Effect of different adjuvants on the immune responses of cattle vaccinated with Mycobacterium tuberculosis culture filtrate proteins

The development of improved vaccines for bovine tuberculosis is urgently required as a cost effective solution for control and eventual eradication of tuberculosis in domestic animals. Studies in small animal models of tuberculosis have shown that vaccination with culture filtrate proteins (CFP), prepared from Mycobacterium tuberculosis or M. bovis, can induce cellular immune responses and confer a level of protection against aerogenic challenge with virulent mycobacteria. As a first step in the development of a mycobacterial CFP vaccine for protection of cattle against bovine tuberculosis, the immune responses of cattle vaccinated with short-term culture filtrate proteins (ST-CFP) from M. tuberculosis and formulated with different adjuvants were compared with those vaccinated with bacille Calmette-Guerin (BCG). The adjuvants included dimethyldioctyldecyl ammonium bromide (DDA), diethylaminoethyl (DEAE)-dextran, and ST-CFP adsorbed onto polystyrene beads. Vaccination with ST-CFP/DEAE-dextran induced high levels of interleukin-2 (IL-2) but low levels of interferon-gamma (IFN-gamma) from whole-blood cultures stimulated with M. tuberculosis ST-CFP in comparison with the strong IFN-gamma and IL-2 responses induced after vaccination with BCG. ST-CFP/DEAE-dextran also induced a strong antigen-specific immunoglobulin antibody response with both immunoglobulin G1 (IgG1) and IgG2 isotypes. Vaccination with ST-CFP/beads induced a weak IgG1-biased antibody response but no IFN-gamma or IL-2 response. DDA did not induce significant immune responses in animals vaccinated with ST-CFP. In comparison to the moderate delayed-type hypersensitivity (DTH) responses induced by vaccination with subcutaneous BCG, none of the ST-CFP vaccines induced a significant DTH response to either M. tuberculosis ST-CFP or bovine purified protein derivative (PPD). While the ST-CFP vaccines used in this study have not induced strong antigen-specific cellular immune responses in cattle comparable to those induced by BCG, they are immunogenic in cattle and it may be possible to overcome this problem by using adjuvants that more effectively promote IFN-gamma responses in this species.     

Vaccination of cattle with Danish and Pasteur strains of Mycobacterium bovis BCG induce different levels of IFNgamma post-vaccination, but induce similar levels of protection against bovine tuberculosis

A number of studies have demonstrated significant protection of cattle against bovine tuberculosis following vaccination with the Pasteur strain of Mycobacterium bovis bacille Calmete-Guerin (BCG). However, it is unclear if other daughter strains of BCG are as effective, which is an important issue to resolve for a variety of regulatory compliance issues. This study compared the protective immune responses to bovine tuberculosis induced in cattle vaccinated with BCG Danish with those induced by BCG Pasteur. Groups of calves (n=10) were vaccinated with 10(6) colony forming units (CFU) BCG Pasteur prepared from a fresh liquid culture, 10(6) CFU BCG Danish prepared from a fresh liquid culture or 0.4 mg of reconstituted freeze-dried culture of BCG Danish. Another group (n=10) served as non-vaccinated controls. BCG Pasteur induced significantly higher and more sustained levels of bovine purified protein derivative (PPD)-specific gamma interferon (IFN-gamma) in whole-blood cultures following vaccination compared to either fresh culture BCG Danish or freeze-dried BCG Danish. Vaccination with a fresh culture of BCG Pasteur, fresh culture BCG Danish and freeze-dried BCG Danish gave a significant enhancement in three, four and three pathological and microbiological parameters of protection, respectively, compared to the non-vaccinated group. These results demonstrate the Danish strain of BCG is a viable alternative to BCG Pasteur for vaccination of cattle as both strains had similar efficacy and there was little difference between freshly cultured and freeze-dried formulation of BCG Danish. The results also show that post-vaccination antigen-specific IFN-gamma levels in whole blood is not always a reliable indicator of protection against a subsequent virulent challenge.     

