Investigation of a pig herd with animals seropositive for classical swine fever and where porcine circovirus-associated disease had been diagnosed.

AIM: To investigate the cause of classical swine fever (CSF) virus-seropositive animals in a nucleus pig-breeding herd in New Zealand, where porcine circovirus-associated disease had been diagnosed. CASE HISTORY AND CLINICAL FINDINGS: An exotic disease investigation was undertaken to exclude CSF and porcine reproductive and respiratory syndrome (PRRS) on a nucleus pig-breeding herd comprising approximately 300 breeding sows, 1,000 weaners, and 650 grower pigs. The herd was experiencing poor reproductive performance in sows, and breeding records showed a declining farrowing rate attributable to a single manager. The growing pigs (10-15 weeks old) were experiencing respiratory disease and wasting, and the mortality rate by pen varied between 9 and 20%. Post-mortem changes in affected grower pigs were consistent with circovirus-associated diseases. DIAGNOSTIC TESTING: Serological screening using an IDEXX-ELISA gave negative results for PRRS virus antibodies, but two grower pigs and one sow tested positive for CSF virus antibodies. These three seropositive animals remained positive to CSF virus, using three commercial ELISA test kits, over 27 weeks. A newly developed virus neutralisation test (VNT), using a New Zealand isolate of border disease (BD) virus, demonstrated that the seropositive pig sera had higher antibody titres to BD virus than to bovine viral diarrhoea (BVD) virus and CSF virus. PCR performed on tonsil, kidney, ileum and spleen gave negative results for CSF virus, and histopathology on lymph nodes, intestine, lung, kidney, liver and brain showed no evidence of the disease. Virus isolation performed on a number of samples was negative. CLINICAL RELEVANCE: The seropositive samples for CSF virus found in this investigation were likely to be a cross reaction to a pestivirus other than CSF virus. The finding of a possible endemic pestivirus capable of being transmitted between sheep and pigs on this farm may explain findings from previous serological survey work in New Zealand, and supports experience elsewhere, where BD virus was found to be the predominant ruminant pestivirus infecting pigs. The results show that pestivirus cross reactivity can result in unexpectedly high titres, and that testing with a full set of (local) pestiviruses is necessary to reach the correct conclusion. The investigation has direct relevance where pig herds with a low seroprevalence are encountered during surveillance for CSF.

Detection of Mycoplasma conjunctivae in sheep affected with conjunctivitis and infectious keratoconjunctivitis.

AIM: To determine the aetiology of a recurring and severe form of infectious keratoconjunctivitis (IKC) in sheep. METHODS: Five sheep flocks that had experienced a severe form of IKC were examined. Clinical history, conjunctival swabs and blood samples were collected from affected animals. Culture for bacteria, and also specifically for Mycoplasma and Chlamydophila spp, and detection of Mycoplasma conjunctivae DNA by polymerase chain reaction (PCR) were attempted. Serum samples were tested for antibodies to M. agalactiae, M. capricolum, M. conjunctivae and Chlamydophila spp. RESULTS: Mycoplasma conjunctivae DNA was detected using PCR in 3/5 flocks, and in all flocks antibodies to M. conjunctivae were detected in sera. A pure growth of Branhamella ovis was cultured from conjunctival swabs from a small proportion of sheep in two flocks. No other pathogens were detected. CONCLUSIONS: This investigation demonstrated that M. conjunctivae was a primary pathogen causing severe IKC in sheep, and is the first report of detection of this organism in sheep in New Zealand. Introduction of clinically normal carrier sheep appeared to have caused the outbreaks. KEYWORDS: Infectious keratoconjunctivitis, Mycoplasma conjunctivae, Chlamydophila pecorum, Branhamella ovis, polymerase chain reaction, ELISA, complement fixation test.

Polymerase chain reaction amplification and gene sequence analysis of a calicivirus from a feral rabbit.

A reverse transcription-polymerase chain reaction (RT-PCR) amplification assay was used to detect calicivirus gene sequences in a liver tissue derived from a feral rabbit which died of a recent outbreak of rabbit haemorrhagic disease (RHD) in New Zealand. Five pairs of primers were designed to amplify five complementary DNA genomic sequence stretching from nucleotide positions 1594 to 7071, yielding amplified fragments of 361, 340, 805,670 and 386 bp for the primer pairs RC-1/RC-2, RC-3/RC-4, RC-5/RC-6, RC-7/RC-8 and RC-9/RC-10 respectively. The identity of the amplified fragments was confirmed by chemiluminescence Southern blot hybridization and direct cycle sequencing. The nucleotide sequences of the five amplified fragments were determined and comparisons of the nucleotide and deduced amino acid sequences revealed a close genetic relationship of the New Zealand isolate 97-10372 with overseas strains of RHD virus.

