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Biomedical subjects

B M Buddle

Publications and source records attributed to B M Buddle.

At least 73 records · Page 4Linked to original sources

Response of the North Island brown kiwi, Apteryx australis mantelli and the lesser short-tailed bat, Mystacina tuberculata to a measured dose of rabbit haemorrhagic disease virus.

Four North Island brown kiwis and six lesser short-tailed bats were inoculated intramuscularly with 300 000 rabbit lethal doses of rabbit haemorrhagic disease (RHD) virus. No clinical abnormalities were observed in the kiwis and bats throughout the study period. Although no viraemia was detected in any of the kiwis, all four birds produced a serological response to RHD virus above the positive cut-off by 14 days after inoculation, and in two of the birds, antibodies persisted for over 5 months. Two kiwis were killed 48 days after inoculation. Their tissues were examined for lesions, and for the presence of persistent virus by both reverse transcription polymerase chain reaction and by inoculation of tissue suspensions into rabbits. No gross or histological lesions suggestive of a viral infection were detected and tests for detection of virus were negative. The serological response in the kiwis was probably due to the birds responding to viral antigen in the inoculum rather than to multiplication of the virus. None of the bats showed a serological response to RHD virus above the positive cut-off by 14 days after inoculation and the results of the pathological and virological examinations were negative.

Journal Article↗

Intraduodenal vaccination of brushtail possums with bacille Calmette-Guérin enhances immune responses and protection against Mycobacterium bovis infection.

SETTING: An effective oral bacille Calmette-Guérin (BCG) vaccine would have advantages for use in humans and as an oral bait vaccine for protecting wild-life against bovine tuberculosis. OBJECTIVE: To compare the level of protection against tuberculosis in intraduodenally BCG-vaccinated possums with those vaccinated intragastrically in order to determine whether degradation of BCG in the stomach lowers vaccine efficacy. DESIGN: Three groups of five possums were vaccinated with BCG by the intraduodenal, intragastric or subcutaneous routes, with a fourth group serving as unvaccinated controls. The animals were later challenged intratracheally with a low dose of virulent Mycobacterium bovis. RESULTS: Possums vaccinated intraduodenally with BCG had significantly greater lymphocyte blastogenic responses to bovine purified protein derivative (PPD) and lower lung bacterial counts in comparison with intragastrically vaccinated animals. In comparison with unvaccinated animals, all of the BCG-vaccinated groups had significant protection against M. bovis infection as assessed by changes in body weight, lung weight and reduction in numbers of mycobacteria and granulomas in the spleen. CONCLUSION: The enhanced immune responses and protection against bovine tuberculosis observed in the intraduodenally BCG-vaccinated possums indicated that if BCG vaccine is protected from degradation in the stomach its efficacy should improve.

Administration, Oral↗

Molecular cloning and characterization of tumor necrosis factor alpha (TNF-alpha) from the Australian common brushtail possum, Trichosurus vulpecula.

Immune responses in the Australian common brushtail possum (Trichosurus vulpecula) and in particular the role of cytokines are poorly understood. We have undertaken to isolate cytokine genes using reverse transcriptase-polymerase chain reaction (RT-PCR) and in this study describe the molecular cloning of TNF-alpha. Primers were designed from consensus sequences at the N-terminus end of eutherian mammalian TNF-alpha and the possum cDNA, derived from spleen RNA, identified by RT-PCR. The complete cDNA encoding possum TNF-alpha was amplified from lymphocyte RNA by 5' and 3' rapid amplification of cDNA ends (RACE). The nucleotide sequence of the protein coding region of this cDNA shared 66-69% identity with other mammalian TNF-alpha genes. The predicted protein of 233 amino acids shared 56-58% identity with eutherian mammalian TNF-alpha was expressed in both Saccharomyces cerevisiae and Escherichia coli by constructing expression plasmid derivatives of the vectors pYES2 and pGEX-2T respectively. Cell extracts prepared from transformants and the purified GST/TNF-alpha fusion protein exhibited cytotoxic activity on the TNF-alpha-sensitive murine fibroblast L929 cells and stimulated proliferation of possum thymocyte cells. The induction of possum TNF-alpha mRNA in alveolar macrophages was analysed by RT-PCR using possum-specific TNF-alpha primers. Macrophages cultured in the presence of LPS showed enhanced transcription of TNF-alpha mRNA. This is the first report of the cloning and sequence analysis of the cDNA encoding a marsupial cytokine gene.

