Effects of culling on badger abundance: implications for tuberculosis control

Culling is often considered as a tool for controlling wildlife diseases that can also infect people or livestock. Culling European badgers Meles meles can cause both positive and negative effects on the incidence of bovine tuberculosis (TB) in cattle. One factor likely to influence the outcome of different badger-culling strategies for cattle TB is the reduction in badger population density achieved. However, this reduction is difficult to measure because badgers, being nocturnal and fossorial, are difficult to count. Here, we use indices of badger abundance to measure the population impacts of two culling strategies tested in Britain. The densities of badger setts and latrines recorded before culling were correlated with the densities of badgers captured on initial culls, suggesting that both were indices of actual badger abundance. Widespread 'proactive' culling was associated with a 73% reduction in the density of badger latrines, a 69% reduction in the density of active burrows and a 73% reduction in the density of road-killed badgers. This population reduction was achieved by a coordinated effort entailing widespread and repeated trapping over several years. However, this strategy caused only modest reductions in cattle TB incidence in culled areas and elevated incidence in neighbouring unculled areas. Localized 'reactive' culling caused a 26% reduction in latrine density, a 32% reduction in active burrow density and a 10% reduction in the density of road-killed badgers, but apparently increased the incidence of cattle TB. These results indicate that the relationship between badger population reduction and TB transmission to cattle is strongly non-linear, probably because culling prompts changes in badger behaviour that influence transmission rates. These findings raise serious questions about the capacity of badger culling to contribute to the control of cattle TB in Britain.     

Immunomagnetic recovery of Mycobacterium bovis from naturally infected environmental samples

AIMS: To adapt an immunomagnetic capture (IMC) technique to concentrate and cultivate Mycobacterium bovis from environmental samples including soil, faeces and urine. METHODS AND RESULTS: Cells of Myco. bovis BCG and wild-type Myco. bovis were successfully isolated and cultured from seeded and naturally infected materials respectively. The IMC cell recovery estimated by colony forming units (CFUs) counts ranged from 0.10% to 0.16% for spiked media, and 0.15-0.36% for naturally infected soil and faeces. Recovery estimated by cell counts calculated using semi-quantitative PCR ranged from 80.3% to 88.6% for spiked and 84.1-88.2% for naturally infected material. The differences in the recovery rates estimated by CFUs compared with pixel intensity is likely to be due to clustering of cells on culture plates, thereby underestimating the true cell count. CONCLUSIONS: The IMC techniques can be applied to isolate viable wild type Myco. bovis from naturally contaminated environmental samples. SIGNIFICANCE AND IMPACT OF STUDY: Cultivation of Myco. bovis from environmental samples using traditional methods is extremely problematic. Here, we demonstrate a novel development of IMC techniques that will greatly facilitate the study of the organism in situ in order to assess its epidemiological importance in bovine tuberculosis persistence.     

Tuberculosis patients co-infected with Mycobacterium bovis and Mycobacterium tuberculosis in an urban area of Brazil

In this cross-sectional study, mycobacteria specimens from 189 tuberculosis (TB) patients living in an urban area in Brazil were characterised from 2008-2010 using phenotypic and molecular speciation methods (pncA gene and oxyR pseudogene analysis). Of these samples, 174 isolates simultaneously grew on Löwenstein-Jensen (LJ) and Stonebrink (SB)-containing media and presented phenotypic and molecular profiles of Mycobacterium tuberculosis, whereas 12 had molecular profiles of M. tuberculosis based on the DNA analysis of formalin-fixed paraffin wax-embedded tissue samples (paraffin blocks). One patient produced two sputum isolates, the first of which simultaneously grew on LJ and SB media and presented phenotypic and molecular profiles of M. tuberculosis, and the second of which only grew on SB media and presented phenotypic profiles of Mycobacterium bovis. One patient provided a bronchial lavage isolate, which simultaneously grew on LJ and SB media and presented phenotypic and molecular profiles of M. tuberculosis, but had molecular profiles of M. bovis from paraffin block DNA analysis, and one sample had molecular profiles of M. tuberculosis and M. bovis identified from two distinct paraffin blocks. Moreover, we found a low prevalence (1.6%) of M. bovis among these isolates, which suggests that local health service procedures likely underestimate its real frequency and that it deserves more attention from public health officials.     

