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.

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.

Serological survey of antibodies to Ehrlichia ruminantium in small ruminants in Tanzania.

Sera from 497 sheep and 555 goats collected in a cross sectional study from different geographical locations in north-eastern Tanzania were examined for antibodies to Ehrlichia ruminantium using MAP 1-B ELISA technique. E. ruminantium antibodies were found in 68.6% (341/497) of sheep and 64.7% (359/555) of goats. Overall seroprevalence was 66.5% (700/1052). Infection rates were higher in sheep than goats (P < 0.05), in pastoral than in agro-pastoral production systems (P < 0.05) and in female sheep than males (P < 0.05). (131/143) 91.6% of the farms/flocks tested revealed sero-positive animals. E.ruminantium infections were found in all the geographical villages and districts tested. The infection rates per administrative district varied from 36.4% (Muheza) to 90% (Mkinga) in goats and from 11.9% (Muheza) to 94.6% (Mkinga) in sheep. The results shows E. ruminantium infection was prevalent and widely but unevenly distributed throughout the eight districts under study. These findings should be taken into consideration when future disease control and livestock upgrading programs are implemented.

The prevalence of serum antibodies to Ehrlichia ruminantium infection in ranch cattle in Tanzania: a cross-sectional study.

Serum samples collected in a cross-sectional survey of grazing cattle on Manyara Ranch, Monduli district, Tanzania, were tested by indirect major antigenic protein 1 fragment B (MAP 1-B) ELISA to determine the seroprevalence of Ehrlichia ruminantium and to assess ranch-level risk factors for heartwater. Heartwater-exposed cattle were widespread on the ranch and overall seroprevalence was 50.3% (95% CI, 44.9-55.6), enough to indicate an endemically unstable situation. Multivariate logistic regression modelling was used to identify risk factors associated with seropositivity. Two factors appeared to increase the herd's risk for contracting heartwater. Seroprevalence increased significantly with age (beta = 0.19 per year of age, P < 0.001) and animals carrying ticks of any species were associated with an increased risk of infection with E. ruminantium (Odds ratio, OR = 3.3, P < 0.001). The force of infection based on the age seroprevalence profile was estimated at 18 per 100 cattle year-risk. The current tick control measures on the ranch were associated with a decreased risk of infection with E. ruminantium (OR = 0.25 for no dipping and OR = 0.31 for low dipping, P < 0.001). Six tick species were identified; in order of frequency these were: Ambylomma variegatum 59.9%, Rhipicephalus evertsi evertsi 13.9%, Rhipicephalus pulchellus 12.5%, Hyalomma truncatum 7.03% and Rhipicephalus appendiculatus 6.07%. The least encountered tick was Rhipicephalus simus, which accounted for 0.38%. The cattle seemed well adapted to their environment and capable of resisting the tick burden under this extensive wildlife/livestock grazing and interaction system.

Immunisation of sheep against heartwater in The Gambia using inactivated and attenuated Ehrlichia ruminantium vaccines.

Heartwater (cowdriosis) is a disease of ruminants caused by a rickettsial pathogen Ehrlichia ruminantium and transmitted by ticks of the genus Amblyomma. The purpose of this work was to evaluate the protective efficacies of inactivated and attenuated vaccines to protect sheep against heartwater in The Gambia. An inactivated vaccine, prepared from E. ruminantium (Gardel stock), and a live attenuated vaccine from E. ruminantium (Senegal stock), were evaluated in two independent on-station trials. A local stock of E. ruminantium (Kerr Seringe) was used as challenge material. Inactivated and live attenuated vaccines provided 43% and 100% protection, respectively, against virulent needle challenge. In a subsequent field trial, the attenuated vaccine protected 75% of sheep against virulent tick challenge, which was fatal for all control sheep. Quantification by real-time PCR showed that an immunising dose of approximately 23,000 attenuated E. ruminantium organisms was sufficient. Moreover, restriction fragment length polymorphism (RFLP) analysis indicated that the local Kerr Seringe genotype caused mortality amongst control sheep, whereas fatalities in the vaccinated group could be attributed to a different genotype.

