Interactions of porcine alveolar macrophages and bone marrow cells with African swine fever virus and virus-infected cells.

Virus yields from porcine alveolar macrophages (AM) infected with African swine fever virus (ASFV) were greater and were achieved more rapidly, when inoculated at a high multiplicity of infection (MOI) than at low MOI. The difference was related to a lower percentage of cells becoming infected after low MOI inoculation. The reduced yields after low MOI were not caused by prolongation of the culture time, by bacterial endotoxins or by production of inhibitory substances by infected AM. Virus-infected AM were not susceptible to lysis in antibody-dependent cell mediated cytotoxicity (ADCC) assays and this was apparently due to a paucity of viral antigen expressed on the cell surface. Uninfected AM did not act as effectors in ADCC. Porcine bone marrow (PBM) cells were effective in mediation of ADCC and their activity was reduced after ASFV infection. Cells separated into adherent and non-adherent populations, depleted by carbonyl iron treatment or separated by Ficoll-Hypaque centrifugation, all showed effector activity in ADCC. The effector cells were not mature neutrophils or lymphocytes and were probably granulocytic precursors.

The role of antibody in protection against African swine fever virus.

Intraperitoneal immunization of pigs with anti-African swine fever virus (ASFV) antibody protected them against the effects of challenge with ASFV. This protection, which was exemplified by a reduction in pyrexia and viraemia plus an increased survival time, appeared to be mediated through the effects of complement-dependent antibody-mediated cytotoxicity (CDAC) or antibody dependent cell mediated cytotoxicity (ADCC). Experiments suggested that the reduction in viraemia was associated with complement lysis whereas protection was conferred by ADCC.

Infection of pigs with the Cameroon isolate (Cam/82) of African swine fever virus.

African swine fever (ASF) was produced in eight pigs by exposure to donors infected with the Cameroon/82 isolate of African swine fever virus. The primary clinical sign was pyrexia of more than 40 degrees C first observed 10 to 13 days post-exposure (dpe) in all pigs; other clinical signs were rarely observed. The most frequent post-mortem lesion was haemorrhage in the visceral lymph nodes. Other lesions included excess fluid in the abdominal cavity and petechial haemorrhages in the kidneys. Viraemia was first observed 1 to 2 days before the onset of pyrexia and maximal titres of more than 10(7.5) HAD50 per ml occurred 11 to 14 dpe. Virus excretion by the pharyngeal route was observed at 2 to 4 days before the onset of pyrexia and continued throughout the course of infection. Susceptible pigs, mixed directly with infected ones, contracted infection within 2 h; transmission time increased to 2 to 6 h when recipient pigs were separated by wire mesh from the infected pigs. The comparatively low mortality, ill-defined clinical signs and clinical recovery of many of the infected pigs show that the Cam/82 ASF virus is of relatively low virulence and thereby resembles recent European, South American and Caribbean isolates.

ADCC and complement-dependent lysis as immune mechanisms against EHV-1 infection in the horse.

Immunity to equine herpesvirus type 1 (EHV-1) was evaluated using sera collected from yearling horses involved in a trial of a commercial vaccine. Measurement of the ability of these sera to mediate antibody-dependent cellular cytotoxicity and complement-dependent lysis revealed that these mechanisms, although potentially important in recovery from EHV-1 infection, do not play a role in protection following vaccination.

Lymphocyte responses to African swine fever virus infection.

The immunological response of pigs infected with African swine fever virus was related to both quantitative and qualitative changes in the lymphocyte population. The cytolytic effect of the virus on lymphocytes caused a proportionally greater drop in B cell than in T cell numbers. A blastogenic test was developed to measure the appearance of specifically sensitised lymphocytes in the circulation. These appeared about 10 days after infection in animals infected with attenuated virus; pigs infected with virulent strains died before the appearance of this response.

Cytotoxic lymphocytes induced by African swine fever infection.

The generation of lymphocytes cytotoxic to African swine fever virus infected testis cells during in vivo infection is described. Peripheral blood lymphocytes from 16 pigs developed cytotoxicity seven to eight days after infection but the lysis was not restricted to autologous cells.

Natural cytotoxicity detected in swine using Aujeszky's disease virus infected targets.

