D. Neil Wedlock

A DNA prime-Mycobacterium bovis BCG boost vaccination strategy for cattle induces protection against bovine tuberculosis.

ABSTRACT The variable efficacy of bacillus Calmette-Guérin (Mycobacterium bovis BCG) in protecting humans and cattle against tuberculosis has prompted a search for a more effective vaccination regimen. A prime-boost strategy was investigated in cattle naturally sensitized to environmental mycobacteria by using a combination of three DNA vaccines coding for Hsp 65, Hsp 70, and Apa for priming, followed by a boost with BCG prior to experimental challenge with virulent M. bovis. Controls were vaccinated with DNA or BCG alone or were not vaccinated. The immune responses were monitored throughout the study, and protection was assessed based on reductions in the numbers of lesions and viable mycobacteria in lymph node samples. Vaccination with BCG alone or with a DNA prime-BCG boost regimen induced high levels of antigen-specific gamma interferon (IFN-γ) in whole-blood cultures. In the prime-boost group there were fewer animals with severe lung lesions, fewer lymph nodes with lesions per animal, a smaller proportion of animals with lesions, lower mean lung and lymph node lesion scores, and less M. bovis isolated from retropharyngeal and thoracic lymph nodes compared to the results obtained for the nonvaccinated animals. The prime-boost regimen induced significant enhancement of protection in six parameters, compared with significant enhancement of protection in only two parameters for BCG alone. In addition, following challenge, in vitro IFN-γ responses against ESAT-6 and CFP-10, as well as bovine tuberculin-induced skin test and in vitro IFN-γ responses, were identified as immunological markers that predicted protection. The use of the prime-boost strategy suggested that a combination of vaccines may be better than a single vaccine for protection against tuberculosis.

Strategies to reduce methane emissions from farmed ruminants grazing on pasture

Methane emissions from livestock are a significant contributor to greenhouse gas emissions and have become a focus of research activities, especially in countries where agriculture is a major economic sector. Understanding the complexity of the rumen microbiota, including methane-producing Archaea, is in its infancy. There are currently no robust, reproducible and economically viable methods for reducing methane emissions from ruminants grazing on pasture and novel innovative strategies to diminish methane output from livestock are required. In this review, current approaches towards mitigation of methane in pastoral farming are summarised. Research strategies based on vaccination, enzyme inhibitors, phage, homoacetogens, defaunation, feed supplements, and animal selection are reviewed. Many approaches are currently being investigated, and it is likely that more than one strategy will be required to enable pastoral farming to lower its emissions of methane significantly. Different strategies may be suitable for different farming practices and systems.

Control of Mycobacterium bovis infections and the risk to human populations

Conventional control methods based on test-and-slaughter policies have, in several countries, led to the successful eradication of bovine tuberculosis in cattle. However, new approaches for control of bovine tuberculosis are required in developing countries and those with a wildlife reservoir of infection. Recent developments include improved diagnostics and evaluation of new vaccination strategies.

The order of prime-boost vaccination of neonatal calves with Mycobacterium bovis BCG and a DNA vaccine encoding mycobacterial proteins Hsp65, Hsp70, and Apa is not critical for enhancing protection against bovine tuberculosis.

ABSTRACT Priming neonatal calves at birth with a Mycobacterium bovis bacillus Calmette-Guérin (BCG) vaccine and boosting with a DNA vaccine consisting of plasmids encoding mycobacterial antigens Hsp65, Hsp70, and Apa or the reverse prime-boost sequence induced similar levels of protection against experimental challenge with Mycobacterium bovis. When M. bovis was isolated from a thoracic lymph node following challenge, the two groups of calves given the prime-boost regimen had significantly lower numbers of M. bovis isolates than those vaccinated with BCG alone. These observations suggest that the exact sequence of administration of a prime-boost vaccination regimen in a neonatal animal model is not critical to the development of immunity.