Amino Acid Sequence↗

Bacterial metabolism, cytokine mRNA transcription and viability of bovine alveolar macrophages infected with Mycobacterium bovis BCG or virulent M. bovis.

Mycobacterium bovis causes tuberculosis in cattle and many other animals including humans while BCG, an attenuated form of M. bovis, has been used widely as a safe vaccine. Both strains infect host macrophages and their fate is determined by their ability to survive within these phagocytic cells. We compared interactions of these two strains with bovine alveolar macrophages in order to gain an understanding of virulence mechanisms involved in the early pathogenesis of M. bovis infection. Macrophages were infected with bacilli at varying multiplicities of infection and cultured for 1-4 days. Bacterial metabolism within macrophages was assessed by [3H]-uracil uptake and bacterial growth was assessed by culture and acid-fast staining. Induction of TNF-alpha, IL-1 beta and IL-6 cytokine mRNA transcription in macrophages was determined by reverse transcriptase-polymerase chain reaction. Infection of macrophages by virulent M. bovis resulted in enhanced bacterial metabolism, enhanced induction of macrophage cytokines and reduced viability of macrophages when compared to M. bovis BCG-infected macrophages. These differences may reflect virulence mechanisms contributing to the early pathogenesis of bovine tuberculosis.

Animals↗

Protection of cattle from bovine tuberculosis by vaccination with BCG by the respiratory or subcutaneous route, but not by vaccination with killed Mycobacterium vaccae.

Groups of cattle were vaccinated either with BCG Pasteur by the intratracheal or subcutaneous route or with killed Mycobacterium vaccae by the intradermal route and challenged intratracheally 54 days later with Mycobacterium bovis. Vaccination with BCG resulted in fewer animals developing tuberculous lesions and in a reduction in the number of lesions in the diseased animals compared with the unvaccinated group and the group vaccinated with M vaccae. None of the nine animals vaccinated intratracheally with BCG developed any tuberculous lung lesions after challenge with M bovis, but two of the nine animals from each of the groups dosed subcutaneously with low and medium doses of BCG developed lung lesions. There was little difference in protection against the M bovis challenge between the animals receiving the low dose (10(3) colony forming units, cfu) or medium dose (10(5) cfu) of subcutaneous BCG, but the medium dose of BCG produced stronger cell-mediated immune responses to bovine purified protein derivative (PPD) after vaccination. Vaccination intradermally with 10(9) heat-killed M vaccae did not protect cattle against an experimental challenge with M bovis and induced only weak cell-mediated immune responses to bovine PPD.

Animals↗

Effectiveness of BCG vaccination in protecting possums against bovine tuberculosis.

Three groups of eight possums were vaccinated with BCG Pasteur by the subcutaneous, intratracheal or intragastric routes, with a fourth group serving as unvaccinated controls. Forty-two days after the start of vaccination, five possums from each group were challenged intratracheally with virulent Mycobacterium bovis. The vaccination by the subcutaneous or intratracheal routes resulted in a marked reduction in the severity of disease compared with the unvaccinated animals or the animals vaccinated intragastrically. The severity of the disease was assessed by changes in bodyweight, pathological changes in the lungs and bronchial nodes and the number of acid-fast bacilli in the lesions. Before the challenge, lymphocyte blastogenic responses to bovine PPD were observed in the eight animals vaccinated subcutaneously and in two of the animals vaccinated intratracheally.

Animals↗

Immunological responses and protection against Mycobacterium bovis in calves vaccinated with a low dose of BCG.

Groups of calves (20 per group) were vaccinated subcutaneously with a single dose of BCG Pasteur (6 x 10(4) or 6 x 10(6) colony forming units) and two months later, 15 calves from each group were challenged intratracheally with virulent Mycobacterium bovis. Vaccination with either dose of BCG induced significant protection against the development of tuberculous lesions compared to non-vaccinated controls. Seven months after BCG vaccination, many of the vaccinated animals which had no lesions and were M. bovis culture-negative at necropsy showed positive reactions for M. bovis in three assays for measuring cellular immune responses (comparative intradermal test, interferon-gamma assay and lymphocyte proliferation assay). This effect was most noticeable in the BCG-vaccinated calves which had been challenged with M. bovis rather than in the non-challenged animals. Antibody responses to M. bovis were very low or absent in the calves during the study. The kinetics of the interferon-gamma response of peripheral blood lymphocytes cultured in vitro with bovine PPD showed that BCG vaccination induced a rapid rise in the response followed by a sharp decline, while infection with virulent M. bovis resulted in an increase in the interferon-gamma response by four weeks after challenge and this response remained high through the study.

