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P F Nettleton

Publications and source records attributed to P F Nettleton.

At least 37 records · Page 2Linked to original sources
PubMed1998

The reactivity of monoclonal antibodies against orf virus with other parapoxviruses and the identification of a 39 kDa immunodominant protein.

A panel of 27 mouse monoclonal antibodies (Mabs) was raised against orf virus. Sixteen of these Mabs reacted with a protein with a molecular mass of 65 kDa, 8 reacted with a protein with a molecular mass of 39 kDa and three remain uncharacterised. Reactivity of the Mabs with a library of recombinant vaccinia viruses expressing various regions of the NZ-2 orf virus genome identified the approximate positions of the genes encoding these 2 immunodominant orf virus proteins. The gene encoding the 39 kDa protein was identified and sequenced. The protein was detected in an envelope fraction of orf virus and was shown to be homologous to the envelope protein encoded by the H3L gene of vaccinia virus. The 65 kDa protein has not been fully chracterised, but the gene encoding it has been localised to a 10 kbp region of the orf virus genome. The Mabs were used to discriminate 4 parapoxviruses derived from sheep, 2 from cattle and 1 each from a seal and squirrel. Eighteen Mabs reacted with all 4 sheep viruses, 19 Mabs reacted with both cattle viruses, 6 recognised seal parapoxvirus and 2 recognised the squirrel parapoxvirus. Only one of the 27 Mabs reacted with all 8 parapoxviruses suggesting it recognises a conserved epitope within the genus.

Amino Acid Sequence↗
PubMed1998

A novel nested reverse transcription PCR detects bovine viral diarrhoea virus in fluids from aborted bovine fetuses.

A nested reverse transcription-PCR (RT-PCR) was developed to detect pestivirus nucleic acid in fetal fluids and to study the number of bovine abortions associated with BVDV infection. Three techniques for the extraction of viral RNA from fetal fluids were compared; phenol:chloroform method, treatment with Catrimox-14 followed by guanidium isothiocyanate buffer and the Qiagen total RNA kit. The Qiagen kit was the most sensitive and reproducible and therefore adopted. After cDNA synthesis, initial amplification of a 288-base pair product using existing primers derived from the highly conserved 5'-untranslated region of the BVDV genome was achieved. Newly designed internal primers yielded a 171-base pair fragment which was visualised after electrophoresis on an ethidium bromide-stained gel. This assay detected 6.0 TCID50 of BVDV per 300 microl of artificially contaminated fetal fluid. One hundred fetal fluids were screened for the presence of BVDV RNA and the results compared with existing virus isolation methods. The BVDV antibody status of each fetus was determined. The nested RT-PCR detected BVDV RNA in eight of the hundred fetal fluids screened, whereas BVD virus was isolated from only one sample. The use of the nested RT-PCR will provide us with a more accurate picture of bovine embryonic infection due to BVDV.

Abortion, Veterinary↗
PubMed1997

A novel strategy for determining protective antigens of the parapoxvirus, orf virus.

We investigated the feasibility of using vaccinia virus (VAC) recombinants containing large multigene fragments of orf virus DNA to identify protective antigens of orf virus (OV). Sixteen OV strain NZ2 DNA fragments with an average size of 11.4 kb were recombined into VAC strain Lister. Each fragment was mapped relative to OV restriction endonuclease maps but was otherwise uncharacterized. Together the recombinants represent 95% of the OV genome in an overlapping manner. Immunofluorescence showed all 16 constructs expressed products recognized by OV antiserum and radioimmune precipitation with the same antiserum allowed the localization of the major antigens of OV to specific recombinants. These data indicated the approximate genomic locations of the genes encoding the OV major antigens and showed that their expression was authentic rather than resulting from read through from VAC sequences adjacent to the site of recombination. Vaccination of OV-naive sheep with the recombinant library provided protection against a subsequent challenge with virulent OV. These data confirm the feasibility of the proposed strategy.

Animals↗
PubMed1997

Molecular characterization of ovine pestiviruses.

