Frank E. Aldwell

Oral vaccination with Mycobacterium bovis BCG in a lipid formulation induces resistance to pulmonary tuberculosis in brushtail possums.

A method was developed for formulating Mycobacterium bovis bacille Calmette-Guerin (BCG) for oral vaccination against tuberculosis. Selected lipid-based formulations of BCG were tested in the brushtail possum for their ability to elicit immune responses and protection against bovine tuberculosis. Formulation of BCG in lipid matrices maintained bacteria in a dormant but viable state. Oral delivery of 2 x 10(8) colony forming units of formulated BCG to possums induced strong lymphocyte proliferation responses to bovine purified protein derivative (PPD) in peripheral blood lymphocytes. Oral vaccination of possums also reduced the severity of disease following aerosol challenge with virulent M. bovis compared with animals vaccinated with non-formulated BCG. In a second experiment, levels of protection with lipid-formulated oral BCG were similar to those seen with subcutaneous BCG vaccination. Our data shows that formulated oral BCG is an efficient means of inducing protection against bovine tuberculosis in possums and should be a practical means of vaccinating wildlife against tuberculosis.

Oral Delivery of Mycobacterium bovis BCG in a Lipid Formulation Induces Resistance to Pulmonary Tuberculosis in Mice

ABSTRACT A lipid-based formulation has been developed for oral delivery of Mycobacterium bovis bacille Calmette-Guérin (BCG) vaccine. The formulated M. bovis BCG was fed to BALB/c mice to test for immune responses and protection against M. bovis infection. The immune responses included antigen-specific cytokine responses, spleen cell proliferation, and lymphocyte-mediated macrophage inhibition of M. bovis. Oral delivery of formulated M. bovis BCG to mice induced strong splenic gamma interferon levels and macrophage inhibition of virulent M. bovis compared with results with nonformulated M. bovis BCG. Formulated oral M. bovis BCG significantly reduced the bacterial burden in the spleen and lungs of mice following aerosol challenge with virulent M. bovis. Our data suggest that oral delivery of formulated M. bovis BCG is an effective means of inducing protective immune responses against tuberculosis. Lipid-based, orally delivered mycobacterial vaccines may be a safe and practical method of controlling tuberculosis in humans and animals.

Oral vaccination of badgers (Meles meles) with BCG and protective immunity against endobronchial challenge with Mycobacterium bovis.

Eurasian badgers (Meles meles) are a reservoir host of Mycobacterium bovis and are implicated in the transmission of tuberculosis to cattle in Ireland and Great Britain. The development of a vaccine for use in badgers is considered a key element of any long-term sustainable campaign to eradicate the disease from livestock in both countries. The aim of this study was to investigate the protective response of badgers vaccinated orally with Bacille Calmette-Guérin (BCG) encapsulated in a lipid formulation, followed by experimental challenge with M. bovis. A group of badgers was vaccinated by inoculating the BCG-lipid mixture containing approximately 10(8)colony forming units (cfu) of BCG into the oesophagus. The control group was sham inoculated with the lipid formulation only. Thirteen weeks after vaccination all the badgers were challenged with approximately 10(4)cfu of M. bovis delivered by endobronchial inoculation. Blood samples were taken throughout the study and the cell mediated immune (CMI) responses in peripheral blood were monitored by the IFN-gamma ELISA and ELISPOT assay. At 17 weeks after infection all the badgers were examined post-mortem to assess the pathological and bacteriological responses to challenge. All badgers in both groups were found to be infected. However, a significant protective effect of BCG vaccination was measured as a decrease in the number and severity of gross lesions, lower bacterial load in the lungs, and fewer sites of infection. The analysis of immune responses showed that vaccination with BCG did not generate any detectable CMI immunological responses, however the levels of the responses increased in both groups following M. bovis infection. The results of the study showed that vaccination with oral BCG in the lipid formulation generated a protective effect in the badgers.

Oral vaccination of brushtail possums (Trichosurus vulpecula) with BCG: immune responses, persistence of BCG in lymphoid organs and excretion in faeces

