Detection by Two Enzyme-Linked Immunosorbent Assays of Antibodies toEhrlichia ruminantiumin Field Sera Collected from Sheep and Cattle in Ghana

Abstract

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.Heartwater, or cowdriosis, caused by the rickettsia Ehrlichia (previously Cowdria) ruminantium (10) and transmitted by ticks of the genus Amblyomma, is an often fatal disease of susceptible domestic ruminants in sub-Saharan Africa and some Caribbean islands (7). Epidemiological surveys of E. ruminantium prevalence in areas of endemicity have been limited by the lack of suitably sensitive and specific serodiagnostic tests (15). Fluorescent antibody tests based on E. ruminantiuminfected mouse macrophages (11), neutrophils and bovine endothelial cell cultures (27), and immunoblotting assays (20, 25) lacked specificity (12,16,25) and were too labor-intensive for mass screening. Several enzyme-linked immunosorbent assay (ELISA) systems have been developed over the last decade, with progressive improvement in performance. Indirect ELISA protocols using crude elementary body-derived antigen (26, 34) and a monoclonal antibody-based competitive ELISA (17) resulted in cross-reactions with other ovine and bovine Ehrlichia species (12,16

Detection of Antibodies to Ehrlichia spp. in Dromedary Camels and Co-Grazing Sheep in Northern Kenya Using an Ehrlichia ruminantium Polyclonal Competitive ELISA

“…The indirect MAP1-B ELISA [ 19 , 27 ] has been widely used in serosurveys of cattle and small ruminants in several African countries and on Caribbean and Indian Ocean islands [ 20 , 28 , 29 , 30 , 31 , 32 , 33 , 34 ] and is considered to be highly specific for E. ruminantium . However, as far as we know, the specific anti-species conjugate required for the MAP1-B ELISA has not been developed for camels.…”

“…The E. ruminantium PC-ELISA employed in the present study has the advantage that it can be applied to sera from any mammal, without the need for host species-specific reagents [ 17 ]. A comparison of the two assays using field sera from sheep and cattle in Ghana found that both were highly specific, and that the PC-ELISA gave more consistent results than the MAP1-B ELISA with bovine sera following seroconversion to E. ruminantium [ 20 ]. The PC-ELISA was then used in extensive longitudinal and point prevalence surveys of cattle, sheep and goats over a three-year period in Ghana [ 18 , 24 ] and a modified version of the assay was used in a serological survey of E. ruminantium exposure in small ruminants in northern Cameroon [ 35 ].…”

“…The PC-ELISA was carried out as described previously [ 17 , 20 ] with minor modifications. Soluble antigens of E. ruminantium (Welgevonden strain) elementary bodies (EB) were isolated from infected bovine pulmonary artery endothelial cells.…”

Longitudinal monitoring of Ehrlichia ruminantium infection in Gambian lambs and kids by pCS20 PCR and MAP1-B ELISA

“…This suggests the possibility of upregulatory-downregulatory effect on the production of antibodies as reported to occur in cattle during persistent infection [14]. However, persistently high antibody levels have been reported in sheep following immunization [31,48], recovery from experimental heartwater [7], and natural field exposure [21,47]. Thus another possible explanation might be that, if vertical transmission is occurring in small ruminants, the resulting infection in the offspring is atypical in some way and does not result in the persistently high antibody levels seen after experimental or tick-transmitted infection.…”

“…In the past, serological tests for detection of antibodies to E. ruminantium suffered from poor specificity due to cross-reactions with other ehrlichial agents [15-17]. Although the MAP1-B ELISA [17] has been reported to detect false-positives in heartwater-free areas attributed to cross-reactions with closely related species [18,19], the assay has higher sensitivity compared to other serological tests to detect E. ruminantium antibodies in ovine and caprine sera [20,21]. This increased sensitivity is attributed to the comparatively longer persistence of MAP1 antibodies in these species [17,20].…”