Animals↗

Cytokine mRNA expressed in tuberculin skin test biopsies from BCG-vaccinated and Mycobacterium bovis inoculated cattle.

To obtain a better understanding of the delayed-type hypersensitivity reaction to Mycobacterium bovis, we measured the expression of cytokine mRNA from tuberculin skin test biopsies of cattle. Non-vaccinated and BCG-vaccinated cattle were inoculated intratracheally with a low dose of virulent M. bovis or sham-inoculated and 20 weeks later were skin tested with tuberculin. At necropsy 1-2 weeks later, tuberculous lesions were found in six of the nine non-vaccinated and three of the nine BCG-vaccinated animals. All of the lesioned and the majority of the non-lesioned M. bovis inoculated cattle showed a distinct skin swelling response to tuberculin, irrespective of vaccination. However, cattle with tuberculous lesions displayed larger skin swelling responses than non-lesioned cattle. Tuberculin-induced expression of IFN-gamma, IL2, IL4, IL10 and TNF-alpha mRNA occurred in the skin biopsies of all of the lesioned, M. bovis inoculated animals except for an absence of tuberculin-induced TNF-alpha mRNA expression in two animals. A lower proportion of the non-lesioned M. bovis inoculated cattle displayed tuberculin-induced expression of the five cytokine mRNA. There was no evidence of a unique pattern of cytokine expression which could be used to distinguish between diseased and protected animals. By 28 weeks after vaccination, the three BCG-vaccinated, sham-inoculated cattle displayed minimal skin swelling response to tuberculin, but tuberculin-induced expression of IFN-gamma, IL2, IL4, IL10 and TNF-alpha mRNA was observed in skin biopsies of all of these animals.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

Route of BCG administration in possums affects protection against bovine tuberculosis.

The Australian brushtail possum (Trichosurus vulpecula) is the major wildlife reservoir of Mycobacterium bovis in New Zealand. Control of bovine tuberculosis in farmed animals requires measures to reduce the transmission of M. bovis from wildlife. Possums were vaccinated with BCG intranasally by aerosol spray, orally or subcutaneously to compare the efficacy of these three routes on protection against challenge with virulent M. bovis. Possums vaccinated with BCG by the intranasal or subcutaneous routes had a marked reduction in severity of disease compared to possums which had been unvaccinated or orally vaccinated. The severity of the disease was assessed by changes in body weight and pathology. BCG vaccination by all three routes resulted in reduced dissemination of M. bovis to the spleen and liver following challenge. Intranasal and oral BCG vaccination induced lower mean peripheral blood lymphocyte blastogenic responses to bovine PPD than subcutaneous vaccination, indicating that these responses did not correlate well with protection from the disease. Given a suitable delivery system, aerosol vaccination of possums, used in conjunction with other control measures, may be a suitable method of reducing the spread of M. bovis from wildlife to domestic animals.

Journal Article↗

Evaluation of three serological assays for the diagnosis of Mycobacterium bovis infection in brushtail possums.

Three serological tests for the diagnosis of Mycobacterium bovis infection were evaluated on 29 possums (Trichosurus vulpecula) with tuberculosis and on 100 possums from a tuberculosis-free area. An indirect ELISA using M. bovis culture filtrate as the antigen had a sensitivity of 45% and a specificity of 96%, while an indirect ELISA using a M. bovis specific antigen (MPB70) had a sensitivity of 21% and a specificity of 98%. A blocking ELISA which utilised a monoclonal antibody against MPB70 had a sensitivity of 28% and a specificity of 99%. Combination of the test results of the three ELISAs resulted in an increase in sensitivity to 51% and a decrease in specificity to 93%. A previous study has shown that possums experimentally infected with M. bovis produced cellular responses to M. bovis antigens relatively early in the infection, but these responses decreased in the terminal stages of the disease. In contrast, analysis of serological responses in the current study from sequentially collected sera of possums experimentally and naturally infected with M. bovis showed that antibody was first detected late in the disease.

Journal Article↗

Experimental Mycobacterium bovis infection in the brushtail possum (Trichosurus vulpecula): pathology, haematology and lymphocyte stimulation responses.

