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Biomedical subjects

D M Haines

Publications and source records attributed to D M Haines.

At least 55 records · Page 3Linked to original sources
PubMed1998

Isolation of porcine circovirus-like viruses from pigs with a wasting disease in the USA and Europe.

Samples of lung, liver, kidney, pancreas, spleen, and lymph node from pigs with postweaning multisystemic wasting syndrome from California (USA) and samples of mesenteric lymph nodes from similarly diseased pigs from Brittany (France) were examined by light microscopy, in situ hybridization (ISH), and/or virus isolation. Whole genomic probes for porcine circovirus (PCV) and chicken anemia virus (CAV) were used for ISH. Tissue homogenate supernatants were inoculated onto PK/15 cells for virus isolation, and the presence of viral antigen and viral particles was verified by indirect immunofluorescence, ISH, and electron microscopy. Histologic examination of lung from pigs from California revealed interstitial pneumonia, alveolar epithelial hyperplasia, and basophilic nuclear and cytoplasmic inclusions in mononuclear cell infiltrates and various pulmonary epithelial cells. Granulomatous lymphadenitis with syncytial cells typified the lesions seen in the pigs from France. PCV-like nucleic acid was detected by ISH in lung, pancreas, lymph node, kidney, and liver in pigs from California. Positive signal was also obtained in lymph node sections from pigs from France. Probes for CAV were consistently negative. PK/15 cell cultures inoculated with lung preparations from diseased California pigs and mesenteric lymph node preparations from pigs from France had positive fluorescence by indirect staining for PCV using pooled polyclonal pig sera and hyperimmune rabbit serum and had variable staining with a panel of 7 monoclonal antibodies specific for cell culture contaminant PCV. PCV-like nucleic acid was also detected by ISH in cell cultures. Cytopathic effect was not observed. Electron microscopic examination of inoculated cell cultures revealed 17-nm viral particles morphologically consistent with PCV. No other virus particles were observed. Although genomic analysis for the definitive identification of these viral isolates remains to be done, the evidence provided strongly suggests that these tissue isolates are closely related to, although antigenically distinct from, the original PCV cell culture contaminant.

Animals↗
PubMed1998

Lesions and distribution of viral antigen following an experimental infection of young seronegative calves with virulent bovine virus diarrhea virus-type II.

During the past several years, acute infections with bovine viral diarrhea virus (BVDV) have been causally linked to hemorrhagic and acute mucosal disease-like syndromes with high mortality. The majority of BVDVs isolated in such cases have been classified as type II on the basis of genetic and antigenic characteristics. It was our objective to examine clinical disease, lesions and potential sites of viral replication, following experimental BVDV type II infection in young calves. On approximately day 35 after birth, calves that had received BVDV-antibody-negative colostrum were infected by intranasal inoculation of 5 x 10(5) TCID50 of BVDV type II isolate 24,515 in 5 mL of tissue culture fluid (2.5 mL/nostril). Calves were monitored twice daily for signs of clinical disease. Approximately 48-72 h after infection, all calves developed transient pyrexia (39.4-40.5 degrees C) and leukopenia. Beginning on approximately day 7 after infection, all calves developed watery diarrhea, pyrexia (40.5-41.6 degrees C), marked leukopenia (> or = 75% drop from preinoculation values), variable thrombocytopenia, and moderate to severe depression. Calves were euthanized on days 10, 11, or 12 after infection due to severe disease. Gross and histological lesions consisted of multifocal bronchointerstitial pneumonia (involving 10%-25% of affected lungs), bone marrow hypoplasia and necrosis, and minimal erosive lesions in the alimentary tract. Immunohistochemical staining for BVDV revealed widespread viral antigen usually within epithelial cells, smooth muscle cells and mononuclear phagocytes in multiple organs, including lung, Peyer's patches, gastric mucosa, thymus, adrenal gland, spleen, lymph nodes, bone marrow, and skin. This BVDV type II isolate caused rapidly progressive, severe multisystemic disease in seronegative calves that was associated with widespread distribution of viral antigen and few gross or histological inflammatory lesions.

Animals↗
PubMed1998

A comparison of diagnostic methods for the detection of bovine respiratory syncytial virus in experimental clinical specimens.

