A pregnancy detection assay using milk samples: evaluation and considerations.

Two experiments were conducted to evaluate a pregnancy-detection assay based on the measurement of pregnancy-associated glycoproteins (PAG) in milk samples. In experiment 1, milk samples were collected on the day of first pregnancy check (33-52 d postinsemination; n=119) or second check (60-74 d postinsemination; n=60). The accuracy in identification of pregnant and nonpregnant cows was 99% at first check. Only 6% of samples were found to be within an intermediate range of PAG concentrations and classified as requiring recheck by the assay. At second check, the accuracy of the assay was 98%. Fifteen percent of these samples were classified as requiring recheck. In experiments 2a (n=17 cows) and 2b (n=16 cows), milk and plasma samples were collected from cows at weekly intervals beginning 2 (experiment 2a) or 4 d (experiment 2b) after insemination. The earliest time point at which pregnant cows were accurately classified as pregnant by the assay was on d 30 postinsemination. A transient decline in PAG levels into the intermediate range was observed on d 46 to 72 postinsemination. This coincides with the time of recheck in experiment 1. Results obtained with the plasma samples were essentially the same. The accuracy of pregnancy identification based on milk samples from nonpregnant and pregnant cows was 99%. Levels of PAG in milk were useful in identifying 6 incidences of embryonic mortality. No consistent relationship was noted between the timing of the decline in PAG levels and the timing of luteal regression in this small number of cows.

Strategic use of serology for the diagnosis of bovine tuberculosis after intradermal skin testing.

Diagnostic tests based on cell-mediated immunity are used in programmes for eradication of bovine tuberculosis (Mycobacterium bovis). Serological assays could be applied as ancillary methods to detect infected animals. Our objective was to evaluate two serological techniques: M. bovis Ab Test (IDEXX, USA) and Enferplex™ TB assay (Enfer, Ireland) in animals tested simultaneously with the single and comparative intradermal tests and the interferon-gamma assay. This work was performed at two stages. First, a preliminary panel of samples collected prior to intradermal tests from tuberculosis-free (n=60) and M. bovis-infected herds (n=78) was assayed, obtaining high specificity: 100% (M. bovis Ab Test) and 98.3% (Enferplex TB assay) but low sensitivity (detection of M. bovis infected animals): 23.9% (M. bovis Ab Test) and 32.6% (Enferplex TB assay). Subsequently, the use of serological techniques was further studied in two herds with M. bovis infection (n=77) using samples collected prior to, and 72 h and 15 days after PPD inoculation. The highest level of detection of infected animals for serology was achieved at 15 days post-intradermal tests taking advantage of the anamnestic effect: 70.4% and 85.2% in herd A, and 66.7% and 83.3% in herd B, using M. bovis Ab Test and Enferplex TB assay, respectively. Quantitative results (average values obtained with M. bovis Ab Test ELISA and degree of positivity obtained with Enferplex TB assay) were higher in animals showing lesions compatible with tuberculosis. No significant differences were observed in the number of confirmed infected animals detected with either serological technique.

Mycobacterium avium subsp. paratuberculosis antibody response, fecal shedding, and antibody cross-reactivity to Mycobacterium bovis in M. avium subsp. paratuberculosis-infected cattle herds vaccinated against Johne's disease.

Vaccination for Johne's disease with killed inactivated vaccine in cattle herds has shown variable success. The vaccine delays the onset of disease but does not afford complete protection. Johne's disease vaccination has also been reported to interfere with measurements of cell-mediated immune responses for the detection of bovine tuberculosis. Temporal antibody responses and fecal shedding of Mycobacterium avium subsp. paratuberculosis, the causative agent of Johne's disease, were measured in 2 dairy cattle herds using Johne's disease vaccine (Mycopar) over a period of 7 years. Vaccination against Johne's disease resulted in positive serum M. avium subsp. paratuberculosis antibody responses in both herds, and the responses persisted in vaccinated cattle up to 7 years of age. Some vaccinated animals (29.4% in herd A and 36.2% in herd B) showed no serological reactivity to M. avium subsp. paratuberculosis. M. avium subsp. paratuberculosis-specific antibody responses were also detected in milk from Johne's disease-vaccinated animals, but fewer animals (39.3% in herd A and 49.4% in herd B) had positive results with milk than with serum samples. With vaccination against M. avium subsp. paratuberculosis, fecal shedding in both dairy herds was reduced significantly (P < 0.001). In addition, when selected Johne's disease-vaccinated and -infected animals were investigated for serological cross-reactivity to Mycobacterium bovis, no cross-reactivity was observed.

Evaluation of a commercial enzyme-linked immunosorbent assay for the diagnosis of bovine tuberculosis from milk samples from dairy cows.

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.