Conjunctival vaccination of the brushtail possum (Trichosurus vulpecula) with bacille Calmette-Guérin

AIM: To determine the efficacy of conjunctival vaccination of captive brushtail possums (Trichosurus vulpecula) with bacille Calmette-Guérin (BCG), as measured by immunological responses to vaccination and response to intratracheal challenge with Mycobacterium bovis. METHODS: Nine adult male brushtail possums were vaccinated by the instillation of a suspension of BCG strain Pasteur 1173P2 into the conjunctival sac of each eye. Each drop contained approximately 2.5 x 105 colony forming units (cfu). At 8 weeks post-vaccination (pv) the vaccinated possums and 10 unvaccinated possums were challenged by intratracheal instillation of approximately 100 cfu of M. bovis. Cellular immune responses to bovine purified protein derivative (PPD) antigen were measured using the lymphocyte proliferation assay (LPA). Possums surviving to 50-51 days after challenge were euthanised and subjected to detailed post-mortem examination, including histopathology, to assess protection against tuberculosis. Sections of lung and spleen were cultured for M. bovis. RESULTS: No conjunctival inflammation or other adverse reactions to the administration of the vaccine were evident macroscopically. The vaccinated group showed a systemic cellular immune response to bovine PPD antigen at 4 and 8 weeks pv, and the response at 8 weeks was significantly greater than at 4 weeks (p<0.05). Conjunctival vaccination induced significant levels of protective immunity, measured as less mass of tuberculous lesions in lung (p<0.05) and less dissemination of disease in vaccinated compared with unvaccinated possums (p<0.05). CONCLUSIONS: Conjunctival vaccination with BCG induced a significant level of protective immunity against M. bovis infection in possums. This route of vaccination, together with intranasal aerosol vaccination, could be utilised in the delivery of an aerosolised vaccine using a device that sprays the vaccine suspension into the eyes and nose of possums.     

Induction of antigen-specific T-cell subset activation to bovine respiratory disease viruses by a modified-live virus vaccine

OBJECTIVE: To determine the efficacy of a modified-live virus vaccine containing bovine herpes virus 1 (BHV-1), bovine respiratory syncytial virus (BRSV), parainfluenza virus 3, and bovine viral diarrhea virus (BVDV) types 1 and 2 to induce neutralizing antibodies and cell-mediated immunity in na?ve cattle and protect against BHV-1 challenge. ANIMALS: 17 calves. PROCEDURES: 8 calves were mock-vaccinated with saline (0.9% NaCl) solution (control calves), and 9 calves were vaccinated at 15 to 16 weeks of age. All calves were challenged with BHV-1 25 weeks after vaccination. Neutralizing antibodies and T-cell responsiveness were tested on the day of vaccination and periodically after vaccination and BHV-1 challenge. Specific T-cell responses were evaluated by comparing CD25 upregulation and intracellular interferon-gamma expression by 5-color flow cytometry. Titration of BHV-1 in nasal secretions was performed daily after challenge. Results-Vaccinated calves seroconverted by week 4 after vaccination. Antigen-specific cell-mediated immune responses, by CD25 expression index, were significantly higher in vaccinated calves than control calves. Compared with control calves, antigen-specific interferon-gamma expression was significantly higher in calves during weeks 4 to 8 after vaccination, declining by week 24. After BHV-1 challenge, both neutralizing antibodies and T-cell responses of vaccinated calves had anamnestic responses to BHV-1. Vaccinated calves shed virus in nasal secretions at significantly lower titers for a shorter period and had significantly lower rectal temperatures than control calves. CONCLUSION AND CLINICAL RELEVANCE: A single dose of vaccine effectively induced humoral and cellular immune responses against BHV-1, BRSV, and BVDV types 1 and 2 and protected calves after BHV-1 challenge for 6 months after vaccination.     

