Detection of atypical porcine pestivirus in semen from commercial boar studs in the United States.

Atypical porcine pestivirus (APPV) has recently been identified as a cause of congenital tremor (CT) in pigs and has been detected in semen and preputial swabs from boars that were known to be clinically affected with CT. Accordingly, the objectives of this study were to 1) detect the presence of APPV in semen, preputial fluids and preputial swabs from adult boars by quantitative reverse transcription PCR (qRT-PCR) and 2) genetically characterize a subset of positive samples to better understand the ecology of APPV in commercial boar studs and the potential risk of transmission of APPV via semen. A total of 597 samples of semen, preputial fluid and preputial swabs each representing a different boar were obtained from four commercial boar studs located in three different states in the United States. Viral RNA was detected by qRT-PCR in 90 samples (15.08%; 90/597), with the greatest per cent positive from preputial swabs (23.81%; 5/21) followed by preputial fluid (22.81%; 26/114) and semen (12.91%; 59/457). The mean cycle quantification (Cq) between sample types was similar while eleven semen samples had Cq values lower than 27.0 corresponding to approximately 2 × 106  copies/ml. Based on phylogenetic analysis of the Npro gene, different viral strains can be on the same farm at the same and different times. This is the first report of detection of APPV in semen from commercial boar studs. Studies investigating the role of semen in the transmission of APPV and production of CT are needed.

Time delay, temperature effects and assessment of positive controls on whole blood for the gamma interferon ELISA to detect paratuberculosis.

Our objective was to evaluate the effects of time and temperature on whole blood used in the gamma interferon enzyme-linked immunosorbent assay (IFN-gamma ELISA) for paratuberculosis along with evaluating four potential positive controls, and four different mycobacterial antigens for the ELISA. Nine adult Holstein cattle naturally infected with Mycobacterium avium ssp. paratuberculosis were used in a randomized complete block design. Forty-nine blood tubes were collected from each animal and held at 48.9, 37.8, 26.7, 21.1, 15.6 and 4.4 degrees C for 0, 4, 8, 12, 18, 24, 32, 48 and 72 h. Each blood tube was tested with four mycobacterial antigens (two johnin PPDs, an avain PPD and a whole cell sonicate) and four potential positive controls [concanavalin A (conA), phytohaemagglutinin A (PHA), pokeweed mitogen (PWM) and Staphylococcus enterotoxin A (SEA)]. After incubation for 24 h, the plasma was assayed with a commercial IFN-gamma ELISA. Blood stored at 21.1 and 15.6 degrees C maintained the highest ELISA optical densities (OD) over time with severe reduction in OD values at or above 37.8 degrees C. None of the potential positive controls exactly mimicked the antigen response. SEA and PWM were able to elicit a response after the whole blood quit responding to the antigen and conA underestimated the responsiveness. Phytohemagglutinin A was similar to the antigens on an average, but there was significant disagreement among samples. The PPDs were more potent at stimulating IFN-gamma production than the whole cell sonicate. In conclusion, whole blood should be stored/transported at ambient room temperature and stimulated within 12 h of collection.