Polymorphisms of 20 regulatory proteins between Mycobacterium tuberculosis and Mycobacterium bovis

Mycobacterium tuberculosis and Mycobacterium bovis are responsible for tuberculosis in humans and animals, respectively. Both species are closely related and belong to the Mycobacterium tuberculosis complex (MTC). M. tuberculosis is the most ancient species from which M. bovis and other members of the MTC evolved. The genome of M. bovis is over >99.95% identical to that of M. tuberculosis but with seven deletions ranging in size from 1 to 12.7 kb. In addition, 1200 single nucleotide mutations in coding regions distinguish M. bovis from M. tuberculosis. In the present study, we assessed 75 M. tuberculosis genomes and 23 M. bovis genomes to identify non‐synonymous mutations in 202 coding sequences of regulatory genes between both species. We identified species‐specific variants in 20 regulatory proteins and confirmed differential expression of hypoxia‐related genes between M. bovis and M. tuberculosis.     

牛分支杆菌与肺结核分支杆菌基因组的比较

通过比较基因组学的方法研究发现,牛分支杆菌与肺结核杆菌基因组的同源性为99．95％,但在牛分枝杆菌基因组中有11个缺失区,大小从1kb到12．7kb,遗传信息的缺失引起牛分枝杆菌的基因组减小;牛分枝杆菌与肺结核分枝杆菌H37Rv间存在着2437个单核苷酸多态性（SNPs）,与肺结核分枝杆菌CDC1551间存在着2423个单核苷酸多态性（SNPs）,牛分支杆菌与肺结核分枝杆菌在编码细胞壁和分泌蛋白上变异程度也是巨大的。研究结果揭示了牛分支杆菌与肺结核分枝杆菌的遗传关系,为研究分支杆菌疫苗和诊断试剂提供理论依据,对牛肺结核病的防治有着非常重要的意义。     

cAMP levels within Mycobacterium tuberculosis and Mycobacterium bovis BCG increase upon infection of macrophages

Adenosine 3',5'-cyclic monophosphate (cAMP)-mediated signal transduction is common in both prokaryotes and eukaryotes, and several bacterial pathogens modulate cAMP signaling pathways of their mammalian hosts during infection. In this study, cAMP levels associated with Mycobacterium tuberculosis and Mycobacterium bovis BCG were measured during macrophage infection. cAMP levels within both bacteria increased c . 50-fold during infection of J774.16 macrophages, relative to the cAMP levels within bacteria incubated in tissue culture media alone. cAMP levels also increased within the macrophage cytoplasm upon uptake of live, but not dead, mycobacteria. The presence of albumin in the absence of oleic acid significantly decreased cAMP secretion and production by both M. tuberculosis and M. bovis BCG. These results suggest that cAMP signaling plays a role in the interaction of tuberculosis-complex mycobacteria with macrophages during infection, and that albumin may be a physiological indicator differentiating host environments during infection.     

The Effect of Long-Term Storage on Mycobacterium bovis

It was established that when stored for many years (10–13 years) in low-temperature conditions (3°C), without sub-culture on a nutrient medium, Mycobacterium bovis grew as visible colonies along the line of inoculation. However, due to long-term storage in conditions of low temperature (3°C) morphology of mycobacteria differed significantly from initial cultures formed by rod-shaped bacteria. Some of them became pigment-forming and smooth on the surface. Unlike the initial strain of mycobacteria, a perennial bacteria stored under hard conditions did not cause the death of guinea pigs or their sensitization to a purified protein derivative for mammals. Morphological forms of the perennial mycobacteria had the following changes: pigment forming, L-forms of the vesicular type, non-acid-fast thread-like (filamentous) bacillary forms, and elementary bodies when compared to the initial strain. There were also some genetic changes in the target DNA due to the long-term storage of M. bovis. It may indicate a mutation in the pathogen’s DNA. These mycobacteria had altered biochemical activity during storage. The number of passages on the solid nutrient medium did not affect their fermentative activity. However, the low cultivation temperature increases mycobacterial catalase activity and the ability to hydrolyze Tween-80.     

