Evalution of the “Iceberg Phenomenon” in Johne's Disease through Mathematical Modelling

Johne''s disease (JD) is a chronic, enteric disease in ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP). Disease progression follows four distinct stages: silent, subclinical, clinical and advanced. Available diagnostic tests have poor sensitivity and cannot detect early stages of the infection; as a result, only animals in the clinical and advanced stages, which represent the tip of the ‘iceberg’, are identified through testing. The Iceberg Phenomenon is then applied to provide estimates for JD prevalence. For one animal in the advanced stage, it is assumed that there are one to two in the clinical stage, four to eight in the subclinical stage, and ten to fourteen in the silent stage. These ratios, however, are based on little evidence. To evaluate the ratios, we developed a deterministic ordinary differential equation model of JD transmission and disease progression dynamics. When duration periods associated with the natural course of the disease progression are used, the above ratios do not hold. The ratios used to estimate JD prevalence need to be further investigated.     

Survival of Mycobacterium avium subsp. paratuberculosis in the intermediate and final digestion products of biogas plants

Aims

To evaluate the survival of Mycobacterium avium subsp. paratuberculosis (MAP) during anaerobic digestion (AD), we studied two different biogas plants loaded with manure and slurry from paratuberculosis‐infected dairy herds.

Methods and Results

Both plants were operating under mesophilic conditions, the first with a single digester and the second with a double digester. Mycobacterium avium subsp. paratuberculosis detection was performed by sampling each stage of the process, specifically the prefermenter, fermenter, liquid digestate and solid digestate stages, for 11 months. In both plants, MAP was isolated from the prefermenter stage. Only the final products, the solid and liquid digestates, of the one‐stage plant showed viable MAP, while no viable MAP was detected in the digestates of the two‐stage plant.

Conclusions

Mycobacterium avium subsp. paratuberculosis showed a significant decrease during subsequent steps of the AD process, particularly in the two‐stage plant. We suggest that the second digester maintained the digestate under anaerobic conditions for a longer period of time, thus reducing MAP survival and MAP load under the culture detection limit.

Significance and Impact of the Study

Our data are unable to exclude the presence of MAP in the final products of the biogas plants, particularly those products from the single digester; therefore, the use of digestates as fertilizers is a real concern related to the possible environmental contamination with MAP.     

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.     

Comparison of linkage disequilibrium and haplotype diversity on macro- and microchromosomes in chicken

Background

Toll like receptors (TLR) play the central role in the recognition of pathogen associated molecular patterns (PAMPs). Mutations in the TLR1, TLR2 and TLR4 genes may change the ability to recognize PAMPs and cause altered responsiveness to the bacterial pathogens.

Results

The study presents association between TLR gene mutations and increased susceptibility to Mycobacterium avium subsp. paratuberculosis (MAP) infection. Novel mutations in TLR genes (TLR1- Ser150Gly and Val220Met; TLR2 – Phe670Leu) were statistically correlated with the hindrance in recognition of MAP legends. This correlation was confirmed subsequently by measuring the expression levels of cytokines (IL-4, IL-8, IL-10, IL-12 and IFN-γ) in the mutant and wild type moDCs (mocyte derived dendritic cells) after challenge with MAP cell lysate or LPS. Further in silico analysis of the TLR1 and TLR4 ectodomains (ECD) revealed the polymorphic nature of the central ECD and irregularities in the central LRR (leucine rich repeat) motifs.

Conclusion

The most critical positions that may alter the pathogen recognition ability of TLR were: the 9^th amino acid position in LRR motif (TLR1–LRR10) and 4^th residue downstream to LRR domain (exta-LRR region of TLR4). The study describes novel mutations in the TLRs and presents their association with the MAP infection.     

Mycobacterium avium subsp. avium and subsp. hominissuis give different cytokine responses after in vitro stimulation of human blood mononuclear cells

Background

Mycobacterium avium is the principal etiologic agent of non-tuberculous lymphadenitis in children. It is also a known pathogen for birds and other animals. Genetic typing of M. avium isolates has led to a proposal to expand the set of subspecies to include M. avium subsp. hominissuis. Isolates associated with disease in humans belong to this subspecies.