Virus shedding was monitored in nasal secretions of 12 calves experimentally infected with bovine respiratory syncytial virus (BRSV) using an antigen capture enzyme-linked immunosorbent assay (ELISA) detecting the nucleoprotein (NP) antigen of BRSV, by a polymerase chain reaction (PCR) amplifying the fusion protein of BRSV, and by a microisolation assay combined with immunoperoxidase staining for the F protein of BRSV. Under the conditions of this study, similar limits of detection and quantitative results were obtained from all three assays. BRSV was detected in nasal secretions of all calves for a minimum of 4 d. Virus shedding began on Day 2 after infection, peaked on Days 3-5, and was cleared in most calves by Day 8. The PCR, and to a lesser extent the ELISA, may detect virus shedding for a longer period after infection than virus isolation, possibly due to neutralization of the virus by rising mucosal antibody. Simulated environmental conditions likely to be experienced during transport of clinical field specimens markedly reduced the sensitivity of virus isolation but had a minimal effect on the results of the NP ELISA. Actual field transport conditions (overnight on ice) had minimal apparent effect on the results of the PCR assay. The less stringent specimen handling requirements, combined with low limits of detection, of both the nucleoprotein ELISA and PCR, indicate either of these assays are more suitable for diagnostic applications than virus isolation.

Animals↗
PubMed1997

Cell-mediated cytolysis of equine herpesvirus-infected cells by leukocytes from young vaccinated horses.

The objective of this study was to determine whether the administration of modified-live equine herpesvirus (EHV-1) to young horses with residual maternal antibodies stimulated EHV-specific cytolytic responses, and whether these responses were crossreactive between EHV-1 and EHV-4. Eighteen clinically normal Belgian cross-foals were used in the study and were commingled in two adjacent pens. Skin biopsies were harvested from 16 foals within 24 h of birth and fibroblast cultures were established, expanded and cryopreserved. Beginning at approximately 10 weeks of age, 10 randomly chosen foals were inoculated on days 0, 21, and 43 of the study with a vaccine containing modified-live EHV-1. Blood mononuclear leukocytes were obtained on days 0, 32, and 50 for the assessment of EHV-specific cytolytic activity using 5 h and 18 h chromium release assays. EHV-1-specific antibodies were assessed by enzyme-linked immunosorbent assay using serum collected on days -21, 0, 32, and 50 of the study. Lymphocyte blastogenic tests and bioassays for interferon activity were conducted on day 50. After two vaccinations, mononuclear leukocytes from seven of ten vaccinated foals had cytolytic activity against autologous EHV-1 cells and leukocytes from six of ten lysed EHV-4-infected cells when tested in an 18 h assay. This activity was enhanced by exogenous interleukin 2 and was markedly reduced using target cells from unrelated horses. Cytotoxicity was not detected in a 5 h assay following in vitro stimulation of leukocytes. After three vaccinations, blood leukocytes from 6/6 vaccinated foals and 0/6 unvaccinated foals had proliferative responses EHV-1. There were no significant differences in interferon production by leukocytes from these foals. Twelve foals tested had low concentrations of (maternal) EHV-1-specific antibody prior to vaccination. Five of eight foals tested had increases in EHV-specific antibodies, while 4/4 commingled unvaccinated foals had a decrease or no change in EHV-specific antibodies. These results demonstrate cytotoxic cellular immune responses can be induced in young horses with maternal antibodies following administration of modified-live vaccine.

Animals↗
PubMed1997

Neurological symptoms associated with sarcocystosis in adult sheep.

Four mature ewes developed mild neurological symptoms. Histological examination revealed a nonsuppurative encephalitis and myelitis associated with protozoan cysts identified as Sarcocystis spp. by immunoperoxidase. The mild clinical signs and apparent recovery of 1 ewe suggest that neurological disease caused by Sarcocystis spp. may be more common than indicated by the infrequency of reports.

Animals↗
PubMed1996

Use of immunohistochemistry and polymerase chain reaction for detection of oncornaviruses in formalin-fixed, paraffin-embedded fibrosarcomas from cats.

