Mycobacterium avium Subspecies Paratuberculosis in the Inflamed Gut Tissues of Patients with Crohn’s Disease in China and its Potential Relationship to the Consumption of Cow’s Milk: A Preliminary Study

Summary Mycobacterium avium subspecies paratuberculosis infection in domestic livestock is widespread in many countries throughout the world. Studies in Europe and the USA show that M. avium subspecies paratuberculosis can be cultured from retail pasteurized cow’s milk and that these organisms are being transmitted to humans by this route. Most people with chronic inflammation of the intestine of the Crohn’s disease type are infected with these chronic enteric pathogens. The production and consumption of cow’s milk has increased in China and so also has the incidence of Crohn’s disease. The present preliminary investigation was carried out to determine whether M. avium subspecies paratuberculosis is present in the intestinal tissues of Chinese patients with Crohn’s disease who have never left China. Archival paraffin-embedded surgical pathology blocks from patients having surgery for Crohn’s disease (CD) or for cancer (nIBD) in China were studied. M. avium subspecies paratuberculosis was detected by nested IS900 PCR with Southern blotting and amplicon sequencing. The intestinal tissues of 9 of 13 (69.2%) CD patients and 2 of 14 (14.3%) nIBD patients were IS900 PCR positive (P = 0.0063; odds ratio = 13.5). These initial studies suggest that people in China are exposed to M. avium subspecies paratuberculosis and that as in other countries, the infection is significantly associated with Crohn’s disease. M. avium subspecies paratuberculosis in dairy herds and retail milk in China needs to be investigated.     

Localization of proteins in the cell wall of <Emphasis Type="Italic">Mycobacterium avium</Emphasis> subsp. <Emphasis Type="Italic">paratuberculosis</Emphasis> K10 by proteomic analysis

Mycobacterium avium subsp. paratuberculosis is a pathogen which causes a debilitating chronic enteritis in ruminants. Unfortunately, the mechanisms that control M. avium subsp. paratuberculosis persistence during infection are poorly understood and the key steps for developing Johne's disease remain elusive. A proteomic analysis approach, based on one dimensional polyacrylamide gel electrophoresis (SDS-PAGE) followed by LC-MS/MS, was used to identify and characterize the cell wall associated proteins of M. avium subsp. paratuberculosis K10 and an cell surface enzymatic shaving method was used to determine the surface-exposed proteins. 309 different proteins were identified, which included 101 proteins previously annotated as hypothetical or conserved hypothetical. 38 proteins were identified as surface-exposed by trypsin treatment. To categorize and analyze these proteomic data on the proteins identified within cell wall of M. avium subsp. paratuberculosis K10, a rational bioinformatic approach was followed. The analyses of the 309 cell wall proteins provided theoretical molecular mass and p I distributions and determined that 18 proteins are shared with the cell surface-exposed proteome. In short, a comprehensive profile of the M. avium subsp. paratuberculosis K10 cell wall subproteome was created. The resulting proteomic profile might become the foundation for the design of new preventive, diagnostic and therapeutic strategies against mycobacterial diseases in general and M. avium subsp. paratuberculosis in particular.     

Immunoreactivity of the <Emphasis Type="Italic">Mycobacterium avium</Emphasis> subsp. <Emphasis Type="Italic">paratuberculosis</Emphasis> 19-kDa lipoprotein

Background  

The Mycobacterium tuberculosis 19-kDa lipoprotein has been reported to stimulate both T and B cell responses as well as induce a number of Th1 cytokines. In order to evaluate the Mycobacterium avium subsp. paratuberculosis (M. avium subsp. paratuberculosis) 19-kDa lipoprotein as an immunomodulator in cattle with Johne's disease, the gene encoding the 19-kDa protein (MAP0261c) was analyzed.     

The autotransporter protein from Bordetella avium, Baa1, is involved in host cell attachment

Bordetella avium is a Gram negative upper respiratory tract pathogen of birds. B. avium infection of commercially raised turkeys is an agriculturally significant problem. Here we describe the functional analysis of the first characterized B. avium autotransporter protein, Baa1. Autotransporters comprise a large family of proteins found in all groups of Gram negative bacteria. Although not unique to pathogenic bacteria, autotransporters have been shown to perform a variety of functions implicated in virulence. To test the hypothesis that Baa1 is a B. avium virulence factor, unmarked baa1 deletion mutants (Δbaa1) were created and tested phenotypically. It was found that baa1 mutants have wild-type levels of serum sensitivity and infectivity, yet significantly lower levels of turkey tracheal cell attachment in vitro. Likewise, semi-purified recombinant His-tagged Baa1, expressed in Escherichia coli, was shown to bind specifically to turkey tracheal cells via western blot analysis. Taken together, we conclude that Baa1 acts as a host cell attachment factor and thus plays a role B. avium virulence.     