Forty-two ovine pestivirus isolates, collected over a period of 18 years, were compared by phylogenetic analysis. The viruses were mostly field isolates from Britain; two others originated from Sweden and two from New Zealand. RT-PCR products were obtained from two genomic regions, one within the 5'-noncoding (5'-NC) region, and the other encompassing parts of the p20 (Npro) and C coding regions. Direct sequencing of the 5'-NC PCR products, followed by computer-assisted phylogenetic analysis, divided the ovine pestiviruses into three main genotypes. The results demonstrated that sheep may naturally be infected not only with border disease virus (BDV), but also with bovine viral diarrhoea virus (BVDV) types I and II. The BDV isolates segregated into two principal subtypes represented by the Moredun strain from Scotland and the 137/4 strain from England. The BVDV-I group was composed of three clusters, two of them represented by BVDV reference strains NADL and Osloss, respectively, and the third by ovine isolates D1120/1 and D1432/P. The grouping of ovine pestiviruses, based on comparative nucleotide sequence analysis of the 5'-NC region, was confirmed by comparative analysis of the p20 (Npro) and C coding regions, performed both at the nucleotide and at the amino acid level. The presence of three genotypes in sheep, including BVDV-I and BVDV-II, indicates the inadequacy of the current hostspecies-based nomenclature and classification of pestiviruses.

Animals↗
PubMed1996

Natural transmission of orf virus from clinically normal ewes to orf-naive sheep.

The apparent natural transmission of orf virus from clinically normal ewes to susceptible sheep was observed during a border disease vaccine experiment. The 14 susceptible sheep were persistently infected with border disease virus and had been reared indoors in isolation from other sheep since birth. Their ages ranged from two to four years and they were housed in two groups; group 1 consisted of four sheep persistently infected with the Moredun strain of border disease virus and group 2 consisted of 10 sheep persistently infected with the Oban strain of the virus. On day 0, six sheep were removed from group 2 and rehoused. To the remaining four sheep in each group were added eight four- to six-year-old pregnant conventionally reared ewes at 48 days gestation. Fourteen days later the four sheep in group 1 were moved to another pen housing eight similar five-year-old pregnant ewes at 48 days' gestation, and the four sheep from group 2 were rehoused with their original stallmates. Twenty-one days later lip lesions typical of orf were first observed on the sheep from both groups and the disease spread to all the sheep persistently infected with border disease virus over the next four weeks. Virological and serological evidence demonstrated that the source of infection for the sheep was almost certainly the conventionally reared ewes, on which no lesions resembling orf were observed at any time during the study.

Animals↗
PubMed1996

Concurrent bovine viral diarrhoea virus and Salmonella typhimurium DT104 infection in a group of pregnant dairy heifers.

Two days after being imported into the United Kingdom one of a group of 30 pregnant dairy heifers showed clinical signs of bovine viral diarrhoea virus (BVDV) infection and subsequently died. Before it died the heifer was BVDV antigen-positive and antibody-negative. The gross post mortem findings were suggestive of mucosal disease but in addition to noncytopathic BVD virus, Salmonella typhimurium DT104 was cultured from tissues and gut contents. The other heifers were screened for S typhimurium by culturing faeces, and serology showed that 13 (45 per cent) of the group seroconverted to BVDV in the three weeks between samplings and the remainder were seropositive, indicating previous exposure. During this period four heifers showed clinical signs of acute BVDV infection but recovered uneventfully. Four animals (14 per cent) were positive for S typhimurium DT104 on faecal culture, and three of these excretors concurrently seroconverted to BVDV. Of the 29 heifers remaining in the group, one aborted in late gestation, 26 bore live calves and two delivered stillborn calves. Pre-colostral blood samples from the calves showed that their dams' pre-existing antibody titres correlated well with in utero fetal protection. In non-immune dams, exposure to BVDV between 69 and 120 days of gestation led to the birth of live persistently viraemic calves. Infection between 120 and 140 days of gestation led to the birth of live calves with evidence of congenital damage to the central nervous system, and infection later than 140 days of gestation led to the birth of live, normal calves with high pre-colostral antibody titres to BVDV. One calf which sucked colostrum was antibody and virus antigen-positive when sampled at 12 hours old but regular blood sampling failed to detect viraemia again until the calf was seven weeks old when it became persistently viraemic.