Study of the immunological profile towards Mycobacterium bovis antigens in naturally infected cattle

A number of studies have determined the contribution of Th1 and Th2 responses to the protective immunity and pathology of Mycobacterium bovis infection. However, much of that information is derived from experimentally infecting cattle with M. bovis and few data from naturally infected animals are available. The aim of this study was to characterize the immunological profile towards M. bovis antigens of naturally infected cattle by measurement of cytokine mRNA expression in PBMC, and to determine which lymphocyte subsets are involved in recall responses of PBMC from M. bovis infected cattle to M. bovis antigens. Consistent with data from cattle experimentally infected with M. bovis , naturally infected animals were found to display a Th1 cytokine profile in response to M. bovis PPDB stimulation. Production of IFN-γ mRNA by PBMC after PPDB stimulation statistically distinguishes between infected and healthy herds, suggesting that this molecule is usable as an M. bovis -infection marker. As happens in experimentally infected cows, CD4, CD8 and γδTCR cells from a herd naturally infected with M . bovis are the predominant T cell subsets expanded in response to PPDB.     

Comparison of decontamination methods for primary isolation of Mycobacterium bovis in paucibacillary bovine tissues

Aims: To compare three decontamination methods applied to paucibacillary samples for primary isolation of Mycobacterium bovis from suspect lesions. Tuberculosis caused by Myco. bovis is an important infectious disease of cattle in Brazil and also has zoonotic potential. Although a national campaign based on testing and slaughtering cattle has achieved good results, there is a strong need to develop better diagnostic methods to identify cattle with recent infections harbouring few bacilli. Methods and Results: A dairy herd (274 adult crossbred cows) located in the state of Rio de Janeiro was tested for tuberculosis with both single intradermal tuberculin test and comparative intradermal tuberculin test. Reactive cows (n = 27, 9·8%) were slaughtered and suspect lesions were collected (one sample per cow). Samples considered paucibacillary (based on microscopy) were decontaminated with 0·75% hexadecylpyridinium chloride (HPC), 4% sodium hydroxide (Petroff) or 6% sulphuric acid. Using these methods, 10, five and six, respectively, of the 27 samples yielded positive cultures. Overall, Myco. bovis was isolated from 14 of 24 cows. Although the HPC method resulted in isolation of more Myco. bovis strains than either Petroff or sulphuric acid methods (P = 0·015), it did not result in the recovery of Myco. bovis from all samples. However, using both HPC and 6% sulphuric acid methods for decontamination was possible to identify 13 of 14 (92·9%) of infected cows. Conclusions: At least two methods should be used concurrently for primary isolation of Myco. bovis from bovine tissues, particularly for paucibacillary samples. Significance and Impact of the Study: Detection of low numbers of Myco. bovis in tissue is an important goal in optimizing the detection of bovine tuberculosis and should assist in identification of infected cattle, in particular, those with few Myco. bovis bacilli. This was apparently the first study comparing three decontamination methods for the detection of Myco. bovis in paucibacillary samples from naturally infected cattle.     

Localized reactive badger culling increases risk of bovine tuberculosis in nearby cattle herds

Human and livestock diseases can be difficult to control where infection persists in wildlife populations. Control of bovine tuberculosis (bTB) in British cattle is complicated by the maintenance of Mycobacterium bovis (the causative agent of bTB) in badgers, acting as reservoirs of infection. Although over 20 000 badgers were culled to control bTB between 1975 and 1997, the incidence of bTB in cattle has substantially increased in parts of Great Britain in recent decades. Our case-control study, involving 1208 cattle herds, provides further evidence of the detrimental effect of localized reactive badger culling in response to the disclosure of a confirmed bTB herd breakdown in cattle. The presence of any reactive badger culling activity and increased numbers of badgers culled in the vicinity of a herd were associated with significantly increased bTB risk, even after adjusting for other important local risk factors. Such findings may partly explain why some earlier localized approaches to bTB control were ineffective.     

The role of setts in badger (Meles meles) group size, breeding success and status of TB (Mycobacterium bovis)

This paper examines the relationship between the number of occupied setts in a badger social group territory and badger group size, breeding success, and status of infection with Mycobacterium bovis (TB). The data used were from a long-term epidemiological and ecological study of a high-density population of badgers Meles meles in south-west England. The number of occupied setts in a social group was significantly and positively related to the number of badgers caught in the social group, so that as a social group increases in size, badgers occupy more of the available setts. This relationship remained significant when numbers of adults, adult males and adult females were examined. The number of breeding females, number of cubs and sex ratio was not related to the number of occupied setts in a social group. It is possible that the advantages to breeding females of a larger number of setts available to breed in might be outweighed by the increased aggression found in larger groups. The TB score for prevalence and for incidence of social groups was significantly and positively related to the number of occupied setts in a social group, such that the more occupied setts there were in a territory, the higher the TB index of the group. Possibly the setts themselves contribute to the persistence of TB within social groups, or badgers infected with TB might show a difference in behaviour from uninfected badgers resulting in their increased use of outlying setts.     