OBJECTIVE: To determine whether there was intralesional infection or expression of FeLV or feline sarcoma virus in suspected vaccine-associated fibrosarcomas in cats. DESIGN: Prospective case series. SAMPLE POPULATION: 130 suspected vaccine-associated fibrosarcomas from cats and 1 multicentric fibrosarcoma from 1 cat. PROCEDURE: Excisional biopsy specimens were fixed in formalin and embedded in paraffin. Expression of FeLV antigen was assessed, using a polyclonal goat anti-FeLV glycoprotein 70 (gp 70) serum and an avidinbiotin immunoperoxidase staining technique. The FeLV genome was detected with a polymerase chain reaction (PCR), using primers targeted to a conserved sequence in the untranslated region of the long terminal repeat (LTR) of the FeLV. RESULTS: FeLV gp 70 and LTR sequence were detected in a multicentric fibrosarcoma. All 130 of the suspected vaccine-associated fibrosarcomas were FeLV gp 70 negative on the basis of immunohistochemical test results: 100 fibrosarcomas also were examined by use of PCR and were negative for FeLV LTR region. CLINICAL IMPLICATIONS: Exogenous retroviruses, FeLV, and feline sarcoma virus were not detected in these suspected vaccine-associated fibrosarcomas, using immunohistochemistry and PCR. Additional testing will be required to determine the nature of genomic alterations that are involved in the oncogenesis of vaccine-associated fibrosarcomas in cats.

Animals↗
PubMed1996

Sequence analysis of the putative viral enhancer in tissues from 33 cats with various feline leukemia virus-related diseases.

Diseases resulting from infection by feline leukemia virus (FeLV) and several other retroviruses relate in part, to non-coding regulatory sequences within the viral long terminal repeat (LTR). Both enhancer repeats and mutations within the LTR have been implicated in FeLV related disease. In order to investigate the relationship between nucleotide sequence of the FeLV LTR and disease, tissues from 33 cats with different types of degenerative and proliferative FeLV-related disease were studied. An FeLV LTR region containing the putative transcriptional enhancer unit was amplified by polymerase chain reaction (PCR) from FeLV-infected tissues. Phylogenetic analysis of FeLV 3'unique (U3) sequences revealed only one meaningful grouping which contained 4 of the 5 antigen-negative lymphosarcomas (LSAs). No sequence duplications were found in any of the 33 FeLV U3 regions. Point mutations relative to the corresponding region of FeLV-A/Glasgow, were identified at 102 positions; 68 of these were accounted for by mutations at 5 locations. Only 1 point mutation was found within the leukemia virus b-simian virus 40-like core (LVb-CORE) site. However, the nuclear factor 1 (NF1) site contained 11 mutations, and the FeLV-specific (FLV-1) site contained 26 mutations. Most of the remaining mutations were upstream of the LVB site between glucocorticoid response element (GRE) and FLV-1. The 10 LSAs, particularly the 5 antigen-negative LSAs, deviated least from the corresponding sequence for FeLV-A/Glasgow. Conclusions were that the spectrum of neoplastic and non-neoplastic FeLV-related diseases investigated in this study, developed in the presence of FeLVs containing the single enhancer unit. The significance of the point mutations is unknown, however, those occurring with high frequency and within nuclear protein binding should be first to be investigated in functional studies.

Animals↗
PubMed1996

Feline leukemia virus detection by ELISA and PCR in peripheral blood from 68 cats with high, moderate, or low suspicion of having FeLV-related disease.

Clinicopathologic criteria were used to group 68 cats according to high, moderate, or low suspicion of having feline leukemia virus (FeLV)-related disease. Peripheral blood samples were tested for FeLV antigen by enzyme-linked immunosorbent assay (ELISA) and for FeLV DNA by polymerase chain reaction (PCR). There was no significant difference between ELISA and PCR results in the 68 cats. In the high-suspicion group, 46%(11/24) of cytopenic cats were test positive (ELISA and PCR) and 87% (13/15) with hemopoietic neoplasms were test-positive. Also within the high suspicion group, test-positive cats were 2.5 times more likely to die within the 1 year follow-up period than were test-negative (ELISA and PCR) cats. Among cats in the moderate-suspicion group, 15% (2/13) were test-positive, and none (0/16) of the cats in the low suspicion group was test positive. The relative risk of a positive test (ELISA and PCR) in the high suspicion group was 3.7 times that for the moderate-suspicion group and 22.8 times that for the low suspicion group. There was no significant difference in the relative risk of a positive test result between the moderate and low suspicion groups. The results indicate that FeLV detection by PCR can be adapted for diagnostic purposes using peripheral blood samples, however, results do not differ significantly from FeLV ELISA results. Also, a proportion of cats with a high suspicion of having FeLV-related cytopenia and hemopoietic tumors are negative for both circulating FeLV antigen and DNA. These cats may not have FeLV-related disease, or FeLV may exist in a disease-producing but nonreplicating form ultimately detectable by PCR in tissues other than peripheral blood.

