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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
PubMed1997

A comparative study on the use of virus and antibody detection techniques for the diagnosis of La Piedad Michoacan paramyxovirus (LPMV) infection in pigs.

La Piedad Michoacan paramyxovirus (LPMV) is newly recognized paramyxovirus that has been associated with neurologic and reproductive disorders in pigs in Mexico. To date, no comparative study of methods for the diagnosis of infection with this virus has been published. In this study, we identified tissues containing maximum virus load to optimize virus isolation procedures, and we compared this method to a rapid diagnostic test employing immunostaining of impression smears for LPMV antigens. In addition, several of the available tests for detecting LPMV antibodies were compared for their sensitivity in detecting seroconversion. Pigs used for the study of virus load in tissues and serologic studies were inoculated at 17 days of age with 10(7.00) TCID50 of LPMV. Serial blood samples were collected from selected pigs, and selected pigs were necropsied over a 14-day period. Pigs used in the investigation comparing standard virus isolation techniques to immunostaining of impression smears were inoculated at 3 days of age as described above and necropsied over an 8-day period. The results demonstrate that in the 17-day-old pigs maximum virus titers were detected in olfactory bulb at 5 days postinoculation (PI) and in midbrain at 9 days PI. In addition, the most consistent recovery of high titer virus was from tonsil (3-9 days PI) and olfactory bulb (4-9 days PI). Immunostaining of impression smears was as sensitive as virus isolation when selected tissues (lung, midbrain, olfactory bulb) were compared, with virus detected by both methods in 11/13 samples and in 1 sample each by immunostaining and virus isolation, respectively. All of the serology tests investigated detected seroconversion in pigs by 8 days PI. The identification of target organs where highest virus titers are found combined with immunofluorescent methods for the detection of LPMV antigens and a comparative study of the available serologic tests should facilitate the selection of techniques suitable for any laboratory to diagnose LPMV infection in pigs.

Animals↗
PubMed1996

Effect of porcine circovirus infection on porcine alveolar macrophage function.

The effect of porcine circovirus (PCV) infection of porcine alveolar macrophage cultures on some of the functional properties of these cells are reported. PCV infection of alveolar macrophages did not effect their ability to phagocytose and kill complement-coated yeast cells or the expression of Fc or complement receptors. A transient increase in major histocompatibility complex (MHC) class I expression in PCV-infected cells were observed 4 days after infection and a decrease in the number of cells expressing MHC class II antigens was observed 8 days after infection. Infection of alveolar macrophages with PCV also resulted in a transient decrease in their ability to act as accessory cells in mitogen-induced lymphocyte proliferation of monocyte-depleted porcine peripheral blood mononuclear cells.

Animals↗
PubMed1996

Ultrastructural study of turkey rhinotracheitis virus infection in turbinates of experimentally infected chickens.

Ultrastructural changes associated with turkey rhinotracheitis virus infection were studied in turbinates of chickens experimentally infected with the isolate CVL 14/86/1. Chickens were sacrificed at 3, 5 and 7 days after inoculation and samples of the middle turbinate were taken, fixed, dehydrated and embedded in an hydrophilic resin. An immunofluorescence technique on semithin sections was carried out and viral antigen was observed in the cytoplasm and associated to cilia of the turbinate epithelial cells, on days 3 and 5 after inoculation. Ultrastructurally, gold stained intracytoplasmic nucleocapsid aggregates of turkey rhinotracheitis virus were observed in ciliated and non-ciliated epithelial cells, as well as budding virus particles, at days 3 and 5 postinoculation. Different ultrastructural abnormalities, including cytoplasmic blebs, clumping and loss of cilia were observed in the apical cell membrane of many infected cells, associated with the presence of intracytoplasmic inclusions. On day 5 after inoculation, substitution of ciliated and non-ciliated epithelial cells was noted and many desquamated epithelial cells were observed within the lumina. Regenerative changes in the ciliated epithelium were observed by day 7 postinoculation. These results indicate that turkey rhinotracheitis virus is able to replicate in ciliated and non-ciliated epithelial cells causing severe alterations to the cell surface and ciliary apparatus of the turbinate epithelium. Viral-induced damage to the turbinate epithelium could enhance the susceptibility of epithelial cells to secondary bacterial infection.

Animals↗
PubMed1996

A sequential study of experimental porcine paramyxovirus (LPMV) infection of pigs: immunostaining of cryostat sections and virus isolation.