Using percoll gradients and standard B and T cell depletion techniques, porcine lymphocytes were investigated for their ability to show natural killing (NK) of many targets, including Aujeszky's disease virus (AJDV) infected cells. Although it was possible to enrich for NK activity using these techniques it was not possible to define a distinct subpopulation of NK cells. It was noted that porcine NK cells consistently showed a preferential lysis of AJDV infected cells in comparison to uninfected cells. Furthermore, pretreatment of these effector cells with virus or interferon increased their lytic ability but did not alter the degree of preference on the lysis of target cells shown by untreated effectors. Although in vitro results suggested that porcine NK activity might be an efficient mechanism for controlling AJDV infections in the animal, experiments designed to monitor NK levels in vivo were unable to demonstrate this. The possible explanations for this are discussed.

Effector mechanisms in the pig. Antibody-dependent cellular cytolysis of African swine fever virus infected cells.

Antibody dependent cellular cytolysis (ADCC) against African swine fever virus infected nucleated cells was investigated in a porcine system. Of the peripheral blood components examined, only neutrophils acted as effectors. Lymph node derived cells displayed no ADCC activity. In vitro yield reduction assays suggested that neutrophil mediated ADCC may play a role in recovery from African swine fever virus infection.

Local humoral and cellular responses in Aujeszky's disease virus infection in pigs.

Mucosal and tracheal washings from pigs vaccinated parenterally and intranasally with Aujeszky's disease virus were tested for specific anti-Aujeszky's disease virus responses. Antibody tests included complement dependent antibody lysis, antibody dependent cellular cytotoxicity, virus neutralisation, and anti-Aujeszky's disease virus IgA and IgG levels. There was no correlation between the levels of these antibodies and protection from subsequent challenge. Direct lymphocyte cytotoxicity against cells infected with Aujeszky's disease virus was found in lymph nodes draining the tonsillar area.

Production of murine cytotoxic T lymphocytes by bluetongue virus following various immunisation procedures.

The induction of bluetongue virus specific cytotoxic T lymphocytes (CTLs) in C3H mice by various live and inactivated bluetongue virus preparations was studied. Live virus preparations were shown to induce good levels of CTLs; however, inactivation of virus preparations either by beta propriolactone or glutaraldehyde induced only a low level response. The use of Freund's adjuvants and double immunisation procedures failed to improve the response of the inactivated preparations. These findings are discussed in relationship to protection from bluetongue disease with various bluetongue virus vaccines.

The pathology and sites of persistence associated with three different strains of feline calicivirus.

The pathology and sites of multiplication associated with three different strains of feline caliciviruses are described. The main pathological lesions were found in the tongue, soft palate and lungs and like the clinical signs were of mild nature. Virus multiplication was associated mainly with the tissues of the mouth and tonsils and in asymptomatic carrier animals the tonsil appeared to be preferred organ of viral persistence.

The clinical disease and patterns of excretion associated with three different strains of feline caliciviruses.

Three groups of cats were infected intranasally with three different feline calicivirus strains: A3, 68/40 and M8. Each strain produced a uniformly muld upper respiratory tract disease, with glossal ulceration being the most prominent clinical sign. Virus was most consistently isolated from the oro-pharyngeal region and, in non-euthanised animals, excretion continued long after clinical signs had disappeared. It is suggested that an asymptomatic phase of excretion may be a normal sequel to FCV infections.

Serological response of pigs infected with Aujeszky's disease virus.

The temporal development of antibody in four groups of pigs infected with different Aujeszky's disease virus isolates was examined. The enzyme-linked immunosorbent assay detected antibody by five to six days after infection and the antibody-dependent cell-mediated cytotoxicity assay detected antibody seven to nine days after infection. Neutralising antibody was first detected nine to 10 days after infection, whereas assays measuring complement mediated antibody lysis did not detect antibody until 10 days after infection. These results are discussed in terms of their importance to the diagnosis of and recovery from Aujeszky's disease.

Functional antibody responses in pigs vaccinated with live and inactivated Aujeszky's disease virus.

Functional antibody tests, including virus neutralising activity of serum, antibody dependent cellular cytotoxicity and complement mediated lysis, were used to measure the response of pigs given either live or inactivated Aujeszky's disease virus vaccines. Pigs were then challenged with virulent Aujeszky's disease virus and antibody responses were analysed and found not to correlate with protection. Reasons for this lack of correlation are discussed and it is suggested that these results indicate that more emphasis should be placed on measuring the local immune response.