Animals↗
PubMed1996

Detection of Mycobacterium paratuberculosis in formalin-fixed paraffin-embedded tissues by the polymerase chain reaction.

The polymerase chain reaction (PCR) utilizing primers specific for the IS900 sequence of Mycobacterium paratuberculosis was applied to tissue sections of formalin-fixed, paraffin-embedded ileum from cattle with Johne's disease and the results compared to those obtained with acid-fast (Ziehl-Neelsen) and immunohistochemical staining. The PCR was positive in 19/21 tissues (90%) while Ziehl-Neelsen staining was positive in 18/21 (86%) and immunohistochemical staining in 21/21 (100%). The Ziehl-Neelsen and immunohistochemical stains are not, however, specific for M. paratuberculosis. The PCR for detection of M. paratuberculosis using the IS900 sequence is a specific and relatively sensitive method for confirmation of Johne's disease and its application to formalin-fixed, paraffin-embedded tissues may be useful for confirmation of dubious cases, for retrospective studies and for epidemiological analyses.

Animals↗
PubMed1996

Identification of monoclonal antibodies for immunohistochemical staining of feline B lymphocytes in frozen and formalin-fixed paraffin-embedded tissues.

Commercially-available monoclonal antibodies to B lymphocytes were evaluated for immunohistochemical staining of feline B lymphocytes in frozen and formalin-fixed, paraffin-embedded tissues using an avidin biotin complex immunoperoxidase immunohistochemical technique. Three monoclonal antibodies: F46A and F72A raised to "carnivore" B lymphocytes and RA3.6B2 raised to murine B lymphocytes, stained B lymphocyte-dependent areas of frozen feline lymphoid tissue. In addition, antibody RA3.6B2 stained B lymphocyte dependent areas in formalin-fixed, paraffin-embedded feline tissues. There was no staining of T lymphocyte-dependent areas in either frozen or formalin-fixed tissues. Dual parameter flow cytometry, using an anti-pan-T lymphocyte antibody, revealed that greater than 99% of the cells stained by RA3.6B2 are a population distinct from T lymphocytes. F46A was shown to stain a sub-population of those cells stained with RA3.6B2. These antibodies may be useful in the identification of feline B lymphocytes using immunohistochemistry and flow cytometry and thereby provide additional tools to study B lymphocyte ontogeny and the significance of lymphocyte phenotype in lymphoid neoplasia in cats.

Animals↗
PubMed1996

Immunohistochemical identification of B and T lymphocytes in formalin-fixed, paraffin-embedded feline lymphosarcomas: relation to feline leukemia virus status, tumor site, and patient age.

The lymphocyte phenotype of 70 formalin-fixed, paraffin-embedded feline lymphosarcomas (LSAs) was determined immunohistochemically using a T cell polyclonal antibody, and a B cell monoclonal antibody. Forty-seven of 70 (67%) tumors were T cell, 19/70 (27%) were B cell, and 4/70 (6%) did not stain with either marker. Thirty-eight of 70 (54%) tumors were positive for feline leukemia virus (FeLV) antigen by immunohistochemistry (IHC), and 52/70 (74%) tumors were positive for FeLV DNA using the polymerase chain reaction (PCR). B cell tumors were as frequently FeLV-positive as T cell tumors using either IHC or PCR. Intestinal tumors were more likely to be B cell than T. The incidence of B and T cell tumors was not different among young (< or = 3 y), middle-aged (> 3 y to < or = 8 y), and old (> 8 y) cats. Both B and T cell tumors from old cats were FeLV-positive more often by PCR than by IHC. Feline leukemia virus DNA but not antigen, was detected in B cell tumors and intestinal tumors from cats > 8 y as often as it was detected in B cell tumors and intestinal tumors from cats < or = 8 y. Previously, most B cell and intestinal tumors from old cats were considered to be negative for FeLV. Here, the results suggest involvement of latent or replication-defective forms of the virus in such tumors from old cats. This study supports a role for FeLV in feline B cell as well as T cell tumorigenesis.