Immunohistochemical markers augment evaluation of vaccine efficacy and disease severity in bacillus Calmette-Guerin (BCG) vaccinated cattle challenged with Mycobacterium bovis

Development of necrotic granulomas in response to Mycobacterium bovis infection in cattle is pathognomonic for bovine tuberculosis. Previously our laboratory reported on M. bovis granuloma classification by stage of lesion advancement within bovine lymph nodes and developed immunohistochemical markers to further characterize these granulomas. In this study of bovine lymph node granulomas we applied this classification system to assess the dynamics of vaccination challenge. Lymph nodes collected from cattle vaccinated with M. bovis bacillus Calmette-Guerin (BCG) and subsequently challenged with virulent M. bovis were compared to lymph nodes from unvaccinated, challenged cattle. Expression of interferon-gamma (IFN-gamma), transforming growth factor-beta (TGF-beta), type I procollagen and cell marker identification of T cells, B cells, macrophages and WC1(+)gammadelta TCR+ cells were assessed. Granulomas formed in vaccinated cattle were greatly reduced in number, area, degree of necrosis and peripheral fibrosis and contained fewer Langhans' giant cells, acid fast bacilli, WC1(+)gammadelta TCR+ cells and less TGF-beta expression in comparison to controls. B cells clustered intensely along the outer granuloma margins within vaccinated calves, with significantly more IFN-gamma producing cells identified in the medullary regions of lymph nodes from BCG-vaccinated animals compared to unvaccinated controls. This may be indicative of immune activation and surveillance in regions not directly associated with ongoing disease. Lymph node evaluation using light microscopy and immunohistochemical markers is useful to assess the immune response and discriminate granulomas to determine vaccine efficacy and disease severity.     

The effect of dose, route and virulence of bovine herpesvirus 1 vaccine on experimental respiratory disease in cattle.

Three experiments were conducted with calves in which, following intramuscular or intranasal vaccination with virulent or attenuated bovine herpesvirus 1, calves were protected against bovine herpesvirus 1 -- Pasteurella haemolytica challenge. Calves receiving low doses of vaccine had lower levels of antibody and greater evidence of virus replication upon challenge than those receiving higher doses. In contrast 11/13 unvaccinated controls had fibrino-purulent pneumonia following challenge. The immune response developed later in younger calves and those given low doses of vaccine. Neutralizing antibodies to bovine herpes-virus 1 were not found in nasal secretions, but were present in serum seven days after vaccination. Bovine herpesvirus 1 was isolated before challenge from nasal secretions of calves vaccinated intranasally or intramuscularly with virulent virus but not those vaccinated intramuscularly with vaccine virus. It was concluded that both routes of vaccination with either virulent or attenuated bovine herpesvirus 1 provided protection from challenge with homologous or heterologous bovine herpesvirus 1 and that live vaccines should contain at least 10(3) plaque forming units/dose for effective immunization.     

Vaccination against infectious bovine keratoconjunctivitis: protective efficacy and antibody response induced by pili of homologous and heterologous strains of Moraxella bovis

The protective effect of 2 Moraxella bovis pili vaccines against infectious bovine keratoconjunctivitis (IBK) experimentally induced by homologous or heterologous strain challenge with virulent, haemolytic M. bovis strain, Dal 2d, was measured in trials using weaned calves aged 3 to 7 months. Purified pili vaccines were prepared from haemolytic strain Dal 2d, (pilus serogroup IV), and haemolytic strain Epp 63, (pilus serogroup III). Calves were challenged by conjunctival instillation of 1 x 10(9) colony forming units of virulent M. bovis strain Dal 2d 14 days after the second of 2 subcutaneous doses of vaccine. Each consisted of 200 micrograms of pili in alum-oil adjuvant administered at an interval of 21 days. In trial 1 the level of protection against challenge with the homologous strain was 46.7% (p less than 0.01). Small, rapidly resolving lesions of IBK occurred in some vaccinates compared with a larger proportion of severe lesions that required treatment in non-vaccinated calves (p less than 0.025). In trial 2, the level of protection against IBK after exposure of vaccinates to the homologous Dal 2d strain was 72.7%, but no significant level of protection or reduction in the size and duration of lesions was apparent in similarly challenged calves vaccinated with Epp 63 pili when contrasted with susceptible, non-vaccinated controls. No marked reduction in the duration of infection with M. bovis Dal 2d following challenge resulted from vaccination with pili of either of the serogroups III or IV. Rising homologous serum IgG antibody titres to serogroups III and IV pili were recorded in response to vaccination with each antigen.(ABSTRACT TRUNCATED AT 250 WORDS)     

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

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.     