Identification of Ehrlichia ruminantium (Gardel strain) IFN-gamma inducing proteins after vaccination with a killed vaccine.

IFN-gamma is considered as a key factor in protection against heartwater of ruminants, caused by the obligate intracellular bacterium Ehrlichia ruminantium. In this study, a better definition of the molecular masses of IFN-gamma inducing proteins of the Gardel strain of E. ruminantium was obtained by the use of continuous flow electrophoresis (CFE) and sensitized polyclonal lymphocytes. Out of 15 E. ruminantium CFE fractions tested within the 14-39 kDa region, eight were commonly reacted to by all goats. Interestingly, half of these fractions fall within the 23-29 kDa region, shown previously to contain polymorphic B-cell epitopes. Thus, the results suggest that this region also contains T-cell epitopes potentially involved in protection. Also, several proteins were found to be more immunogenic than the serologically immunodominant MAP1 protein. Finally, high activity within the 15-19 kDa region was observed, which confirms previous work done with CD4+ T-cell lines obtained from cattle immunized with a South African strain of E. ruminantium. The proteins falling within the molecular weight ranges defined in this study may have potential as vaccine antigens.

Comparing the detection of exposure to Ehrlichia ruminantium infection on a heartwater-endemic farm by the pCS20 polymerase chain reaction assay and an indirect MAP1-B enzyme linked immunosorbent assay.

Detection of heartwater is not always easy especially because all the serological assays so far available either have poor sensitivity or specificity. The indirect MAP-1B ELISA has been reported to be the most specific test for heartwater, although it does also detect antibodies to some closely related ehrlichial agents. This study was undertaken to compare two methods for the detection of heartwater infection caused by the ehrlichial agent Ehrlichia (Cowdria) ruminantium. Fifteen cattle on a heartwater-endemic farm infested with high numbers of Amblyomma hebraeum ticks, and hence exposure to E. ruminantium infection were monitored over an 8-week period by pCS20 PCR and an indirect MAP-1B ELISA. Infection was detected by pCS20 PCR in most animals with the highest number of positives (60%) in week 6 of the study. Similarly, exposure to E. ruminantium was detected by indirect MAP-1B ELISA in some animals, with the highest number of seropositives (27%) at weeks 2-6 of the study. The data demonstrated a fluctuating rickettsaemia in cattle in a heartwater-endemic area. Comparison of the two tests indicated that the pCS20 PCR assay was more reliable because it detected more infections than the indirect MAP-1B ELISA and would therefore be the method of choice for detection of E. ruminantium infection.

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.

Seasonal epidemiology of ticks and aspects of cowdriosis in N'Dama village cattle in the Central Guinea savannah of Côte d'Ivoire.

In the Central Guinea savannah of Côte d'Ivoire, cattle breeding started only approximately 30 years ago. The impact of parasitism on the overall health status and productivity of the trypanotolerant N'Dama cattle in this area is unknown. In close collaboration with national veterinary institutions and local farmers, we studied spectrum, burden and seasonal dynamics of ticks (including aspects of cowdriosis) on N'Dama village cattle. In a longitudinal study, three randomly selected cattle herds (traditional farming type) of one village were examined repeatedly for ticks. Spectrum, burden, seasonal epidemiology of ticks were assessed. In these traditional herds (which lack (ecto)parasite management), all animals were infested by ticks at monthly counts. Five different tick species were identified; the four genera in order of frequency were: Amblyomma (overall prevalence 96%), Boophilus (47%), Hyalomma (<1%) and Rhipicephalus (<1%). Amblyomma variegatum was the most-abundant tick on cattle in all seasons. Seroprevalence of Cowdria ruminantium was 31% (95% CI: 26, 36%). Most of the animals typically carried low tick burdens. N'Dama cattle seem well adapted to their environment and can resist the tick burdens under this traditional farming system.