Aging↗
PubMed1995

Cellular and antibody responses to equine herpesviruses 1 and 4 following vaccination of horses with modified-live and inactivated viruses.

The ability of monovalent and bivalent equine herpesvirus (EHV) vaccines to stimulate cellular and antibody responses to EHV-1 and EHV-4 was compared in healthy horses. Comparison of data from lymphocyte blastogenesis tests in which live viruses were used as antigens and that were conducted prior to vaccination and after 2 vaccinations revealed that horses given modified-live EHV-1 had significant increases in proliferative responses to EHV-1 (P = 0.03) and EHV-4 (P = 0.04). Responses to EHV-1 and EHV-4 in horses given the inactivated-virus bivalent vaccine were less; however, significant differences were not noticed when postvaccinal lymphocyte blastogenesis tests were compared between the groups of vaccinees. Interleukin-2 activity was not detected in leukocyte cultures from either group of vaccines following stimulation with live EHV-1 or EHV-4; however, interferon activity was found in similar cultures from both groups of vaccinees. For EHV-4, interferon activity in cultures from both groups of vaccinees was significantly (P < 0.05) greater than that in leukocyte cultures from unvaccinated controls. Both vaccines induced significant (P < 0.05) increases in serum antibodies that neutralized EHV-1 infectivity. The ELISA for EHV-1 and EHV-4 antibodies revealed that both vaccines induced significant (P < 0.05) increases (compared with preinoculation values) in antibodies reactive with these 2 types of EHV. Total serum antibody responses, as measured by ELISA, to EHV-1 and EHV-4 were significantly (P < 0.05) higher in horses that received the bivalent inactivated-virus vaccine, compared with that in horses that received monovalent vaccine. Evaluation of these data revealed that vaccination with modified-live EHV-1 can stimulate cellular and antibody responses that cross-react with EHV-4.

Analysis of Variance↗
PubMed1995

The relationship between single radial hemolysis, hemagglutination inhibition, and virus neutralization assays used to detect antibodies specific for equine influenza viruses.

Antibodies specific for equine influenza viruses are usually quantified using single radial hemolysis (SRH), hemagglutination inhibition (HI) or virus neutralization (VN). Neutralizing antibodies are thought to provide optimum protection to challenged animals. The purpose of this study was to determine the extent to which SRH and HI assays detect antibodies which neutralize equine influenza viruses. Acute and convalescent sera from 41 horses were analyzed using VN, SRH, and HI assays. These horses were present in a population of Thoroughbred racehorses during an epidemic of upper respiratory tract disease associated with influenza A/equine/Saskatoon/1/91 (H3N8), infections. Concentrations of antibodies binding to influenza A/equine/Kentucky/1/81 (H3N8), A/equine/Miami/1/63 (H3N8), and A/equine/Prague/1/56 (H7N7) were determined. Results of the VN assay were compared with results from the SRH and HI assays for acute antibody levels, changes in antibody concentrations between acute and convalescent sampling, and the occurrence of seroconversion. The correlation between assays for pre-exposure antibody levels ranged from 88% to 96%. The correlation between assays for change in antibody concentration ranged from 83% to 90% for the H3N8 viruses. This study shows that antibody concentrations specific for equine influenza virus, measured using SRH and HI assays, are highly correlated with concentrations detected using a VN assay.

Animals↗
PubMed1995

The effect of changing single radial hemolysis assay method when quantifying influenza A antibodies in serum.

Single radial hemolysis (SRH) is a simple method for measuring concentrations of antibody specific for influenza A viruses. There are many differences in reported assay methods and these changes may significantly affect results. This study evaluated the effect of changing erythrocyte concentrations, influenza virus concentrations used to sensitize erythrocytes, incubation times, and incubation temperatures. These factors were shown to significantly affect results of the SRH assay. A method for standardizing the assay and expressing results as relative antibody concentrations is described. Standardized relative antibody concentrations show significantly less variability than do hemolysis zone measurements when the SRH assay method is changed. This paper demonstrates that use of this method would allow for more consistent results to be obtained within laboratories. Use of this method with quantified, internationally standardized sera would allow results of the single radial hemolysis assay to be directly compared between laboratories.