The Mycobacterium bovis homologous protein of the Mycobacterium tuberculosis serine/threonine protein kinase Mbk (PknD) is truncated

We previously identified a 70-kDa serine/threonine protein kinase (MbK or PknD) from Mycobacterium tuberculosis Erdman containing a transmembrane domain and bearing a 270-amino acid N-terminal kinase domain. With the use of a polyclonal serum, Mbk has now been identified by Western blotting in protein extracts from M. tuberculosis and confirmed to be localised in the envelope. An identical mbk gene has been found by sequencing different M. tuberculosis and M. africanum strains. Surprisingly, in two virulent M. bovis strains and four different strains of M. bovis BCG, an additional adenine after position 829 of the open reading frame was found that produces a frame shift resulting in a predicted truncated, presumably free cytoplasmic protein, encoding only the N-terminal 30-kDa Mbk kinase domain. This sequence polymorphism has been confirmed by Western blot analysis of M. bovis BCG protein extracts.     

Sterilization as an alternative strategy to control wildlife diseases: bovine tuberculosis in European badgers as a case study

Sterilization has rarely been considered as an alternative to culling or vaccination to control wildlife diseases. Disease control by sterilization, as by culling, has most promise when the host'ss ability for compensatory growth following the removal of density-dependent inhibitions is limited, and when moderate reductions in population density cause disproportionately large reductions in disease prevalence, or even eliminate the disease. For many host/disease examples this will not be the case and vaccination may have overwhelming advantages or may be the only practical option. The impact of sterilization on host density and disease prevalence will develop relatively slowly because sterilization can prevent the recruitment of only one age-cohort at a time. Moreover, unless there is vertical transmission, this age-cohort will consist only of susceptibles. Culling, on the contrary, removes infected as well as susceptible animals. However, for certain disease/host examples, the r elative effectiveness of the different control strategies may be altered considerably if their variable effects on the probability of disease transmission are taken into account. Social perturbation or stress could render certain culling strategies ineffective or even counter-productive. Depending on how disease dynamics are influenced by the host'ss age-structure and reproductive investment, fertility control could offer epidemiological advantages that have been ignored by most disease/host models. We illustrate some of these principles by investigating the theoretical and practical feasibility of an hypothetical sterilization campaign to control bovine tuberculosis in badgers (and hence cattle) in Britain.     

Polymorphisms of the SLC11A1 gene and resistance to bovine tuberculosis in African Zebu cattle

Bovine tuberculosis (BTB) is a considerable health threat to livestock keepers and general communities in many developing countries. Information on genetic resistance or susceptibility because of polymorphisms of candidate genes could be used in making selection decisions for breeding disease tolerant/resistant animals. Here, we investigated associations between polymorphisms at the solute carrier family 11 (proton‐coupled divalent metal ion transporters), member 1 gene (SLC11A1, previously known as natural resistant associated macrophage protein 1, NRAMP1), with BTB phenotypes in Chadian cattle. Phenotypes were (i) single intradermal comparative cervical tuberculin test (SICCT) outcome, (ii) presence of gross visible lung lesions, (iii) a bacteriological culture test outcome and (iv) a predicted true BTB infection status using a Bayesian model. All traits were recorded as binary (presence or absence) traits. A total of 211 cattle were genotyped for a microsatellite within the SLC11A1 candidate gene. Standard linear and threshold‐liability models regressing BTB traits on copy number of SLC11A1 alleles revealed statistically significant effects of SLC11A1 alleles (P < 0.001) on most BTB traits. Polymorphisms (alleles 211, 215 and 217) are significantly related to lower incidence of BTB traits in Chadian cattle. This is the first study to report the association of SLC11A1 gene polymorphisms with BTB traits in Chadian or any other African cattle breeds.