Methodology/Principal Findings

Peripheral blood mononuclear cells from six healthy blood donors were stimulated in vitro with ten isolates of M. avium avium and 11 isolates of M. avium hominissuis followed by multiplex bead array quantification of cytokines in supernatants. M. avium hominissuis isolates induced significantly more IL-10 and significantly less IL-12p70, TNF, IFN-γ and IL-17 when compared to M. avium avium isolates. All strains induced high levels of IL-17, but had very low levels of IL-12p70.

Conclusion/Significance

The strong association between M. avium subsp. hominissuis and disease in humans and the clear differences in the human immune response to M. avium subsp. hominissuis compared to M. avium subsp. avium isolates, as demonstrated in this study, suggest that genetic differences between M. avium isolates play an important role in the pathogenicity in humans.     

Proteomic analysis of purified protein derivative of Mycobacterium tuberculosis

Background

Purified protein derivative (PPD) has been used for more than half a century as an antigen for the diagnosis of tuberculosis infection based on delayed type hypersensitivity. Although designated as “purified,” in reality, the composition of PPD is highly complex and remains ill-defined. In this report, high resolution mass spectrometry was applied to understand the complexity of its constituent components. A comparative proteomic analysis of various PPD preparations and their functional characterization is likely to help in short-listing the relevant antigens required to prepare a less complex and more potent reagent for diagnostic purposes.

Results

Proteomic analysis of Connaught Tuberculin 68 (PPD-CT68), a tuberculin preparation generated from M. tuberculosis, was carried out in this study. PPD-CT68 is the protein component of a commercially available tuberculin preparation, Tubersol, which is used for tuberculin skin testing. Using a high resolution LTQ-Orbitrap Velos mass spectrometer, we identified 265 different proteins. The identified proteins were compared with those identified from PPD M. bovis, PPD M. avium and PPD-S2 from previous mass spectrometry-based studies. In all, 142 proteins were found to be shared between PPD-CT68 and PPD-S2 preparations. Out of the 354 proteins from M. tuberculosis–derived PPDs (i.e. proteins in either PPD-CT68 or PPD-S2), 37 proteins were found to be shared with M. avium PPD and 80 were shared with M. bovis PPD. Alignment of PPD-CT68 proteins with proteins encoded by 24 lung infecting bacteria revealed a number of similar proteins (206 bacterial proteins shared epitopes with 47 PPD-CT68 proteins), which could potentially be involved in causing cross-reactivity. The data have been deposited to the ProteomeXchange with identifier PXD000377.

Conclusions

Proteomic and bioinformatics analysis of different PPD preparations revealed commonly and differentially represented proteins. This information could help in delineating the relevant antigens represented in various PPDs, which could further lead to development of a lesser complex and better defined skin test antigen with a higher specificity and sensitivity.     

Decreased Expression of Matrix Metalloproteinase-9 and Increased Expression of Tissue Inhibitors of Matrix Metalloproteinase-1 in Paratuberculosis-Infected Cattle in the ELISA-Negative Subclinical Stage

We investigated the gene expression of matrix metalloproteinases-9 (MMP-9) and tissue inhibitors of matrix metalloproteinases-1 (TIMP-1) in peripheral blood cells from infected cattle with Mycobacterium avium subsp. paratuberculosis (Map) in the ELISA-negative subclinical stage compared with uninfected control cattle. Significant decreased MMP-9 expression and increased TIMP-1 expression were found in peripheral blood cells from Map-infected cattle after stimulation with Map lysate and Map purified protein derivative (PPD) than in control cattle by real-time RT-PCR analysis. In contrast to the uninfected controls, the activity of MMP-9 was also decreased in peripheral blood cell culture supernatants from Map-infected cattle at 24 hr after Map lysate and MapPPD stimulation by gelatin zymography analysis. As a result, the MMP-9 may play an important role in the development of Mycobacterium avium subsp. paratuberculosis disease.     

Isolation of Mycobacterium avium Subspecies paratuberculosis Reactive CD4 T Cells from Intestinal Biopsies of Crohn's Disease Patients

Background

Crohn''s disease (CD) is a chronic granulomatous inflammation of the intestine. The etiology is unknown, but an excessive immune response to bacteria in genetically susceptible individuals is probably involved. The response is characterized by a strong Th1/Th17 response, but the relative importance of the various bacteria is not known.

