Antibody Responses to MAP 1B and Other Cowdria ruminantium Antigens Are Down Regulated in Cattle Challenged with Tick-Transmitted Heartwater

Abstract

Serological diagnosis of heartwater orThe rickettsia Cowdria ruminantium is the causative agent of heartwater, an acute, fatal infectious disease of domestic and wild ruminants (5, 40) which is transmitted by ticks of the genus Amblyomma (45). Efforts to study the epidemiology of heartwater and to implement disease control have been hampered by the lack of reliable serodiagnostic tests. Available tests are based on cultured organisms or antigen extracts (7,9,13,15,23,26,34,36) and on the major antigenic protein of C. ruminantium, MAP 1 (2), which has been targeted as a diagnostic antigen because of its immunodominance (2,11,14). These tests are, however, limited by the extensive antigenic similarity between C. ruminantium and closely related agents of the genus Ehrlichia (1,8,12,14,17,35,41), some of which also infect ruminants. Recently, a partial fragment of MAP 1, MAP 1B, which spans amino acids 47 to 92 of the mature protein (42, 43), has been shown to have high specificity for C. ruminantium in an indirect enzyme-linked immunosorbent assay (ELISA) (24,25,43). The assay does not detect antibodies to ehrlichial agents infecting domestic ruminants, such as E. bovis, E. ovina, and E. phagocytophila. It does detect antibodies to E. canis (which infects dogs) and antibodies to E. chaffeensis (a pathogen of humans). In the United States, E. chaffeensis infects white-tailed deer (6, 16), a species that is highly susceptible to heartwater. In areas of Zimbabwe (22,43) and the Caribbean (24, 25) that are designated heartwater free by the absence of Amblyomma ticks and clinical heartwater, the MAP 1B indirect ELISA demonstrated a high specificity with cattle, sheep and goat sera. This assay is also reliable for the detection of experimental infections in small ruminants, and it detects antibodies to geographically diverse C. ruminantium isolates from different countries (24, 43). Hence, its use has been proposed for diagnosis and surveillance of heartwater.In a preliminary serological survey of heartwater in Zimbabwe using the MAP 1B indirect ELISA, only 33% of cattle sera from areas with endemic heartwater infection tested positive (22). The low seroprevalence was unexpected, given the high infection pressure in these regions and the consequent likely high prevalence of infection (27). Epidemiological studies conducted on some of these farms over several years demonstrated a tick infection rate of 10% and a vector attachment rate of between one and four ticks every 2 days (31). At this tick attack rate, it was estimated that cattle were exposed to

Development of a Polyclonal Competitive Enzyme-Linked Immunosorbent Assay for Detection of Antibodies to Ehrlichia ruminantium

“…An appropriate diagnostic sensitivity is also very important to reduce the proportion of false negatives in import-export screening. The sensitivity of tests for detection of cattle recovered from heartwater appears to be poor, with a decline of antibodies to negative or minimal levels within 3 to 7 months of infection or of removal from tick infestation, respectively, of calves exposed experimentally to or field challenged with E. ruminantium (18,29). Down-regulation of antibody responses to E. ruminantium antigens was postulated to explain low seropositivity rates detected by MAP1-B ELISA in cattle sera from farms in Zimbabwe where heartwater is endemic (28,29).…”

“…Although improvement of specificity has been the principal concern in recent heartwater serodiagnostic test development, the lack of sensitivity is an equally if not more important concern for the use of serology in epidemiological studies in areas of endemicity (28,29). An appropriate diagnostic sensitivity is also very important to reduce the proportion of false negatives in import-export screening.…”

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

“…Variable and generally lower antibody levels detected in immune cattle by the PC-ELISA in the present study and by other serological tests (11,25) indicate that there may be a reduced host antibody response to E. ruminantium in general following first exposure. Indeed, a recent study in Zimbabwe of field-exposed and ex- (33). With the use of the MAP1-B ELISA on groups of field-exposed NЈDama and Gobra zebu cattle in The Gambia, variation in monthly seroprevalences between 7.5 and 98.1% was found, depending on season, tick control regime, and consequent level of A. variegatum challenge (28).…”

“…Overall consideration of the results from comparison of the two tests suggests that the ideal cutoff point for both ruminant species would lie somewhere between the value calculated for Ghanaian sera and initially recommended values, with the aim of improving sensitivity without loss of specificity. MAP1-B ELISA cutoff points for bovine sera between 15.6 and 50 PP, calculated in a variety of ways, have been used in field surveys and experimental studies in areas of Africa and the Caribbean where heartwater is endemic (8,19,28,30,33).…”

“…Serological tests utilizing recombinant antigens include an indirect ELISA based on a specific fragment of the E. ruminantium major antigenic protein (MAP1), the MAP1-B ELISA (38), and a competitive ELISA using recombinant MAP1 antigen and anti-MAP1 monoclonal antibody competitor (18). These have been shown to have increased specificity, though both antigens cross-react with antibodies to Ehrlichia chaffeensis, which infects deer and domestic goats in the southern United States (9), and sensitivity for bovine sera appears to be poor (24,33).…”