Standardisation and comparison of serial dilution and single dilution enzyme linked immunosorbent assay (ELISA) using different antigenic preparations of the Babesia (Theileria) equi parasite

Abstract

-Serial dilution and single dilution enzyme linked immunosorbent assays (ELISA) were standardised and their sensitivity and specificity were compared for serodiagnosis of Babesia equi infection. The antibody titres of 24 donkey sera of known identity were determined separately by serial dilution ELISA using three different B. equi antigens namely whole merozoite (WM), cell membrane (CM) and high speed supernatant (HSS). The ratios of the optical density (OD) of known positive and known negative sera at different serum dilutions were calculated and termed as the positive/negative (P/N) ratio. The coefficients of correlation (r) were calculated between the P/N ratios at different dilutions of sera and the log 10 antibody titres of the same sera were ascertained by serial dilution ELISA. The highest value of 'r' was obtained at a serum dilution of 1:200. From log 10 antibody titre of sera (y) and their P/N ratio at a dilution of 1:200 (x), regression equations (y = a + bx) were calculated separately for the three antigens. Test sera were diluted to 1:200, their OD were read in duplicate wells and were converted to the P/N ratio. Antibody titres were predicted from the P/N ratio using a regression equation separately for the three antigens. Titres obtained by both ELISAs were not significantly different from each other, thus confirming that single dilution ELISA could be successfully used to replace conventional serial dilution ELISA. The sensitivity, specificity and predictive value of single dilution ELISA was validated statistically using 42 B. equi diseasepositive sera and 106 B. equi disease-negative sera. The WM antigen was found to be the most sensitive with a higher predictive value for negative test sera as compared to the CM or HSS antigens. Sera positive for other equine infections including Babesia caballi showed no cross-reaction with the three B. equi antigens in ELISA, thus the test was immunologically specific. Antibody titres of 109 unknown field donkey/horse sera obtained by serial and single dilution ELISA using the WM antigen did not show any significant difference. Since the single dilution ELISA was found to be

<i>In-vivo</i> Therapeutic Efficacy Trial with Artemisinin Derivative, Buparvaquone and Imidocarb Dipropionate against <i>Babesia equi</i> Infection in Donkeys

“…The importance lies in the fact that infected animals become carriers for life and potential intrauterine infection can occur throughout the breeding life of the mare. A high seroprevalence of B. equi antibodies have been reported in equids from many tropical countries specially India [15,16], African continent and Madagaskar [8] and clinical cases are very often. Losses due to intrauterine infection leading to abortion and strong regulation on export of B. equi carrier equine species are relatively common.…”

Serodiagnosis of Babesia equi in horses submitted to exercise stress

“…Additionally, various procedures of ELISA have been standardized and shown to be more sensitive and specific than CFT and IFAT (Knowles et al 1991, Kumar et al 2003. Therefore, the advantages and disadvantages of each test should be considered at the moment of choice of the best test for the diagnosis of equine piroplasmosis.…”

Immunokinetics of Theileria equi specific antibodies: a comparison in serial and single dilution ELISA antibody end titres

“…Application of conventional serial dilution for detection of antibodies against T. equi parasite is now well established (6,10,12,20). The diagnostic specificity and sensitivity of single dilution ELISA for detection of T. equi antibodies in equine serum has previously been reported (6).…”

“…The diagnostic specificity and sensitivity of single dilution ELISA for detection of T. equi antibodies in equine serum has previously been reported (6). This study was aimed to determine the immunokinetics of T. equi antibody response in experimentally infected donkeys so as to ascertain the diagnostic sensitivity of single dilution ELISA vis-à-vis routinely used serial dilution ELISA.…”

“…Single dilution ELISA considered the P/N ratio instead of the OD value (serial dilution ELISA) for ascertaining the status of unknown samples (positive or negative). The OD value of a particular sample depends largely on assay conditions and time of incubation (particularly in the colour development step), whereas the P/N ratio is more stable (6). These factors influence the status of samples having low titres and may be a plausible explanation for the high sensitivity of single dilution ELISA on these samples.…”