La Piedad Michoacan Paramyxovirus (LPMV) is a recently recognized paramyxovirus infecting pigs throughout Mexico. Disease syndromes observed in field cases associated with LPMV infection include neurologic, respiratory, and reproductive disorders. Clinical signs and the distribution of LPMV virus and antigen in tissue samples from pigs experimentally infected with LPMV by natural routes were studied. Severe neurologic disease and death occurred following experimental inoculation of 3- and 17-day-old pigs. All of the pigs inoculated at 3 days of age were either dead or moribund by 8 days after inoculation, whereas 30% of the pigs inoculated at 17 days of age were affected. Virus was consistently recovered from or demonstrated in tissues from the respiratory tract of both groups of pigs. LPMV and antigen were also demonstrated in central nervous system (CNS) tissues from these pigs; however, differences in virus distribution within the CNS were demonstrated in the 2 groups. In the pigs inoculated at 17 days of age, isolation of LPMV was restricted to the olfactory bulb and midbrain. In contrast, in the pigs inoculated at 3 days of age, isolation of LPMV was more widespread throughout the CNS tissue examined. Virus excretion studies indicated that nasal spread of LPMV was more important than fecal spread. Comparatively large quantities of infectious LPMV were consistently recovered from urine samples of experimentally infected pigs.

Animals↗
PubMed1995

Pathogenesis of porcine circovirus; experimental infections of colostrum deprived piglets and examination of pig foetal material.

The results of virus and antigen distribution following experimental infection of colostrum deprived pigs with pig circovirus (PCV) by oral/nasal and intravenous routes are reported. PCV and antigen were detected using virus isolation and indirect immunofluorescence on cryostat sections respectively. PCV antigen was detected in tissues throughout the body but primarily in spleen thymus, and lung. No PCV antigen or virus was detected in tissue samples from the central nervous system. Examination of pig foetal material from field cases of abortion/stillbirth resulted in 3 PCV isolates from 2 sera and a spleen sample from 2 groups of stillborn piglets from the same farm. No antibody to PCV alone was detected in 160 foetal sera tested. These results suggest that transplacental infection with PCV does occur, possibly prior to foetal immunocompetance. However, it is probably not a significant cause of reproductive disorders in pigs in Northern Ireland.

Animals↗
PubMed1995

A comparison of digoxigenin and biotin labelled DNA and RNA probes for in situ hybridization.

A number of in situ hybridization protocols using digoxigenin or biotin labelled probes were assessed for viral nucleic acid detection in formalin fixed, paraffin embedded tissue. Single-step detection protocols for biotin labelled probes produced low sensitivity; however, enzyme based one-step detection protocols for digoxigenin probes produced high sensitivity for both RNA and DNA systems. For both probe types, multistep detection protocols produced equally high sensitivity. Use of an enhanced APAAP procedure for digoxigenin labelled probes achieved maximal sensitivity without use of biotin-strep-tavidin reactions. The sensitivity of nucleic acid detection obtained with a digoxigenin labelled probe is comparable to that obtained using biotin. Digoxigenin labelled probes for nucleic acid detection are recommended for tissues with endogenous biotin.

Animals↗
PubMed1994

Infection of leucocyte cell cultures derived from different species with pig circovirus.

Cultures of leucocyte cells were prepared from pig bone marrow, peripheral blood, lung washings, thymus and lymph nodes. Cell cultures were also prepared from peripheral blood from sheep, cattle and a human. Immunofluorescent (IF) staining of all these cultures, following inoculation with pig circovirus (PCV), detected virus replication in all the cell cultures derived from pigs and in the cell cultures derived from cattle. Virus replication in pig leucocyte cell cultures was confirmed by demonstrating the production of infectious virus. Double immunostaining of PCV infected cells using monoclonal antibodies specific for cell membrane markers indicated infection was confined to monocyte/macrophage cell types. No PCV antigen was detected in T or B cells in infected cell cultures.

Animals↗
PubMed1994

Production, preliminary characterisation and applications of monoclonal antibodies to porcine circovirus.

The preparation of monoclonal antibodies (mAbs) to porcine circovirus is described. Preliminary characterisation was carried out on nine mAbs obtained from two fusions and included isotyping, virus neutralisation assays and indirect immunofluorescence staining patterns obtained following immunostaining of both a porcine circovirus (PCV)-persistently infected pig kidney (PK/15/W) and Vero (Vero-PCV) cell line. Significant differences in the staining patterns were observed in both cell lines which appeared to be dependent on the subculture status of the Vero-PCV cultures. The development of a mAb-based antigen capture enzyme linked immunosorbent assay (ELISA) as an aid to virus purification is also described. The use of mAbs for the detection of PCV antigen in cryostat sections from a pig experimentally infected with the virus leading to identification of the sites of replication of PCV is also reported.

Animals↗
PubMed1994

Some biological and physico-chemical properties of porcine circovirus.

Some important biological and physico-chemical characteristics of porcine circovirus are reported. These include a study on the host distribution in nature, levels of colostrum-derived antibodies in piglets from sero-positive sows and the susceptibility of a range of cell cultures to infection with this virus. The results of haemagglutination studies, resistance to pH 3, chloroform and heat are also reported as are comparative buoyant densities and sedimentation coefficients of porcine circovirus and chicken anaemia virus.

Animals↗
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↗