Abstract AIMS: To determine immune responses, and the localisation and persistence of Mycobacterium bovis bacille Calmette-Guérin (BCG) in gut-associated lymphoid tissues (GALT) and other organs in possums vaccinated orally with lipid-formulated BCG vaccine. To determine the duration of excretion and longevity of survival of BCG in the faeces of vaccinated animals. METHODS: Possums (n=28) were vaccinated with lipid-formulated BCG (1 x 10 8 colony forming units (cfu) of formulated BCG) by the oral route. Control possums (n=17) were fed oral bait pellets containing formulation medium only. Possums were sacrificed at 3 days and at 1, 3, 6 and 8 weeks after vaccination or ingestion of bait. Proliferation responses to bovine purified protein derivative (PPD) were measured in lymphocytes from blood and mesenteric lymph nodes (MLN) and samples of lung, spleen, liver, MLN and Peyers patches (PP) were cultured for the presence of BCG. The number of BCG organisms excreted in faeces and the duration of excretion were determined in eight vaccinated possums and eight control possums over a 3-week period. In a separate experiment, a further six possums were vaccinated with oral BCG vaccine (5–10 x 10 8 cfu BCG/possum) and their faeces collected over 48–72 h, for culture of BCG. The longevity of survival of BCG in these faeces was determined by storing faecal samples (n=12) under three different conditions: in an incubator (22.5°C), and conditions which simulated the forest floor and open pasture. A proportion (1–2 g) of these faecal samples was collected after storage for 1, 3, 5, 8 or 20 weeks, and cultured for BCG. RESULTS: Possums vaccinated orally with BCG vaccine showed strong proliferation responses to bovine PPD in peripheral blood lymphocytes at 6–8 weeks post-vaccination (p.v.). Positive lymphocyte proliferation assay (LPA) responses to bovine PPD were first evident in MLN at 3 weeks p.v. BCG was cultured from MLN and PP in a proportion of animals at 3–8 weeks p.v. BCG was not cultured from sections of spleen, lung or liver at any time p.v. BCG was recovered in low to moderate numbers from the faeces of vaccinated possums for up to 7 days, and maximal numbers were cultured in faeces collected 48–72 h p.v. After storage for 1 week, BCG was cultured from all faecal samples placed in the incubator and from a proportion of faeces exposed to conditions similar to those on the forest floor and pasture. With the exception of one faecal sample stored under forest floor conditions which was culture-positive for BCG at 3 and 5 weeks, BCG was not cultured from any other faecal sample stored for more than 1 week. CONCLUSIONS: Ingestion of oral BCG vaccine by possums was associated with the development of strong cell-mediated immunity in both blood and MLN. Following oral vaccination with BCG, the organisms were localised and persisted in GALT but did not spread to the spleen, liver or lungs. BCG was shed in low to moderate numbers in the faeces for up to 7 days p.v. The viability of BCG excreted in faeces decreased rapidly, particularly when faeces were exposed to an open pasture environment. Oral vaccination of possums with formulated BCG is unlikely to result in undue contamination of the environment with BCG.

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‐α, IL‐1β 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.

Oral vaccination of brushtail possums with BCG: Investigation into factors that may influence vaccine efficacy and determination of duration of protection

Abstract AIMS: To determine factors that may influence the efficacy of an oral pelleted vaccine containing Mycobacterium bovis bacille Calmette-Guérin (BCG) to induce protection of brushtail possums against tuberculosis. To determine the duration of protective immunity following oral administration of BCG. METHODS: In Study 1, a group of possums (n=7) was immunised by feeding 10 pellets containing dead Pasteur BCG, followed 15 weeks later with a single pellet of live Pasteur BCG. At that time, four other groups of possums (n=7 per group) were given a single pellet of live Pasteur BCG orally, a single pellet of live Danish BCG orally, 10 pellets of live Pasteur BCG orally, or a subcutaneous injection of live Pasteur BCG. For the oral pelleted vaccines, BCG was formulated into a lipid matrix, and each pellet contained approximately 107 colony forming units (cfu) of BCG, while the vaccine injected subcutaneously contained 106 cfu of BCG. A sixth, non-vaccinated, group (n=7) served as a control. All possums were challenged by the aerosol route with a low dose of virulent M. bovis 7 weeks after vaccination, and killed 7–8 weeks after challenge. Protection against challenge with M. bovis was assessed from pathological and bacteriological findings. In Study 2, lipid-formulated live Danish BCG was administered orally to three groups of possums (10–11 per group), and these possums were challenged with virulent M. bovis 8, 29 or 54 weeks later. The possums were killed 7 weeks after challenge, to assess protection in comparison to a non-vaccinated group. RESULTS: The results from Study 1 showed that vaccine efficacy was not adversely affected by feeding dead BCG prior to live BCG. Feeding 10 vaccine pellets induced a level of protection similar to feeding a single pellet. Protection was similar when feeding possums a single pellet containing the Pasteur or Danish strains of BCG. All vaccinated groups had significantly reduced pathological changes or bacterial counts when compared to the non-vaccinated group. In Study 2, oral administration of Danish BCG induced protection against challenge with M. bovis, which persisted for at least 54 weeks after vaccination. Some protection was observed in possums challenged 54 weeks after vaccination, but this protection was significantly less than that observed in groups vaccinated 29 or 8 weeks prior to challenge. There was a strong relationship between the proportion of animals producing positive lymphocyte proliferation responses to M. bovis antigens and protection against challenge with M. bovis. CONCLUSIONS: Factors considered potentially capable of interfering with vaccination, including feeding dead BCG to possums prior to feeding live BCG, feeding multiple doses of BCG at one time, and changing strains of BCG, were shown not to interfere with the acquisition of protective immune responses in possums. Protection against tuberculosis was undiminished up to 29 weeks after vaccination with BCG administered orally. It is concluded that vaccination of possums by feeding pellets containing BCG is a robust and efficient approach to enhance the resistance of these animals to tuberculosis.