Detection of antibodies against the core protein p24 of the bovine leukaemia virus in cattle for confirmatory serological testing.

An electrophoretic immunoblotting technique which was developed recently was evaluated for the identification of serum antibodies against the bovine leukaemia virus core protein p24 by using 167 sera from a bovine leukaemia virus-negative herd, and 144 sera from herds naturally infected with the virus. The sensitivity of the immunoblot was 97.4%, relative to sera which were positive in the polymerase chain reaction and in a commercial EBL-ELISA. The specificity of the immunoblot was 99.4%, for the sera from a cattle herd in which all animals were negative by a commercial EBL-ELISA, and it was 96.7% relative to sera which were negative by the polymerase chain reaction and by the agar gel immunodiffusion test from bovine leukaemia virus-infected cattle herds. A p24-specific ELISA was developed, using a monoclonal anti-p24 antibody for coating microtitre plates, a crude antigen preparation, and a monoclonal anti-bovine IgG-horse radish peroxidase conjugate as components. All reagents were commercially available. While the p24-ELISA worked well with sera from serial bleeds from calves infected experimentally with the bovine leukaemia virus and its sensitivity with sera from the naturally-infected cattle was 96.5%, its specificity was relatively low at 85.0 or 53.3%, respectively for the two negative sera groups.

Isolation and identification of canine adenovirus type-2 from the upper respiratory tract of a dog.

AIM: To report on the isolation and identification of canine adenovirus type-2 (CAV-2) from a greyhound dog with tracheitis/tonsillitis. METHODS: Virus isolation was performed with Madin and Darby canine kidney (MDCK) cell monolayers using standard virological techniques. The isolated virus was identified by haemagglutination inhibition and serum neutralisation tests. Viral DNA was extracted from infected MDCK cells and subjected to restriction endonuclease analysis using the endonuclease enzymes Bam HI, Bgl II, Eco RI and Hind III. RESULTS: A virus, designated 5 113-87, was isolated in MDCK cells yielding typical cytopathic effect. The virus could be neutralised with a CAV-2 specific reference antiserum and also showed some cross neutralisation with CAV-1 specific reference antiserum. The virus 5 113-87 had a high haemagglutination inhibition titre with CAV-2 antiserum using human group 0 red blood-cells and CAV-1 and CAV-2 reference antisera. This virus also had DNA restriction profiles identical to those of the reference CAV-2 (Toronto A26/61), whereas previously isolated strains of adenovirus from dogs in New Zealand had DNA restriction patterns identical to the prototype CAV-1 strain (Utrecht). CONCLUSION: The findings show that the virus 5 113-87 isolated from the upper respiratory tract of a dog in New Zealand is CAV-2.

Evaluation of alternative methods for the detection of bovine leukaemia virus in cattle.

AIM: To evaluate commercially available enzyme-linked immunosorbent assays (ELISAs) and the polymerase chain reaction (PCR) for their ability to detect antibodies against or nucleic acid of the bovine leukaemia virus (BLV), the causal agent of enzootic bovine leukosis (EBL), and to assess their usefulness in a national eradication programme. METHODS: Eighty-two well-defined sera (including 18 from an OIE reference laboratory) and 399 field sera from New Zealand cattle were tested in five ELISAs and the results compared with the agar gel immunodiffusion (AGID) test and electrophoretic immunoblotting (EIB) results. A polymerase chain reaction-based technique, which could detect BLV-RNA and proviral-DNA, was also evaluated on a subsample of the field cases. RESULTS: Two commercial ELISAs classified 99% of the defined sera correctly, with the other three ranging in their correct classification between 88% and 95%. The ELISAs agreed in their general classification on the majority of the 399 blood samples (91.7%), and with the AGID for more than 95 % of the sera. In a dilution series of the international reference serum E4, the highest dilution with a positive (or suspicious) result ranged from 1:80 to 1:5120. A dilution series of 202 field positive samples tested in the preferred ELISA detected 98% of positive sera at a 15 and 1: 10 dilution, reducing to 78% at a 1:80 dilution of the sera. Agreement between serological tests and PCR was poor, mainly due to failure of the PCR to detect a number of serologically positive animals. CONCLUSION: ELISA tests detected about 10% more reactors than the AGID and the EIB combined. Some ELISA-positive animals were not detected by PCR, raising doubts about the usefulness of PCR-based technology in EBL eradication programmes.

