequences by the polymerase chain reaction (PCR). Amplification of two DNA products (181 and 279 bp) from pCS20 DNA and C. ruminantium genomic DNA of heartwater strains was demonstrated. In contrast, amplification of these products or any other products was not possible from genomic DNAs of Anaplasma marginale, Babesia bigemina, Trypanosoma brucei brucei, Escherichia coli, and bovine endothelial cells. The cross-reactivities of the 32P-labeled PCR products with genomic DNAs from several heartwater strains were similar to those with the pCS20 DNA probe. A nucleic acid-based test that uses hybridization assays and PCR provides a sensitive method for the detection of heartwater in both animals and ticks and has applications in epidemiological studies for the disease, which may allow for improved disease control.
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PMCID: PMC265197  PMID: 1572987
6.  An immunoblotting diagnostic assay for heartwater based on the immunodominant 32-kilodalton protein of Cowdria ruminantium detects false positives in field sera. 
Journal of Clinical Microbiology  1993;31(10):2729-2737.
Heartwater, a major constraint to improved livestock production in Zimbabwe, threatens to invade areas which have been previously unaffected. To monitor its spread in Zimbabwe, an immunoblotting diagnostic assay based on the responses of animals to the immunodominant, conserved 32-kDa protein of Cowdria ruminantium was evaluated. In this assay, no false reactions were detected with sera known to be positive and negative, but sera from some cattle, sheep, and goats from heartwater-free areas of Zimbabwe reacted strongly with the 32-kDa protein, suggesting that either these animals had previous exposure to heartwater or they were false positives. To investigate the possibility of previous exposure to heartwater, 11 immunoblot-positive and 6 immunoblot-negative sheep from heartwater-free areas of Zimbabwe were compared regarding their susceptibilities to challenge with C. ruminantium. Prior to challenge, C. ruminantium could not be detected in any sheep by transmission to Amblyomma hebraeum ticks or by the polymerase chain reaction (PCR) conducted with plasma samples. All sheep were equally susceptible to the challenge, and infection was confirmed by brain biopsy, necropsy, PCR, and transmission of C. ruminantium to ticks. Our data suggest that the immunoblot-positive reactions of sera from heartwater-free areas were due not to previous C. ruminantium infection but rather to antigenic cross-reactivity between C. ruminantium and another agent(s) such as Ehrlichia species. In conclusion, the immunodominant 32-kDa protein is not antigenically specific to C. ruminantium and its use in serological diagnosis of heartwater requires reevaluation.
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PMCID: PMC265996  PMID: 8253974
7.  Antibody Responses to MAP 1B and Other Cowdria ruminantium Antigens Are Down Regulated in Cattle Challenged with Tick-Transmitted Heartwater 
Serological diagnosis of heartwater or Cowdria ruminantium infection has been hampered by severe cross-reactions with antibody responses to related ehrlichial agents. A MAP 1B indirect enzyme-linked immunosorbent assay that has an improved specificity and sensitivity for detection of immunoglobulin G (IgG) antibodies has been developed to overcome this constraint (A. H. M. van Vliet, B. A. M. Van der Zeijst, E. Camus, S. M. Mahan, D. Martinez, and F. Jongejan, J. Clin. Microbiol. 33:2405–2410, 1995). When sera were tested from cattle in areas of endemic heartwater infection in Zimbabwe, only 33% of the samples tested positive in this assay despite a high infection pressure (S. M. Mahan, S. M. Samu, T. F. Peter, and F. Jongejan, Ann. N.Y. Acad. Sci 849:85–87, 1998). To determine underlying causes for this observation, the kinetics of MAP 1B-specific IgG antibodies in cattle after tick-transmitted C. ruminantium infection and following recovery were investigated. Sera collected weekly over a period of 52 weeks from 37 cattle, which were naturally or experimentally infected with C. ruminantium via Amblyomma hebraeum ticks, were analyzed. MAP 1B-specific IgG antibody responses developed with similar kinetics in both field- and laboratory-infected cattle. IgG levels peaked at 4 to 9 weeks after tick infestation and declined to baseline levels between 14 and 33 weeks, despite repeated exposure to infected ticks and the establishment of a carrier state as demonstrated by PCR and xenodiagnosis. Some of the serum samples from laboratory, and field-infected cattle were also analyzed by immunoblotting and an indirect fluorescent-antibody test (IFAT) to determine whether this observed seroreversion was specific to the MAP 1B antigen. Reciprocal IFAT and immunoblot MAP 1-specific antibody titres peaked at 5 to 9 weeks after tick infestation but also declined between 30 and 45 weeks. This suggests that MAP 1B-specific IgG antibody responses and antibody responses to other C. ruminantium antigens are down regulated in cattle despite repeated exposure to C. ruminantium via ticks. Significantly, serological responses to the MAP 1B antigen may not be a reliable indicator of C. ruminantium exposure in cattle in areas of endemic heartwater infection.
doi:10.1128/CDLI.8.2.388-396.2001
PMCID: PMC96068  PMID: 11238227
8.  Transcription Analysis of the Major Antigenic Protein 1 Multigene Family of Three In Vitro-Cultured Ehrlichia ruminantium Isolates 
Journal of Bacteriology  2005;187(14):4782-4791.