Oral vaccination of mice with lipid-encapsulated Mycobacterium bovis BCG: Anatomical sites of bacterial replication and immune activity

Lipid microencapsulation of Mycobacterium bovis bacille Calmette–Guérin (BCG) produces an oral delivery vaccine that can establish systemic cell‐mediated immune reactivity and protection against aerosol mycobacterial challenge in mice. Here, we describe the lymphatic and mucosal sites of bacterial replication, and location of Mycobacterium‐specific IFN‐γ‐secreting cell populations, following oral vaccination of BALB/c mice. Eight weeks following a single oral dose of lipid‐encapsulated BCG, viable BCG organisms were recovered from the mesenteric lymph nodes (MLN) of 11/12 mice investigated (93%). Live bacteria were also occasionally recovered from the cervical lymph nodes (17%) and Peyers patches (8%), but not from homogenates of the lungs or spleen. Strong Mycobacterium‐specific IFN‐γ production was recorded among isolated splenocytes, but not among populations of mononuclear cells derived from the MLN or lungs. Oral vaccination of mice with lipid‐encapsulated BCG thus appears to promote a state of systemic immunological reactivity more akin to that observed following parenteral rather than conventional oral vaccination, despite the fact that replicating bacilli are restricted to lymphatic tissues of the alimentary tract. Possible patterns of lymphocyte sensitization and trafficking are discussed.

Sequential activation of alveolar macrophages by IFN-gamma and LPS is required for enhanced growth inhibition of virulent Mycobacterium bovis but not M. bovis BCG.

Alveolar macrophages (AM) form the first line of defence against most respirators’ pathogens and, unlike tissue macrophages, are constantly exposed to a wide variety of antigenic stimuli. In this study we investigated the in vitro effects of IFN‐γ and LPS on growth of virulent Mycobacterium bovis and M. bovis bacille Calmette‐Guérin (BCG) in bovine AM. Bovine AM were purified from bronchial lavage fluid and cultured in serum‐free medium. Pretreatment of bovine AM with IFN‐γ resulted in growth inhibition of M. bovis BCG but only partially inhibited growth of virulent M. bovis. Enhanced inhibition of virulent M. bovis by bovine AM required sequential stimulation with IFN‐γ and LPS and was associated with increased induction of nitric oxide (NO) and IL‐12 mRNA. Growth inhibition of M. bovis was not affected by treatment of macrophages with the L‐arginine analogue. NG‐monomethyl‐L‐arginine although this treatment decreased NO production. These results suggest that a second activation signal in the form of TNF‐α or LPS may be required to induce bacteriostasis of virulent M. bovis by bovine AM in vivo. The ability of bovine AM to respond to activation stimuli in vitro suggests that these cells may play an important role in preventing establishment of intracellular bacterial infections in the lung.

Molecular cloning and characterization of tumour necrosis factor alpha (TNF-α) 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‐α. Primers were designed from consensus sequences at the N‐terminus end of eutherian mammalian TNF‐α and the possum cDNA, derived from spleen RNA, identified by RT‐PCR. The complete cDNA encoding possum TNF‐α 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‐α genes. The predicted protein of 233 amino acids shared 56‐58% identity with eutherian mammalian TNF‐α. Possum TNF‐α was expressed in both Saceharomyces 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‐α fusion protein exhibited cytotoxic activity on the TNF‐α‐sensitive murine fibroblast L929 cells and stimulated proliferation of possum thymocyte cells. The induction of possum TNF‐α mRNA in alveolar macrophages was analysed by RT‐PCR using possumspecific TNF‐α primers. Macrophages cultured in the presence of LPS showed enhanced transcription of TNF‐α mRNA. This is the first report of the cloning and sequence analysis of the cDNA encoding a marsupial cytokine gene.

Mice Fed Lipid-Encapsulated Mycobacterium bovis BCG Are Protected against Aerosol Challenge with Mycobacterium tuberculosis

ABSTRACT Mice that consumed a single dose of 107 lipid-encapsulated Mycobacterium bovis BCG bacilli showed significant pulmonary and systemic protection against aerosol challenge with M. tuberculosis H37Rv. As an extension of previous challenge studies with virulent strains of M. bovis, this report describes a reduction in M. tuberculosis infection in mice vaccinated orally with lipid-encapculated BCG comparable to that observed in mice vaccinated subcutaneously with BCG. These results are consistent with the induction of tuberculin-specific cell-mediated immune responses.