Molecular and clinicopathological diagnosis of malignant catarrhal fever in cattle, deer and buffalo in New Zealand.

Fresh and formalin-fixed tissues and blood samples in ethylenediaminetetraacetate were collected from cattle, deer and buffalo with clinical signs suggestive of malignant catarrhal fever (MCF). In addition, formalin-fixed, paraffin-embedded tissue blocks collected from these animals and retrospectively from field cases of MCF were examined. DNA samples extracted from these samples were analysed by polymerase chain reaction (PCR) assay using primers specific for the sheep-associated (SA)- and wildebeest-associated (WA)-MCF viruses. Both the SA-MCF virus and WA-MCF virus PCR yielded positive results which were in nearly complete agreement with the histopathological diagnoses of MCF in fresh and formalin-fixed, paraffin-embedded tissue from 29 cattle, 24 deer and three buffaloes. Some blood samples tested by the two assays indicated that some of the infected cattle were possible carriers.

Polymerase chain reaction amplification of the thymidine kinase and protein kinase-related genes of channel catfish virus and a putative pilchard herpesvirus.

Polymerase chain reaction (PCR) amplification assays were developed for the detection of thymidine kinase (TK) and protein kinase (PK)-related genes of the channel catfish virus (CCV). Two pairs of primers were constructed based on the published nucleotide sequences of CCV and were used to amplify the expected fragments of 584 and 755 bp for the TK and PK-related genes, respectively. The amplified fragments were shown to be specific for each of the target genes by chemiluminescence Southern blot hybridisation with a digoxigenin-labelled 20-base internal probe. The optimised CCV PCR assay can be used to amplify TK- and PK-related genes in other mammalian and avian herpesviruses. The CCV TK PCR assay also amplified a TK-like gene in some pilchard's gills and tissues obtained in the recent epizootic of pilchard kills in New Zealand.

Apoptosis in cell cultures induced by infectious bursal disease virus following in vitro infection.

Chicken embryo fibroblasts (CEFs) and Vero cells infected with infectious bursal disease virus (IBDV) exhibited the biochemical feature of apoptosis. Agarose gel electrophoresis of DNA extracted from IBDV-infected cells revealed the characteristic laddering pattern of DNA fragmentation, which was more intense in infected CEFs than in Vero cells. The appearance of apoptotic nucleosomal DNA fragments in IBDV-infected CEFs was independent of virus replication and occurred at an early stage following in vitro infection.

Detection of infectious bursal disease virus by reverse transcription-polymerase chain reaction amplification of the virus segment A gene.

A reverse transcription-polymerase chain reaction (RT-PCR) amplification assay was developed to detect infectious bursal disease virus (IBDV) gene sequences in clinical samples, infected cell cultures and chicken embryos. Two pairs of primers were designed to amplify the 5'- and 3'-termini of segment A genes that partially code for the IBDV proteins VP2 and VP3, respectively. One primer pair specifies a 309-bp fragment, the other a 520-bp fragment. Direct RT-PCR analysis of 5 bursal samples of chickens derived from a suspected first outbreak of infectious bursal disease in New Zealand yielded the 309-bp and 520-bp by fragments. The identity of both amplified fragments was confirmed by restriction endonuclease analysis, chemiluminescence Southern blot hybridization and direct cycle sequencing. RT-PCR amplification of RNAs extracted from 4 out of 5 IBDV isolates propagated in Vero cells, chicken embryo fibroblasts and specific pathogen-free chicken embryos yielded IBDV-specific fragments of unpredicted small sizes.

Comparison of an antigen capture enzyme-linked assay with reverse transcription--polymerase chain reaction and cell culture immunoperoxidase tests for the diagnosis of ruminant pestivirus infections.