Vaccination of Cattle with a CpG Oligodeoxynucleotide-Formulated Mycobacterial Protein Vaccine and Mycobacterium bovis BCG Induces Levels of Protection against Bovine Tuberculosis Superior to Those Induced by Vaccination with BCG Alone

ABSTRACT The development of a subunit protein vaccine for bovine tuberculosis which could be used either in combination with Mycobacterium bovis BCG (to improve the efficacy of that vaccine) or alone would offer significant advantages over currently available strategies. A study was conducted with cattle to determine the protective efficacy of a strategy based on concurrent immunization with an M. bovis culture filtrate (CFP) vaccine and BCG compared to vaccination with either vaccine alone. One group of calves (10 animals per group) was vaccinated subcutaneously with CFP formulated with Emulsigen and combined with a CpG oligodeoxynucleotide (ODN). A second group was vaccinated with both the CFP vaccine and BCG injected at adjacent sites (CFP-BCG). One further group was vaccinated subcutaneously with BCG, while another group served as nonvaccinated control animals. Vaccination with CFP-BCG induced levels of antigen-specific gamma interferon (IFN-γ) and interleukin-2 (IL-2) in whole-blood cultures that were higher than those induced by vaccination with BCG alone. The combination of CFP and BCG did not enhance the production of antibodies to M. bovis CFP compared to vaccination with CFP alone. Vaccination with CFP alone led to delayed antigen-specific IFN-γ and IL-2 responses. Vaccination with CFP-BCG induced a high level of protection against an intratracheal challenge with virulent M. bovis, based on a significant enhancement of six pathological and microbiological parameters of protection compared with the nonvaccinated group. In contrast, vaccination with BCG alone induced a significant enhancement of protection in only one parameter, while CFP alone induced no protection. These results suggest that a combination of a CpG ODN-formulated protein vaccine and BCG offers better protection against bovine tuberculosis than does BCG alone.

Vaccination of Cattle with Mycobacterium bovis Culture Filtrate Proteins and Interleukin-2 for Protection against Bovine Tuberculosis

ABSTRACT In this study vaccines prepared from culture filtrate proteins (CFP) of Mycobacterium bovis and interleukin-2 (IL-2) were tested in cattle for their capacity to stimulate immune responses and to protect against an intratracheal challenge with virulent M. bovis. Nine groups of cattle were vaccinated with combinations of different doses of CFP and bovine IL-2 mixed with a monophosphoryl lipid A (MPL) adjuvant. An additional group was vaccinated withM. bovis BCG. Immune responses in CFP–IL-2-vaccinated animals differed from those seen in BCG-vaccinated animals by inducing high antigen-specific antibody responses and low levels of gamma interferon and IL-2 released from purified protein derivative-stimulated whole-blood cultures. In a concurrent experiment, additional animals were added to the high-dose CFP–IL-2, MPL control, and BCG groups and these expanded groups of animals were challenged intratracheally with virulent M. bovis. Although the lung lesion scores were significantly lower for both the CFP–IL-2-and BCG-vaccinated groups compared to the MPL control group, the overall level of protection was greatest for the BCG-vaccinated animals. There were more animals with extrathoracic spread of disease in the CFP–IL-2 group than in the other groups. While vaccination of cattle withM. bovis CFP gave an encouraging reduction in tuberculous lesions and did not induce a delayed-type hypersensitivity response to PPD, future CFP vaccines must prevent any extrathoracic spread of disease.

Bacterial Polyester Inclusions Engineered To Display Vaccine Candidate Antigens for Use as a Novel Class of Safe and Efficient Vaccine Delivery Agents

ABSTRACT Bioengineered bacterial polyester inclusions have the potential to be used as a vaccine delivery system. The biopolyester beads were engineered to display a fusion protein of the polyester synthase PhaC and the two key antigens involved in immune response to the infectious agent that causes tuberculosis, Mycobacterium tuberculosis, notably antigen 85A (Ag85A) and the 6-kDa early secreted antigenic target (ESAT-6) from Mycobacterium tuberculosis. Polyester beads displaying the respective fusion protein at a high density were successfully produced (henceforth called Ag85A-ESAT-6 beads) by recombinant Escherichia coli. The ability of the Ag85A-ESAT-6 beads to enhance mouse immunity to the displayed antigens was investigated. The beads were not toxic to the animals, as determined by weight gain and absence of lesions at the inoculation site in immunized animals. In vivo injection of the Ag85A-ESAT-6 beads in mice induced significant humoral and cell-mediated immune responses to both Ag85A and ESAT-6. Vaccination with Ag85A-ESAT-6 beads was efficient at stimulating immunity on their own, and this ability was enhanced by administration of the beads in an oil-in-water emulsion. In addition, vaccination with the Ag85A-ESAT-6 beads induced significantly stronger humoral and cell-mediated immune responses than vaccination with an equivalent dose of the fusion protein Ag85A-ESAT-6 alone. The immune response induced by the beads was of a mixed Th1/Th2 nature, as assessed from the induction of the cytokine gamma interferon (Th1 immune response) and increased levels of immunoglobulin G1 (Th2 immune response). Hence, engineered biopolyester beads displaying foreign antigens represent a new class of versatile, safe, and biocompatible vaccines.