Visualization of Mycobacterium avium in Crohn's tissue by oil-immersion microscopy

Studies seeking Mycobacterium avium subsp. paratuberculosis in Crohn's disease by PCR have generated inconsistent findings. As an alternative, microscopy offers a number of advantages, including direct visualization of organisms in tissue. Experimental infections have demonstrated that M. avium organisms can be seen by both acid-fast staining and species-specific in situ hybridization, but because they are smaller than M. tuberculosis, oil-immersion microscopy (×1000 magnification) is needed. We performed a blinded search for M. avium in paraffin-embedded surgical resections from Crohn's and control subjects at two centres. Specimens were coded and subjected to acid-fast staining and ribosomal RNA in situ hybridization for M. avium rRNA. Agreement between these two methods was good (42/52 patients, κ = 0.60) and similar results were observed for patients from two centers. Together, both methods provided positive results in 10 of 17 Crohn's subjects (59%, 95% CI: 36–78), contrasting with only 5 of 35 control subjects (Odds ratio for Crohn's vs. controls = 8.6, p = 0.002). M. avium organisms had an intracellular localization within inflammatory lesions, but were often observed as lone organisms outside of granulomas. Using two assays in two settings, presence of M. avium organisms was strongly associated with Crohn's disease.     

Serological survey of selected infectious diseases in mouflon (Ovis aries musimon) from south-central Spain

Serum samples from 101 mouflons (Ovis aries musimon) collected from July 2002 to January 2006 were tested for antibodies against Anaplasma spp., Brucella spp., bovine viral diarrhea virus, Chlamydophila abortus, Coxiella burnetii, Mycobacterium avium ssp. paratuberculosis, and Maedi-Visna virus. Mouflon came either from extensive farms or high ungulate density fenced hunting estates. Antibodies were detected against Anaplasma spp. (22.2%), C. burnetii (4.0%), M. avium ssp. paratuberculosis (1.0%) and C. abortus (1.0%). According to our results, mouflons could participate as a wild reservoir in the epidemiology of Anaplasma spp. infection and maybe Q fever, but they do not seem to contribute in the epidemiology of the rest of the studied infectious diseases in south-central Spain.     

Killing of Mycobacterium avium subspecies paratuberculosis within macrophages

Background  

Mycobacterium avium subspecies paratuberculosis (M. paratuberculosis) is a facultative intracellular pathogen that resides within host macrophages during infection of ruminant animals. We examined survival of M. paratuberculosis infections within cultured macrophages to better understand the interplay between bacterium and host.     

Cytokine responses of bovine macrophages to diverse clinical Mycobacterium avium subspecies paratuberculosis strains

Background  

Mycobacterium avium subsp. paratuberculosis (MAP), the causative agent of Johne's disease (JD) persistently infects and survives within the host macrophages. While it is established that substantial genotypic variation exists among MAP, evidence for the correlates that associate specific MAP genotypes with clinical or sub-clinical disease phenotypes is presently unknown. Thus we studied strain differences in intracellular MAP survival and host responses in a bovine monocyte derived macrophage (MDM) system.     

Occurrence of Mycobacterium avium subsp. paratuberculosis in Untreated Water in Northern Ireland

Mycobacterium avium subsp. paratuberculosis is the known cause of Johne's disease of both domestic and wild ruminants and has been implicated as a possible cause of Crohn's disease in humans. The organism is shed in the feces of infected animals and can survive for protracted periods in the environment and hence could be present in catchment areas receiving agricultural runoff. A limited survey was undertaken in Northern Ireland to test for M. avium subsp. paratuberculosis in untreated water entering nine water treatment works (WTWs) over a 1-year period. Three detection methods were employed, viz., immunomagnetic separation-PCR and culture on Herrold's egg yolk medium (HEYM) and BACTEC 12B medium, the latter both supplemented with mycobactins. Of the 192 untreated water samples tested, 15 (8%) tested M. avium subsp. paratuberculosis positive by one or more of the three detection methods. M. avium subsp. paratuberculosis was successfully isolated from eight untreated water samples, three by BACTEC culture and five by culture on HEYM. Although the highest incidence of M. avium subsp. paratuberculosis was found in spring, overall, there was no statistically significant difference between the seasons. No significant correlation was found between numbers of coliforms or fecal coliforms and the presence of M. avium subsp. paratuberculosis. In general, a higher incidence of M. avium subsp. paratuberculosis was found in untreated water entering those WTWs that had a high mean water pH value over the sampling period. This work indicates the need to determine the efficacy of water treatment processes to either kill or remove M. avium subsp. paratuberculosis from untreated water and the possible risks posed by contact with recreational water sources.     