Effects of a single intranasal dose of modified-live bovine respiratory syncytial virus vaccine on resistance to subsequent viral challenge in calves

OBJECTIVE: To determine whether a single intranasal dose of modified-live bovine respiratory syncytial virus (BRSV) vaccine protects calves from BRSV challenge and characterize cell-mediated immune response in calves following BRSV challenge. ANIMALS: 13 conventionally reared 4- to 6-week-old Holstein calves. PROCEDURES: Calves received intranasal vaccination with modified live BRSV vaccine (VC-group calves; n = 4) or mock vaccine (MC-group calves; 6) 1 month before BRSV challenge; unvaccinated control-group calves (n = 3) underwent mock challenge. Serum virus neutralizing (VN) antibodies were measured on days -30, -14, 0, and 7 relative to BRSV challenge nasal swab specimens were collected for virus isolation on days 0 to 7. At necropsy examination on day 7, tissue specimens were collected for measurement of BRSV-specific interferon gamma (IFN-gamma) production. Tissue distribution of CD3+ T and BLA.36+ B cells was evaluated by use of immunohistochemistry. RESULTS: The MC-group calves had significantly higher rectal temperatures, respiratory rates, and clinical scores on days 5 to 7 after BRSV challenge than VC-group calves. No difference was seen between distributions of BRSV in lung tissue of VC- and MC-group calves. Production of BRSV-specific IFN-gamma was increased in tissue specimens from VC-group calves, compared with MC- and control-group calves. Virus-specific IFN-gamma production was highest in the mediastinal lymph node of VC-group calves. Increased numbers of T cells were found in expanded bronchial-associated lymphoid tissue and airway epithelium of VC-group calves. CONCLUSIONS AND CLINICAL RELEVANCE: An intranasal dose of modified-live BRSV vaccine can protect calves against virulent BRSV challenge 1 month later.     

The correlation of virus-specific interferon-gamma production and protection against classical swine fever virus infection.

The level of antigen-specific interferon-gamma (IFN-gamma) production can be used as an indicator of cellular immunity. In this study, we investigated the role of cellular immune response in protection against classical swine fever virus (CSFV). Pigs were vaccinated once with CSFV vaccine and challenged 6 days post-vaccination (dpv). Vaccinated animals had significantly higher CSFV-specific IFN-gamma secreting cells than the unvaccinated pigs (p<0.05) at the time of challenge and were protected against CSFV infection, whereas the control pigs died within 14 days post-infection (dpi). In the second experiment, pigs were vaccinated once with either CSFV vaccine or CSFV vaccine combined with Aujeszky's disease (AD) vaccine and challenged at 140 dpv. All vaccinated pigs developed both CSFV-specific, cellular and antibody responses and were protected against CSFV infection. However, differences in cellular, but not antibody, responses were observed in the two vaccinated groups. The group vaccinated with CSFV vaccine developed a significantly higher number of CSFV-specific, IFN-gamma secreting cells (p<0.05), exhibited a shorter fever period and less pathological changes, when compared with the group vaccinated with the combined vaccine. The kinetics of IFN-gamma production, following challenge in the two vaccinated groups, were also different. Taken together, our results indicated that CSFV-specific, IFN-gamma production could be detected early after antigen exposure and correlated with protection against CSFV challenge. Our findings highlight the role of cellular immune responses in porcine anti-viral immunity.     

