An inactivated parainfluenza virus type 3 vaccine: the influence of vaccination regime on the response of calves and their subsequent resistance to challenge.

Calves maintained in insolated pens were vaccinated with an inactivated parainfluenza virus type (3) (pi3) vaccine usingparenteral and local route singly and in combination. The calves were subsequently monitored for serum antibody response and challenged intranasally with live virus to assess the protection derived from vaccination. Calves receiving one subcutaneous dose of vaccine in oil adjuvant produced a marked antibody response and were partially protected against challenge. Those receiving two successive subcutaneous doses produced a much greater antiboyd response and were completely protected against challenge. One intranasal dose of aqueous vaccine failed elicit a significant serum antibody response or protection against challenge. However, there was some evidence that intranasal vaccination following a single subcutaneous vaccination produced more effective immunity than one subcutaneous dose alone. Thus a vaccination regime was established which protected calves against experimental challenge and which could thefore be used in the field to assess the role of Pi3 virus in calf respiratory disease.

The growth of respiratory syncytial virus in organ cultures of bovine foetal trachea.

Respiratory syncytial (RS) virus grown in organ cultures of bovine foetal trachea at 37 degrees C and pH 7.2 reached maximum titres of up to 1 X 10(5) PFU/ml between 11 and 21 days after inoculation. Virus yield was increased three fold by incubation at 33 degrees C, but depressed by the addition of RS virus antiserum, with or without bovine complement, or by the addition of alveolar macrophages. Variation in pH or the concentration of foetal calf serum and magnesium chloride did not affect the virus yield. Virus growth did not affect ciliary activity of the cultures. Histological changes involved slight flattening of the epithelium and the appearance of phloxinophilic inclusion bodies. Fluorescent antibody staining showed more virus antigen in the peri-tracheal connective tissue than in the ciliated epithelium. The presence of non-cytopathic mucosal disease (MD) virus in RS virus infected organ cultures slightly depressed RS virus growth but did not influence ciliary activity. These in vitro experiments suggest that the tracheal epithelium may not be an important target in the pathogenesis of RS virus infection in vivo.

Infection of gnotobiotic calves with a bovine and human isolate of respiratory syncytial virus. Modification of the response by dexamethasone.

A bovine and a human strain of RSV both adapted to bovine cell culture, have been inoculated separately into 13 and 7 gnotobiotic calves respectively by 3 different methods. Both strains infected calves and showed similar growth patterns. Virus was recovered from the nasopharynx between one and 11 days with peak titres between 3 and 8 days following inoculation. With the exception of 4 calves treated with dexamethasone no clinical signs and only minimal macroscopic lesions of the lung were induced, which histologically comprised a mononuclear infiltration of alveolar walls and of the peribronchiolar tissue. The serological response to both strains was similar. Antibody was detected by virus neutralisation or single radial haemolysis from 12 days after inoculation. Specific anti-RSV IgM was detected from 10 days and IgG from 16 days after inoculation. Treatment with dexamethasone (0.5 mgm/Kg daily for 10 days) enhanced lung lesions produced by the bovine strain, prolonged the period of virus shedding and increased peak titres. The specific IgM response was suppressed.

Experimental pneumonia in gnotobiotic calves produced by respiratory syncytial virus.

A bovine isolate of respiratory syncytial virus (RSV), when inoculated intranasally into eight gnotobiotic calves produced significant macroscopic lesions of the lung (2-25% consolidation) but failed to produce any clinical signs of disease. The microscopic lesions comprised proliferative and exudative bronchiolitis with accompanying alveolar collapse and infiltration by mononuclear cells of the peribronchiolar tissue and alveolar walls. Virus was recovered from the nasopharynx between days 2 and 11 after infection with peak titres between days 4 and 7. Demonstration of viral antigen by immunofluorescence in nasopharyngeal cells followed a similar detection pattern. Virus was recovered from lung or detected by immunofluorescence in the bronchiolar epithelium up to 11 days following inoculation. A serological response to RSV was demonstrated both by virus neutralization and single radial haemolysis (SRH) tests, in serum of calves from 11 days following inoculation. Specific anti-RSV IgM was detected from 9 days following infection. It is suggested that the close resemblance between the experimental disease in calves and the pathology of acute bronchiolitis in children make cattle a particularly relevant model for the human disease.

