Purification and Characterisation of Badger IgA and Its Detection in the Context of Tuberculosis

European badgers are a wildlife reservoir of bovine tuberculosis in parts of Great Britain. Accurate diagnosis of tuberculosis in badgers is important for the development of strategies for the control of the disease. Sensitive serological tests for badger TB are needed for reasons such as cost and simplicity. Assay of mucosal IgA could be useful for diagnosing respiratory pathogens such as Mycobacterium bovis and for monitoring the response to mucosal vaccination. To develop an IgA assay, we purified secretory IgA from badger bile, identifying secretory component (SC), heavy chain (HC) and light chain (LC), at 66, 46 and 27 Kda, respectively, on the basis of size comparison with other species. Monoclonal antibodies (mAbs) were generated to purified IgA.We selected two for ELISA development. The detection limit of the IgA-specific mAbs was found to be approximately 20 ng/mL when titrated against purified badger bile. One monoclonal antibody specific for badger IgA was used to detect IgA in serum and tracheal aspirate with specificity to an immunodominant antigen of M. bovis. An M. bovis infection dose-dependent IgA response was observed in experimentally infected badgers. IgA was also detected by immunohistochemistry in the lungs of bTB-infected badgers. With further characterisation, these represent new reagents for the study of the IgA response in badgers

Lactic acid Bacteria isolated from European badgers (Meles meles) reduce the viability and survival of Bacillus Calmette-Guerin (BCG) vaccine and influence the immune response to BCG in a human macrophage model

Abstract Background Bovine tuberculosis (bTB) caused by Mycobacterium bovis is the most serious endemic disease affecting livestock in the UK. The European badger (Meles meles) is the most important wildlife reservoir of bTB transmission to cattle, making eradication particularly difficult. In this respect, oral vaccination with the attenuated M. bovis vaccine Bacillus Calmette-Guerin (BCG) has been suggested as a wide-scale intervention to reduce bTB infection in badgers. However, experimental studies show variable protection. Among the possibilities for this variation is that the resident gut bacteria may influence the success of oral vaccination in badgers; either through competitive exclusion and/or inhibition, or via effects on the host immune system. In order to explore this possibility, we have tested whether typical gut commensals such as Lactic Acid Bacteria (LAB) have the capacity to impact on the viability and survival rate of BCG and to modulate the immune response to BCG using an in vitro model. Results Twelve LAB isolated from badger faeces displayed inhibitory activity to BCG that was species-dependent. Weissella had a bacteriostatic effect, whereas isolates of enterococci, lactobacilli and pediococci had a more bactericidal activity. Furthermore, BCG-induced activation of the pro-inflammatory transcription factor NF-κB in human THP-1 macrophages was modulated by LAB in a strain-dependent manner. Most pediococci enhanced NF-κB activation but one strain had the opposite effect. Interestingly, isolates of enterococci, lactobacilli and weissella had different effects as immunomodulators of BCG-induced macrophage responses as some had no significant influence on NF-κB activation, but others increased it significantly. Conclusions Our in vitro results show that LAB isolated from badgers exhibit significant inhibitory activity against BCG and influence the immune activation mediated by BCG in a human macrophage assay. These findings suggest that gut commensal bacteria could play a role in influencing the outcome of oral BCG vaccination. Inactivated cells of LAB, or LAB that are bacteriostatic but have a synergistic immunostimulatory effect with BCG, could be potential adjuvants to be used for oral vaccination in badgers. Further work is needed to take into account the complex nature of the gut microbiome, specific immunity of the badger and the in vivo context

Additional file 1: of Lactic acid Bacteria isolated from European badgers (Meles meles) reduce the viability and survival of Bacillus Calmette-Guerin (BCG) vaccine and influence the immune response to BCG in a human macrophage model