Analysis of Variance↗
PubMed1995

Evaluation of Directigen Flu A assay for detection of influenza antigen in nasal secretions of horses.

The Directigen Flu A assay (Becton Dickinson, Microbiology Systems, Mississauga, Ontario, Canada) is a commercially available immunoassay designed for rapid in vitro recognition of influenza A nucleoprotein. The purpose of this study was to evaluate this assay for detection of influenza virus in nasal secretions of naturally infected horses. The assay was shown to react with representative strains of influenza virus which cause disease in horses and did not react with nasal secretions from uninfected horses kept in isolation. Between 33% and 45% of nasal secretions specimens obtained from clinically diseased horses during influenza epidemics reacted positively in the assay and 95% to 98% of horses not showing signs of disease during influenza epidemics tested negative. In contrast, influenza virus was isolated from only 7% of diseased horses using conventional techniques. Diseased horses which were positive in the Directigen assay had lower pre-exposure influenza antibody concentrations and showed more clinical signs than diseased Directigen-negative horses. This evaluation demonstrates that the Directigen Flu A assay detects influenza virus in nasal secretions of infected horses and is more sensitive than virus isolation.

Animals↗
PubMed1995

Recombinant type 5 adenoviruses expressing bovine parainfluenza virus type 3 glycoproteins protect Sigmodon hispidus cotton rats from bovine parainfluenza virus type 3 infection.

Cotton rats were used to study the replication and pathogenesis of bovine parainfluenza virus type 3 (bPIV3) and to test the efficacy of the F and HN glycoproteins in modulating infection. In vitro cultures of cotton rat lung cells supported the growth of bPIV3 as shown by virus recovery, immunofluorescence, immunoprecipitation, and syncytium induction. Intranasal (i.n.) inoculation of cotton rats with 10(7) PFU resulted in peak recovery of virus after 2 days (8 x 10(4) PFU/g of lung tissue) and significant bronchiolitis with lymphocyte infiltration 5 to 7 days postinfection. Immunohistochemical staining of lungs and trachea demonstrated that virus antigen-positive cells increased in frequency over the course of infection to a maximum on day 5. Serum antibody responses were evaluated by enzyme-linked immunosorbent assays (ELISA), hemagglutination inhibition (HAI), and serum neutralization (SN). Following a single i.n. inoculation, serum antibody levels were 1/40,960, 1/32, and 1/80, as detected by ELISA, HAI, and SN, respectively. When an intramuscular inoculation of 10(7) PFU was administered 10 days prior to the i.n. inoculation, a secondary response which resulted in an ELISA titer of 1/163,000, an HAI titer of 1/640, and an SN titer of 1/512 was induced. IN inoculation of recombinant adenoviruses type 5 containing the bPIV3 F or HN protein or a combination of the two viruses protected cotton rats from bPIV3 challenge. Protection was evaluated serologically by ELISA, HAI, and SN titers, histopathology, immunohistochemistry, and virus recovery.

Adenoviruses, Human↗
PubMed1995

Local production of tumor necrosis factor-alpha in corynebacterial pulmonary lesions in sheep.

Corynebacterium pseudotuberculosis infection is a common cause of pyogranulomas in ovine lungs and often occurs as a dual infection with lentiviruses. This coinfection usually leads to the development of chronic pneumonia and cachexia that is similar to the clinical syndrome seen in human beings with AIDS-related pneumonias. Recent in vitro studies indicate that monokines such as tumor necrosis factor-alpha (TNF alpha) are induced by C. pseudotuberculosis, suggesting that TNF alpha is involved in the pathogenesis of corynebacterial lesions in vivo. To substantiate in vitro observations concerning bacterial induction of TNF alpha in ovine pulmonary macrophages, immunohistochemical labeling techniques were used in combination with in situ hybridization to identify TNF-producing cells in corynebacterial lesion sites in vivo. TNF alpha message and translation product were found in macrophages comprising pyogranulomas that were induced by naturally acquired and experimental pulmonary C. pseudotuberculosis infections.

Animals↗