Evaluation of a Commercial Enzyme-Linked Immunosorbent Assay for the Diagnosis of Bovine Tuberculosis from Milk Samples from Dairy Cows

Abstract

Milk samples from dairy cows provide a ready source of material for measuring antibody responses to Mycobacterium bovis antigens. In this study, we evaluated the IDEXX enzyme-linked immunosorbent assay (ELISA) for the measurement of antibody responses to M. bovis antigens MPB70 and MPB83 in milk samples from New Zealand cattle. Test sensitivities for individual milk and serum samples were assessed in samples collected from 44 M. bovis-infected cows, and test specificities were assessed in milk samples collected from 356 cows from tuberculosis (TB)-free herds. Milk vat samples were collected from 505 herds from regions with relatively high or low prevalences of infection. The ELISA had a sensitivity of 50% and a specificity of 97.5% for milk samples, and the test sensitivities for milk and serum samples were the same. Dilution of the positive test milk samples in milk from noninfected cows at 1/10, 1/20, and 1/50 dilutions reduced the proportions of positive responses to 13/21, 9/21, and 4/21, respectively. Small differences were observed in the ELISA responses of milk samples from individual TB-free cows collected at different times during lactation. No significant differences were detected in the ELISA responses of milk vat samples collected from infected and noninfected herds. This study shows that milk samples can be substituted for serum samples for screening individual cows for M. bovis infection, and pooling of milk samples from 10 to 20 animals can result in a reduction in the sensitivity by approximately 50%. However, screening of milk vat samples is unlikely to be useful in countries with low prevalences of M. bovis in cattle and large herd sizes. Bovine tuberculosis (TB) caused by Mycobacterium bovis is a major economic problem in many countries, and control of this chronic disease in domestic livestock has often proved difficult. Although the disease has been eradicated from cattle in a number of countries through the implementation of a "test-andslaughter" control program, this strategy has been less effective in countries which have a wildlife reservoir of M. bovis infection and has been impractical in countries where the strategy is unaffordable or socially unacceptable (1). The test-and-slaughter programs have been based on screening of animals using the tuberculin skin test, which measures delayed hypersensitivity responses to purified protein derivative (PPD) prepared from M. bovis or which, in some countries, compares PPDs prepared from M. bovis and Mycobacterium avium. More recently, the whole-blood gamma interferon (IFN-␥) test has been used in conjunction with the skin test to improve specificities by retesting skin test reactor cattle or to improve sensitivities by retesting skin test-negative animals from chronically infected herds (2, 3). Although the IFN-␥ test has made a valuable contribution in improving the accuracy of diagnosing TB in cattle, additional types of assays are needed, particularly assays which are inexpensive and which can potentially be used on pooled samples from group...

The clinical and serological diagnosis of Mycobacterium bovis in blood and milk serums of lactating cows by IDEXX ELISA test in Wasit and Dhi-Qar provinces/Iraq

“…31 Also, it would be missed; apparently, their benefit through an examination of a few diseased cattle that persist at large groups. 20 The results of Tables 2 and 3 revealed that in Wasit province, the morbidity rate was more than this reported in Dhi-Qar province. Although, the role of local spread of bTB in some areas is not well understood, bTB testing had a significant impact on the expansion and long distance spread of disease, especially on transmission to areas with relatively low incidence.…”

“…Thus, IDEXX ELISA had a sensibility to detect the antibodies in blood more than in milk, and several causes may be concluded in the interpretation of this difference such as the individual differences in animal's antibody responses, lactation stage, time of milk collection, herd size, proportion of milk dilution, cutoff level changing, and the producing of nonspecific antibody responses to test antigens. 20 However, the test sensitivity is increased markedly with severity of the disease. 27 Also, the high relationship among an antibody's reactions in milk and blood had been reported, at same animal, in bTB and John's disease.…”

Investigation of the cause of geographic disparities in IDEXX ELISA sensitivity in serum samples from Mycobacterium bovis-infected cattle

“…Due to the economic consequences associated with M. bovis infections in cattle, as well as its zoonotic potential, methods for quickly and accurately identifying infected cattle are of considerable importance. One such method is an enzyme-linked immunosorbent assay (ELISA) developed by IDEXX Laboratories (Westbrook, ME), which allows for the rapid detection of M. bovis -specific antibodies in bovine serum or milk samples 5 6 . This assay detects antibodies to the antigenic proteins MPB70 (where “MPB” is an acronym for p rotein from M .…”

“…The IDEXX ELISA has been tested on serum and milk samples from cattle that are not infected with M. bovis , and has been found to have a specificity of approximately 98%—that is, the test gives a positive result for only 2% of uninfected cattle 5 6 . The sensitivity of the ELISA has been evaluated using cattle that were verified to be infected with M. bovis using independent tests.…”

Temporal Pattern of Circulating Antigens and Antibody Responses in Cats Experimentally Infected with Dirofilaria immitis

“…After a wash step, detection of anti-heartworm specific antibody was performed using a secondary anti-species antibody horseradish peroxidase conjugate (1:2000 dilution, Jackson ImmunoResearch, West Grove, PA). Following a wash step and addition of substrate, plate absorbance optical density (OD) values were recorded and sample-to-positive ratios (S/P; sample OD divided by positive control OD) were calculated for each sample (sample OD-negative control OD]/positive control OD-negative control OD) (Buddle et al 2013). Samples with S/P ratios ≥ 0.5 were positive.…”