Comparison of real-time PCR and culture isolation in colostrum-deprived pigs immunized and challenged with Haemophilus parasuis.

AIMS: A real-time PCR (RT-PCR) based on the detection of the infB gene of Haemophilus parasuis is compared with culture isolation (Frandoloso et al., (2011) Clin Vaccine Immunol 18, 50-58.), evaluating different subunit or commercial vaccines. METHODS AND RESULTS: Samples from different tissues of 24 experimentally infected and challenged colostrum-deprived piglets were tested. The RT-PCR gave globally a 23·3% more of positive results than culture, and all samples being positive by culture were positive by RT-PCR also. H. parasuis could not be cultured from any of the samples of the piglets included in the three vaccinated groups resulting in a strong protection, but it could be detected by RT-PCR in six samples in the group immunized with the commercial vaccine, in three in that vaccinated with native proteins with affinity to porcine transferrin (NPAPT) administered intramuscularly and in only two in that immunized with NPAPT intratracheally. CONCLUSIONS: The RT-PCR was more sensitive than culture for H. parasuis detection in the organs compared. SIGNIFICANCE AND IMPACT OF THE STUDY: The RT-PCR evidenced that NPAPT vaccines were those yielding the best protection results in terms of H. parasuis clearance.

Comparison between Haemophilus parasuis infection in colostrums-deprived and sow-reared piglets.

The aim of this study was to compare the development of Glasser's disease in sow-reared and colostrum-deprived piglets. Ninety piglets from a commercial pig farm in Spain were used. The farm was positive for Haemophilus parasuis. Fifty-two pigs were sow-reared (SR) and 38 were colostrum-deprived (CD) piglets. The animals were intratracheally inoculated with H. parasuis serovar 5 and sacrificed at 1, 2 and 3 days post-infection. To assess the development of disease, antibody titers, clinical signs, pathological lesions, microbiological isolation and PCR amplification were compared between the groups. Inoculation of SR pigs did not cause clinical signs or lesions of Glasser's disease. In SR pigs, H. parasuis isolation and specific PCR amplification from tissues showed a very low number of positive samples. In contrast, in CD pigs, inoculation resulted in the typical signs and lesions of Glasser's disease. Positive microbiological isolation and specific PCR products were obtained from the majority of the tissues tested, and no antibodies against H. parasuis were detected. The experimental infection using CD pigs describes a successful method to study this microorganism and confirms the important role that maternal antibodies play in protection against clinical signs and disease.

Development of polymerase chain reaction and comparison with in situ hybridization for the detection of Haemophilus parasuis in formalin-fixed, paraffin-embedded tissues.

DNA extraction and nested polymerase chain reaction (PCR) were developed for the detection of Haemophilus parasuis from formalin-fixed, paraffin-embedded tissues. The results for nested PCR were compared with those determined by in situ hybridization. The optimal results obtained show that use of xylene deparaffinization, digestion with proteinase K followed by nested PCR is a reliable detection method. A distinct positive signal was detected in 20 pigs naturally infected with H. parasuis by in situ hybridization. The rate of agreement between nested PCR and in situ hybridization for the detection of H. parasuis in formalin-fixed, paraffin-embedded tissues was 100%. The nested PCR could be applied successfully to formalin-fixed, paraffin-embedded tissues for the detection of H. parasuis with bacterial isolation.