Methodology/Principal Findings

In an attempt to address this issue, we made T-cell lines from intestinal biopsies of patients with CD (n = 11), ulcerative colitis (UC) (n = 13) and controls (n = 10). The T-cell lines were tested for responses to various bacteria. A majority of the CD patients with active disease had a dominant response to Mycobacterium avium subspecies paratuberculosis (MAP). The T cells from CD patients also showed higher proliferation in response to MAP compared to UC patients (p<0.025). MAP reactive CD4 T-cell clones (n = 28) were isolated from four CD patients. The T-cell clones produced IL-17 and/or IFN-γ, while minimal amounts of IL-4 were detected. To further characterize the specificity, the responses to antigen preparations from different mycobacterial species were tested. One T-cell clone responded only to MAP and the very closely related M. avium subspecies avium (MAA) while another responded to MAP, MAA and Mycobacterium intracellulare. A more broadly reactive T-cell clone reacted to MAP1508 which belongs to the esx protein family.

Conclusions/Significance

The presence of MAP reactive T cells with a Th1 or Th1/Th17 phenotype may suggest a possible role of mycobacteria in the inflammation seen in CD. The isolation of intestinal T cells followed by characterization of their specificity is a valuable tool to study the relative importance of different bacteria in CD.     

Virulence-related <Emphasis Type="Italic">Mycobacterium avium</Emphasis> subsp <Emphasis Type="Italic">hominissuis</Emphasis> MAV_2928 gene is associated with vacuole remodeling in macrophages

Background  

Mycobacterium avium subsp hominissuis (previously Mycobacterium avium subsp avium) is an environmental organism associated with opportunistic infections in humans. Mycobacterium hominissuis infects and replicates within mononuclear phagocytes. Previous study characterized an attenuated mutant in which the PPE gene (MAV_2928) homologous to Rv1787 was inactivated. This mutant, in contrast to the wild-type bacterium, was shown both to have impaired the ability to replicate within macrophages and to have prevented phagosome/lysosome fusion.     

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.     

Disruption of Mycobacterium avium subsp. paratuberculosis-specific genes impairs in vivo fitness

Background

Mycobacterium avium subsp. paratuberculosis (MAP) is an obligate intracellular pathogen that infects many ruminant species. The acquisition of foreign genes via horizontal gene transfer has been postulated to contribute to its pathogenesis, as these genetic elements are absent from its putative ancestor, M. avium subsp. hominissuis (MAH), an environmental organism with lesser pathogenicity. In this study, high-throughput sequencing of MAP transposon libraries were analyzed to qualitatively and quantitatively determine the contribution of individual genes to bacterial survival during infection.

Results

Out of 52384 TA dinucleotides present in the MAP K-10 genome, 12607 had a MycoMarT7 transposon in the input pool, interrupting 2443 of the 4350 genes in the MAP genome (56%). Of 96 genes situated in MAP-specific genomic islands, 82 were disrupted in the input pool, indicating that MAP-specific genomic regions are dispensable for in vitro growth (odds ratio = 0.21). Following 5 independent in vivo infections with this pool of mutants, the correlation between output pools was high for 4 of 5 (R = 0.49 to 0.61) enabling us to define genes whose disruption reproducibly reduced bacterial fitness in vivo. At three different thresholds for reduced fitness in vivo, MAP-specific genes were over-represented in the list of predicted essential genes. We also identified additional genes that were severely depleted after infection, and several of them have orthologues that are essential genes in M. tuberculosis.

Conclusions

This work indicates that the genetic elements required for the in vivo survival of MAP represent a combination of conserved mycobacterial virulence genes and MAP-specific genes acquired via horizontal gene transfer. In addition, the in vitro and in vivo essential genes identified in this study may be further characterized to offer a better understanding of MAP pathogenesis, and potentially contribute to the discovery of novel therapeutic and vaccine targets.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-415) contains supplementary material, which is available to authorized users.     

Development of a New, Combined Rapid Method Using Phage and PCR for Detection and Identification of Viable Mycobacterium paratuberculosis Bacteria within 48 Hours

The FASTPlaqueTB assay is an established diagnostic aid for the rapid detection of Mycobacterium tuberculosis from human sputum samples. Using the FASTPlaqueTB assay reagents, viable Mycobacterium avium subsp. paratuberculosis cells were detected as phage plaques in just 24 h. The bacteriophage used does not infect M. avium subsp. paratuberculosis alone, so to add specificity to this assay, a PCR-based identification method was introduced to amplify M. avium subsp. paratuberculosis-specific sequences from the DNA of the mycobacterial cell detected by the phage. To give further diagnostic information, a multiplex PCR method was developed to allow simultaneous amplification of either M. avium subsp. paratuberculosis or M. tuberculosis complex-specific sequences from plaque samples. Combining the plaque PCR technique with the phage-based detection assay allowed the rapid and specific detection of viable M. avium subsp. paratuberculosis in milk samples in just 48 h.     