A study to compare the merits of three different tests for the diagnosis of ruminant pestivirus infections was carried out. Sensitivity studies using reference strains of bovine viral diarrhoea virus (BVDV) and buffy coat samples from persistently infected (PI) carriers showed the reverse transcription-polymerase chain reaction (RT-PCR) had a greater sensitivity than the other tests. The antigen capture enzyme-linked immunosorbent assay (ELISA) was least sensitive and could only be used on samples containing cells (tissue or blood). When 169 clinical samples were examined, the RT-PCR detected the most positives (42) compared to the ELISA (32) and the immunoperoxidase test (IPT) (20). The RT-PCR was more successful when specific antibody was also present in the sample. The lower sensitivity of the IPT was related to the use of a 1 passage (4-day) test and the testing of toxic or contaminated samples. The ELISA was found to be most suitable for large-scale testing for the diagnosis and control of pestivirus infections.

Polymerase chain reaction amplification of latent Aujeszky's disease virus in dexamethasone treated pigs.

A polymerase chain reaction (PCR) amplification assay was developed for the detection of Aujeszky's disease virus (ADV) DNA in cell cultures and clinical samples. Pigs vaccinated with commercial ADV vaccines and challenged with a field isolate of ADV were immunosuppressed by dexamethasone treatment. Nasal swabs collected from the pigs at various times post-immunosuppression showed that ADV was excreted for at least four to six days starting from day 8 or day 10 following dexamethasone treatment, by virus isolation and/or PCR. However, PCR only detected latent ADV in the trigeminal ganglia, mandibular lymph node, spleen and tonsils, but not in the brain stem, pons and olfactory lobe of two pigs following dexamethasone treatment, whereas tissue explanation and cocultivation failed to demonstrate the presence of the virus.

Polymerase chain reaction amplification of wildebeest-associated and cervine-derived malignant catarrhal fever virus DNA.

A polymerase chain reaction (PCR) assay was developed for the detection of alcelaphine herpesvirus 1 (AHV1), a causative agent of malignant catarrhal fever (MCF) of ruminants. A pair of 20-base primers was constructed based on the published nucleotide sequence of gene A of the WC11 isolate of AHV1 and was used to amplify a DNA fragment of 413 base pairs. The optimised PCR assay was highly sensitive, i.e. it detected 10 fg of genomic DNA of AHV1 (WC11 isolate). The amplified fragment was shown to be specific for AHV1 DNA by (i) cleavage with XbaI which yielded 2 subfragments of approximately 140 and 280 base pairs and (ii) chemiluminescence Southern blot hybridisation with a digoxigenin-labelled 25-base internal probe. The PCR assay also amplified AHV1 gene sequences in tissue samples from deer and rabbits experimentally infected with materials derived from deer with clinical sheep-associated MCF.

A comparison of the efficacy of two commercial Aujeszky's disease vaccines with glycoprotein-I deletion in pigs.

Two commercial Aujeszky's disease vaccines, a modified killed vaccine and a sub-unit vaccine, both carrying a deletion of glycoprotein-I, were evaluated in pigs. Each vaccine was administered to two groups of four pigs, twice at 4-week intervals, with two pigs held as unvaccinated controls. All pigs were challenged with a New Zealand field isolate of Aujeszky's disease virus 3 weeks after the second vaccination. The results indicate that the sub-unit vaccine was able to protect pigs against clinical Aujeszky's disease much better than the pigs vaccinated with the modified killed vaccine when challenged with a virulent virus. However, the amount and the duration of virulent virus excretion following challenge was greater with the sub-unit vaccine than the modified killed vaccine. Pigs vaccinated with the sub-unit vaccine were shown to be latently infected following challenge. Latent infection was demonstrated by excretion of Aujeszky's disease virus from the nasal cavity after dexamethasone treatment and seroconversion of a sentinel in contact pigs to Aujeszky's disease virus.

Polymerase chain reaction amplification for direct detection of chicken anemia virus DNA in tissues and sera.

Direct detection of chicken anemia virus (CAV) DNA in tissues and sera was investigated by a polymerase chain reaction (PCR) assay. Using a pair of primers constructed to amplify the coding sequence of the CAV DNA genome, the PCR assay was shown to be extremely sensitive, being able to detect 1 fg of CAV replicative form DNA. The oligonucleotide primers used for the PCR yielded 583 base-pair (bp) amplified product, which was sized by ethidium bromide-agarose gel electrophoresis. Tissue samples from seven cases of suspected chicken infectious anemia were obtained for CAV isolation. DNA extracted from the homogenized suspension of pooled tissues of each case was analyzed by the PCR assay. Results showed that five of the seven cases were positive for CAV DNA by PCR, whereas CAV was isolated from four cases only. The PCR assay also detected CAV DNA in two of 37 serum samples from disease-free chickens. The specificity of PCR was confirmed by chemiluminescence dot-blot analysis of the amplified products.