Cross reactions between porcine, bovine and ovine interleukin-2 preparations.

Optimum conditions for the production of porcine interleukin-2 were found to include a delay of 24 hours before the addition of mitogen. Porcine and bovine interleukin-2 responded optimally in homologous systems whereas bovine interleukin-2 gave a better response in the ovine system than homologous ovine interleukin-2. Interleukin-2 produced from a continuous gibbon cell line reacted well with porcine, ovine and bovine T cell blasts indicating that it could act as a universal growth factor for T cell clones produced from these species.

Role of neutralising antibody in passive immunity to bluetongue infection.

A group of sheep inoculated with serum obtained from sheep which had recovered from bluetongue virus type 3 infection were protected from challenge with the homologous virus type but not from heterologous challenge. Twin lambs which had received colostrum containing virus antibodies were shown to be only partially protected against homologous challenge. A monoclonal antibody directed against the type-determining protein of the virus was also shown to give partial protection against challenge. From this series of experiments it was concluded that antibody has a significant role in protection from bluetongue but that the outcome of challenge will depend on several interacting factors.

Clinical and serological outcome following the simultaneous inoculation of three bluetongue virus types into sheep.

The simultaneous inoculation of sheep with three different bluetongue virus types resulted in the replication of only two of the virus types and the formation of neutralising antibodies to only those two types and a failure in the production of heterotypic antibodies. This suggests that the present system of control, using multivalent vaccines in areas in which a number of bluetongue serotypes exist, should be reappraised.

Serial inoculation of sheep with two bluetongue virus types.

Groups of sheep inoculated with bluetongue virus type 4 were challenged at various intervals after inoculation (from seven to 70 days) with bluetongue virus type 3. Examination of the clinical and serological response showed that animals were protected from challenge with a second bluetongue virus for up to 14 days after the inoculation of the first virus type. An adoptive transfer experiment in monozygotic sheep involving both antibody and T lymphocytes was carried out. Only partial protection was observed against heterologous virus challenge, indicating that although the T cell response has a cross-protective component, antibody is not involved. These observations indicate that current vaccination procedures should be reappraised, particularly in terms of revaccination with multiple bluetongue virus type.

Antibody and complement mediated lysis of felid herpesvirus 1 infected cells in vitro.

The lysis of cells infected by felid herpesvirus 1 (FHV) by feline anti-FHV antibody and complement was demonstrated. Lytic activity was sensitive to dilution of both antibody and, especially, complement. It was first detected within 10 to 20 minutes, increased rapidly during the next 30 minutes of incubation and then rose more slowly in a linear manner. Using standard antibody and complement concentrations and assay duration, it was shown that FHV infected cells underwent significant (P less than 0.05) lysis from eight hours after infection in a system in which FHV-specific membrane antigen was first detected at three hours after infection and spread of FHV by the intracellular route began eight to nine hours after infection. The ability of antibody and complement to reduce FHV spread in this system was demonstrated by a significant (P less than 0.05) reduction in FHV plaque numbers, although the restriction of spread was not absolute. Chelation of divalent cations and heat inactivation of complement factor B revealed that the lytic system was dependent on factor B and Mg2+ but not Ca2+, suggesting involvement of the alternative pathway of complement activation.

Immune response in pigs to Aujeszky's disease viruses defective in glycoprotein g1 or gX.

Two Aujeszky's disease virus glycoprotein genes, gX and g1, have been used to produce deletion mutants which have then been developed into vaccines. These deletions then allow differentiation between pigs infected with wild type virus and those given the vaccine. It is not clear whether the glycoproteins encoded for by these genes are needed to induce a full protective immune response, in which case deletion mutants would suffer from lack of potency. To test this, commercially available Aujeszky's virus vaccines which lacked either gX or g1 were compared and isogenic constructs were made which differed only in the absence or presence of gX and, or, g1. These constructs and vaccines were used to vaccinate the natural host of Aujeszky's disease, the pig, and potency was measured using challenge with wild type virus. In all cases vaccines which lacked g1 performed significantly less well than those in which g1 was present, whereas deletions of gX had no significant effect on vaccine performance.