Fluorescence In Situ Hybridization Using Peptide Nucleic Acid Probes for Rapid Detection of Mycobacterium avium subsp. avium and Mycobacterium avium subsp. paratuberculosis in Potable-Water Biofilms

Here, we present for the first time a high-affinity peptide nucleic acid (PNA) oligonucleotide sequence for detecting Mycobacterium avium bacteria, including the opportunistically pathogenic subspecies M. avium subsp. avium, M. avium subsp. paratuberculosis, and M. avium subsp. silvaticum, by the fluorescence in situ hybridization (FISH) method. There is evidence that M. avium subsp. avium especially is able to survive and grow in drinking-water biofilms and possibly transmit via drinking water. The designed PNA probe (MAV148) specificity was tested with several bacterial species, including other mycobacteria and mycolic acid-containing bacteria. From the range of bacterial strains tested, only M. avium subsp. avium and M. avium subsp. paratuberculosis strains were hybridized. The PNA FISH method was applied successfully to detect M. avium subsp. avium spiked in water samples and biofilm established within a Propella biofilm reactor fed with potable water from a distribution supply.     

Replication and Long-Term Persistence of Bovine and Human Strains of Mycobacterium avium subsp. paratuberculosis within Acanthamoeba polyphaga

Free-living protists are ubiquitous in the environment and form a potential reservoir for the persistence of animal and human pathogens. Mycobacterium avium subsp. paratuberculosis is the cause of Johne's disease, a systemic infection accompanied by chronic inflammation of the intestine that affects many animals, including primates. Most humans with Crohn's disease are infected with this chronic enteric pathogen. Subclinical infection with M. avium subsp. paratuberculosis is widespread in domestic livestock. Infected animals excrete large numbers of robust organisms into the environment, but little is known about their ability to replicate and persist in protists. In the present study we fed laboratory cultures of Acanthamoeba polyphaga with bovine and human strains of M. avium subsp. paratuberculosis. Real-time PCR showed that the numbers of the pathogens fell over the first 4 to 8 days and recovered by 12 to 16 days. Encystment of the amoebic cultures after 4 weeks resulted in a 2-log reduction in the level of M. avium subsp. paratuberculosis, which returned to the original level by 24 weeks. Extracts of resection samples of human gut from 39 patients undergoing abdominal surgery were fed to cultures of A. polyphaga. M. avium subsp. paratuberculosis detected by nested IS900 PCR with amplicon sequencing and visualized by IS900 in situ hybridization and auramine-rhodamine staining was found in cultures derived from 13 of the patients and was still present in the cultures after almost 4 years of incubation. Control cultures were negative. M. avium subsp. paratuberculosis has the potential for long-term persistence in environmental protists.     

How accurately can we detect Mycobacterium avium subsp. paratuberculosis infection?

Mycobacteria have thwarted detection by scientists for centuries. Mycobacterium paratuberculosis is one of the most fastidious of the Mycobacteriaceae, and has been implicated in both animal and human diseases. In domestic livestock, M. paratuberculosis has been associated with Johne's disease, which given its increasing incidence, is currently a cause for concern, due to the potential for M. paratuberculosis to enter our food chain. In addition, a tenuous link has been reported between M. paratuberculosis and Crohn's disease, however evidence to support this link is hampered by the lack of accurate methodologies for detection of M. paratuberculosis in humans. This review compares the sensitivity and specificity of traditional and more recent techniques to the culture and molecular detection of M. paratuberculosis. While serology and culture are popular choices for the livestock industry they have not produced useful data for human infection. Although the advent of molecular biology has enabled faster diagnosis of M. paratuberculosis in human infection, there is currently no gold standard such as culture on which to validate these findings. Even with DNA/RNA detection methods, there is the ever present issue of the genetic relatedness of M. paratuberculosis to other mycobacteria of the Mycobacterium avium complex, some of which also infect humans with very different pathological outcomes. Recent developments in this field include more rapid methods of M. paratuberculosis culture as well as the development of more accurate and sensitive PCR assays. The application of these techniques should offer a greater insight as to the role of M. paratuberculosis in human gastrointestinal diseases.     