Patterns and implications of subclinical vulval human papillomavirus infection: the impact of PCR analysis.

This study was performed to elucidate the presence of human papillomavirus (HPV) infection of the vulva by colposcopy, histology and the polymerase chain reaction (PCR). Colposcopy defined 5 patterns of vulval epithelial lesions inconspicuous to the naked eye. Of these 75 subclinical vulval lesions, HPV infection was diagnosed by histology in 20.0% of minor epithelial changes with faint acetowhitening, 52.2% of conspicuous acetowhite lesions, 63.0% of acetowhite areas with satellite lesions, 84.6% of villous lesions, and 85.7% of villous lesions with surrounding acetowhitening. The corresponding HPV DNA positivity rates by PCR were 60.0%, 73.9%, 70.4%, 84.6% and 100% respectively. The oncogenic HPV type 16 was detected by PCR in 37.3% of the samples. These results provide firm evidence for the prevalent existence of subclinical HPV lesions of the vulva. Some of these infections may not produce significant morphologic changes detectable by colposcopy or histology. Subclinical vulval lesions are common and may constitute a reservoir for repeated cervical HPV infections, as well as a source of contamination of cervical samples for HPV DNA detection by sensitive molecular techniques.

Detection of chicken anaemia agent DNA sequences by the polymerase chain reaction.

A polymerase chain reaction (PCR) assay was developed for detection of chicken anaemia agent (CAA) DNA. The assay used a single set of 20-base primers complementary to sequences located in the coding regions of the CAA replicative form (RF) DNA genome at positions 485 to 504 and 1048 to 1067. The observed amplification product had the expected size of 583 bp and was confirmed to derive from CAA RF DNA by a unique Hind III restriction enzyme cleavage pattern. The amplified fragment was shown to be specific for CAA RF DNA after chemiluminescence dot blot hybridisation with a digoxigenin-labelled 25-base internal probe. The optimised PCR assay was specific for CAA and highly sensitive, being able to detect a single CAA-infected MDCC-MSB1 cell and at least 100 fg of CAA RF DNA. Preliminary results also showed that the PCR assay can detect CAA DNA in clinical specimens from chicks experimentally infected with CAA.

Diagnostic sensitivity of polymerase chain reaction and Southern blot hybridization for the detection of human papillomavirus DNA in biopsy specimens from cervical lesions.

Human papillomaviruses (HPVs) are associated with benign and malignant neoplasms of the cervix. One of the criteria for their etiologic role requires an assessment of whether virtually all or only a small fraction of lesions contain viral genomes. DNA preparations from colposcopically directed punch biopsies of cervical lesions were analyzed by Southern blot hybridization and the polymerase chain reaction (PCR) for the presence of HPV DNA. The biopsy specimens represented different pathologic entities (koilocytosis, condyloma, cervical intraepithelial neoplasia, and invasive carcinoma). In Southern blot hybridization with radioactive probes for HPV 11, 16, 18, 31, and 33, HPV DNA was detected in 74% of the biopsy specimens (42 of 57 cases), with the predominant types being HPV 16 and HPV 18. In contrast, after PCR amplification with primers yielding fragments of characteristic size for HPV 11, 16, and 18, the analysis of the same 57 biopsy specimens revealed that all samples were positive for at least one HPV type. To exclude false-positive PCR results, controls without HPV DNA were interspersed at regular intervals, and results were evaluated only if these controls remained HPV negative. To exclude false-negative results due to failure of the reaction, a target sequence within the c-Ha-ras-1 gene was used as an internal control. All HPV typing results obtained by Southern blot hybridization were in agreement with HPV typing by PCR. The higher number of positive samples in the latter analysis stems from the increased sensitivity of PCR, which was which was effective in identifying as few as 10-100 HPV DNA molecules; in contrast, the sensitivity of Southern blot hybridization was 1 pg, or approximately 10(5) molecules of HPV DNA. The authors conclude that, with sufficiently sensitive diagnostic methods, HPV DNA can be detected in most, if not all, neoplastic cervical lesions.