An intra-laboratory cultural and real-time PCR method comparison and evaluation for the detection of subclinical paratuberculosis in dairy herds

Mycobacterium avium subsp. paratuberculosis (MAP) is a vigorous microorganism which causes incurable chronic enteritis, Johne’s disease (JD) in cattle. A target of control programmes for JD is to accurately detect MAP-infected cattle early to reduce disease transmission. The present study evaluated the efficacy of two different cultural procedures and a TaqMan real-time PCR assay for detection of subclinical paratuberculosis in dairy herds. Therefore, sixty-one faecal samples were collected from two Dutch dairy herds (n = 40 and n = 21, respectively) which were known to be MAP-ELISA positive. All individual samples were assessed using two different cultural protocols in two different laboratories. The first cultural protocol (first laboratory) included a decontamination step with 0.75% hexadecylpyridinium chloride (HPC) followed by inoculation on Herrold’s egg yolk media (HEYM). The second protocol (second laboratory) comprised of a decontamination step using 4% NaOH and malachite green-oxalic acid followed by inoculation on two media, HEYM and in parallel on modified Löwenstein-Jensen media (mLJ). For the TaqMan real-time PCR assay, all faecal samples were tested in two different laboratories using TaqMan® MAP (Johne’s) reagents (Life Technologies). The cultural procedures revealed positive reactions in 1.64% of the samples for cultivation protocol 1 and 6.56 and 8.20% of the samples for cultivation protocol 2, respectively. The results of the TaqMan real-time PCR performed in two different laboratories yielded 13.11 and 19.76% positive reaction. The kappa test showed proportional agreement 0.54 between the mLJ media (second laboratory) and TaqMan® real-time PCR method (second laboratory). In conclusion, the TaqMan real-time PCR could be a strongly useful and efficient assay for the detection of subclinical paratuberculosis in dairy cattle leading to an improvement in the efficiency of MAP control strategies.     

Assessing the Inactivation of Mycobacterium avium subsp. paratuberculosis during Composting of Livestock Carcasses

Mycobacterium avium subsp. paratuberculosis causes Johne''s disease (JD) in ruminants, with substantial economic impacts on the cattle industry. Johne''s disease is known for its long latency period, and difficulties in diagnosis are due to insensitivities of current detection methods. Eradication is challenging as M. avium subsp. paratuberculosis can survive for extended periods within the environment, resulting in new infections in naïve animals (W. Xu et al., J. Environ. Qual. 38:437-450, 2009). This study explored the use of a biosecure, static composting structure to inactivate M. avium subsp. paratuberculosis. Mycobacterium smegmatis was also assessed as a surrogate for M. avium subsp. paratuberculosis. Two structures were constructed to hold three cattle carcasses each. Naturally infected tissues and ground beef inoculated with laboratory-cultured M. avium subsp. paratuberculosis and M. smegmatis were placed in nylon and plastic bags to determine effects of temperature and compost environment on viability over 250 days. After removal, samples were cultured and growth of both organisms was assessed after 12 weeks. After 250 days, M. avium subsp. paratuberculosis was still detectable by PCR, while M. smegmatis was not detected after 67 days of composting. Furthermore, M. avium subsp. paratuberculosis remained viable in both implanted nylon and plastic bags over the composting period. As the compost never reached a homogenous thermophilic (55 to 65°C) state throughout each structure, an in vitro experiment was conducted to examine viability of M. avium subsp. paratuberculosis after exposure to 80°C for 90 days. Naturally infected lymph tissues were mixed with and without compost. After 90 days, M. avium subsp. paratuberculosis remained viable despite exposure to temperatures typically higher than that achieved in compost. In conclusion, it is unlikely composting can be used as a means of inactivating M. avium subsp. paratuberculosis associated with cattle mortalities.     