Ehrlichia ruminantium, an obligate intracellular bacterium transmitted by ticks of the genus Amblyomma, causes heartwater disease in ruminants. The gene coding for the major antigenic protein MAP1 is part of a multigene family consisting of a cluster containing 16 paralogs. In the search for differentially regulated genes between E. ruminantium grown in endothelial and tick cell lines that could be used in vaccine development and to determine if differences in the map1 gene cluster exist between different isolates of E. ruminantium, we analyzed the map1 gene cluster of the Senegal and Gardel isolates of E. ruminantium. Both isolates contained the same number of genes, and the same organization as found in the genome sequence of the Welgevonden isolate (H. Van Heerden, N. E. Collins, K. A. Brayton, C. Rademeyer, and B. A. Allsopp, Gene 330:159-168, 2004). However, comparison of two subpopulations of the Gardel isolate maintained in different laboratories demonstrated that recombination between map1-3 and map1-2 had occurred in one subpopulation with deletion of one entire gene. Reverse transcription-PCR on E. ruminantium derived mRNA from infected cells using gene-specific primers revealed that all 16 map1 paralogs were transcribed in endothelial cells. In one vector (Amblyomma variegatum) and several nonvector tick cell lines infected with E. ruminantium, transcripts were found for between 4 and 11 paralogs. In all these cases the transcript for the map1-1 gene was detected and was predominant. Our results indicate that the map1 gene cluster is relatively conserved but can be subject to recombination, and differences in the transcription of map1 multigenes in host and vector cell environments exist.
doi:10.1128/JB.187.14.4782-4791.2005
PMCID: PMC1169525  PMID: 15995193
9.  Potential Value of Major Antigenic Protein 2 for Serological Diagnosis of Heartwater and Related Ehrlichial Infections 
Cowdria ruminantium is the etiologic agent of heartwater, a disease causing major economic loss in ruminants in sub-Saharan Africa and the Caribbean. Development of a serodiagnostic test is essential for determining the carrier status of animals from regions where heartwater is endemic, but most available tests give false-positive reactions with sera against related Erhlichia species. Current approaches rely on molecular methods to define proteins and epitopes that may allow specific diagnosis. Two major antigenic proteins (MAPs), MAP1 and MAP2, have been examined for their use as antigens in the serodiagnosis of heartwater. The objectives of this study were (i) to determine if MAP2 is conserved among five geographically divergent strains of C. ruminantium and (ii) to determine if MAP2 homologs are present in Ehrlichia canis, the causative agent of canine ehrlichiosis, and Ehrlichia chaffeensis, the organism responsible for human monocytic ehrlichiosis. These two agents are closely related to C. ruminantium. The map2 gene from four strains of C. ruminantium was cloned, sequenced, and compared with the previously reported map2 gene from the Crystal Springs strain. Only 10 nucleic acid differences between the strains were identified, and they translate to only 3 amino acid changes, indicating that MAP2 is highly conserved. Genes encoding MAP2 homologs from E. canis and E. chaffeensis also were cloned and sequenced. Amino acid analysis of MAP2 homologs of E. chaffeensis and E. canis with MAP2 of C. ruminantium revealed 83.4 and 84.4% identities, respectively. Further analysis of MAP2 and its homologs revealed that the whole protein lacks specificity for heartwater diagnosis. The development of epitope-specific assays using this sequence information may produce diagnostic tests suitable for C. ruminantium and also other related rickettsiae.
PMCID: PMC95689  PMID: 10066656
10.  Comparative Genomic Analysis of Three Strains of Ehrlichia ruminantium Reveals an Active Process of Genome Size Plasticity†  
Journal of Bacteriology  2006;188(7):2533-2542.
Ehrlichia ruminantium is the causative agent of heartwater, a major tick-borne disease of livestock in Africa that has been introduced in the Caribbean and is threatening to emerge and spread on the American mainland. We sequenced the complete genomes of two strains of E. ruminantium of differing phenotypes, strains Gardel (Erga; 1,499,920 bp), from the island of Guadeloupe, and Welgevonden (Erwe; 1,512,977 bp), originating in South Africa and maintained in Guadeloupe in a different cell environment. Comparative genomic analysis of these two strains was performed with the recently published parent strain of Erwe (Erwo) and other Rickettsiales (Anaplasma, Wolbachia, and Rickettsia spp.). Gene order is highly conserved between the E. ruminantium strains and with A. marginale. In contrast, there is very little conservation of gene order with members of the Rickettsiaceae. However, gene order may be locally conserved, as illustrated by the tuf operons. Eighteen truncated protein-encoding sequences (CDSs) differentiate Erga from Erwe/Erwo, whereas four other truncated CDSs differentiate Erwe from Erwo. Moreover, E. ruminantium displays the lowest coding ratio observed among bacteria due to unusually long intergenic regions. This is related to an active process of genome expansion/contraction targeted at tandem repeats in noncoding regions and based on the addition or removal of ca. 150-bp tandem units. This process seems to be specific to E. ruminantium and is not observed in the other Rickettsiales.