The characterization and uses of monoclonal antibodies to respiratory syncytial virus.

Fifteen hybridomas secreting monoclonal antibodies against respiratory syncytial virus (RSV) have been produced. Three react with nucleoprotein (MW 42 000 daltons), 1 with phosphoprotein (MW 36 000 daltons), 1 with a larger protein (MW 60 000 daltons) and 10 with the fusion glycoprotein (MW 46 000 + 22 000 daltons). By immunofluorescent staining of infected cells, 5 monoclonal antibodies bind to cytoplasmic inclusions and the remainder give diffuse cytoplasmic fluorescence. Virus infectivity is neutralized by two monoclonal antibodies. Thirteen react with both bovine and human strains of RSV, two fail to react with three human strains. The antibodies have been used to induce passive protection in mice against challenge with RSV and to purify viral components on immunoadsorbent columns.

A comparison of three vaccines against respiratory syncytial virus in calves.

An inactivated vaccine against respiratory syncytial virus (RSV) was compared with two live vaccines. The inactivated (GC) vaccine consisted of glutaraldehyde-fixed bovine nasal mucosa cells persistently infected with RSV and emulsified with oil adjuvant. The live vaccines were a modified virus (MV) derived from a bovine strain of RSV and a temperature-sensitive mutant (ts-1) derived from a human strain. The GC vaccine was inoculated subcutaneously into 12 calves and the live vaccines intramuscularly into eight calves each. Nine unvaccinated calves acted as controls. The vaccines were administered in two doses 3 weeks apart and all calves were challenged intranasally with 2 X 10(7) p.f.u. of bovine RSV 3 weeks after the second dose. At the time of challenge calves given GC, MV and ts-1 vaccines had mean serum neutralizing antibody titres of 25, 19 and 2 respectively; mean titres of IgG1 antibody by radioimmunoassay were log10 4.5, 1.3 and 2.6 respectively and mean zone areas by single radial haemolysis (SRH) were 107, 27 and 36 mm2 respectively. Eleven of 12 calves given GC vaccine were completely protected against challenge but all control animals and those given the two live vaccines were infected. The mean peak titre of virus in nasal swabs of control calves was 3.0 log10 p.f.u./ml and the mean duration of virus shedding was 6.8 days. Both these parameters were significantly reduced in animals given MV and ts-1 vaccines: mean peak titres were 2.1 and 2.4 log10 p.f.u./ml and mean duration of shedding was 3.4 and 3.3 days respectively. Thus, protection correlated better with RSV antibody detected by radioimmunoassay and SRH than with neutralizing antibody. These results are discussed in relation to the possible mechanism by which protection was mediated.

A survey of virus infections of the respiratory tract of cattle and their association with disease.

A total of 1590 caves were investigated between May 1972 and December 1975. Twenty-two per cent were treated for respiratory disease and 2 . 5% died of pneumonia. Almost 80% of the respiratory illness occurred in six sharp outbreaks. Samples of virology were collected routinely from 127 healthy calves and from 354 calves treated for respiratory signs and comprised 1143 nasopharyngeal swabs and 1069 sera. Virus infections were detected on 540 occasions including 135 by parainfluenzavirus type 3 (Pi-3), 78 by respiratory syncytial virus (RSV), 103 by rhinovirus, 49 by bovine virus diarrhoea virus (BVDV), 29 by adenoviruses, 53 by reoviruses and 88 by enteroviruses. The seasonal and age distribution of infections differed between viruses. Only infections by RSV, Pi-3 and BVDV were significantly associated with disease.

Monoclonal antibodies protect against respiratory syncytial virus infection in mice.

Twenty-five monoclonal antibodies (Mab) to respiratory syncytial virus (RSV) and two to hepatitis B virus were inoculated intravenously into mice. Twenty-four hours later the mice were challenged intranasally with RSV. Eleven of 14 Mab against fusion protein and four out of six Mab against a larger glycoprotein (GP84) significantly reduced the titre of RSV in the lungs when mice were killed 5 days later. Five Mab against three other RSV proteins and two Mab against hepatitis B virus had no significant effect on RSV infection. These results indicated that serum IgG against one epitope on the fusion protein and another on the larger glycoprotein (GP84) will completely protect mice against challenge. These epitopes are primary candidates for an RSV vaccine produced by techniques of gene cloning and peptide synthesis.