Vaccination of cattle with Mycobacterium bovis culture filtrate proteins and CpG oligodeoxynucleotides induces protection against bovine tuberculosis

Culture filtrate protein (CFP) vaccines have been shown to be effective in small animal models for protecting against tuberculosis while immunisation with these types of vaccines in cattle has been less successful. A study was conducted in cattle to evaluate the ability of selected adjuvants and immunomodulators to stimulate protective immune responses to tuberculosis in animals vaccinated with Mycobacterium bovis CFP. Seven groups of cattle (n=5) were vaccinated with M. bovis CFP formulated with either Emulsigen or Polygen adjuvant alone or in combination with a specific oligodeoxynucleotides (ODN), polyinosinic acid: polycytidylic acid (poly I:C) or poly I:C and recombinant granulocyte-macrophage colony stimulating factor. Two additional groups were vaccinated subcutaneously with BCG or non-vaccinated. In contrast to the strong interferon-gamma (IFN-gamma) responses induced by BCG, the CFP vaccines induced strong antibody responses but weak IFN-gamma responses. The addition of CpG ODN to CFP significantly enhanced cell-mediated responses and elevated antibody responses to mycobacterial antigens. Of the CFP vaccinated groups, the strongest IFN-gamma responses to CFP vaccines were measured in animals vaccinated with CFP/Emulsigen+CpG or CFP/Polygen+CpG. The animals in these two groups, together with those in the BCG and non-vaccinated groups were challenged intratracheally with virulent M. bovis at 13 weeks after the first vaccination and protection was assessed, by examination for presence of tuberculous lesions in the lungs and lymph nodes, 13 weeks later at postmortem. While BCG gave the best overall protection against tuberculosis, significant protection was also seen in animals vaccinated with CFP/Emulsigen+CpG. These results establish an important role for CpG ODN in stimulating protective Th1 responses to tuberculosis in cattle and indicate that a sub-unit protein vaccine can protect these animals against tuberculosis.     

IFN-gamma diagnostic tests in the context of bovine mycobacterial infections in Belgium

In countries where cattle tuberculosis caused by Mycobacterium bovis (Mbov) and paratuberculosis caused by Mycobacterium avium subsp. paratuberculosis (Mptb) are present, testing strategies for the Mbov eradication have to discriminate between these two infections. Present indirect tests are based on the analysis of the specific cellular immune response (DTH, IFN-gamma) against crude mycobacterial antigens (avian and bovine PPD). In this study, we compared the evolution of the IFN-gamma responses of animals experimentally infected with Mbov, Mptb, or inoculated with Mycobacterium phlei. Mbov inoculation induced a strong IFN-gamma response that allows rapid classification of the status of the animals following interpretation criteria set up by us. Experimental inoculation with M. phlei induced sensitisation to mycobacterial antigens as detected by the IFN-gamma test but these reactions were of short duration, therefore, repeated testing allows us to define these animals as aspecific reactors. IFN-gamma response induced after oral inoculation of calves with Mptb was of low intensity and ratio of responses measured against avian versus bovine PPD did not allow a clear diagnostic at least for the six first month of infection.     

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.     

BCG vaccination of neonatal calves: potential roles for innate immune cells in the induction of protective immunity

Bovine tuberculosis is a disease of increasing incidence in the UK causing major economic losses and with significant impact on bovine and, potentially human health: the causative agent Mycobacterium bovis is a zoonotic pathogen. Neonatal vaccination with the attenuated M. bovis Bacille Calmette Guerin (BCG) vaccine confers a significant degree of protection in cattle, and is a widely used control strategy for human TB. The adaptive immune system is relatively immature in neonates and increased numbers of innate effector cells present in young animals and human infants may compensate for this, enabling effective immune responses to vaccination. Natural killer cells and subsets of γδ TCR+ T lymphocytes secrete high levels of interferon gamma and can interact with antigen presenting cells to promote both innate and adaptive immune responses. These cell populations may be pivotal in determining immune bias following neonatal vaccination with BCG.     

