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Polymerase chain reaction (PCR) and dot blot hybridisation (DBH) tests for detecting pigeon circovirus (PiCV) DNA were developed and evaluated using tissue samples obtained from diseased and clinically normal pigeons, which originated in Belgium and Northern Ireland. When PCR product was visually detected, the limit of detection of the PCR test was 31 fg, while that of the DBH was 1.6p g. For evaluation purposes, the results of the PCR and DBH tests, performed with DNAs extracted from samples of bursa of Fabricius (BF), were compared with those of in situ hybridisation (ISH) and histology. In 32 samples tested by all four tests, 27 (84%) were positive by PCR, 24 (75%) were positive by ISH, 20 (63%) were positive by DBH, and 13 (41%) were positive by histology. Additional PCR testing showed that in some disease-affected birds, PiCV DNA could be detected in a range of tissues including thymus, spleen, liver, kidney and brain. The PCR detection of PiCV DNA in BF samples from clinically normal birds indicated that PCR can detect infections in the absence of disease, a finding that mitigates against its use as a disease diagnostic. In addition, nucleotide sequence determinations indicated that PCR test performance was adversely affected by the sequence diversity exhibited by selected PiCVs. The application of the DBH test to dilutions of test samples indicated that the BF from some diseased pigeons contained very large amounts of virus DNA, as much as 10(13)genome copies/g tissue, and suggested that this test may be a convenient method of providing a semi-quantitative estimate of virus load.

A population of porcine isolates of Camplobacter jejuni (n = 11) and C. coli (n = 17) were examined for genotypic relatedness employing ribotyping, as well as polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of the flagellin (fla)A gene locus. PCR was employed to amplify a 533 bp fragment from the flaA gene, including the previously described short variable region (SVR), employing the novel primers, A2 and Al and successfully generated this amplicon for all wild-type strains examined (n = 28) of both C. jejuni and C. coli, as well as with both type strains, i.e. C. jejuni NCTC 11351 and C. coli NCTC 11366. Individual genotypes were assigned to each isolate typed employing the four typing methods (flaA-RFLP(Hae) III, flaA-RFLP(Pst) I ribotyping(Hae) III and ribotyping(Pst) I) and were assigned an arbitrary genotype code in ascending alphabetical order in comparison with a database of established genotypes for each of the methods employed. This study showed that several flaA-RFLP and ribopatterns existed within C. jejuni and C. coli, and demonstrated a heterogeneous diversity of strains occurring in the pigs examined. Ribotyping of strains with 16S and 23S rRNA with Pst I and Hae III digested chromosomal DNA allowed subdivision of strains into nine and eight groups, respectively. RFLP analyses with Pst I and Hae III digests probed with the flaA gene probe allowed subdivision of strains into eight and eleven subtypes, respectively. Employment of RFLP with the flaA nucleic acid probe and Hae III digests produced the greatest amount of variation of any genotyping scheme employed. Although there was a high degree of variability demonstrated by both typing methods, most isolates ( > 60%) clustered into four main genotypes, i.e. genotypes A-D. FlaA-PCR-RFLP typing demonstrated that the majority of isolates, 67.9 and 60.7%, were included in these four main genotypes for Pst I and Hae III restriction digests, respectively, although there was a high prevalence (7/11; 63.6%) of fla(Hae) III genotype A occurring within the C. jejuni isolates. Likewise, ribotyping studies demonstrated that most isolates were clustered into these four main genotypes, accounting for 81.5 and 60.7% of isolates for Pst I and Hae III restriction digests, respectively. This may indicate that the clonal population of campylobacters within this pig population is largely composed of persistent and dominant types, with a smaller number of hypervariable subtypes. Such data may useful in determining epidemiological routes of transmission of campylobacters from animal to animal, as well as helping to identify virulence determinants in persistent subtype populations.