Animals↗
PubMed1996

Tissue culture-propagated orf virus vaccine protects lambs from orf virus challenge.

Twenty, eight-day-old specific pathogen-free (SPF) lambs were vaccinated by a single scarification approximately 4 cm in length on the inner right thigh with a double-pronged applicator. The titre of live virus in the vaccine was 10(7.2) TCID50/ml and the estimated dose per lamb was 0.04 ml. Three months and six months later 10 of the vaccinated lambs and five age-matched unvaccinated control specific pathogen free lambs were challenged by a single scarification with virulent virus on the inner left thigh in the same way. After the vaccination all 20 lambs developed lesions characteristic of orf virus infection that had largely resolved four weeks later, when they all had reciprocal ELISA antibody titres > or = 3200 that persisted in all but one of them until they were challenged. After the challenge, the development of lesions in the vaccinated and unvaccinated sheep was compared daily for four weeks by means of a clinical scoring system. Both groups of vaccinated lambs had significantly lower (P < 0.01) total clinical scores after challenge at three months and six months than the unvaccinated lambs.

Animals↗
PubMed1996

The distinction of serologically related ruminant alphaherpesviruses by the polymerase chain reaction (PCR) and restriction endonuclease analysis.

The amplification and analysis of a 468bp fragment from the gB gene of the serologically related ruminant alphaherpesviruses bovine herpesvirus-1.1 and 1.2 (BHV-1.1 and BHV-1.2), caprine herpesvirus-1 (CapHV-1), cervine herpesvirus-1 (CerHV-1) and rangiferine herpesvirus-1 (RanHV-1) by PCR and restriction endonuclease analysis is described. As primers, 22bp oligomers selected from the BHV-1 gB gene sequences were used for the amplification of the DNA from the five viruses. The amplification product from each virus was analysed by the restriction endonuclease enzymes BglI, HinfI, SmaI and AvaI. The specific amplification obtained demonstrate the existence of the gB gene sequences for each of the five alphaherpesviruses. However, sequences from some of the fragments were found to be different from those predicted from the gB gene following restriction endonuclease analysis. All five amplification products generated the same number of fragments after digestion with HinfI except for two additional bands evident in CapHV-1. The CerHV-1 and RanHV-1 fragments contained slightly different BglI restriction sites from those of the other three. While BHV-1.1, BHV-1.2, CapHV-1 and CerHV-1 contained SmaI and AvaI restriction sites, the RanHV-1 amplification product lacked both SmaI and AvaI restriction sites.

Animals↗
PubMed1995

Isolation of a parapoxvirus from a grey seal (Halichoerus grypus).

A grey seal (Halichoerus grypus) developed cutaneous pocks which progressed to involve the skin extensively, necessitating euthanasia. Macroscopically and histologically the lesions resembled previous descriptions of parapoxvirus infections of seals and virus particles were observed in preparations of a scab and a skin lesion. Suspensions of the scab and skin lesion were prepared and inoculated on to monolayer cultures of grey seal kidney cells. After 25 days in culture and three passages, cytopathic effects were observed and parapoxvirus particles were detected by electron microscopy in the supernatant fluid. Both isolates were adapted to cultures of fetal lamb muscle cells and shown to be antigenically related to orf virus.

Animals↗
PubMed1995

A double monoclonal antibody ELISA for detecting pestivirus antigen in the blood of viraemic cattle and sheep.

A panel of monoclonal antibodies (mAbs) has been produced to the p125/p80 non-structural polypeptide of border disease virus (BDV) and bovine virus diarrhoea virus (BVDV). This polypeptide appears to be highly conserved among BDV and BVDV isolates and consequently the mAbs directed against it have a broad cross-reactivity with pestivirus isolates. The epitope specificities of these mAbs were determined by competitive binding and four of the mAbs with mutually exclusive epitope specificities were selected for the development of a diagnostic ELISA. Two mAbs were used to capture virus antigen prepared from the blood of infected cattle and sheep, then two different mAbs used to detect the captured antigen. This double mAb ELISA was compared to existing ELISAs which rely on polyclonal antibodies (pAbs) for detecting captured antigen. The mAb detection ELISA was more sensitive than the pAb detection ELISAs for both cattle and sheep and resulted in higher optical densities for positive samples without an increase in background readings of negative controls.

