Abstract

The Johne’s disease serum antibody enzyme-linked immunosorbent assay (ELISA) has been a useful herd infection screening tool for domestic agriculture species. This protein G conjugate test provides valuable diagnostic information and is both fast and inexpensive. Since the ELISA assay has been validated only for cattle in the United States, screening for M. paratuberculosis infection in U.S. captive and free-ranging non-domestic species has not benefitted from ELISA technology.¹ Johne’s disease surveillance thus has been limited in most cases to fecal culture.

The ability of protein G to bind immunoglobulin in well-characterized laboratory species (rat, mouse, human, etc.) has been shown,² but its ability to bind immunoglobulin from most non-domestic hoofstock species has not been well-described.³ To determine if this antibody detection system can be used effectively for multiple non-domestic species, the binding characteristics of the protein G conjugate was assessed for 12 non-domestic hoofstock species and two domestic control species (bovine [positive control], chicken [negative control], elk, muntjac, white-tail deer, bison, sheep, bontebok, impala, llama, kudu/nyala, addax, antelope, oryx).

Partially purified immunoglobulin was obtained through high-performance liquid chromatography (HPLC) of sera from six to eight healthy adult individuals belonging to each of the 14 species . Two methods (size-exclusion chromatography and SDS-PAGE) were used to confirm the presence of antibody and to quantify recovery. The binding characteristics of the protein G-horseradish peroxidase detection system utilized in the ELISA were compared for each sample. This was accomplished by measuring the optical density of a uniform amount of immunoglobulin from each of the 106 individual samples when tested against seven concentrations (2.0–5.0 g/ml) of solid-phase protein G (the antigen).

Discussion and Conclusions

Preliminary data shows protein G is capable of binding immunoglobulins from each of the non-domestic species assessed, although the binding patterns were not uniform. Table 1 demonstrates immunoglobulin binding (expressed in mean optical density units for a species) for a uniform amount of immunoglobulin as the protein G antigen was increased from 2.0–5.0 g/ml. As expected, protein G did not bind immunoglobulin from any samples for the negative-control species (chicken) at any antigen concentrations. The pattern for the positive-control species (bovine) was also as expected; there was a step-wise increase in bound immunoglobulin as antigen concentration was increased. Interspecies immunoglobulin binding variability can be seen most clearly for the muntjac samples. When muntjac results are compared with, for example, white-tail deer (another cervid species) the latter showed even greater binding in the same step-wise pattern than what was seen with bovine sera, while immunoglobulin from muntjac samples bound poorly at each antigen concentration. Binding within species varied as well, a finding that will need to be considered when the algorithms used to interpret assay results are developed. For instance, statistically significant intraspecies variation was found for all species except bison, elk, and white-tail deer (p=0.05). The data did not support an ability to predict binding patterns based on taxonomic categories.

Table 1. Antibody binding (mean optical densities)

The number following the species name indicates number of individual samples (e.g., for chicken [negative control], eight were tested, for bovine [positive control], five were tested).
The OD value is the mean of the individual samples per species (e.g., 0.14 is the mean OD for five bovine samples tested at an antigen [protein G] concentration of 2.0).

These findings suggest that the protein G conjugate may be useful in detecting antibody from many hoofstock species. This conjugate may, therefore, be useful not just for detecting antibody due to M. paratuberculosis infection but as a surveillance tool for finding antibody to many types of infection that produce antibody (by substituting different antigens in the solid-phase portion of the assay). The performance of the ELISA is now being assessed using a repository of sera from confirmed cases of M. paratuberculosis infection representing a variety of species. Methods for interpretation and testing protocols are also being developed.

Acknowledgments

The authors gratefully acknowledge Busch Gardens-Tampa, Turner Enterprises, Disney’s Animal Kingdom, and University of Wisconsin SVM-Pathobiological Sciences for their support of this research. The contributions of serum kindly made by many zoologic facilities have been much appreciated.

Literature Cited

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2.  Akerstrom B, Brodin T, Reis K, Bjork L. Protein G: a powerful tool for binding and detection of monoclonal and polyclonal antibodies. J Immuno. 1985;135(4):2589–2592.

3.  Kelly PJ, Tagwira M, Mattewman L, Mason P, Wright E. Reactions of sera from laboratory, domestic, and wild animals in Africa with protein A and a recombinant chimeric protein AG. Comp Immun Microbiol Infect Dis. 1993;16(4);299–305.