Humoral and cell-mediated immune responses of old horses following recombinant canarypox virus vaccination and subsequent challenge infection

Equine influenza virus is a leading cause of respiratory disease in the horse population; however, the susceptibility of old horses to EIV infection remains unknown. While advanced age in horses (>20 years) is associated with age-related changes in immune function, there are no specific recommendations regarding the vaccination of older horses even though a well-characterized effect of aging is a reduced antibody response to standard vaccination. Therefore, we evaluated the immunological and physiological response of aged horses to a live non-replicating canarypox-vectored EIV vaccine and subsequent challenge infection. Vaccination of the aged horses induced EIV-specific IgGb and HI antibodies. No specific increase in cell-mediated immune (CMI) response was induced by the vaccine as determined by EIV-specific lymphoproliferation and the detection of EIV-specific IFNγ⁺ CD5⁺T cells, IFNγ, IL-2, IL-4 and IL-13 mRNA expression. Non-vaccinated aged horses exhibited clinical signs of the disease (coughing, nasal discharge, dyspnea, depression, anorexia) as well as increased rectal temperature and viral shedding following challenge. Concomitant with the febrile episodes, we also observed increased production of pro-inflammatory cytokine mRNA production in vivo using RT-PCR. Naïve horses were included in this study for vaccine and challenge controls only. As expected, the canarypox-vectored EIV vaccine stimulated significant CMI and humoral immune responses and provided significant protection against clinical signs of disease and reduced virus shedding in naive horses. Here, we show that aged horses remain susceptible to infection with equine influenza virus despite the presence of circulating antibodies and CMI responses to EIV and vaccination with a canarypox-vectored EIV vaccine provides protection from clinical disease.     

An inactivated parainfluenza virus type 3 vaccine: the influence of vaccination regime on the response of calves and their subsequent resistance to challenge.

Calves maintained in insolated pens were vaccinated with an inactivated parainfluenza virus type (3) (pi3) vaccine usingparenteral and local route singly and in combination. The calves were subsequently monitored for serum antibody response and challenged intranasally with live virus to assess the protection derived from vaccination. Calves receiving one subcutaneous dose of vaccine in oil adjuvant produced a marked antibody response and were partially protected against challenge. Those receiving two successive subcutaneous doses produced a much greater antiboyd response and were completely protected against challenge. One intranasal dose of aqueous vaccine failed elicit a significant serum antibody response or protection against challenge. However, there was some evidence that intranasal vaccination following a single subcutaneous vaccination produced more effective immunity than one subcutaneous dose alone. Thus a vaccination regime was established which protected calves against experimental challenge and which could thefore be used in the field to assess the role of Pi3 virus in calf respiratory disease.     

A DNA vaccine encoding MPB83 from Mycobacterium bovis reduces M. bovis dissemination to the kidneys of mice and is expressed in primary cell cultures of the European badger (Meles meles)

Nucleic acid (DNA) vaccination against tuberculosis in the European badger (Meles meles) is one approach to addressing the escalating problem of bovine tuberculosis in Great Britain. The aim of vaccination is to reduce the burden of tuberculosis within the badger population and the shedding of Mycobacterium bovis to levels that would break the transmission of infection to cattle. To this end, the vaccine would be required to limit the amount of disseminated tuberculosis in the badger, especially dissemination to the kidney from where M. bovis can be shed in the urine. A promising candidate DNA vaccine encoding a 26 kDa major antigen (MPB83) of M. bovis was evaluated in a mouse model of disseminated M. bovis infection. Using the DNA vaccine, protection against infection of the kidney was found to be greater than that achieved with the current live vaccine, Bacille Calmette-Guerin (BCG). Kidney tissue and skeletal muscle from the badger was used to derive primary cell cultures in which to examine the expression of MPB83 following transfection with the DNA vaccine. Kidney cortex gave rise to a monotypic culture of epithelial cells whilst the muscle gave rise to a mixed culture of fibroblasts and myoblasts. During culture the myoblasts differentiated into multinucleated myotubes, verified by immunofluorescent detection of mammalian desmin. Successful expression of MPB83 by transfected epithelial and myotube cells was confirmed by immunofluorescence using a monoclonal antibody specific to the protein. These observations fulfil the early requirements for the development of a DNA vaccine for badger tuberculosis.     

