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Biomedical subjects

P F Nettleton

Publications and source records attributed to P F Nettleton.

At least 19 recordsLinked to original sources

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

The production and survival of lambs persistently infected with border disease virus.

From 1985 to 1989 lambs persistently infected with border disease virus (BDV) were produced for comparative immunological studies by infecting 57 susceptible pregnant ewes between 50 and 60 days' gestation with Moredun or Oban strains of BDV. Ewes were infected either by injection with virus grown in cell culture or by housing with lambs excreting BDV. There was no significant difference in the outcomes of these different methods of infection. There was a significant difference in the number of viable lambs born to ewes receiving the two viruses. Of 41 ewes infected with Moredun virus 21 produced 32 live lambs of which 17 were reared to 1 month old (53% viability). Of 16 ewes receiving Oban virus 10 gave birth to 17 live lambs of which 15 were reared to 1 month old (88% viability). All the lambs born to ewes infected with Moredun BDV had varying signs of tremor and increased hairiness ("hairy-shakers") while those born to ewes infected with the Oban virus had no obvious clinical signs. Survival of the lambs was poor. Up until February 1991, 14 Moredun and 10 Oban sheep between the ages of 4 months and 5.5 yr had died from a variety of causes. The two commonest causes were a chronic wasting syndrome and a mucosal disease-like syndrome which was associated with the recovery of cytopathic BDV. Mating of unrelated persistently infected sheep was largely unproductive although 2 lambs were reared.

Animals

Cell phenotypes in the efferent lymph of sheep persistently infected with Border disease virus.

The prefemoral efferent lymphatics of sheep persistently infected (PI) with Border disease virus (BDV) were cannulated in order to study the effects of the virus on cells of the immune system. Efferent lymphocytes recovered from PI sheep were phenotyped using a panel of monoclonal antibodies (MoAb) specific for ovine cell-surface markers and compared to lymphocytes recovered from normal, healthy controls. PI sheep had an increased percentage of cells expressing the T cell-associated molecules CD5, CD4, CD8 and T19, also an increase in cells expressing CD1 and a population of cells expressing low levels of the T19 molecule which was not present in control sheep. The lymphocytes were examined for the presence of BDV using virus-specific MoAb. On average 8.5% of the efferent lymphocytes from PI sheep carried virus antigen. BDV antigen was also found in the mononuclear cells and connective tissue of lymph nodes indicating widespread virus dissemination within the lymphoid system of PI sheep.

Animals

Production and characterization of monoclonal antibodies to cervine herpesvirus-1.

Fourteen hybridoma cell lines secreting monoclonal antibodies (Mab) to cervine herpesvirus-1 (CerHV-1) produced following the fusion of NSO myeloma cells with splenocytes of BALB/c mice previously immunized with gradient purified CerHV-1 were selected using an indirect enzyme-linked immunosorbent assay (ELISA) employing CerHV-1 antigen and tested by the ELISA against four other ruminant alphaherpesviruses from cattle (bovine herpesvirus type 1.1 and 1.2) goat (caprine herpesvirus-2) and reindeer (rangiferine herpesvirus-1). Comparison of all five ruminant alphaherpesviruses with these Mabs confirmed their close antigenic relationships, with two Mabs reacting against all viruses. Ten Mabs which were able to differentiate between the viruses reacted with a 64 kDa polypeptide in a western blot. Four Mabs including two specific only for CerHV-1 with neutralizing activity against the virus used for immunization were directed against a 74 kDa viral protein.

Animals

A monoclonal antibody capture ELISA to detect antibody to border disease virus in sheep serum.

An enzyme-linked immunosorbent assay (ELISA) has been developed to detect antibody to border disease virus (BDV) in sheep serum. A monoclonal antibody bound to 96-well microplates was used to capture antigen from detergent-solubilised BDV-infected cells. Single dilutions of test sera were then added to wells containing bound BDV antigen and control wells containing uninfected cell lysates. Specific antibody to BDV was detected by an anti-ovine IgG antiserum conjugated with horseradish peroxidase and the results expressed as ELISA units with reference to a standard curve. Sequential sera from 16 experimentally infected sheep and single sera from 103 sheep involved in a field outbreak were tested in the ELISA and for neutralising antibody. There was good qualitative correlation between the two tests.

Animals

An ELISA for detecting pestivirus antigen in the blood of sheep persistently infected with border disease virus.