doi:10.1128/JB.188.7.2533-2542.2006
PMCID: PMC1428390  PMID: 16547041
11.  Tick infestation patterns in free ranging African buffalo (Syncercus caffer): Effects of host innate immunity and niche segregation among tick species☆ 
Graphical abstract
Highlights
► Rhipicephalus evertsi evertsi and Amblyomma hebraeum are abundant on wild African buffalo. ► The two tick species do not affect each other’s abundance. ► Strong differences in attachment site preference suggest spatial niche segregation. ► Host traits (immunity, age, pregnancy status, body condition) drive tick abundance. ► Buffalo with stronger innate immunity have fewer ticks.
Ticks are of vast importance to livestock health, and contribute to conflicts between wildlife conservation and agricultural interests; but factors driving tick infestation patterns on wild hosts are not well understood. We studied tick infestation patterns on free-ranging African buffalo (Syncercus caffer), asking (i) is there evidence for niche segregation among tick species?; and (ii) how do host characteristics affect variation in tick abundance among hosts? We identified ticks and estimated tick burdens on 134 adult female buffalo from two herds at Kruger National Park, South Africa. To assess niche segregation, we evaluated attachment site preferences and tested for correlations between abundances of different tick species. To investigate which host factors may drive variability in tick abundance, we measured age, body condition, reproductive and immune status in all hosts, and examined their effects on tick burdens.
Two tick species were abundant on buffalo, Amblyomma hebraeum and Rhipicephalus evertsi evertsi. A. hebraeum were found primarily in the inguinal and axillary regions; R. e. evertsi attached exclusively in the perianal area. Abundances of A. hebraeum and R. e. evertsi on the host were unrelated. These results suggest spatial niche segregation between A. hebraeum and R. e. evertsi on the buffalo. Buffalo with stronger innate immunity, and younger buffalo, had fewer ticks. Buffalo with low body condition scores, and pregnant buffalo, had higher tick burdens, but these effects varied between the two herds we sampled.
This study is one of the first to link ectoparasite abundance patterns and immunity in a free-ranging mammalian host population. Based on independent abundances of A. hebraeum and R. e. evertsi on individual buffalo, we would expect no association between the diseases these ticks transmit. Longitudinal studies linking environmental variability with host immunity are needed to understand tick infestation patterns and the dynamics of tick-borne diseases in wildlife.
doi:10.1016/j.ijppaw.2012.11.002
PMCID: PMC3862501  PMID: 24533310
Amblyomma hebraeum; Co-infestation; Immunity; Host traits; Rhipicephalus evertsi evertsi
12.  Multi-locus sequence typing of Ehrlichia ruminantium strains from geographically diverse origins and collected in Amblyomma variegatum from Uganda 
Parasites & Vectors  2011;4:137.
Background
The rickettsial bacterium Ehrlichia ruminantium is the causative agent of heartwater in ruminants. A better understanding of the population genetics of its different strains is, however, needed for the development of novel diagnostic tools, therapeutics and prevention strategies. Specifically, the development of effective vaccination policies relies on the proper genotyping and characterisation of field isolates. Although multi-locus sequence typing (MLST) has been recently developed, only strains from geographically restricted collections have been analysed so far. The expansion of the MLST database to include global strains with different geographic origins is therefore essential. In this study, we used a panel of reference strains from geographically diverse origins and field samples of E. ruminantium detected from its vector, Amblyomma variegatum, in heartwater-endemic areas in Uganda.
Results
A total of 31 novel alleles (six, four, six, three, two, five, three, and two for gltA, groEL, lepA, lipA, lipB, secY, sodB, and sucA loci, respectively) and 19 novel sequence types (STs) were identified. Both neighbour-joining and minimum spanning tree analyses indicated a high degree of genetic heterogeneity among these strains. No association was observed between genotypes and geographic origins, except for four STs from West African countries. When we performed six different tests for recombination (GeneConv, Bootscan, MaxChi, Chimaera, SiScan, and 3Seq) on concatenated sequences, four possible recombination events were identified in six different STs. All the recombination breakpoints were located near gene borders, indicating the occurrence of intergenic recombination. All four STs that localized to a distinct group in clustering analysis showed evidence of identical recombination events, suggesting that recombination may play a significant role in the diversification of E. ruminantium.
