Comparing recombinant MPB70/SahH and native 20-kDa protein for detecting bovine tuberculosis using ELISA.

Bovine tuberculosis (bTB) is a zoonosis caused by Mycobacterium bovis. Test-and-cull protocols and gross pathological examinations of abattoir animals as well as milk pasteurization have been implemented to prevent the spread of tuberculosis from animals to humans worldwide. Despite the importance of precise and rapid diagnostic tests, conventional methods including intradermal skin tests and γ-interferon assays are limited by the high rate of false-negative results for cattle in the late infectious stage and due to laborious and time-consuming procedures. Therefore, antibody detection methods such as enzyme-linked immunosorbent assay (ELISA) are urgently needed to supplement the established approaches and expand the diagnostic window. This study was conducted to develop a bTB ELISA by evaluating recombinant and native proteins and various assay parameters. We produced recombinant MPB70 and SahH (M70S) and a native 20-kDa protein (20K) and optimized the ELISA protocol. The 20K ELISA showed 94.4% sensitivity and 98.2% specificity with an optimal sample-to-positive ratio cut-off of 0.531. The sensitivity and specificity of M70S ELISA were 94.4% and 97.3%, respectively, with an optimal sample-to-negative ratio cut-off of 1.696. Both assays showed acceptable diagnostic efficiency and could be used for bTB diagnosis in combination with established methods for herd screening and to expand the diagnostic window.

Seroprevalence of low pathogenic avian influenza (H9N2) and associated risk factors in the Gyeonggi-do of Korea during 2005-2006.

Between November 2005 and March 2006, a total of 253 poultry flocks in the Gyeonggi-do of Korea were examined for seroprevalence against avian influenza (AI) using a hemagglutination inhibition (HI) test and an agar gel precipitation test. No low pathogenic avian influenza (LPAI) virus was isolated from 47 seropositive flocks that lacked clinical signs during sampling. The unadjusted percentage of seroprevalence rates of layer and broiler flocks were not significantly different, i.e., 26% (25/96) and 23% (22/97), respectively. The HI titer of the layers (mean = 89) was higher than the broilers (mean = 36; p < 0.001). A cross-sectional study was conducted for the seroprevalence of LPAI in the layers. Of 7 risk factors, farms employing one or more workers had a higher seropositive prevalence as compared to farms without hired employees (adjusted prevalence OR = 11.5, p = 0.031). Layer flocks older than 400 d had higher seropositivity than flocks younger than 300 d (OR = 4.9, p = 0.017). The farmers recognized at least one of the clinical signs in seropositive flocks, such as decreased egg production, respiratory syndromes, and increased mortality (OR = 2.3, p = 0.082). In a matched case-control study, 20 pairs of case and control flocks matched for type of flock, hired employees, age, and flock size were compared. Frequent cleansing with disinfectants was associated with a decreased risk of seropositivity (OR = 0.2, p = 0.022). Although there was a low statistical association, using a foot disinfectant when entering the building led to a decreased rate of seropositivity (OR = 0.3, p = 0.105).

Evaluation of enzyme-linked immunosorbent assay using milk samples as a potential screening test of bovine tuberculosis of dairy cows in Korea.

An enzyme-linked immunosorbent assay (ELISA) using bulk tank milk samples was evaluated as a screening test for bovine tuberculosis (TB), a contagious chronic disease of cattle. An ELISA with MPB70, a major antigen of Mycobacterium bovis was performed using paired sets of milk and sera samples from 33 tuberculin-positive and 43 tuberculin-negative cattle. Anti-MPB70 antibodies were detected in milk samples and there was a significant correlation between seroreactivities of milk and sera samples (R(2)=0.83). Using the tuberculin skin test as the reference test, the sensitivities of ELISA using milk and sera samples were 87.8% and 81.8%, respectively, and the specificities were 97.7% and 100%, respectively. In the screening test using bulk tank milk samples from 931 dairy herds in Whasung, Gyeonggi-do, Korea, the positive rate for anti-MPB70 antibody was 4.5% (42/931) and the tuberculin-positive rate was 2.8% (26/931). Individual milk samples (n=253) were collected from randomly selected 8 problematic and 3 negative herds (positive and negative in the screening test by MPB70 ELISA using bulk tank milk samples, respectively) and tested by MPB70 milk ELISA. In the problematic herds, positive rates were 10.5% (20/190) for anti-MPB70 antibodies in milk ELISA and 2.1% (4/190) in the tuberculin skin test. More than one dairy cows were positive by milk ELISA among the problematic herds, and all tuberculin-positive dairy cows were positive in the milk ELISA. Further, no positive cows were detected in negative herds both by milk ELISA and tuberculin skin test. These results suggest that an ELISA, using bulk tank milk samples, might be a potential efficient screening test for bovine TB of dairy cows.