Protection against bovine tuberculosis induced by oral vaccination of cattle with Mycobacterium bovis BCG is not enhanced by co-administration of mycobacterial protein vaccines

Mycobacterium bovis bacille Calmette-Guérin (BCG) delivered to calves by the oral route in a formulated lipid matrix has been previously shown to induce protection against bovine tuberculosis. A study was conducted in cattle to determine if a combination of a low dose of oral BCG and a protein vaccine could induce protective immunity to tuberculosis while not sensitising animals to tuberculin. Groups of calves (10 per group) were vaccinated by administering 2 × 10(7)colony forming units (CFU) of BCG orally or a combination of 2 × 10(7)CFU oral BCG and a protein vaccine comprised of M. bovis culture filtrate proteins (CFP) formulated with the adjuvants Chitin and Gel 01 and delivered by the intranasal route, or CFP formulated with Emulsigen and the TLR2 agonist Pam(3)CSK(4) and administered by the subcutaneous (s.c.) route. Two further groups were vaccinated with the CFP/Chitin/Gel 01 or CFP/Emulsigen/Pam(3)CSK(4) vaccines alone. Positive control groups were given 10(8)CFU oral BCG or 10(6)CFU s.c. BCG while a negative control group was non-vaccinated. All animals were challenged with M. bovis 15 weeks after vaccination and euthanized and necropsied at 16 weeks following challenge. Groups of cattle vaccinated with s.c. BCG, 10(8)CFU or 2 × 10(7)CFU oral BCG showed significant reductions in seven, three and four pathological or microbiological disease parameters, respectively, compared to the results for the non-vaccinated group. There was no evidence of protection in calves vaccinated with the combination of oral BCG and CFP/Emulsigen/Pam(3)CSK(4) or oral BCG and CFP/Chitin/Gel 01 or vaccinated with the protein vaccines alone. Positive responses in the comparative cervical skin test at 12 weeks after vaccination were only observed in animals vaccinated with s.c. BCG, 10(8)CFU oral BCG or a combination of 2 × 10(7)CFU oral BCG and CFP/Chitin/Gel 01. In conclusion, co-administration of a protein vaccine, administered by either systemic or mucosal routes with oral BCG did not enhance the protection conferred by administration of oral BCG alone.     

Evaluation of the long-term immune response in cattle after vaccination against paratuberculosis in two Dutch dairy herds

In the past decades, vaccination against paratuberculosis in cattle was performed in The Netherlands only on a limited scale. Because of its interference with the diagnosis of bovine tuberculosis, vaccination was restricted to herds with a high prevalence of clinical cases of paratuberculosis and was meant to aid in the economical survival of the farm. Recently, a voluntary paratuberculosis certification program has started, based in part on serological screening of cattle of at least 3 years of age. Herds that have been vaccinated against paratuberculosis are, therefore, likely to encounter problems when entering this program.The aim of this study was to evaluate the immune response resulting from vaccination with a heat-killed paratuberculosis vaccine. Over a period of 12-14 years, new-born calves were vaccinated in two herds. The B-cell response was evaluated using both the complement-fixation test (CFT) and an enzyme-linked immunosorbent assay (ELISA) and the cell-mediated immune response was evaluated using the gamma-interferon assay. Data obtained show a marked and prolonged effect of the vaccination on both cellular and humoral immune responses, in particular to the paratuberculosis antigen but also to the bovine tuberculosis antigen, using the respective tests. These responses were detected rapidly after vaccination. The individual responses were highly variable between animals with respect to both the level and to the duration of the evoked immune response. No relation between the results obtained with the ELISA and the CFT was observed. In conclusion, for a large number of vaccinated cattle, a long lasting interference is to be expected with the presently available immunodiagnostic methods for both bovine tuberculosis and paratuberculosis.