Conclusions
The compilation of MLST data set across the African continent will be particularly valuable for the understanding of the existing genetic diversity of field isolates in African countries. Comprehensive information on the degree of cross-protection between strains and further understanding of possible relationships between genotypes and phenotypes such as vaccine efficacy are expected to lead to the development of region-specific vaccination strategies.
doi:10.1186/1756-3305-4-137
PMCID: PMC3151223  PMID: 21762509
13.  Longitudinal monitoring of Ehrlichia ruminantium infection in Gambian lambs and kids by pCS20 PCR and MAP1-B ELISA 
Background
The epidemiology of E. ruminantium infection in extensively managed young animals is not adequately understood. Thus in this study, we monitored the onset (age at first infection) and kinetics of E. ruminantium infection and antibody response in extensively managed newborn lambs and kids at three sites in The Gambia.
Methods
We used a nested pCS20 PCR and MAP1-B ELISA in a longitudinal study to monitor the onset (age at first infection) and kinetics of E. ruminantium infection and antibody response respectively, in 77 newborn lambs and kids under a traditional husbandry system at three sites (Kerr Seringe, Keneba, Bansang) in The Gambia where heartwater is known to occur. The animals were monitored for field tick infestation and the comparative performance of the two assays in detecting E. ruminantium infection was also assessed.
Results
The infection rate detected by pCS20 PCR varied between 8.6% and 54.8% over the 162-day study period. Nineteen per cent of the animals in week 1 post-partum tested positive by pCS20 PCR with half of these infections (7/14) detected in the first 3 days after birth, suggesting that transmission other than by tick feeding had played a role. The earliest detectable A. variegatum infestation in the animals occurred in week 16 after birth. Antibodies detected by MAP1-B ELISA also varied, between 11.5% and 90%. Although there is considerable evidence that this assay can detect false positives and due to this and other reasons serology is not a reliable predictor of infection at least for heartwater. In contrast to the pCS20 PCR, the serological assay detected the highest proportion of positive animals in week 1 with a gradual decline in seropositivity with increasing age. The pCS20 PCR detected higher E. ruminantium prevalence in the animals with increasing age and both the Spearman's rank test (rs = -0.1512; P = 0.003) and kappa statistic (-0.091 to 0.223) showed a low degree of agreement between the two assays.
Conclusion
The use of pCS20 PCR supported by transmission studies and clinical data could provide more accurate information on heartwater epidemiology in endemic areas and single-occasion testing of an animal may not reveal its true infection status. The view is supported because both the vector and vertical transmission may play a vital role in the epidemiology of heartwater in young small ruminants; the age range of 4 and 12 weeks corresponds to the period of increased susceptibility to heartwater in traditionally managed small ruminants.
doi:10.1186/1471-2334-7-85
PMCID: PMC1949406  PMID: 17662144
14.  Occurrence of tick-transmitted pathogens in dogs in Jos, Plateau State, Nigeria 
Parasites & Vectors  2014;7:119.
Background
Canine babesiosis caused by Babesia rossi, transmitted by Haemaphysalis elliptica in South Africa, has also been reported from Nigeria. Although H. leachi (sensu lato) is widespread in sub-Saharan Africa, published literature on the occurrence of canine babesiosis is meagre. It has been postulated that the genotype of Babesia rossi Erythrocyte Membrane Antigen 1 (BrEMA1) may be linked to virulence of the specific isolate. The primary objective of this study was to detect and characterise tick-borne pathogens in dogs presented to a veterinary hospital using molecular techniques. In B. rossi-positive specimens, we aimed to determine whether the BrEMA1 gene occurred and to compare genotypes with those found in other isolates. Lastly, we wished to identify the tick species that were recovered from the sampled dogs.
Methods
Blood specimens (n = 100) were collected during January to March 2010 from domestic dogs presented at an animal hospital in Jos, Plateau State, Nigeria. They were screened for the presence of Babesia/Theileria and Ehrlichia/Anaplasma genomic DNA using PCR and Reverse Line Blot (RLB) assays. Positive B. rossi specimens were tested for the presence of the BrEMA1gene using an RT-PCR. In addition, ticks were collected from dogs found to be infested during sampling.
Results
On RLB, 72 (72%) of the specimens were positive for one or more haemoparasites. Of the positive specimens, 38 (53%) were infected with B. rossi; 9 (13%) with Theileria sp. (sable); 5 (7%) with either Ehrlichia canis or Anaplasma sp. Omatjenne, respectively; 3 (4%) with Theileria equi; and 1 (1%) with B. vogeli and E. ruminantium, respectively. Co-infections were detected in 13 (18%) of the specimens. Results of RT-PCR screening for the BrEMA1 gene were negative. A total of 146 ticks belonging to 8 species were collected and identified: Rhipicephalus sanguineus 107 (73%), Haemaphysalis leachi (sensu stricto) 27 (18%), R. turanicus 3 (2%), and Amblyomma variegatum, H. elliptica, R. lunulatus, R. muhsamae and R. senegalensis 1 (1%), respectively.