Groups of adult male brushtail possums (5 per group) were inoculated intratracheally with a high (2 x 10(5) colony forming units (cfu)), medium (2 x 10(3) cfu) or low (approximately 20 cfu) dose of Mycobacterium bovis. Two sham-inoculated groups acted as in-contact controls or controls kept in a separate room. Possums in the high and medium dose groups became clinically affected 3-5 weeks post-inoculation (PI) and all possums were euthanased between 5-9 weeks PI. Grossly visible tuberculous lesions were found in the lungs and associated lymph nodes of all possums from the high, medium and low dose groups. No lesions were observed in possums from the two control groups. Histopathologically, two characteristic types of lesions were observed; microscopic aggregates of macrophages with few acid-fast organisms, and larger lesions with limited granulomatous reaction, extensive necrosis and the presence of numerous acid-fast organisms. M. bovis was isolated from the lungs and lymph nodes of all of the possums from the high, medium and low dose groups and from the lungs of one of the in-contact controls. Changes in the haematological profile of the M. bovis-inoculated possums included lymphocytopaenia and eosinopaenia, together with raised fibrinogen levels. The onset of these changes was dependent on the size of the challenge dose. Lymphocyte stimulation responses to M. bovis tuberculin purified protein derivative were detected in 14 of 15 M. bovis-inoculated possums.

Animals↗

Tuberculosis in the brushtail possum (Trichosurus vulpecula) after intratracheal inoculation with a low dose of Mycobacterium bovis.

Six possums were each inoculated with approximately 125 colony-forming units of Mycobacterium bovis via a cannula inserted per os into the trachea. Five other possums were sham inoculated and housed separately. At postmortem examination 55 to 57 days after inoculation, all six infected possums showed extensive macroscopical lesions of tuberculosis in the lungs and bronchial lymph nodes and some also had lesions in the liver, kidney, spleen and hepatic lymph nodes. Mycobacterium bovis was isolated from all of these possums. No evidence of M. bovis infection was detected in the five control animals. In the M. bovis-infected possums, microscopical examination of organs and lymph nodes demonstrated a much wider distribution of lesions than did macroscopical examination. The location of early lesions indicated that the paracortical region of the lymph nodes, the marginal zone and periphery of lymphoid follicles in the spleen, and the cortex of the kidney were predilection sites for lesions resulting from haematogenous spread of infection. This method for reproducing bovine tuberculosis in the possum was more suitable for experimental studies than previously published methods.

Animals↗

Experimental Mycobacterium bovis infection of cattle: effect of dose of M. bovis and pregnancy on immune responses and distribution of lesions.

Groups of 18-month-old cattle were inoculated intratracheally with 5 x 10(5) colony forming units (high dose) or 500 colony forming units (low dose) of Mycobacterium bovis to determine an appropriate dose to induce lesions similar to those seen in the natural disease. An additional group of 21-28 weeks pregnant cattle were inoculated with the high dose of M. bovis to determine if pregnancy increased the susceptibility of cattle to M. bovis infection. By 23-24 weeks after challenge, the high dose of M. bovis had induced extensive lung lesions, and tuberculous lesions were observed in the lymph nodes of the head, neck, and thoracic and abdominal cavities. In contrast, the low dose of M. bovis induced predominantly small lesions (< 1 cm diameter) which were localised to the lungs and pulmonary lymph nodes. The lesions induced by the low dose were similar to those seen in the natural disease in cattle. The majority of the high dose group cattle produced strong antibody responses to M. bovis culture filtrate, while only one low dose animal produced a detectable response. All of the M. bovis-inoculated cattle produced strong cellular immune responses to bovine PPD (skin test and interferon-gamma responses). Pregnancy did not appear to affect the susceptibility to M. bovis infection, and immune responses of the cattle in this group at the end of the study were similar to those in the high dose non-pregnant group. However, from the first test after calving, the interferon-gamma responses of peripheral blood cultures to bovine PPD were low compared with the responses prior to calving.

Journal Article↗

Association of blood eosinophilia with the expression of resistance in Romney lambs to nematodes.

The dynamics of blood eosinophilia was studied in Romney lambs dosed twice weekly with 5000 infective larvae of Trichostrongylus colubriformis. A marked rise in blood eosinophil counts of the dosed lambs coincided with the start of the decline of faecal egg counts (FEC). Eosinophilia was also studied in grazing Romney lambs from a breeding programme, based on selection for high or low FEC. Comparison of the sire groups suggested that the magnitude of the eosinophil response was under genetic control. The significant negative correlations between blood eosinophil counts and FECs confirmed the association between eosinophilia and the expression of resistance in Romney lambs to nematodes. However, comparison of the mean blood eosinophil counts and FECs for both the ram and ewe lambs of the resistance sire groups suggested that the association of eosinophilia and resistance may be greater for some sire groups than others.