A monoclonal antibody capture enzyme-linked immunosorbent assay (ELISA) has been developed to detect a pestivirus-specific antigen in leucocytes of sheep persistently infected with border disease virus. A blind trial was conducted to compare the specificity of the ELISA with conventional tissue culture virus isolation on blood samples from 58 sheep, aged 3 to 48 months. There was total agreement between the two tests; 27 sheep were shown to be BDV-infected. The ELISA OD values of the positive samples ranged from 0.12 to 0.86 and were not related to age, strain of virus with which they were infected or presence of serum neutralising antibody. Negative samples had OD values between 0 and 0.02.

Animals

A quantitative enzyme-linked immunosorbent assay for bovine herpesvirus type 1 (BHV-1) antibody.

A quantitative enzyme-linked immunosorbent assay (ELISA) was developed to detect and measure antibody to bovine herpesvirus type 1 (BHV-1) in cattle sera. The optical density produced from a single dilution of test serum was compared with a standard curve and the results were read and printed out from a computer interfaced to a multichannel ELISA reader. The printed results were expressed in ELISA units. The ELISA results obtained on 370 cattle sera were compared with those of the serum neutralisation test (SNT). An agreement of 90.5% was obtained when reciprocal SNT titres equal to or greater than 4 and IgG ELISA units equal to or greater than 50 were taken as indicative of a specific reaction. Of the 370 sera, 35 gave discrepant results of which 21 were SNT positive/IgG ELISA negative and 14 were SNT negative/IgG ELISA positive. When the SNT positive sera negative in the IgG ELISA were tested in an IgM ELISA, 19 were found to be positive. Thus, when the IgG and IgM ELISA results were combined the overall agreement between the ELISA and SNT increased to 95.7%. The IgG ELISA had a sensitivity of 82.4% and specificity of 94.4% relative to the SNT, whereas the combined IgG and IgM ELISA results gave a sensitivity and specificity of 98.3% and 94.4% respectively. There was a good positive correlation between the two tests (r = 0.86).

Animals

Cytopathic and non-cytopathic biotypes of border disease virus induce polypeptides of different molecular weight with common antigenic determinants.

Ten monoclonal antibodies have been raised against lysates of cells infected with cytopathic border disease virus (BDV). These antibodies all recognize non-cytopathic BDV and react with a number of different strains of bovine viral diarrhoea virus (BVDV). Studies with radiolabelled cell lysates show that all the antibodies precipitate two polypeptides of apparent Mr 80,000 and 130,000 from cells infected with cytopathic virus and a single polypeptide of apparent Mr 120,000 from cells infected with non-cytopathic virus. Two of the monoclonal antibodies react on immunoblots and show the same pattern of reactivity indicating that these three polypeptides are antigenically related.

Animals

Pestivirus infections in ruminants other than cattle.

Pestiviruses infect a wide range of domestic, captive and free-living ruminants. Among domestic livestock, Border disease virus is a well recognised cause of an important congenital disease of sheep in virtually all sheep-rearing countries of the world. The clinical signs, pathogenesis, diagnosis, epidemiology and control of this disease are described in detail. One natural outbreak of Border disease in domestic goats has been described and there is serological and virological evidence that pestiviruses occur widely in this species. A pestivirus has been isolated from a farmed red deer (Cervus elaphus) and there is serological evidence of a widespread low prevalence of infection among this new domestic species. Pestiviruses have been associated also with outbreaks of disease among captive ruminants in zoological collections. Among free-living ruminants, pestiviruses have been recovered from dead roe deer (Capreolus capreolus), fallow deer (Dama dama), African buffalo (Syncerus caffer), giraffe (Giraffa camelopardalis) and wildebeest (Connochaetes spp.) but in all these instances the contribution of the virus to the cause of the disease was uncertain. Serological surveys have shown that many species of free-living ruminants in North America, Europe and Africa have varying prevalence rates of antibodies to pestiviruses.

Animals

Lymphocyte subpopulations in the blood of sheep persistently infected with border disease virus.

The surface phenotypes of peripheral blood lymphocytes in groups of lambs and adult sheep persistently infected with Border disease virus (P-I BD) were compared with those of healthy controls. The proportion and number of lymphocytes bearing surface immunoglobulin (sIg+) and expressing class II MHC antigen (B cells) were significantly increased. A significant increase in CD1+ lymphocytes was also evident. Conversely, the proportion of T lymphocytes in P-I BD lambs was reduced. A marked reduction in the proportion of circulating lymphocytes expressing class I MHC antigen was also observed. These findings were not affected by differences in the strain of the virus responsible for the persistent infection.