IFN-γ enhances bovine macrophage responsiveness to Mycobacterium bovis : Impact on bacterial replication, cytokine release and macrophage apoptosis

We sought to determine the impact of bovine IFN‐γ on the interaction between Mycobacterium bovis and bovine macrophages. Bovine macrophages released small amounts of nitric oxide (NO), TNF‐α, IL‐1β and IL‐12 upon infection with bacille Calmette−Guérin (BCG). Prior pulsing of cells with IFN‐γ significantly enhanced the release of NO and IL‐12. Infection of bovine macrophages with virulent M. bovis led to the release of higher levels of pro‐inflammatory mediators, compared to levels released upon BCG infection. IFN‐γ treatment of macrophages enhanced the release of pro‐inflammatory mediators, but did not modify bacterial replication in M. bovis‐infected macrophages. Treatment of macrophages with a combination of IFN‐γ and LPS led to a reduction in bacterial replication. Infected cells treated with IFN‐γ/LPS progressed mostly through an apoptotic pathway, whereas untreated infected cells eventually died by necrosis. Agents that prevented the acquisition of bacteriostatic activity by activated macrophages also prevented the induction of apoptosis in infected macrophages (IL‐10 and neutralizing anti‐TNF‐α). We conclude that virulent M. bovis is a major determinant of release of pro‐inflammatory cytokines by macrophages. IFN‐γ amplifies the macrophage cytokine release in response to M. bovis. Induction of apoptosis is closely linked to the emergence of macrophage resistance to M. bovis replication, which is dependent on endogenous TNF‐α release.

Vaccines Displaying Mycobacterial Proteins on Biopolyester Beads Stimulate Cellular Immunity and Induce Protection against Tuberculosis

ABSTRACT New improved vaccines are needed for control of both bovine and human tuberculosis. Tuberculosis protein vaccines have advantages with regard to safety and ease of manufacture, but efficacy against tuberculosis has been difficult to achieve. Protective cellular immune responses can be preferentially induced when antigens are displayed on small particles. In this study, Escherichia coli and Lactococcus lactis were engineered to produce spherical polyhydroxybutyrate (PHB) inclusions which displayed a fusion protein of Mycobacterium tuberculosis, antigen 85A (Ag85A)–early secreted antigenic target 6-kDa protein (ESAT-6). L. lactis was chosen as a possible production host due its extensive use in the food industry and reduced risk of lipopolysaccharide contamination. Mice were vaccinated with PHB bead vaccines with or without displaying Ag85A–ESAT-6, recombinant Ag85A–ESAT-6, or M. bovis BCG. Separate groups of mice were used to measure immune responses and assess protection against an aerosol M. bovis challenge. Increased amounts of antigen-specific gamma interferon, interleukin-17A (IL-17A), IL-6, and tumor necrosis factor alpha were produced from splenocytes postvaccination, but no or minimal IL-4, IL-5, or IL-10 was produced, indicating Th1- and Th17-biased T cell responses. Decreased lung bacterial counts and less extensive foci of inflammation were observed in lungs of mice receiving BCG or PHB bead vaccines displaying Ag85A–ESAT-6 produced in either E. coli or L. lactis compared to those observed in the lungs of phosphate-buffered saline-treated control mice. No differences between those receiving wild-type PHB beads and those receiving recombinant Ag85A–ESAT-6 were observed. This versatile particulate vaccine delivery system incorporates a relatively simple production process using safe bacteria, and the results show that it is an effective delivery system for a tuberculosis protein vaccine.

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.