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Biomedical subjects

G M Allan

Publications and source records attributed to G M Allan.

At least 19 recordsLinked to original sources
PubMed1992

Detection protocols for biotinylated probes: optimization using multistep techniques.

Recent studies using biotinylated in situ hybridization (ISH) have utilized a wide range of detection protocols for the biotinylated hybrids, leading to conflicting reports in the literature regarding sensitivity. In this study we compared 11 different detection protocols for biotinylated ISH using a measles virus-specific RNA probe on formalin-fixed, paraffin-embedded central nervous system tissue infected with measles virus. Maximum sensitivity was achieved with five-step detection protocols incorporating the use of a monoclonal antibody to biotin. Single-step detection protocols were found to be insensitive, as shown by their failure to detect viral nucleic acid in infected white-matter cells. Only by increasing the number of steps in the detection protocols were these infected cells demonstrable. Unless pre-hybridization, hybridization, and detection protocols are optimized, the results obtained in pathogenicity studies using ISH could be misinterpreted, leading to false conclusions about nucleic acid distribution. This also applies to the ever-increasing use of ISH for diagnostic purposes.

Antibodies, Monoclonal↗
PubMed1991

Comparison of three animal viruses with circular single-stranded DNA genomes.

No common antigenic determinants and no DNA sequence homologies were detected when three animal viruses, chicken anaemia agent (CAA), porcine circovirus (PCV), and psittacine beak and feather disease virus (PBFDV), all of which possess circular single-stranded DNA genomes, were compared. Negative contrast electron microscopy showed that PCV and PBFDV particles were 30% smaller than CAA particles and lacked the surface structure of CAA.

Animals↗
PubMed1991

Comparison of reporter molecules for viral in situ hybridization.

A number of streptavidin-linked reporter molecules at the endpoint of a five-step detection protocol for viral in situ hybridization using biotinylated probes were examined. DNA-DNA and RNA-RNA model systems were used. Streptavidin linked to either peroxidase or fluorescein was found to be optimal in terms of sensitivity and resolution within individual cells. All other reporter molecules labelled similar numbers of cells with low background reaction. However, streptavidin-5 nm gold followed by silver enhancement gave very high background staining making interpretation of positive signals very difficult.

Alkaline Phosphatase↗
PubMed1990

Use of immunocytochemistry and biotinylated in situ hybridisation for detecting measles virus in central nervous system tissue.

Optimised immunocytochemical (ICC) and in situ hybridisation (ISH) protocols for long term, formalin fixed, central nervous system tissue infected with measles virus were developed. The effectiveness of 10 proteases for the enzymatic unmasking of formalin fixed antigen and nucleic acid was investigated. Protease VIII gave maximal signal generation with optimal tissue preservation and no background staining for both techniques. The use of a microwave oven as an additional pre-hybridisation step for RNA-RNA in situ hybridisation produced a significant increase in the number of cells labelled for genomic RNA. The ability to show the presence of antigen and nucleic acid in long term, formalin fixed tissue facilitates the use of stored necropsy material available in pathology departments for ICC and ISH investigations.

Antigens, Viral↗
PubMed1989

Histopathologic and immunocytochemical studies of distemper in seals.

Thousands of harbor seals (Phoca vitulina) died in European seas during 1988. Respiratory distress and oculonasal discharge were common clinical signs. We necropsied 76 affected seals. The main necropsy finding was severe pneumonia. Microscopic lung changes were characterized by proliferation of type II pneumocytes, filling of alveolar lumina with serofibrinous exudate, leukocytes, and macrophages, and necrosis of bronchial and bronchiolar epithelium. Intracytoplasmic and intranuclear acidophilic inclusion bodies characteristic of morbillivirus infection were seen in bronchial and bronchiolar epithelial cells. Microscopic lesions of non-suppurative demyelinating encephalitis were seen in the brain. There was degeneration and necrosis of neurons, focal gliosis, perivascular cuffing, and patchy demyelination. Many neurons and astrocytes contained intracytoplasmic and intranuclear inclusions. Using an immunoperoxidase technique, we detected morbillivirus antigen in many tissues including lung, brain, spleen, and urinary bladder. The origin of the seal morbillivirus is unknown.

Animals↗
PubMed1989

Demonstration of bovine virus diarrhoea virus antigen in formalin fixed, paraffin embedded tissue using a streptavidin/biotin technique.

The detection of bovine virus diarrhoea virus (BVDV) antigen in sections from formalin fixed, paraffin embedded tissue is described. Pre-digestion of the sections with 0.02 per cent protease XIV for 18 hours at 4 degrees C is necessary to unmask formalin fixed antigen. A hyperimmune antiserum prepared in a pig, using a combination of BVDV and hog cholera virus inoculations, linked to a biotinylated anti-pig/streptavidin peroxidase detection system demonstrated antigen in a wide range of tissues from cases of mucosal disease and persistently viraemic animals. The inclusion of a monoclonal anti-pig immunoglobulin linked to a biotinylated anti-mouse/streptavidin peroxidase detection system greatly reduced non-specific staining.

Animals↗
PubMed1988

An immunoperoxidase method of detecting respiratory syncytial virus antigens in paraffin sections of pneumonic bovine lung.

