Development of an indirect Tams1 enzyme-linked immunosorbent assay for diagnosis of Theileria annulata infection in cattle

Abstract

An enzyme-linked immunosorbent assay (ELISA) was developed based on a recombinant major Theileria annulata merozoite surface antigen, Tams1. Four different recombinant proteins derived from two different Tams1 alleles, both in two different truncated forms, were tested for their performance in the ELISA. Furthermore, antigen concentration, various buffers, washing protocol, and the choice of anti-total-immunoglobulin G (IgG), anti-IgG1, or anti-IgG2 as second antibody were evaluated. The performance of the resulting ELISA was analyzed by measuring the coefficient of variation (CV). A total of 22 sera were analyzed over the measurement range, resulting in a CV of ca. 10%, whereas 30% variation is the maximum acceptable. The cutoff value was determined by the two-graph receiver operating characteristic (TG-ROC), using the indirect fluorescent antibody test (IFAT) as a reference. It was shown that up to 3 months postinfection (p.i.) IFAT is more sensitive and specific, whereas beyond 3 months p.i. ELISA performed as well as IFAT. The cutoff was determined at maximal sensitivity, based on the TG-ROC after 3 months p.i. Nine calves experimentally infected with four different T. annulata stocks remained positive in the ELISA for at least 1 year p.i. Finally, limited cross-reaction was found only with T. parva antisera, but not with any other Theileria or Babesia species. Since the T. parva endemic area hardly overlaps with T. annulata, the Tams1 ELISA has the potential to become a useful tool in the epidemiology of tropical theileriosis.

FIG. 1

Influence of different NaCl concentrations on the specificity of the Tams1 ELISA. The NaCl concentration was varied in buffers used for blocking, serum incubation, and second antibody incubation. False-positive reacting sera were compared with negative, low-positive, moderate-positive, and high-positive control sera. ELISA results are expressed as the PP of the high-positive control serum for each different NaCl concentration. Asterisks indicate the three false-positive sera that could not be sufficiently reduced. The cutoffs were determined for each NaCl concentration by doubling the PP value of the standard negative serum.

FIG. 2

Precision profile plots of the Tams1 ELISA for 22 cattle sera expressed as the CV as a function of PP. Each point represents the mean of an individual serum measured 128 times on eight different plates.

FIG. 3

TG-ROC analysis of the Tams1 ELISA results for <3 months p.i. (A) and >3 months p.i. (B). The intermediate range (IR) is determined by the cutoff values at 95% sensitivity (Se) and 95% specificity (Sp).

FIG. 4

ROC plots of Tams1 ELISA results for <3 months p.i. (A) and >3 p.i. (B). The graph in panel B falls precisely over the left y axis and the top x axis. Se, sensitivity; Sp, specificity.

FIG. 5

Youden's index (J) and efficiency (Ef) of the Tams1 ELISA as a function of the selected cutoff value at <3 months p.i. (A) and >3 months p.i. (B).

FIG. 6

Logarithm of negative (LR−) and positive (LR+) LRs as a function of the selected cutoff value at <3 months p.i. (A) and >3 months p.i. (B).

FIG. 7

Antibody profiles of four different T. annulata isolates in nine different animals as measured by Tams1 ELISA (●) and piroplasm IFAT (○). Panels: A, animal 119 infected with T. annulata from Bahrain; B, animal 287, Spain; C, animal 124, Bahrain; D, animal 292, Spain; E, animal 178, Mauritania; F, animal 293, Spain; G, animal 343, Mauritania; H, animal 341, Ankara; I, animal 165, Bahrain. The ELISA cutoff is 12.6 PP; the IFAT is considered positive at titers of >80.