Conclusions
Up to 8 tick-borne pathogens possibly occur in the dog population at Jos, with B. rossi being the most prevalent. The absence of the BrEMA1 gene suggests that B. rossi occurring in that area may be less virulent than South African isolates.
doi:10.1186/1756-3305-7-119
PMCID: PMC3974742  PMID: 24661795
Babesia rossi; BrEMA1; Haemaphysalis leachi; Haemaphysalis elliptica; Rhipicephalus sanguineus; Domestic dogs; Nigeria
15.  Diversity of Ehrlichia ruminantium Major Antigenic Protein 1-2 in Field Isolates and Infected Sheep▿  
Infection and Immunity  2009;77(6):2304-2310.
Proteins expressed from the map1 multigene family of Ehrlichia ruminantium are strongly recognized by immune T and B cells from infected animals or from animals that were infected and have recovered from heartwater disease (although still remaining infected carriers). Analogous multigene clusters also encode the immunodominant outer membrane proteins (OMPs) in other ehrlichial species. Recombinant protein analogs of the expressed genes and DNA vaccines based on the multigene clusters have been shown to induce protective immunity, although this was less effective in heterologous challenge situations, where the challenge strain major antigenic protein 1 (MAP1) sequence differed from the vaccine strain MAP1. Recent data for several ehrlichial species show differential expression of the OMPs in mammalian versus tick cell cultures and dominant expression of individual family members in each type of culture system. However, many genes in the clusters appear to be complete and functional and to generate mRNA transcripts. Recent data also suggest that there may be a low level of protein expression from many members of the multigene family, despite primary high-level expression from an individual member. A continuing puzzle, therefore, is the biological roles of the different members of these OMP multigene families. Complete genome sequences are now available for two geographically divergent strains of E. ruminantium (Caribbean and South Africa strains). Comparison of these sequences revealed amino acid sequence diversity in MAP1 (89% identity), which is known to confer protection in a mouse model and to be the multigene family member primarily expressed in mammalian cells. Surprisingly, however, the greatest sequence diversity (79% identity) was in the less-studied map1-2 gene. We investigated here whether this map1-2 diversity was a general feature of E. ruminantium in different cultured African strains and in organisms from infected sheep. Comparison of MAP1-2s revealed amino acid identities of 75 to 100% (mean of 86%), compared to 84 to 100% (mean of 89%) for MAP1s. Interestingly, MAP1-2s varied independently of MAP1s such that E. ruminantium strains with similar MAP1s had diverse MAP1-2s and vice versa. Different MAP1-2s were found in individual infected sheep. Different regions of a protein may be subjected to different evolutionary forces because of recombination and/or selection, which results in those regions not agreeing with a phylogeny deduced from the whole molecule. This appears to be true for both MAP1 and MAP1-2, where statistical likelihood methods detect heterogeneous evolutionary rates for segments of both molecules. Sera from infected cattle recognized a MAP1-2 variable-region peptide in enzyme-linked immunosorbent assay, but less strongly and consistently than a MAP1 peptide (MAP1B). Heterologous protective immunity may depend on recognition of a complex set of varying OMP epitopes.
doi:10.1128/IAI.01409-08
PMCID: PMC2687337  PMID: 19307215
16.  Detection by Two Enzyme-Linked Immunosorbent Assays of Antibodies to Ehrlichia ruminantium in Field Sera Collected from Sheep and Cattle in Ghana 
Two serological tests for detection of antibodies to Ehrlichia (previously Cowdria) ruminantium, the causative agent of heartwater, were compared by using field sera collected from sheep and cattle as part of serosurveys in Ghana. Sera selected as either negative or positive by a new polyclonal competitive enzyme-linked immunosorbent assay (PC-ELISA) were tested by the indirect MAP1-B ELISA. Cutoff values of 14 percent positivity (14 PP) for both ruminant species were obtained for the MAP1-B ELISA by using preseroconversion Ghanaian sera and were compared with previously recommended cutoff values of 29 PP for sheep and 38 PP for cattle. With the 14-PP cutoff, of 151 sheep sera which tested negative by PC-ELISA, 89% were also negative by MAP1-B ELISA, while of 419 sheep sera positive by PC-ELISA, 98% were also positive by MAP1-B ELISA. Of 261 bovine sera negative by PC-ELISA, 82% were also negative by MAP1-B ELISA. Of 511 bovine sera positive by PC-ELISA, only 47% were positive by MAP1-B ELISA; these included 168 sera collected from cattle following first seroconversion as detected by both tests, with 125 of these sera positive by PC-ELISA but only 59 and 5 positive by MAP1-B ELISA with the 14- and 38-PP cutoff levels, respectively. These results indicate that both assays are highly sensitive and specific for detection of E. ruminantium exposure in sheep but that the MAP1-B ELISA lacks sensitivity for postseroconversion bovine sera in comparison to the PC-ELISA. Both tests confirm E. ruminantium seroprevalence of at least 70% in Ghanaian sheep; levels of exposure among Amblyomma variegatum-infested Ghanaian cattle are likely to be higher than the seroprevalence value of 66% obtained with the PC-ELISA.
doi:10.1128/CDLI.10.5.917-925.2003
PMCID: PMC193896  PMID: 12965927
17.  Protective immunity to heartwater (Cowdria ruminantium infection) is acquired after vaccination with in vitro-attenuated rickettsiae. 