Clustering of Mycobacterium avium subsp. paratuberculosis in Rabbits and the Environment: How Hot Is a Hot Spot?

Clustering of pathogens in the environment leads to hot spots of diseases at local, regional, national, and international levels. Scotland contains regional hot spots of Johne's disease (caused by Mycobacterium avium subsp. paratuberculosis) in rabbits, and there is increasing evidence of a link between paratuberculosis infections in rabbits and cattle. The spatial and temporal dynamics of paratuberculosis in rabbits within a hot spot region were studied with the overall aim of determining environmental patterns of infection and thus the risk of interspecies transmission to livestock. The specific aims were to determine if prevalence of paratuberculosis in rabbits varies temporally between seasons and whether the heterogeneous spatial environmental distribution of M. avium subsp. paratuberculosis on a large scale (i.e., regional hot spots) is replicated at finer resolutions within a hot spot. The overall prevalence of M. avium subsp. paratuberculosis in rabbits was 39.7%; the temporal distribution of infection in rabbits followed a cyclical pattern, with a peak in spring of 55.4% and a low in summer of 19.4%. Spatially, M. avium subsp. paratuberculosis-infected rabbits and, thus, the risk of interspecies transmission were highly clustered in the environment. However, this is mostly due to the clustered distribution of rabbits. The patterns of M. avium subsp. paratuberculosis infection in rabbits are discussed in relation to the host's socioecology and risk to livestock.     

Demonstration of Allelic Exchange in the Slow-Growing Bacterium Mycobacterium avium subsp. paratuberculosis, and Generation of Mutants with Deletions at the pknG, relA, and lsr2 Loci

Mycobacterium avium subsp. paratuberculosis is the causative pathogen of Johne's disease, a chronic inflammatory wasting disease in ruminants. This disease has been difficult to control because of the lack of an effective vaccine. To address this need, we adapted a specialized transduction system originally developed for M. tuberculosis and modified it to improve the efficiency of allelic exchange in order to generate site-directed mutations in preselected M. avium subsp. paratuberculosis genes. With our novel optimized method, the allelic exchange frequency was 78 to 100% and the transduction frequency was 1.1 × 10⁻⁷ to 2.9 × 10⁻⁷. Three genes were selected for mutagenesis: pknG and relA, which are genes that are known to be important virulence factors in M. tuberculosis and M. bovis, and lsr2, a gene regulating lipid biosynthesis and antibiotic resistance. Mutants were successfully generated with a virulent strain of M. avium subsp. paratuberculosis (M. avium subsp. paratuberculosis K10) and with a recombinant K10 strain expressing the green fluorescent protein gene, gfp. The improved efficiency of disruption of selected genes in M. avium subsp. paratuberculosis should accelerate development of additional mutants for vaccine testing and functional studies.     

Detection of Mycobacterium avium subspecies paratuberculosis specific IS900 insertion sequences in bulk-tank milk samples obtained from different regions throughout Switzerland

Background  

Since Mycobacterium avium subspecies paratuberculosis (MAP) was isolated from intestinal tissue of a human patient suffering Crohn's disease, a controversial discussion exists whether MAP have a role in the etiology of Crohn's disease or not. Raw milk may be a potential vehicle for the transmission of MAP to human population. In a previous paper, we have demonstrated that MAP are found in raw milk samples obtained from a defined region in Switzerland. The aim of this work is to collect data about the prevalence of MAP specific IS900 insertion sequence in bulk-tank milk samples in different regions of Switzerland. Furthermore, we examined eventual correlation between the presence of MAP and the somatic cell counts, the total colony counts and the presence of Enterobacteriaceae.     