Aging

Border disease without nervous signs or fleece changes.

A natural infection with border disease virus occurred in a flock on low ground in Argyll in the spring of 1984. The outbreak was unusual in that the typical clinical signs of border disease, ie, tremor and, or, fleece changes were not present; manifestations of disease were restricted to abortion and the birth of small weak lambs. The disease was shown to have been introduced to the flock by four healthy ewes persistently infected with border disease virus among a group of 39 purchased in October 1983. Further investigations in late August 1984 detected viraemia in six of seven ill-thriven lambs and four of 24 apparently healthy lambs. Attempted 'natural vaccination' of susceptible sheep by mixing them at grass for three months with groups of ewes and lambs known to contain virus excretors was largely unsuccessful as only four of 22 'sentinel' sheep seroconverted. In October 1984 the persistently infected purchased animals and all that year's lamb crop were removed from the farm. No disease occurred in 1985 when the lambing percentage was 129 per cent compared with 100 per cent in 1984. Two of the four persistently infected purchased ewes were mated at Moredun Research Institute in December 1984 and both produced healthy but persistently infected lambs.

Abortion, Veterinary

The ultrastructure of cell cultures infected with border disease and bovine virus diarrhoea viruses.

The morphology of border disease virus and bovine virus diarrhoea virus in infected bovine embryonic testis cells was examined by electron microscopy. Particles which appeared to be mature virions of both viruses were similar, being roughly circular and approximately 46 nm in diameter with a 20 to 25 nm core. Virus replication took place totally within the cytoplasm in association with structures formed from modified endoplasmic reticulum.

Animals

Pathogenesis and epidemiology of border disease.

The pathogenesis of BD virus infection of sheep is reviewed briefly. The most serious consequences occur when susceptible pregnant sheep are infected. The virus crosses the placenta readily and can cause foetal death with resorption, mummification or stillbirths. Other lambs survive, however, and are born with varying degrees of tremor and/or hairy fleeces. Many such "hairy-shaker" lambs die shortly after birth but survivors gradually recover. These survivors plus other apparently normal lambs can be persistently infected with virus and excrete it constantly often for the rest of their lives. It is these persistently infected sheep that are the key to the epidemiology of the virus and may be responsible for its introduction into a susceptible flock. In addition, cattle grazed with sheep are potentially an important source of pestiviruses capable of causing BD since in some herds the prevalence of cattle persistently infected with pestivirus is one to three per cent. Results are presented of studies on antigenic relation ships among Scottish isolates of cattle pestiviruses (BVD) and BD viruses, and the conclusion drawn that while a single strain of BVDV may be suitable for use as a cattle vaccine this would not be very efficacious in preventing BD since two antigenically distinguishable strains of BD virus have been identified. An effective vaccine against BD would have to protect sheep against both these strains and ideally a cattle vaccine should also contain both strains.

Animals

A bovine respiratory virus vaccination trial.

A respiratory virus vaccination trial was carried out in a commercial calf-rearing unit with a history of virus pneumonia. The effects of vaccination on the incidence of virus respiratory disease and growth rate were assessed. Forty-four bought-in calves were allocated to groups and treated as follows: A, unvaccinated controls; B, intranasal temperature-sensitive infectious bovine rhinotracheitis (IBR) vaccine at three and 10 weeks; C, intranasal temperature-sensitive combined IBR and parainfluenza-3 (PI3) vaccine at three and 10 weeks; D, intranasal temperature-sensitive combined IBR and PI3 vaccine at three and 10 weeks plus live attenuated bovine respiratory syncytial (BRS) virus vaccine intramuscularly at seven, 10 and 16 weeks. Two outbreaks of virus pneumonia occurred, one at three to four months of age associated with BRS virus and the other at four to five months of age with PI3 virus. During these outbreaks the incidence of pneumonia was lower and the number of days of elevated temperature and the number of treatments were significantly less in groups vaccinated against the associated virus. Despite these findings there were no significant differences between the growth rates of the groups either during the outbreaks of virus pneumonia or during the 10 month period to slaughter.

Animals