Elsevier

Veterinary Parasitology

Volume 79, Issue 1, 1 September 1998, Pages 19-34
Veterinary Parasitology

A multiple antigen ELISA to detect Neospora-specific antibodies in bovine sera, bovine foetal fluids, ovine and caprine sera

https://doi.org/10.1016/S0304-4017(98)00156-3Get rights and content

Abstract

Neospora caninum is a cyst-forming coccidian parasite recently identified as a cause of abortion in cattle. The epidemiology of neosporosis is poorly understood, partly because accurate diagnosis of infection is difficult. In this paper we describe the development of a multiple antigen-based enzyme-linked immunosorbent assay (ELISA) to detect antibodies to N. caninum in sera from cattle, sheep and goats as well as from bovine foetal fluids. A water-soluble fraction (wsf) of sonicated NC-1 strain tachyzoites was used as the antigen in the ELISA. Minimum optical density (OD) values that were considered to be Neospora antibody-positive, that is, the cut-off OD values were determined separately for bovine maternal sera, bovine foetal fluids, ovine sera and caprine sera; they were 0.40, 0.17, 0.23 and 0.41 OD, respectively. The ELISA gave a high signal/noise ratio, giving good sensitivity and specificity, correlating well with the indirect fluorescent antibody test (IFAT) currently used to diagnose Neospora infection in cattle, sheep and goats. In both the ELISA and immunoblot analysis using the same antigen, there was no significant cross-reactivity with sera from cattle, sheep or goats that had been infected with Toxoplasma gondii. The ELISA also showed no cross-reactivity in sera from cattle infected with Sarcocystis cruzi, Babesia divergens, B. bovis and B. bigemina. The wsf fraction of sonicated N. caninum tachyzoites used in this ELISA can be easily prepared and may be more sensitive than a single antigen ELISA, whilst still retaining good specificity.

Introduction

Neospora caninum is a cyst-forming coccidian parasite that was originally identified in the tissues of paralyzed dogs (Bjerkås and Presthus, 1988; Dubey et al., 1988a). The parasite has since been identified in a wide range of animal species (Dubey and Lindsay, 1996) and has been recognised as a significant cause of abortion in cattle (Anderson et al., 1991; Barr et al., 1991; Trees et al., 1994). Diagnosis of N. caninum infection can be confirmed by immunohistology, or by the polymerase chain reaction (PCR) (Dubey and Lindsay, 1996). However, diagnosis by direct detection is often difficult because the number of parasites in tissues is low and is only achieved post-mortem. Ante-mortem serological tests which can identify N. caninum-infected animals provide valuable tools for diagnosis and epidemiological surveys as well as for experimental investigations. An indirect fluorescent antibody test (IFAT) has been widely used for the sero-diagnosis of neosporosis. It is, however, time-consuming when dealing with large numbers of samples and requires trained personnel for the interpretation of slides resulting in subjective data. Moreover, the nature of data obtained using IFAT is discrete, making it less amenable to statistical analysis. Due to the inconveniences inherent in the IFAT, there has been increasing interest in the development of enzyme-linked immunosorbent assays (ELISAs) for the sero-diagnosis of neosporosis which enables rapid processing of large numbers of samples. Much of the development has concentrated on the use of different antigen preparations in the ELISA, such as iscom-antigen (Björkman et al., 1994, Björkman et al., 1997), recombinant antigens (Lally et al., 1996; Jenkins et al., 1997), or formalin-fixed whole tachyzoite antigen (Williams et al., 1997). The variety of antigen preparations used reflects concern over the potential problem of antigenic cross-reactivity with other closely related parasites. Whilst the use of highly selected antigens may minimise the risk of cross-reactivity with other parasite species, a limited repertoire of antigens may restrict individual recognition of sera from different animals. The use of multiple antigens in an ELISA such as those present in a sonicated whole tachyzoite preparation, may overcome the potential problem of antigenic diversity in Neospora isolates and have a greater sensitivity (Paré et al., 1995). Moreover, since the complete life-cycle of the parasite is not yet known, an ELISA using multiple Neospora antigens may be more useful in diagnosis as well as in epidemiological studies, since there is less chance of basing the assay on stage-specific antigens.

Although detection of maternal antibody is useful in determining the seroprevalence of N. caninum, detection of antibodies in foetal fluids may give a better indication of the involvement of the parasite in bovine abortion (Barr et al., 1995; Buxton et al., 1997a, Buxton et al., 1997b; Wouda et al., 1997). For accurate diagnosis as well as a better understanding of the epidemiology of the parasite, the ability to measure antibody in foetal fluids is essential.

There have been many reports worldwide linking Neospora infection with bovine abortion. However, problems due to naturally acquired Neospora infection have been reported in other species including sheep (Dubey et al., 1990) and goats (Barr et al., 1992; Dubey et al., 1992, Dubey et al., 1996a). The prevalence of Neospora infection in sheep and goats is not known, and the significance of infection in these animals is yet to be determined. The development of an indirect ELISA for Neospora sp. in sheep and goats as well as cattle is required to allow a large-scale serological survey on Neospora infection in these species. The aim of this study was to develop such a multiple antigen-based ELISA to detect Neospora-specific antibodies in maternal sera and foetal fluid of cattle and the sera of sheep and goats.

Section snippets

Cultivation of N. caninum tachyzoites

Antigen was prepared using tachyzoites of the NC-1, Neospora caninum isolate, originally isolated from a congenitally infected dog (Dubey et al., 1988b). Tachyzoites were cultured in vero cells in Iscove's Modified Dulbecco's Medium (IMDM) supplemented with 50 U/ml of penicillin, 50 μg/ml of streptomycin (Gibco, UK) and 2% (v/v) heat-inactivated normal equine serum (Advanced Protein Products, Brierley Hill, UK), at 37°C in a 5% CO2-humidified incubator using the method described by Innes et al.

Optimum concentrations of reagents

The optimum concentration of antigen, dilution of sera and dilution of conjugate for each species-specific ELISA were determined by criss-cross serial-dilution analysis (Table 1). Combinations which gave the highest signal–noise ratios were determined as optimum.

Optimum cut-off points for the ELISA

To determine the optimal cut-off OD value for the bovine assay to allow discrimination between positive and negative samples, the ELISA OD values of 115 bovine maternal sera were compared with the IFAT titres obtained from the same sera

Discussion

In this paper we describe the development of an ELISA to detect Neospora-specific antibodies in the maternal sera and foetal fluids of cattle and in the sera of sheep and goats. The multiple water-soluble antigens used as a basis for the assay were easy to prepare from Neospora tachyzoites and gave consistent results. The assay was both sensitive and specific with a high signal-to-noise ratio. The high OD values obtained may be due to the large number of antigens selected in the wsf and

Acknowledgements

The authors would like to thank Prof. Duncan Brown and colleagues at the Centre for Tropical Veterinary Medicine, University of Edinburgh, UK and Mr. Hilary Edgar, Veterinary Science Department, Belfast, UK, for the provision of Babesia sp. positive sera, Dr. Joakim Holmdahl, National Veterinary Institute, Uppsala, Sweden, for the provision of Sarcocystis cruzi positive serum, Dr. Anja Heckeroth, Institut für Parasitologie, Tieraerztliche Hochschule Hannover, Hannover, Germany, for the

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