圈养条件下新疆马鹿两个亚种的肠道菌群比较分析

肠道菌群组成复杂，与宿主肠道内环境动态平衡和健康息息相关。马鹿是鹿科动物中分布最广的种类，亚种分化众多，但现有马鹿肠道菌群研究较少。为丰富马鹿肠道菌群数据，以马鹿的两个亚种为阶元，运用高通量16S rRNA基因测序技术检测了7头雄性天山马鹿和7头雄性塔里木马鹿的肠道微生物，对其核心菌群、结构组成、多样性及肠道微生物的差异进行分析，旨在了解不同马鹿亚种肠道微生物组成差异，为两亚种间肠道菌群的差异提供参考。研究结果表明，塔里木马鹿菌群多样性及丰度低于天山马鹿。厚壁菌门（Firmicutes）、放线菌门（Actinobacteria）、变形菌门（Proteobacteria）及拟杆菌门（Bacteroidetes）为两组马鹿的优势菌门。此外，天山马鹿肠道中分解蛋白质、脂质的拟杆菌门和变形菌门相对丰度高于塔里木马鹿，而分解植物纤维的双歧杆菌（Bifidobacterium）、瘤胃球菌科UCG 014（Ruminococcaceae UCG 014）的相对丰度低于塔里木马鹿。本研究对天山马鹿及塔里木马鹿肠道菌群组成进行初步了解，可根据研究结果调整其饲料配比，为马鹿饲养管理的改善提供参考。     

Comparative genomics between human and animal associated subspecies of the Mycobacterium avium complex: a basis for pathogenicity

Background

A human isolate of Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis 43525) was sequenced and compared genomically to other mycobacterial pathogens. M. paratuberculosis 43525 was recently isolated from a patient with ulcerative colitis and belongs to the M. avium complex, a group known to infect both humans and animals. While M. paratuberculosis is a known pathogen of livestock, there are only 20 human isolates from the last 20 years, therefore we took the opportunity to perform a whole genome comparison between human and animal mycobacterial pathogens. We also compared virulence determinants such as the mycobactin cluster, PE/PPE genes and mammalian cell entry (mce) operons between MAC subspecies that infect animals and those that infect humans. M. tuberculosis was also included in these analyses given its predominant role as a human pathogen.

Results

This genome comparison showed the PE/PPE profile of M. paratuberculosis 43525 to be largely the same as other M. paratuberculosis isolates, except that it had one PPE and one PE_PGRS protein that are only present in human MAC strains and M. tuberculosis. PE/PPE proteins that were unique to M. paratuberculosis 43525, M. avium subsp. hominissuis and a caprine M. paratuberculosis isolate, were also identified. In addition, the mycobactin cluster differed between human and animal isolates and a unique mce operon flanked by two mycobactin genes, mbtA and mbtJ, was identified in all available M. paratuberculosis genomes.

Conclusions

Despite the whole genome comparison placing M. paratuberculosis 43525 as closely related to bovine M. paratuberculosis, key virulence factors were similar to human mycobacterial pathogens. This study highlights key factors of mycobacterial pathogenesis in humans and forms the basis for future functional studies.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1889-2) contains supplementary material, which is available to authorized users.     

New probes used for IS1245 and IS1311 restriction fragment length polymorphism of Mycobacterium avium subsp. avium and Mycobacterium avium subsp. hominissuis isolates of human and animal origin in Norway

Background  

Mycobacterium avium is an environmental mycobacterium that can be divided into the subspecies avium, hominissuis, paratuberculosis and silvaticum. Some M. avium subspecies are opportunistic pathogens for animals and humans. They are ubiquitous in nature and can be isolated from natural sources of water, soil, plants and bedding material. Isolates of M. avium originating from humans (n = 37), pigs (n = 51) and wild birds (n = 10) in Norway were examined by IS1245 and IS1311 RFLP using new and specific probes and for the presence of IS901 and ISMpa1 by PCR. Analysis and generation of a dendrogram were performed with the software BioNumerics.     

New PCR systems to confirm real-time PCR detection of Mycobacterium avium subsp. paratuberculosis

Background  

Johne's disease, a serious chronic form of enteritis in ruminants, is caused by Mycobacterium avium subsp. paratuberculosis (MAP). As the organism is very slow-growing and fastidious, several PCR-based methods for detection have been developed, based mainly on the MAP-specific gene IS900. However, because this gene is similar to genes in other mycobacteria, there is a need for sensitive and reliable methods to confirm the presence of MAP. As described here, two new real-time PCR systems on the IS900 gene and one on the F57 gene were developed and carefully validated on 267 strains and 56 positive clinical faecal samples.