Figure S1. pH of BCG co-cultures with LAB isolates or E. coli and Salmonella after 0 (white bars), 24 (grey bars) and 48 (black bars) h of incubation. Different letters signify statistical differences between mean values using ANOVA followed by LSD (p < 0.05). Figure S2. Viability (A) and survival rate (B) of BCG when grown as a monoculture at pH 5 or pH 7 after 0 (white bars), 24 (grey bars) and 48 (black bars) h of incubation. Viability and survival rate are expressed as log10 Fluorescence Units (FU) and log10 CFU/mL, respectively. Figure S3. BCG viability when grown in cell-free supernatants from 48 h-cultures of enterococci (i), weissella (ii), lactobacilli (iii), pediococci (iv) and gut-associated Gram-negative bacteria (v) after 0 (white bars), 24 (grey bars) and 48 (black bars) h of incubation. BCG viability is expressed as log10 Fluorescence Units (FU). Figure S4. Survival rate of BCG when grown in cell-free supernatants from 48 h-cultures of enterococci (i), weissella (ii), lactobacilli (iii), pediococci (iv) and gut-associated Gram-negative bacteria (v) after 0 (white bars), 24 (grey bars) and 48 (black bars) h of incubation. BCG survival rate is expressed as log10 CFU/mL.Figure S5. Significant positive correlation between mean values of NF-κB activation and TNFα production in PMA-differentiated THP-1 macrophages exposed to inactivated bacterial strains and LPS, at a ratio of 1:100 (macrophage::bacteria) and 0.2 μg/μl, respectively (p < 0.0001). Figure S6. Growth rate of enterococci (i), weissella (ii), lactobacilli (iii), pediococci (iv) and gut-associated Gram-negative bacteria (v) after 0 (white bars), 24 (grey bars) and 48 (black bars) h of incubation when co-cultured with BCG. Growth rate is expressed as log10 CFU/mL. (DOCX 217 kb

Protection of Eurasian badgers (Meles meles) from tuberculosis after intra-muscular vaccination with different doses of BCG

a b s t r a c t Mycobacterium bovis infection is widespread in Eurasian badger (Meles meles) populations in Great Britain and the Republic of Ireland where they act as a wildlife reservoir of infection for cattle. Removal of infected badgers can significantly reduce the incidence of bovine tuberculosis (TB) in local cattle herds. However, control measures based on culling of native wildlife are contentious and may even be detrimental to disease control. Vaccinating badgers with bacillus Calmette-Guerin (BCG) has been shown to be efficacious against experimentally induced TB of badgers when administered subcutaneously and orally. Vaccination may be an alternative or complementary strategy to other disease control measures. As the subcutaneous route is impractical for vaccinating wild badgers and an oral vaccine bait formulation is currently unavailable, we evaluated the intramuscular (IM) route of BCG administration. It has been demonstrated that the IM route is safe in badgers. IM administration has the practical advantage of being relatively easy to perform on trapped wild badgers without recourse to chemical immobilisation. We report the evaluation of the efficacy of IM administration of BCG Danish strain 1331 at two different doses: the dose prescribed for adult humans (2-8 × 10 5 colony forming units) and a 10-fold higher dose. Vaccination generated a dose-dependent cell-mediated immune response characterised by the production of interferon-␥ (IFN␥) and protection against endobronchial challenge with virulent M. bovis. Protection, expressed in terms of a significant reduction in the severity of disease, the number of tissues containing acid-fast bacilli, and reduced bacterial excretion was statistically significant with the higher dose only. Crow

Bacillus Calmette-Guérin vaccination reduces the severity and progression of tuberculosis in badgers

Control of bovine tuberculosis (TB) in cattle has proven particularly challenging where reservoirs of infection exist in wildlife populations. In Britain and Ireland, control is hampered by a reservoir of infection in Eurasian badgers (Meles meles). Badger culling has positive and negative effects on bovine TB in cattle and is difficult, costly and controversial. Here we show that Bacillus Calmette-Guérin (BCG) vaccination of captive badgers reduced the progression, severity and excretion of Mycobacterium bovis infection after experimental challenge. In a clinical field study, BCG vaccination of free-living badgers reduced the incidence of positive serological test results by 73.8 per cent. In common with other species, BCG did not appear to prevent infection of badgers subjected to experimental challenge, but did significantly reduce the overall disease burden. BCG vaccination of badgers could comprise an important component of a comprehensive programme of measures to control bovine TB in cattle