Serological and virological detection of canine herpesvirus-1 in adult dogs with and without reproductive disorders.

Canine herpesvirus 1 (CaHV-1) is known to cause reproductive disorders in adult dogs and neonatal mortality in puppies. The seroprevalence of CaHV-1 has not been documented in Italy. Sera from 865 dogs were screened for CaHV-1 using a serum neutralization assay (SN). All CaHV-1 positive sera and 100 CaHV-1 negative sera were also tested using an in-house immunofluorescence (IF) test. Thirteen bitches with reproductive disorders and three bitches with no history of reproductive diseases were also examined clinically so that lesions associated with CaHV-1 and CaHV-1 DNA could be identified using PCR analysis of vaginal swabs. An overall seroprevalence of 14.6% was observed using SN, and 18.6% using IF. The correlation between SN and IF was moderate. The SN assay demonstrated a greater sensitivity than IF, with a few exceptions. None of the vaginal swabs tested positive for CaHV-1 DNA. The differences in the seropositivity rates between SN and IF were not statistically significant (P = 0.16). Using the SN test as the reference standard, the sensitivity and specificity of IF were 29% and 95%, respectively. These results suggest that CaHV-1 is common in canine populations and could pose a threat to neonatal survival and canine fertility in breeding kennels in Italy. Vaccination of breeding bitches should be recommended if there is a history of reproductive disorders.

Evaluation of an in-clinic assay for the diagnosis of canine parvovirus.

The results of a study designed to evaluate the performance of an in-clinic test for the detection of canine parvovirus (CPV) are reported. A total of 150 faecal samples collected from dogs with acute diarrhoea were tested using the in-clinic test, a haemagglutination assay (HA) and a real-time PCR assay for CPV detection, quantification and characterisation. CPV was detected in 66, 73, and 101 faecal samples by in-clinic, HA and PCR testing, respectively. The relative sensitivity and specificity of the in-clinic test were 86.3% and 96.1%, respectively, when the test was compared to HA, and 65.3% and 100%, respectively, when compared to real-time PCR. The sample distribution according to the virus type was CPV-2a, n=44; CPV-2b, n=11; CPV-2c, n=44, CPV-2, n=2, as determined by minor groove binder probe assays and/or sequence analysis. The percentage of positive in-clinic tests was 70.5% for CPV-2a, 72.7% for CPV-2b and 75.0% for CPV-2c (P>0.05). Using real-time PCR as the reference standard for CPV detection, the in-clinic test was more specific than HA and had comparable sensitivity to HA, demonstrating detection rates similar to those previously observed for other rapid in-clinic tests. The in-clinic test was also able to detect all CPV types at equivalent rates.

Agreement between the cell culture titrations of canine minute virus determined by two susceptibility-testing methods.

The correct diagnosis of canine minute virus is critical in dog breeding. In this study, the Bland Altman test was used to compare the performance of two susceptibility-testing methods, namely polymerase chain reaction (PCR) and indirect immunofluorescence assay (IFA). The agreement between IFA and PCR in monocytes revealed a mean difference of -1752.16 with 95% confidence and an interval ranging from -3229.80 to -274.53 (SD=2325.62). The agreement between IFA and PCR in Walter Reed canine cells (WRCC) revealed a mean difference of -2396.55 with 95% confidence and an interval ranging from -3774.63 to -1018.48 (SD=2168.93). The Bland Altman test confirmed the overall accuracy of PCR vs IFA and the plot showed that all points were not randomly arranged in the range of average ± 1.96 × SD of the differences.

Detection of Vibrio parahaemolyticus in shellfish using polymerase chain reaction-enzyme-linked immunosorbent assay.

AIMS: This study evaluated the application of polymerase chain reaction-enzyme-linked immunosorbent assay (PCR-ELISA) for the detection of Vibrio parahaemolyticus in shellfish. METHODS AND RESULTS: The PCRs were selected to amplify a species-specific sequence region. In particular, internal tl biotin-labelled oligonucleotide probe was used to capture the DIG-labelled PCR products. Next, the probe PCR product hybrids, immobilized on a streptavidin-coated microtiter plate, were detected with peroxidase-conjugated anti-digoxigenin antibody (anti-DIG-POD) and the colorimetric peroxidase substrate ABTS [2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid)] using an ELISA plate reader. CONCLUSIONS: The PCR-ELISA system described is a feasible, sensitive method for the direct and specific detection of V. parahaemolyticus in shellfish samples. Compared with gel-based detection methods, PCR-ELISA in this study increased sensitivity by 100-fold for V. parahaemolyticus. SIGNIFICANCE AND IMPACT OF THE STUDY: The PCR-ELISA described may be used for potential rapid detection in routine shellfish analysis for the seafood industry. The sector requires simultaneous large-scale sample screenings to monitor contamination levels in processing plants and evaluate the performance of the hazard analysis and critical control point (HACCP) system. PCR-ELISA also proved to be economical, with a cost of about 9 Euros per sample, and the quick assay taking 8 h to complete starting from DNA extraction.