Infection and Immunity  1991;59(2):729-731.
A Senegalese (S) stock of Cowdria ruminantium was passaged on bovine umbilical endothelial cells with an average interval of 13.9 days (range, 8 to 34 days) between passages. The virulence of infected bovine umbilical endothelial cultures was tested in susceptible goats and sheep by intravenous inoculation of culture supernatant from passages 2 (51 days in vitro), 3 (69 days), 11 (229 days), 14 (264 days), and 16 (291 days). Both animals inoculated with passages 2 and 3 died of heartwater. However, clinical reactions were completely absent in goats and sheep that were inoculated with C. ruminantium from passages 11, 14, and 16. High antibody titers were detected, with immunofluorescence in all vaccinated animals, and a strong signal was found against a 32-kDa Cowdria protein in Western blots (immunoblots). Moreover, the vaccinated animals proved solidly immune when challenged with virulent Cowdria sp.-infected blood stabilate (S strain), whereas all control goats died. No attenuation of a second Cowdria stock (W) was achieved after 226 days in culture, at which time passage 17 was tested in a recipient goat which died of typical heartwater. This is the first report of vaccination with live attenuated C. ruminantium. These attenuated organisms may replace vaccination with virulent blood currently in use in areas where heartwater is endemic.
Images
PMCID: PMC257822  PMID: 1987089
18.  Influence of the Biotope on the Tick Infestation of Cattle and on the Tick-Borne Pathogen Repertoire of Cattle Ticks in Ethiopia 
PLoS ONE  2014;9(9):e106452.
Background
The majority of vector-borne infections occur in the tropics, including Africa, but molecular eco-epidemiological studies are seldom reported from these regions. In particular, most previously published data on ticks in Ethiopia focus on species distribution, and only a few molecular studies on the occurrence of tick-borne pathogens or on ecological factors influencing these. The present study was undertaken to evaluate, if ticks collected from cattle in different Ethiopian biotopes harbour (had access to) different pathogens.
Methods
In South-Western Ethiopia 1032 hard ticks were removed from cattle grazing in three kinds of tick biotopes. DNA was individually extracted from one specimen of both sexes of each tick species per cattle. These samples were molecularly analysed for the presence of tick-borne pathogens.
Results
Amblyomma variegatum was significantly more abundant on mid highland, than on moist highland. Rhipicephalus decoloratus was absent from savannah lowland, where virtually only A. cohaerens was found. In the ticks Coxiella burnetii had the highest prevalence on savannah lowland. PCR positivity to Theileria spp. did not appear to depend on the biotope, but some genotypes were unique to certain tick species. Significantly more A. variegatum specimens were rickettsia-positive, than those of other tick species. The presence of rickettsiae (R. africae) appeared to be associated with mid highland in case of A. variegatum and A. cohaerens. The low level of haemoplasma positivity seemed to be equally distributed among the tick species, but was restricted to one biotope type.
Conclusions
The tick biotope, in which cattle are grazed, will influence not only the tick burden of these hosts, but also the spectrum of pathogens in their ticks. Thus, the presence of pathogens with alternative (non-tick-borne) transmission routes, with transstadial or with transovarial transmission by ticks appeared to be associated with the biotope type, with the tick species, or both, respectively.
doi:10.1371/journal.pone.0106452
PMCID: PMC4172431  PMID: 25248165
19.  Innovative approach for transcriptomic analysis of obligate intracellular pathogen: selective capture of transcribed sequences of Ehrlichia ruminantium 
BMC Molecular Biology  2009;10:111.
Background
Whole genome transcriptomic analysis is a powerful approach to elucidate the molecular mechanisms controlling the pathogenesis of obligate intracellular bacteria. However, the major hurdle resides in the low quantity of prokaryotic mRNAs extracted from host cells. Our model Ehrlichia ruminantium (ER), the causative agent of heartwater, is transmitted by tick Amblyomma variegatum. This bacterium affects wild and domestic ruminants and is present in Sub-Saharan Africa and the Caribbean islands. Because of its strictly intracellular location, which constitutes a limitation for its extensive study, the molecular mechanisms involved in its pathogenicity are still poorly understood.