Animals↗
PubMed1995

Detection of bovine herpesvirus 1 in clinical samples by the polymerase chain reaction.

A PCR assay with primers selected from the gl gene and flanking a 468 bp DNA fragment was tested on clinical samples. Of 27 samples (nasal swabs, lung, lymph nodes, tracheal mucosa) collected from 16 different outbreaks in Scotland, 18 were positive by PCR and 13 by virus isolation. Some samples of isolated DNA had to be diluted by a factor 50-100 to obtain a positive PCR result. PCR assay could detect the BHV-1 positive samples collected from different European regions, namely from Slovakia, Italy, England, Scotland as well as the IPV sample.

Animals↗
PubMed1994

Pestiviruses isolated from pigs, cattle and sheep can be allocated into at least three genogroups using polymerase chain reaction and restriction endonuclease analysis.

A polymerase chain reaction-based assay capable of detecting a broad range of pestiviruses from pigs, cattle, or sheep was developed. Of six sets of primers selected from different parts of the pestivirus genome, the best results were provided by a pair from the highly conserved 5' non-coding region which gave amplification with all 129 isolates tested. This panel consisted of 33 isolates from pigs, 79 from cattle, and 17 from sheep. Differentiation between the viruses was achieved by cutting the PCR-amplified products with the restriction endonucleases AvaI and Bg1I. Using this procedure it was possible to distinguish at least 3 genogroups; group 1 (HCV) contained 32 of the pig isolates, group II (BVDV) contained all the cattle isolates tested plus 6 sheep isolates and group III (BDV) contained 11 sheep isolates and 1 pig isolate.

Animals↗
PubMed1994

Evaluation of a 'one tube' reverse transcription-polymerase chain reaction for the detection of ruminant pestiviruses.

A 'one tube' reverse transcription-polymerase chain reaction ('one tube' RT-PCR) using rTth DNA polymerase was compared with an existing RT-PCR using Taq DNA polymerase (Taq RT-PCR) to detect ruminant pestiviruses in infected cell cultures. The technically simpler and more convenient 'one tube' method was relatively insensitive detecting only 11 of the 34 samples tested, all of which were positive by Taq RT-PCR.

Animals↗
PubMed1994

Rapid detection of bovine herpesvirus 1 (BHV 1) using the polymerase chain reaction.

A polymerase chain reaction (PCR) assay based on primers from the viral gI glycoprotein gene detected 3 fg pure BHV-1 DNA, 0.1-1.0 TCID50 or a single infected cell. No amplification was observed with DNA from BHV-2, BHV-3, BHV-4, OHV-1 or OHV-2. However, a fragment of the correct size was amplified using DNA from herpesviruses isolated from reindeer, red deer and goats. The PCR assay was able to detect virus in nasal swabs up to 14 days after experimental infection of cattle and there was a good correlation when PCR was compared with virus isolation for the detection of BHV-1 in clinical field samples. Detection of BHV-1 in fetal bovine serum and semen samples was also successful.

Animals↗
PubMed1992

A comparison of serological relationships among five ruminant alphaherpesviruses by ELISA.

Using enzyme-linked immunosorbent assays the cross reactivity of bovine herpesvirus-1.1, bovine herpesvirus-1.2, caprine herpesvirus-2, cervine (red deer) herpesvirus-1 and rangiferine (reindeer) herpesvirus-1 has been examined using rabbit hyperimmune antisera and convalescent cattle and red deer field sera. Significant cross-reactivity among all the five viruses was demonstrated. A detailed analysis showed that: (1) the two bovine herpesviruses are most closely related, (2) the cervine, caprine and rangiferine viruses are more closely related to the bovine viruses than they are to each other, (3) the cervine herpesvirus is more related to the bovine herpesvirus than to the rangiferine or caprine herpesviruses and (4) the rangiferine virus is more related to the cervine virus than to the bovine and caprine viruses. Cattle and red deer sera reacted most strongly with the bovine and cervine viruses respectively.

Analysis of Variance↗