Linking Chronic Infection and Autoimmune Diseases: Mycobacterium avium Subspecies paratuberculosis,SLC11A1 Polymorphisms and Type-1 Diabetes Mellitus

Background

The etiology of type 1 diabetes mellitus (T1DM) is still unknown; numerous studies are performed to unravel the environmental factors involved in triggering the disease. SLC11A1 is a membrane transporter that is expressed in late endosomes of antigen presenting cells involved in the immunopathogenic events leading to T1DM. Mycobacterium avium subsp. paratuberculosis (MAP) has been reported to be a possible trigger in the development of T1DM.

Methodology/Principal Findings

Fifty nine T1DM patients and 79 healthy controls were genotyped for 9 polymorphisms of SLC11A1 gene, and screened for the presence of MAP by PCR. Differences in genotype frequency were evaluated for both T1DM patients and controls. We found a polymorphism in the SLC11A1 gene (274C/T) associated to type 1 diabetic patients and not to controls. The presence of MAP DNA was also significantly associated with T1DM patients and not with controls.

Conclusions/Significance

The 274C/T SCL11A1 polymorphism was found to be associated with T1DM as well as the presence of MAP DNA in blood. Since MAP persists within macrophages and it is also processed by dendritic cells, further studies are necessary to evaluate if mutant forms of SLC11A1 alter the processing or presentation of MAP antigens triggering thereby an autoimmune response in T1DM patients.     

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.     

Genome-Wide Diversity and Phylogeography of Mycobacterium avium subsp. paratuberculosis in Canadian Dairy Cattle

Mycobacterium avium subsp. paratuberculosis (MAP) is the causative bacterium of Johne’s disease (JD) in ruminants. The control of JD in the dairy industry is challenging, but can be improved with a better understanding of the diversity and distribution of MAP subtypes. Previously established molecular typing techniques used to differentiate MAP have not been sufficiently discriminatory and/or reliable to accurately assess the population structure. In this study, the genetic diversity of 182 MAP isolates representing all Canadian provinces was compared to the known global diversity, using single nucleotide polymorphisms identified through whole genome sequencing. MAP isolates from Canada represented a subset of the known global diversity, as there were global isolates intermingled with Canadian isolates, as well as multiple global subtypes that were not found in Canada. One Type III and six “Bison type” isolates were found in Canada as well as one Type II subtype that represented 86% of all Canadian isolates. Rarefaction estimated larger subtype richness in Québec than in other Canadian provinces using a strict definition of MAP subtypes and lower subtype richness in the Atlantic region using a relaxed definition. Significant phylogeographic clustering was observed at the inter-provincial but not at the intra-provincial level, although most major clades were found in all provinces. The large number of shared subtypes among provinces suggests that cattle movement is a major driver of MAP transmission at the herd level, which is further supported by the lack of spatial clustering on an intra-provincial scale.     

The Thioamides Methimazole and Thiourea Inhibit Growth of M. avium Subspecies paratuberculosis in Culture

Background

Thyrotoxicosis is conceptualized as an “autoimmune” disease with no accepted infectious etiology. There are increasingly compelling data that another “autoimmune” affliction, Crohn disease, may be caused by Mycobacterium avium subspecies paratuberculosis (MAP). Like M. tb, MAP is systemic. We hypothesized that some cases of thyrotoxicosis may be initiated by a MAP infection. Because other thioamides treat tuberculosis, leprosy and M. avium complex, we hypothesized that a mode of action of some thioamide anti-thyrotoxicosis medications may include MAP growth inhibition.

Methods

The effect of the thioamides, thiourea, methimazole and 6-propo-2-thiouracil (6-PTU) were studied in radiometric Bactec® culture, on ten strains of three mycobacterial species (six of MAP, two of M. avium and two of M. tb. complex). Data are presented as “cumulative growth index,” (cGI) or “percent decrease in cumulative GI” (%-ΔcGI).

Principal Findings

Methimazole was the most effective thioamide at inhibiting MAP growth. At 128µg/ml: MAP UCF-4; 65%-ΔcGI & MAP ATCC 19698; 90%-ΔcGI. Thiourea inhibited MAP “Ben” maximally; 70%-ΔcGI. Neither methimazole nor thiourea inhibited M. avium or M. tb. at the doses tested. 6-PTU has no inhibition on any strain studied, although a structurally analogous control, 5-PTU, was the most inhibitory thioamide tested.

Significance

We show inhibition of MAP growth by the thioamides, thiourea and methimazole in culture. These data are compatible with the hypothesis that these thioamides may have anti-prokaryotic in addition to their well-established eukaryotic actions in thyrotoxic individuals.