Results
We successfully adapted the SCOTS method (Selective Capture of Transcribed Sequences) on the model Rickettsiales ER to capture mRNAs. Southern Blots and RT-PCR revealed an enrichment of ER's cDNAs and a diminution of ribosomal contaminants after three rounds of capture. qRT-PCR and whole-genome ER microarrays hybridizations demonstrated that SCOTS method introduced only a limited bias on gene expression. Indeed, we confirmed the differential gene expression between poorly and highly expressed genes before and after SCOTS captures. The comparative gene expression obtained from ER microarrays data, on samples before and after SCOTS at 96 hpi was significantly correlated (R2 = 0.7). Moreover, SCOTS method is crucial for microarrays analysis of ER, especially for early time points post-infection. There was low detection of transcripts for untreated samples whereas 24% and 70.7% were revealed for SCOTS samples at 24 and 96 hpi respectively.
Conclusions
We conclude that this SCOTS method has a key importance for the transcriptomic analysis of ER and can be potentially used for other Rickettsiales. This study constitutes the first step for further gene expression analyses that will lead to a better understanding of both ER pathogenicity and the adaptation of obligate intracellular bacteria to their environment.
doi:10.1186/1471-2199-10-111
PMCID: PMC2806407  PMID: 20034374
20.  Role of cattle in the epidemiology of tick-bite fever in Zimbabwe. 
Journal of Clinical Microbiology  1991;29(2):256-259.
Almost 100% of 52 cattle tested from the southern areas of Zimbabwe were found to have antibodies reactive with Rickettsia conorii compared with less than 30% of 120 cattle from the north. Steers artificially infected with R. conorii isolated from Amblyomma hebraeum were found to show no hematological or biochemical signs of disease but did seroconvert. Clinical signs of infection were restricted to regional lymphadenopathy and dermal erythema, edema, and tenderness at the inoculation site. Rickettsemia was detectable for at least 32 days postinfection. Our findings indicate that cattle could be involved in the transmission of rickettsias by A. hebraeum and may serve as a reservoir of human tick-bite fever in southern Africa.
PMCID: PMC269749  PMID: 2007631
21.  Development of Loop-Mediated Isothermal Amplification (LAMP) Assays for Rapid Detection of Ehrlichia ruminantium 
BMC Microbiology  2010;10:296.
Background
The rickettsial bacterium Ehrlichia ruminantium is the causative agent of heartwater, a potential zoonotic disease of ruminants transmitted by ticks of the genus Amblyomma. The disease is distributed in nearly all of sub-Saharan Africa and some islands of the Caribbean, from where it threatens the American mainland. This report describes the development of two different loop-mediated isothermal amplification (LAMP) assays for sensitive and specific detection of E. ruminantium.
Results
Two sets of LAMP primers were designed from the pCS20 and sodB genes. The detection limits for each assay were 10 copies for pCS20 and 5 copies for sodB, which is at least 10 times higher than that of the conventional pCS20 PCR assay. DNA amplification was completed within 60 min. The assays detected 16 different isolates of E. ruminantium from geographically distinct countries as well as two attenuated vaccine isolates. No cross-reaction was observed with genetically related Rickettsiales, including zoonotic Ehrlichia species from the USA. LAMP detected more positive samples than conventional PCR but less than real-time PCR, when tested with field samples collected in sub-Saharan countries.
Conclusions
Due to its simplicity and specificity, LAMP has the potential for use in resource-poor settings and also for active screening of E. ruminantium in both heartwater-endemic areas and regions that are at risk of contracting the disease.
doi:10.1186/1471-2180-10-296
PMCID: PMC3000401  PMID: 21087521
22.  Use of a specific immunogenic region on the Cowdria ruminantium MAP1 protein in a serological assay. 
Journal of Clinical Microbiology  1995;33(9):2405-2410.
