Antibody response to a sterile filtered PPD tuberculin in M. bovis infected and M. bovis sensitized cattle.

BACKGROUND: Bovine tuberculosis, caused by Mycobacterium bovis, afflicts approximately 50 million cattle worldwide and is detected by the tuberculin skin test (TST). While it has long been recognized that purified protein derivative (PPD) tuberculin is composed of a mixture of M. bovis derived protein components, little is known about the quality, relative quantity and identity of the proteins that make up PPD tuberculin. We manufactured a sterile filtered PPD tuberculin (SF-PPD) from a nine-week-old M. bovis culture supernatant in order to characterise the culture filtrate proteins (CFP) which make up M. bovis PPD tuberculin and to compare the antibody response of M. bovis infected versus M. bovis sensitized cattle. RESULTS: SF-PPD resolved into approximately 200 discrete spots using two-dimensional polyacrylamide gel electrophoresis (2-DE) while fewer than 65 spots could be discerned from 2-DE gels of tuberculin derived from autoclaved culture supernatant. Two dimensional Western blot analyses indicated that sera from M. bovis sensitized cattle recognized additional SF-PPD antigens as compared to M. bovis infected cattle at seven weeks post infection/sensitization. However, application of a comparative tuberculin skin test resulted in an antibody boosting response to the same set of M. bovis CFPs in both the M. bovis infected and M. bovis sensitized cattle. CONCLUSIONS: We concluded that it is the heat sterilization of the M. bovis CFPs that causes severe structural changes to the M. bovis proteins. This work suggests that M. bovis infected cattle and cattle artificially sensitized to M. bovis with an injection of heat killed cells exhibit similar antibody responses to M. bovis antigens.

Genetic analysis of Mycobacterium avium complex strains used for producing purified protein derivatives.

For over a century, purified protein derivatives (PPD) have been used to detect mycobacterial infections in humans and livestock. Among these, reagents to detect infections by Mycobacterium avium complex organisms have been produced, but the utility of these reagents has not been clearly established due in part to limited biologic and immunologic standardization. Because there is little information about the strains used to produce these reagents (avian PPD, intracellulare PPD, scrofulaceum PPD, and Johnin), we have performed genetic characterizations of strains used to produce these products. Sequence analysis of 16S rRNA and the hsp65 gene provided results concordant with species designations provided for M. avium, Mycobacterium intracellulare, and Mycobacterium scrofulaceum organisms. For M. avium strains, comparative genomic hybridization was performed on a whole-genome DNA microarray, revealing one novel 7.9-kilobase genomic deletion in certain Johnin-producing strains, in addition to genomic variability inherent to the particular M. avium subspecies. Our findings indicate that considerable genomic differences exist between organisms used for reagents and the infecting organism being studied. These results serve as a baseline for potency studies of different preparations and should aid in comparative studies of newly discovered antigens for the diagnosis of infection and disease by M. avium complex organisms.

The effect of sampling time and sample handling on the detection of Staphylococcus aureus in milk from quarters with subclinical mastitis.

Staphylococcus aureus mastitis is an important cause of economic loss for the dairy industry. Control programs rely on the timely and accurate identification of positive quarters. The effects of sampling time and sample handling were examined in an attempt to improve the accuracy of detection of S. aureus. Premilking and postmilking milk samples were collected from 55 lactating quarters with subclinical S. aureus infection. Each sample was divided into 2 aliquots; one of which was cultured fresh, the other was frozen at -20 degrees C for 14 days before being cultured. Analysis of variance was used to determine the effect of sampling time (premilking vs postmilking) and sample handling (fresh vs frozen) on the detection of S. aureus, as measured by the mean category for colony-forming units per millilitre (cfu/mL). A stratified analysis was required, due to interaction between sampling time and sample handling. Only a fresh postmilking sample was inferior, yielding a lower mean category for cfu/mL (P < 0.05). The ability to detect S. aureus in quarters with subclinical intramammary infection, as measured by the mean category of cfu/mL, was maximized in fresh or frozen premilking samples and in frozen postmilking samples.