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Standardization and validation of an agar gel immunodiffusion test for the diagnosis of equine infectious anemia using a recombinant p26 antigen

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Abstract

We developed and validated an agar gel immunodiffusion test (AGID) test for the diagnosis of equine infectious anemia (EIA) using as antigen the p26 protein of equine infectious anemia virus (EIAV) produced in the Escherichia coli expression system. The developed rp26-AGID test showed an excellent diagnostic relative sensitivity (100%) and specificity (100%) compared to a commercial AGID assay when 1855 field serum samples were analyzed. In addition, the rp26-AGID demonstrated to be a precise assay with excellent repeatability and reproducibility. In the analytical sensitivity trial, positive sera showed nearly the same endpoint dilutions for both compared tests. No positive-reactions were observed with 35 serum samples with antibodies related to other endemic agents and also with severely hemolysed samples, demonstrating that the rp26-AGID has an excellent analytical specificity. Complete concordance with blind previous results from five proficiency test panels confirmed the capability of the assay of accurate detection of EIAV antibodies. This is the first time that a recombinant AGID assay able to identify EIAV infections has been standardized and validated in Argentina according to international guidelines. Taking into account the results obtained, the p26-AGID could be adopted as an official test method for the diagnosis and control of EIA in this country.

Introduction

Because of the severe economic impact that Equine infectious anemia (EIA) has on the equine industry this disease falls under a regulatory control program in many countries. In Argentina the national animal health authority (SENASA) states that all horses imported, moving within the country, or congregating at public assemblies must have a negative EIA report conducted within the previous 2 months (Secretaria de Agricultura, Ganadería, Pesca y Alimentos, 2005, Directive 617/05). Although no national survey programme has been carried out, local reports let presume that the infection rate in Argentina is relatively low (Jacobo et al., 2004). The infection has been reported mainly in the north-east area of the country, where the transmission risk is higher because of the great number of large biting insects as well as a great number of non-tested horses.

Seropositivity is the best indication of EIA infection since the integration of equine infectious anemia virus (EIAV) into the host-cell genome conducts the EIAV-infected animals to mount a persistent and vigorous immune response to the viral infection, characterized by the presence of high-titer antibodies directed to the three major viral proteins: the envelope glycoproteins, surface unit (gp90) and transmembrane unit (gp45), and the capsid or core protein (p26) (Cheeves and McGuire, 1985, Coggins et al., 1972).

The agar gel immunodiffusion test (AGID) was designed by Coggins in 1970 as an effective tool for the detection of specific EIA antibodies with high correlation with the horse inoculation test (Coggins and Norcross, 1970, Coggins et al., 1972). Although other serologic tests have been developed and approved the “Coggins Test” is still internationally recognized as the “gold standard” test by the World Organization for Animal Health (OIE) (OIE, 2004a). The detection of EIA antibodies by ELISA has also been described and test kits has been approved in some countries, with excellent agreement with the AGID assay, better turnaround time, and higher sensitivity in some particular cases (Suzuki et al., 1982, Shane et al., 1984, Shen et al., 1984). Nevertheless, especially in some developing countries as Argentina, the most used diagnostic tool continues to be the AGID test. This test detects antibodies against the p26 core viral protein, is inexpensive, simple and highly specific to identify EIAV-infected animals.

According to the OIE recommendations the antigen used for AGID test can be prepared from spleens of acute EIAV-infected horses, from persistently infected cell cultures or from a recombinant expression system. The classic AGID EIAV antigen preparation derived from spleen of infected animals or equine dermal cultures cells has an important disadvantage since it might be contaminated with other cellular or host-derived proteins that could produce non-specific precipitation lines with antibodies against non-EIAV antigens present in the tested serum. These kind of non-specific reactions are frequently found with field serum samples and sometimes the serologic status must be confirmed by ELISA (OIE, 2004a). Additionally, the EIAV is hard to culture; consequently large-scale antigen production is expensive, laborious and time-consuming.

The objective of this study was to express a recombinant EIAV p26 (rp26) protein in Escherichia coli together with the standardization and validation of an AGID test using the rp26 as antigen. We analysed its performance characteristics according to international guidelines and compared the results of a large number of field serum samples against those obtained with an imported commercially available AGID test kit, officially approved by SENASA. Also we have proposed to determine if, this new recombinant test could be adopted as an official test for the diagnosis and control of EIA in our country.

Section snippets

Expression of recombinant p26 of EAIV

Total RNA was extracted with Trizol (Invitrogen) from spleen tissue of an EIAV experimentally infected horse according to manufacturer's instructions and used as template for the RT-PCR amplification. The primers of the nucleic acid sequence encoding the complete EIAV p26 gene were designed according to the complete EIAV Wyoming strain genomic sequence (Table 1). The amplified DNA was cloned into the pBAD/Thio-TOPO expression vector (Invitrogen) according to the manufacturer's instructions. The

Results

A 675bp fragment of the EIAV gag gene corresponding to nucleotides 906–1571 encoding the protein p26 of EIAV was successfully amplified from total RNA extracted from spleen tissue of an experimentally infected horse with a native strain of EIAV by RT-nested PCR. The amino acid sequence variation in the translated of p26 gene consisted of 15 amino acid changes (6%) randomly distributed with respect to the amino acid sequence of the prototype Wyoming strain of EIAV (data not shown). The

Discussion

The AGID test is still internationally recognized as the gold standard test for the diagnosis of EIA (OIE, 2004a) and continues to be the most commonly used diagnostic tool in developing countries as Argentina because it is an inexpensive, simple and highly specific assay to identify EIAV-infected animals. Approximately 300,000 Coggins tests are done each year in our country using a national test kit consisting of a tissue culture-derived antigen or alternatively a USA imported test kit

Acknowledgements

We thank G. Brejof for supplying spleen tissues from an experimentally infected horse; T. Becu, C. Bel, S. Meyra and J. Reynal from SENASA qualified laboratories for supplying serum samples. We also thank C. Gonzalez and A. Varone for excellent technical assistance. This work was partially supported by the INTA/Haras technical agreement. Irene Alvarez was supported by a fellowship from INTA.

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