Diagnosis of tuberculosis due to Mycobacterium bovis in New Zealand red deer (Cervus elaphus) using a composite blood test and antibody assays

Abstract

A blood test for tuberculosis in deer was developed as an ancillary test to clarify the status of skin test-positive deer, with non-specific sensitisation following exposure to saprophytic mycobacteria. The blood test incorporates the measurement of the relative humoral and cellular immunological responses to Mycobacterium bovis and M. avium antigens to provide a composite test with high levels of sensitivity (>95%) and specificity (>98%). The specificity of the test has allowed it to be used in parallel with the skin test to salvage thousands of tuberculosis-free deer with non-specific skin test-positive reactions, while its high sensitivity has consistently identified M. bovis-specific reactivity in tuberculous skin test-positive animals. The rules for establishing the diagnostic parameters for the cellular and antibody assays were developed by retrospective analysis of the laboratory results using blood samples from many thousand tuberculous or disease-free deer. The sensitivity of the blood test was tested in this study using 150 animals with tuberculosis diagnosed by the isolation of M. bovis. It had sensitivity values of 95.7-95.9% in herds with a low (<2.0%) or a high (>30.0%) incidence of tuberculosis. The test had a specificity of 98.0% when tested on 218 disease-free animals, 118 of which were skin test-positive. An antibody test was developed to diagnose M. bovis in skin test-negative anergic deer from tuberculosis infected herds. When this test was used with deer blood taken 10 days after reading the skin test, it had a sensitivity of 85.3% for 102 M. bovis-positive deer. When used in combination with skin test, the antibody test complemented the skin test to raise the sensitivity of the combined tests to 95.0%, when antibody-positive or skin test-positive tests were used to diagnose tuberculosis. The specificity of the antibody test was 100% when used to evaluate 218 disease-free deer from non-infected herds.

Immunoglobulin G1 Enzyme-Linked Immunosorbent Assay for Diagnosis of Johne's Disease in Red Deer ( Cervus elaphus )

“…After incubation overnight at 4°C, unbound antigen was removed from the plates by washing them six times in phosphate-buffered saline (14) was then added and incubated for 1 h at 37°C, and unbound antibody was removed by washing six times. Antibody binding was visualized using a polyclonal goat antimouse IgG horseradish peroxidase-conjugated tertiary antibody (Biosource International, Camarillo, CA) and an o-phenylenediamine dihydrochloride substrate system, as described previously (11,14). The reaction was stopped by the addition of H 2 SO 4 , and the absorbance was read at 490 nm using an automated microplate reader (model 3550; Bio-Rad).…”

Experimental Infection Model for Johne's Disease in Sheep

“…An enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies was adapted from a method previously described by Griffin et al (18). Flat-bottomed microtiter plates (Nunc Maxisorp immunoplate) were coated with 50 l of the antigen PPDj (12.5 g/ml).…”

Immunological and Molecular Characterization of Susceptibility in Relationship to Bacterial Strain Differences inMycobacterium aviumsubsp.paratuberculosisInfection in the Red Deer (Cervus elaphus)

“…Antibody binding was visualized using a polyclonal goat antimouse IgG horseradish peroxidase-conjugated tertiary antibody (Biosource International, Camarillo, CA) and an O-phenylenediamine dihydrochloride (Sigma, St. Louis, Mo.) substrate system, as described previously (13). The reaction was stopped by addition of H 2 SO 4 and the absorbance read at 490 nm using an automated microplate reader (Bio-Rad model 3550).…”

“…Leukocytes were plated at 2.5 ϫ 10 5 viable cells per well into multiple wells of 96-well tissue culture plates (Nunc Products, Denmark). Cells were stimulated in the presence or absence of 2.5 g of PPDj or purified protein derivative of Mycobacterium bovis (PPD-B) (CSL Ltd., Melbourne, Australia) for 5 days, prior to measurement of lymphocyte transformation via nuclear incorporation of [ 3 H]thymidine as described previously (13). Beta emission was measured as mean counts per minute using a liquid scintillation counter.…”