Animals↗

Influence of stress of capture on haematological values and cellular immune responses in the Australian brushtail possum (Trichosurus vulpecula).

Possums (Trichosurus vulpecula) captured for the first time (single-capture) had significantly lower blood lymphocyte and eosinophil counts, but higher neutrophil counts than possums which had been captured frequently. Single-capture possums also had depressed lymphocyte responses to concanavalin A (males and females) and pokeweed mitogen (males only). The haematological changes and the depressed lymphocyte reactivity to the T-cell mitogens were consistent with the animals being in a stressed state following capture for the first time. The haematological profile of single-capture possums changed to a profile similar to that of the frequently captured possums following housing in cages for 1 week, and high lymphocyte responses to T-cell mitogens were recorded following housing for 3-5 weeks. This information, together with body weight data, indicates that a minimum of 4 weeks adaptation to housing in cages should be allowed before possums are used in experimental studies.

Journal Article↗

Experimental respiratory infection of goats with caprine herpesvirus and Pasteurella haemolytica.

Groups of six male goats were inoculated intratracheally and intranasally with either caprine herpesvirus followed 6 days later by Pasteurella haemolytica, canine herpesvirus alone or P. haemolytica alone. Pneumonic lesions were observed in five of the six goats inoculated with caprine herpesvirus followed by P. haemolytica and in three of the six goats inoculated with P. haemolytica alone, but were not observed in goats inoculated with caprine herpesvirus alone or in non-infected controls. Pasteurella haemolytica was isolated from seven of eight lungs with pneumonia, but only from one of sixteen lungs without pneumonia. The lesions ranged from fatal acute exudative necrotising pneumonia to predominantly proliferative pneumonia. Half of the caprine herpesvirus-inoculated goats developed a clinical catarrhal rhinitis five days post-inoculation and the only virus-specific histopathological lesion was a mild tracheitis. Canine herpesvirus was recovered from the nasal swabs of all caprine herpesvirus- inoculated goats developed a clinical catarrhal rhinitis five days post-inoculation and the only virus-specific histopathological lesion was a mild tracheitis. Canine herpesvirus was recovered from the nasal swabs of all canine herpesvirus-inoculated goats and from the lungs of three goats inoculated with caprine herpesvirus alone. The experimental inoculations demonstrated that P. haemolytica alone can produce pneumonia in goats. In addition, the study showed that caprine herpesvirus readily proliferates in the upper respiratory tract and lungs of goats but the role of caprine herpesvirus in the aetiology of pneumonia remains uncertain.

Journal Article↗

A caprine pneumonia outbreak associated with caprine herpesvirus and Pasteurella haemolytica respiratory infections.

In a field disease outbreak, 60 female goats died over a 2-3 week period. Necropsies of seven of these does revealed that six had an acute exudative necrotising broncho-pneumonia, and moderate to high numbers of Pasteurella haemolytica were isolated from their lungs. Caprine herpesvirus, identified as Bovid herpesvirus type 6, was isolated from the lungs of two of these does, including one with pneumonia, and from nasal swabs of in-contact goats. Clinical disease was only observed in does, and deaths began 3 weeks after the introduction of a mob of goat hoggets from another farm.

Journal Article↗

Protective effect of glucan against experimentally induced staphylococcal mastitis in ewes.

Glucan, an immunostimulant, was evaluated for its ability to modify a staphylococcal mammary challenge in ewes. Glucan was administered subcutaneously to ewes prior to lactation or during lactation, and all ewes, including a control group, were subsequently challenged intramammarily with Staphylococcus haemolyticus 40 days after the mean lambing date. The glucan treatment was shown to modify the staphylococcal mammary infection as the milk bacterial counts from all of the glucan-treated groups were significantly reduced compared to controls. For the glucan-treated groups, the highest mean somatic cell counts were recorded 1 day post-challenge, while for the control group, the mean cell count rose more gradually to peak by 3 days post-challenge. Glucan did not increase serum lysozyme levels or blood neutrophil bactericidal activity. However, there was a negative correlation between the bactericidal activity of blood neutrophils collected from the glucan-treated ewes prior to challenge and their mean milk bacterial counts post-challenge. Glucan was observed to stimulate ovine mammary macrophages in vitro, while the addition of zymosan or opsonised killed Staphylococcus aureus to macrophage cultures had no effect. These studies indicate that, in sheep, glucan can enhance some elements of the immune system against staphylococcal infections.

Adjuvants, Immunologic↗