Currently available serological tests for cowdriosis (Cowdria ruminantium infection) in domestic ruminants are hampered by their low specificities because of cross-reactivity with Ehrlichia spp. The use of recombinant major antigenic protein (MAP1) of C. ruminantium for serodiagnosis was investigated. Overlapping fragments of the MAP1 protein were expressed in Escherichia coli and were reacted with sera from sheep infected with either C. ruminantium or Ehrlichia ovina. Two immunogenic regions on the MAP1 protein, designated MAP1-A and MAP1-B, were identified. MAP1-A was reactive with C. ruminantium antisera, E. ovina antisera, and three MAP1-specific monoclonal antibodies, whereas MAP1-B reacted only with C. ruminantium antisera. An indirect enzyme-linked immunosorbent assay (ELISA) based on MAP1-B was further developed and validated with sera from animals experimentally infected with C. ruminantium or several Ehrlichia spp. Antibodies raised in sheep, cattle, and goats against nine isolates of C. ruminantium reacted with MAP1-B. Cross-reactivity with MAP1-B was limited to Ehrlichia canis and Ehrlichia chaffeensis, two rickettsias which do not infect ruminants. Antibodies to Ehrlichia spp. which do infect ruminants (E. bovis, E. ovina, and E. phagocytophila) did not react with MAP1-B. Antibody titers to C. ruminantium in sera from experimentally infected cattle, goats, and sheep were detectable for 50 to 200 days postinfection. Further validation of the recombinant MAP1-B-based ELISA was done with sera obtained from sheep raised in heartwater-free areas in Zimbabwe and from several Caribbean islands. A total of 159 of 169 samples which were considered to be false positive by immunoblotting or indirect ELISA did not react with MAP1-B.(ABSTRACT TRUNCATED AT 250 WORDS)
PMCID: PMC228424  PMID: 7494037
23.  Heartwater (Cowdria ruminantium Infection) as a Cause of Postrestocking Mortality of Goats in Mozambique 
A serological survey in Mozambique to detect antibodies to Cowdria ruminantium, the etiologic agent of heartwater, revealed a seroprevalence of 8.1% (n = 332) for goats in the northern province of Tete and of 65.6% (n = 326) for goats in the southern provinces. Translocation of 10 serologically negative goats from Tete to farms in the south resulted in two clinical cases of heartwater that were fatal. In addition, four goats seroconverted within the study period of 5 weeks. One goat showed no symptoms. Two goats died of other causes, whereas the remaining goat went missing after 1 week. Experimental needle infections of goats and sheep were conducted to confirm results and to isolate different strains of C. ruminantium. These data indicate that translocation of goats from the north to the south of Mozambique bears a high risk of C. ruminantium infection, which can cause fatal disease.
doi:10.1128/CDLI.8.4.843-846.2001
PMCID: PMC96156  PMID: 11427440
24.  Investigation of tick-borne viruses as pathogens of humans in South Africa and evidence of Dugbe virus infection in a patient with prolonged thrombocytopenia. 
Epidemiology and Infection  1996;116(3):353-361.
In the course of investigating suspected cases of viral haemorrhagic fever in South Africa patients were encountered who had been bitten by ticks, but who lacked evidence of infection with Crimean-Congo haemorrhagic fever (CCHF) virus or non-viral tick-borne agents. Cattle sera were tested by enzyme-linked immunoassay to determine whether tick-borne viruses other than CCHF occur in the country. The prevalence of antibody in cattle sera was 905/2116 (42.8%) for CCHF virus, 70/1358 (5.2%) for Dugbe, 21/1358 (1.5%) for louping ill, 6/450 (1.3%) for West Nile, 7/1358 (0.5%) for Nairobi sheep disease, 3/625 (0.5%) for Kadam and 2/450 (0.4%) for Chenuda. No reactions were recorded with Hazara, Bahig, Bhanja, Thogoto and Dhori viruses. The CCHF findings confirmed previous observations that the virus is widely prevalent within the distribution range of ticks of the genus Hyalomma, while antibody activity to Dugbe antigen was detected only within the distribution range of the tick Amblyomma hebraeum. Cross-reactivity for the nairoviruses, Hazara, Nairobi sheep disease and Dugbe, was detected in serum samples from 3/72 human patients with confirmed CCHF infection, and serum from 1/162 other patients reacted monospecifically with Dugbe antigen. The latter patient suffered from febrile illness with prolonged thrombocytopenia.
PMCID: PMC2271429  PMID: 8666081
25.  The Influence of Interspecific Competition and Host Preference on the Phylogeography of Two African Ixodid Tick Species 
PLoS ONE  2013;8(10):e76930.
A comparative phylogeographic study on two economically important African tick species, Amblyomma hebraeum and Hyalomma rufipes was performed to test the influence of host specificity and host movement on dispersion. Pairwise AMOVA analyses of 277 mtDNA COI sequences supported significant population differentiation among the majority of sampling sites. The geographic mitochondrial structure was not supported by nuclear ITS-2 sequencing, probably attributed to a recent divergence. The three-host generalist, A. hebraeum, showed less mtDNA geographic structure, and a lower level of genetic diversity, while the more host-specific H. rufipes displayed higher levels of population differentiation and two distinct mtDNA assemblages (one predominantly confined to South Africa/Namibia and the other to Mozambique and East Africa). A zone of overlap is present in southern Mozambique. A mechanistic climate model suggests that climate alone cannot be responsible for the disruption in female gene flow. Our findings furthermore suggest that female gene dispersal of ticks is more dependent on the presence of juvenile hosts in the environment than on the ability of adult hosts to disperse across the landscape. Documented interspecific competition between the juvenile stages of H. rufipes and H. truncatum is implicated as a contributing factor towards disrupting gene flow between the two southern African H. rufipes genetic assemblages.
doi:10.1371/journal.pone.0076930
PMCID: PMC3793905  PMID: 24130813

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