Application and field validation of a PCR assay for the detection of Mycoplasma hyopneumoniae from swine lung tissue samples.

A PCR assay was validated for the detection of Mycoplasma hyopneumoniae in porcine lung tissue. The detection limit of the assay was 0.18 colony-forming units/g of lung sample spiked with M. hyopneumoniae. In field validation, 426 pigs from 220 cases were examined for M. hyopneumoniae infection by M. hyopneumoniae PCR and a fluorescent antibody (FA) test. In total, 103 pig lungs (24.2%) were positive in the PCR test, and 69 pig lungs (16.2%) were positive in the FA test, among which, 62 pigs were positive for both PCR and FA test. Most of the PCR-positive but FA test-negative cases had lesions compatible with M. hyopneumoniae infection. With Bayesian modeling, the diagnostic sensitivity and specificity of the PCR were determined to be 97.3% and 93.0%, respectively.

Restriction fragment length polymorphism of porcine reproductive and respiratory syndrome viruses recovered from Ontario farms, 1998-2000.

From January 1998 to July 2000, 2,456 clinical samples, including lung, tonsil, lymph node, and serum, from 760 cases submitted to the Animal Health Laboratory, Ontario, Canada, were tested for porcine reproductive and respiratory syndrome viruses (PRRSV) using reverse transcriptase polymerase chain reaction (RT-PCR) and RT-PCR product restriction fragment length polymorphism (RFLP) analysis. A total of 516 samples from 284 cases were RT-PCR positive for the PRRSV open reading frame (ORF) 7 sequence. The RT-PCR RFLP typing assay was performed using 2 different sets of primers, amplifying 716 or 933 base pairs of ORF 4, 5, and 6 of PRRSV. Samples from 254 cases were typeable, yielding 34 different RFLP types. Of these, 164 cases had 32 different RFLP types of field or intermediate strains, 86 had a pattern similar to a commercial PRRSV vaccine or VR 2332 strain of the virus, 4 had a RFLP type shared by another commercial vaccine and a field strain. In 4 cases, 2 different RFLP types were identified from tissues from different pigs that were submitted at the same time from the same farm. Of the 195 farms that submitted PRRSV PCR-positive samples, 48 submitted samples on more than 1 occasion during the specified time frame. In 23 of those 48 farms, RFLP patterns of PRRSV differed between submissions, whereas in the other 25 farms, the RFLP pattern remained unchanged. There were 34 different PRRSV patterns identified from 236 cases using the primer set amplifying 716 base pairs of PRRSV. There were 18 cases, consisting of 9 different patterns, typeable only by using the primers amplifying a 933-base pair fragment of the virus.

Field validation of a commercial blocking ELISA to differentiate antibody to transmissible gastroenteritis virus (TGEV) and porcine respiratory coronavirus and to identify TGEV-infected swine herds.

A commercially available blocking ELISA was analyzed for its ability to identify antibodies to porcine coronaviruses (transmissible gastroenteritis virus [TGEV] or porcine respiratory coronavirus [PRCV]), to differentiate antibodies to TGEV and PRCV, and to identify TGEV-infected herds. Nine sera from uninfected pigs, 34 sera from 16 pigs experimentally infected with TGEV, and sera from 10 pigs experimentally infected with PRCV were evaluated using both the TGEV/PRCV blocking ELISA and a virus neutralization (VN) assay. The ELISA was not consistently effective in identifying pigs experimentally infected with TGEV until 21 days postinfection. Sera from 100 commercial swine herds (1,783 sera; median 15 per herd) were similarly evaluated using both tests. Thirty of these commercial herds had a clinical history of TGEV infection and a positive TGEV fluorescent antibody test recorded at necropsy within the last 35 months, while 70 herds had no history of clinical TGEV infection. The blocking ELISA and the VN showed good agreement (kappa 0.84) for the detection of porcine coronavirus antibody (TGEV or PRCV). The sensitivity (0.933) of the ELISA to identify TGEV-infected herds was good when considered on a herd basis. The ELISA was also highly specific (0.943) for the detection of TGEV-infected herds when the test results were evaluated on a herd basis. When sera from specific age groups were compared, the ELISA identified a greater proportion (0.83) of pigs in herds with TGEV antibody when suckling piglets were used. In repeatability experiments, the ELISA gave consistent results when the same sera were evaluated on different days (kappa 0.889) and when sera were evaluated before and after heating (kappa 0.888). The blocking ELISA was determined to be useful for herd monitoring programs and could be used alone without parallel use of the VN assay for the assessment of large swine populations for the detection of TGEV-infected herds.

Relationship between group A porcine rotavirus and management practices in swine herds in Ontario.

A case-control epidemiological study was conducted to determine whether an increased diagnostic rate for group A rotavirus in swine herds in Ontario was associated with specific management factors. The number of new herds tested per year and the proportion of new positive herds increased between 1994 and 1997. Herd size was larger and weaning age was younger in rotavirus-positive herds compared with rotavirus-negative herds. Pigs raised in all-in all-out nurseries were 3.4 times more likely to have a positive group A rotavirus diagnosis than pigs in continuous flow facilities. This study demonstrates that the changes seen in group A rotavirus disease herd status in Ontario are associated with changes in farm management practices, including farm expansion, early weaning, and all-in all-out production.