Population-based evaluation of the Ehrlichia ruminantium MAP 1B indirect ELISA.

The indirect MAP 1B ELISA based on the recombinant MAP 1B fragment of the immunodominant MAP I protein of Ehrlichia ruminantium is considered to be the most sensitive and specific assay for the serodiagnosis of heartwater. In this study, we evaluated its reliability in detecting exposure to E. ruminantium in field populations of domestic ruminants in Zimbabwe. Cattle and goat herds in endemically stable areas with high infection pressure and an expected close to 100% prevalence of E. ruminantium exposure were sampled. Bovine sera (858) and caprine sera (706) collected at seven locations representative of the two main production systems (communal lands and large scale commercial farms) in the two main agroecological zones of Zimbabwe (highveld and lowveld) were analysed. The prevalence of MAP 1B-specific antibodies in goats was similar and high, ranging from 67 to 100%, at all except one site (43%). Age-specific differences in goats (1, 2, 3, 4, 5 years) were not observed. In contrast, MAP 1B seroprevalence in cattle was significantly lower (P < 0.001), ranging from 46 to 61% in the lowveld communal area and from 24 to 33% in the remaining areas (P < 0.001). Age-specific differences in seroprevalence (1, 2, 3, 4, 5-7 + years) were similarly not evident in cattle (P < 0.15). Hence, the indirect MAP 1B ELISA may be an unreliable indicator of past exposure to heartwater in field-infected cattle in Zimbabwe. Although the reasons for this low response in field cattle are not fully understood, this study illustrates the need for field validation of the performance of new diagnostic tests prior to their use for epidemiological purposes.

Recombinant expression and use in serology of a specific fragment from the Cowdria ruminantium MAP1 protein.

The major antigenic protein (MAP1) of Cowdria ruminantium was screened for immunogenic regions by expression of overlapping recombinant DNA clones of the gene encoding the MAP1 protein. Two regions, designated MAP1-A and MAP1-B, were recognized by all antisera to 9 different isolates of C. ruminantium. MAP1-A contained one or more epitopes responsible for false-positive reactions with Ehrlichia antisera in several serological tests for cowdriosis. Cross-reactivity with MAP1-B was limited to antisera to Ehrlichia chaffeensis and Ehrlichia canis. Antisera to Ehrlichia species that infect ruminants (E. bovis, E. ovina, and E. phagocytophila) did not recognize MAP1-B. The sensitivity of an indirect ELISA based on MAP1-B was found to be excellent, since all sera from animals experimentally infected with C. ruminantium (64 out of 64) reacted with MAP1-B. Validation of this ELISA was carried out with field sera obtained from sheep raised in heartwater-free areas in Zimbabwe and from several Caribbean islands. Only 9 out of 111 samples from Zimbabwe, and 1 out of 58 samples from the Caribbean islands, which were considered to be false positives by immunoblot or indirect ELISA, reacted with MAP1-B. Thus, the ELISA based on MAP1-B is at present the most specific and sensitive serological test for cowdriosis.

Use of a specific immunogenic region on the Cowdria ruminantium MAP1 protein in a serological assay.

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. In conclusion, recombinant MAP1-B may be a suitable antigen for a sensitive serological test for cowdriosis, with dramatically improved specificity.

The clinical pathology of heartwater. III. Changes in blood clotting, blood calcium, blood protein, haematocrit and white-cell counts in sheep with experimentally induced heartwater.

Studies to evaluate changes in blood clotting, blood calcium and protein, the haematocrit and white-cell counts were undertaken in seven sheep with experimentally induced heartwater. A marked decline in thrombocyte count was recorded during the acute stage of the disease. This was associated with increases in both prothrombin time (PT) and activated partial thromboplastin time (APTT); fibrinogen increased while there was no detectable increase in fibrinogen degradation products (FDP). At the same time total serum protein (TSP), albumin and globulin dropped very sharply; total calcium showed a progressive drop but ionized calcium rose initially and was followed by a terminal decline. The total leucocyte count showed a terminal increase while the haematocrit dropped progressively.