Using an avidin-biotin-peroxidase complex immunoperoxidase staining method, respiratory syncytial virus (RSV) antigen was demonstrated in glutaraldehyde-fixed, paraffin-processed lung sections from calves with induced RSV pneumonia. The virus also was detected in formalin-fixed, paraffin-processed lung sections from calves with naturally occurring RSV pneumonia. Specific immunoperoxidase staining was detected within the cytoplasm of epithelial cells and syncytia in small bronchi, bronchioli, and alveoli. Staining also was detected within exudates in airway lumina and in mononuclear and multinucleate cells within alveolar lumina. Optimal intensity of staining was achieved by proteolytic enzyme treatment of lung sections, using 0.1% pronase and overnight incubation in diluted primary antiserum. The distribution of antigen had a close correlation with presence of lesions. Antigen-staining patterns were similar in lung tissue from calves with naturally occurring and induced RSV disease.

Animals↗
PubMed1985

The rapid detection of Aujeszky's disease virus in pigs by direct immunoperoxidase labelling.

Direct immunoperoxidase labelling on impression smears of brain and pharynx was compared with virus isolation and direct immunofluorescence for the detection of Aujeszky's disease virus in experimentally-infected pigs. Immunoperoxidase labelling was as sensitive as immunofluorescence and more sensitive than virus isolation for tissue that had been stored at room temperature (approximately 20 degrees C) for up to 144 h.

Animals↗
PubMed1984

Epidemiology of rotavirus infection in broiler chickens: recognition of four serogroups.

In a longitudinal survey of 11 broiler flocks, rotavirus excretion was detected by direct electron microscopic examination of faeces in 10. In most of these flocks, rotavirus excretion was first detected during the third week of life. In some flocks, infection with 2 antigenically distinct serogroups of rotavirus was demonstrated. In a more detailed survey of a 34,000 bird broiler crop, rotavirus excretion was detected intermittently from 9-50 days. Infection with 4 different RNA electropherotypes of rotavirus occurred in waves with each wave of infection lasting about 1 week. Analysis of representatives of the 4 different electropherotypes by cross-immunofluorescence indicated that each electropherotype represented an antigenically distinct serogroup. Two of these serogroups were represented by the previously characterised Ch 1 and 132 chicken rotavirus isolates. The other 2 serogroups have not been previously recognised.

Animals↗
PubMed1984

Coronavirus infection of the bovine respiratory tract.

Two viruses, morphologically resembling coronaviruses and antigenically indistinguishable from bovine enteric coronavirus, were isolated in bovine tracheal organ cultures from the lungs and trachea of young calves with respiratory disease. Intranasal and intratracheal inoculation of these viruses into neonatal calves resulted in a predominantly upper respiratory tract infection, which was associated with the development of mild respiratory symptoms.

Animals↗
PubMed1984

Rapid diagnosis of Aujeszky's disease in pigs by immunofluorescence.

Direct immunofluorescence on impression smears of brain and pharynx was compared with virus isolation in cell culture for the diagnosis of Aujeszky's disease in experimentally and naturally infected pigs. Pharyngeal impression smears were more sensitive than virus isolation in two pigs killed 10 and 12 days after experimental infection. Both methods were of similar sensitivity in the detection of virus from field cases of disease. Smears of brain and pharynx were more sensitive than virus isolation for tissue which had been stored at room temperature (approximately 20 degrees C) for up to 48 hours. Some reduction in the amounts of virus recovered from tissues and the intensity of fluorescent staining occurred in these samples.

Animals↗
PubMed1983

Experimental respiratory syncytial virus pneumonia in young calves: microbiologic and immunofluorescent findings.

Young calves were inoculated with respiratory syncytial virus (RSV) intranasally or by a combined intranasal and intratracheal route and were killed between postinoculation (initial) days (PID) 1 and 14. Viral antigens were detected by immunofluorescence in nasopharyngeal cells from calves killed between PID 2 and 10. Evidence of infection of the trachea and lungs with RSV was obtained by immunofluorescence and virus isolation in calves inoculated by the combined route, but not in calves inoculated intranasally. Within the lungs, RSV antigens were observed in epithelial cells of bronchioli and alveoli. The only virus detected in inoculated calves was RSV. With the exception of 1 calf, bacteria or mycoplasmas were not isolated from the lower respiratory tracts of inoculated calves. Antibody to RSV was not detected in calves killed up to PID 5, but 4 of 5 colostrum-deprived calves killed between PID 10 and 13 had antibodies to RSV. Preexisting, maternally derived antibody to RSV did not protect the calves from infection. Seemingly, the clinical signs of pneumonia and pathologic lesions observed in inoculated calves were caused by RSV infection.

Animals↗
PubMed1981

Isolation from chickens of a rotavirus lacking the rotavirus group antigen.

A virus, designated 132 virus, was isolated from the faeces of chickens in chick embryo liver cell cultures. The morphology and morphogenesis of 132 virus were indistinguishable from that of rotaviruses. The nucleic acid of 132 virus had the nuclease resistance of double-stranded RNA, and was separated by polyacrylamide gel electrophoresis into 11 segments with mol. wt. ranging from 2.07 x 10(6) to 0.20 x 10(6). SPF chickens were susceptible to oral infection with 132 virus, which replicated in the villous epithelial cells of the small intestine. 132 virus was therefore a rotavirus by morphological, biochemical and biological criteria. However, by immunofluorescence it was not possible to demonstrate an antigenic relationship between 132 virus and known avian and mammalian rotaviruses, indicating that 132 virus does not possess the group antigen shared by all previously characterized rotaviruses. This finding has implications for the diagnosis of rotavirus infections by serological tests.

Animals↗