Mannheimia haemolytica serovars associated with respiratory disease in cattle in Great Britain.

BACKGROUND: Mannheimia haemolytica is commonly associated with respiratory disease in cattle worldwide as a cause of fibrinous pneumonia, bronchopneumonia and pleuritis. M. haemolytica is further subdivided into 12 serovars, however not all are considered to be pathogenic in cattle. The study aim was to determine the most common serovars of M. haemolytica associated with respiratory disease in cattle in Great Britain, which is currently unknown and could be useful information for clinicians when considering preventative strategies. RESULTS: One hundred four M. haemolytica isolates isolated from bovine clinical pathology and post-mortem samples from pneumonia cases between 2016 and 2018 were tested using a multiplex PCR assay to identify M. haemolytica serovars A1, A2 and A6. 46 isolates (44.2%) typed as M. haemolytica serovar A1, 31 (29.8%) as M. haemolytica serovar A2 and 18 isolates (17.3%) as M. haemolytica serovar A6. Nine isolates (8.7%) were not A1, A2 or A6 so were considered to belong to other serovars or were not typable. CONCLUSION: This study highlights the importance of M. haemolytica serovars other than A1 which may be responsible for respiratory disease in cattle and could help guide the veterinarian when making choices on preventative vaccination programmes.

Use of tissue swabbing as an alternative to tissue dissection and lysis prior to nucleic acid extraction and real-time polymerase chain reaction detection of Bovine viral diarrhea virus and Porcine reproductive and respiratory syndrome virus.

The use of swabbing to sample tissue samples, prior to nucleic acid extraction and performance of a real-time polymerase chain reaction (PCR) assay, was investigated for the detection of the viral pathogens Bovine viral diarrhea virus (BVDV) and Porcine reproductive and respiratory syndrome virus (PRRSV). The tissue swabbing method involved swabbing recently cut tissues, eluting the swabbed material, and extracting nucleic acid from the eluate prior to PCR amplification. Parallel testing of this method with traditional nucleic acid extraction from tissues, where small pieces of tissue are dissected and digested (typically overnight) in lysis buffer prior to nucleic acid extraction, was carried out for 50 samples for each virus. The results demonstrated that equivalent PCR results were obtained with both methods. It was also shown on a smaller number of samples that equivalent PCR results were also obtained when the lysis step of the swabbing method was reduced to only 2 hr. The ability to remove the overnight step typically associated with processing tissue samples for PCR analysis offers the potential for same-day testing of tissue. Although the current study is preliminary in nature and further validation will be required before adoption for routine use, the results show that tissue swabbing is a promising approach. It offers a convenient, simpler, and less time-consuming alternative to tissue dissection and lysis and has potential advantages for routine laboratory operation and outbreak testing, including easier pooling and sampling of large areas of tissue and carcasses.

Porcine reproductive and respiratory syndrome virus: interlaboratory ring trial to evaluate real-time reverse transcription polymerase chain reaction detection methods.

To compare the real-time reverse transcription quantitative polymerase chain reaction (RT-qPCR) assays used for the diagnosis of Porcine reproductive and respiratory syndrome virus (PRRSV), a Europe-wide interlaboratory ring trial was conducted. A variety of PRRSV strains including North American (NA) and European (EU) genotype isolates were analyzed by the participants. Great differences regarding qualitative diagnostics as well as analytical sensitivity were observed between the individual RT-qPCR systems, especially when investigating strains from the EU genotype. None of the assays or commercial kits used in the ring trial could identify all different PRRSV strains with an optimal analytical and diagnostic sensitivity. The genetic variability of the PRRSV strains, which is supposed to hinder the diagnostic of the RT-PCR because of mutations at the primer binding sites, was also confirmed by sequencing and subsequent phylogenetic analysis. In summary, a major problem in PRRSV diagnostics by RT-qPCR is false-negative results. To achieve maximum safety in the molecular diagnosis of PRRSV, the combined usage of different assays or kits is highly recommended.

Porcine reproductive and respiratory syndrome virus: antigenic and molecular diversity of British isolates and implications for diagnosis.

Porcine reproductive and respiratory syndrome (PRRS) is an endemic disease of pigs, caused by PRRS virus, a member of the Arteriviridae family. First seen in Britain in 1991, the disease continues to be a significant economic and welfare problem for pig producers. To date, only PRRSV genotype 1 has been found in Britain. At the genetic level, a considerable increase has been reported in the diversity of PRRS viruses isolated in Britain between 2003 and 2007, versus the early 1990 s. In this study, the diversity has been shown to extend to the antigenic level too, with potential consequences for diagnostic methods. Antigenic diversity was assessed using a panel of twelve monoclonal antibodies, only one of which reacted with all isolates tested. Nine diverse viruses were compared as potential antigens in immunoperoxidase monolayer assays, where each one produced quite different results for a common panel of sera. As a single virus is used in each diagnostic assay, results must therefore be interpreted cautiously. For a real-time RT-PCR assay, published oligonucleotide primer and probe sequences were evaluated against available genetic sequences of British and European viruses, and were re-designed where considerable mismatches were found. The multiplex assay incorporating these modified primers to detect genotype 1 and 2 PRRS viruses was then validated for use with diagnostic sera and tissues. As the increasing degree of diversity exhibited by British strains is mirrored in other countries, PRRSV will continue to provide an ongoing challenge to diagnosis at a global, as well as national level.