Studies on the presence of endotoxin activity and inflammatory products (leukotrienes) in cases of experimentally induced heartwater in sheep.

The presence of endotoxin was examined in seven sheep with experimentally induced heartwater. Elevations in endotoxin levels were recorded in one sampling in three of the seven sheep during the acute stage of the disease. The elevations in endotoxin levels were of short duration and decreased in the 24-h follow-up samples. There was no elevation of leukotrienes (B4, C4 and D4) in the blood, or the thoracic or pericardial fluid of any of the sheep.

Changes in the blood-gas status of sheep with experimentally induced heartwater.

The blood-gas status of seven sheep with experimentally induced heartwater during the acute and terminal stages was investigated. Changes in blood gas included a decline in arterial oxygen tension (pO2) combined with a respiratory alkalosis. Although the sheep became hypoxaemic, blood-gas changes associated with respiratory failure were not observed.

Histologic and immunochemical study of the pathogenesis of heartwater (Cowdria ruminantium infection) in goats and mice.

Eleven adult goats and 32 adult outbred mice were inoculated IV with Cowdria ruminantium-infected blood (Kwanyanga isolate), monitored clinically, then serially euthanatized. Predominant clinical signs of disease in goats were depression, head tremors, seizures, and dyspnea. In mice, dyspnea and depression were the only clinical signs of disease noticed. Tissues were examined histologically and immunohistochemically for C ruminantium colonies or antigen. In goats, C ruminantium was detected only in endothelial cells of the brain, even though gross and microscopic lesions were confined to the thorax. In mice, C ruminantium was detected only in endothelial cells of the heart and lungs.

Protection of goats against Caribbean and African heartwater isolates by the Ball 3 heartwater vaccine.

Two groups of castrated male adult goats (three goats/group) were infected intravenously with the Ball 3 vaccine strain of Cowdria ruminantium and treated with long-acting oxytetracycline at the onset of clinical disease 10 days later. Five weeks post-vaccination one group was challenged with a Caribbean isolate (Gardel) and the other group with a West African isolate (Mali) of C. ruminantium. Non-vaccinated controls infected with either the Gardel or Mali isolate died. All of the vaccinated animals challenged with the Gardel strain reacted and survived. The three vaccinated animals challenged with the Mali isolate also reacted and two died of heartwater.

A mouse lethal dose assay for detection and titration of Cowdria ruminantium (Kwanyanga strain) in goats and ticks.

A mouse lethal dose assay was used to detect a mouse pathogenic strain (Kwanyanga) of Cowdria ruminantium, the etiological agent of heartwater in goats and ticks. The titer of the rickettsial organisms in goat blood was directly related to the febrile response of the goat and the rickettsia were undetectable after the fever subsided. The maximum rickettsial titer in goat blood was 10(3) mouse LD50 ml-1. Cowdria-infected goat blood was shown to retain infectivity when held on ice for up to 2 h, but when held at room temperature infectivity declined by greater than 50% in 2 h. The mouse assay detected Cowdria in feeding female Amblyomma variegatum only on the eighth day of feeding and in feeding males on the second and eleventh days of feeding. Cowdria was shown to persist in the hemolymph of the soft tick Ornithodoros coriaceus for a period of at least 2 years.

Susceptibility to heartwater of calves born to non-immune cows.

The resistance to artificial infection with Cowdria ruminantium of calves born to cows fully susceptible to heartwater is no different from that of calves bred in heartwater endemic areas where the tick challenge is negligible to considerable. The sub-inoculation into mice of blood collected 14-26 days after infection proved the presence of the heartwater agent in the blood of 8 out of 10 calves with no other clinical signs than mild to moderate fever. The combined use of a mouse model and the indirect fluorescent antibody test revealed considerable variation in the degrees to which calves become infected and react to artificial infection.