IL-18 and related function proteins associated with tuberculosis severity and screening for active TB among patients with non-mycobacterial community-acquired pneumonia (CAP)

BackgroundDifferentiation of active pulmonary tuberculosis (TB) from non-mycobacterial community-acquired pneumonia (CAP) still remains a diagnostic challenge.ObjectiveThe study aimed to quantify the IL-18, IFN-γ, IL-18BP, IL-37, and IP-10 levels in serum and Mycobacterium tuberculosis (M.tb) antigens-stimulated blood cultures from TB or CAP patients and explore if the proteins can be a useful basis for discriminating these diseases.MethodsIn total, 124 Polish adults, including mild/moderate (M/MTB) or advanced (ATB) TB patients, and CAP patients, were enrolled in the study. The concentrations of IL-18, IL-18BP, IFN-γ, IL-37, and IP-10 in sera and M.tb-stimulated cultures were measured by ELISA.ResultsThe most specific and sensitive serum proteins discriminating TB from CAP were IP-10 and IL-18BP; however, IP-10 had the highest AUC in the ROC curve for the diagnosis. Serum IP-10 and IL-18BP levels increased significantly in M/MTB or ATB groups. The IL-18BP elevation in ATB group was accompanied by an increase in IL-18. No single protein measured in M.tb-stimulated cultures differed TB from CAP patients.ConclusionsThe combined analysis of serum IL-18BP and IP-10 might be considered as an auxiliary tool in the differentiation of TB from CAP.     

Association of IFN-gamma and IL-10 gene variants with the risk of extrapulmonary tuberculosis

Tuberculosis (TB) caused by Mycobacterium tuberculosis (MTB) is a chronic infectious disease. Interferon-gamma (IFN-γ) is an important cytokine imparting resistance to mycobacterial diseases. It is believed that IFN-γ and Interleukin-10 (IL-10) play divergent roles in the host immune system against MTB infection. IL-10 is an important inhibitory cytokine and helps balancing the inflammatory and immune responses. IL-10 is involved in down regulation of Th1 cytokines, MHC class II antigen and co-stimulatory molecular expression on macrophages, while IFN-γ results in macrophage activation allowing them to exert the microbicidal role. The objectives were to find out the association of IL-10 (?1082 A/G) and IFN-γ (+874 A/T) single nucleotide polymorphisms (SNPs) with extrapulmonary tuberculosis in ethnic Kashmiri population. A total of 100 extrapulmonary tuberculosis cases and 102 healthy controls were analyzed for IL-10 (?1082 A/G) and IFN- γ (+874 A/T) SNPs using Allele-Specific PCR. We found a significant association of IFN-γ + 874 ‘TT’ genotype with extrapulmonary tuberculosis (p = 0.006) and in case of IL-10 (?1082 A/G) we found a significant association with extrapulmonary tuberculosis under recessive model (GG vs GA + AA) (p = 0.03) in Kashmiri population. IL-10 (?1082 A/G) and IFN-γ (+874 A/T) have a significant association with extrapulmonary tuberculosis in ethnic Kashmiri population.     

Cloning,expression, and immunogenicity of novel fusion protein of Mycobacterium tuberculosis based on ESAT-6 and truncated C-terminal fragment of HSP70

Tuberculosis (TB) remains as a major public health problem worldwide. Identification and selection of immunodominant antigens of Mycobacterium tuberculosis (MTB), capable of efficiently inducing a protective immune response is the ultimate goal of TB vaccine development studies.Accordingly, this study was designed to produce a novel M. tuberculosis fusion protein consisted of MTB ESAT-6 (early secreted antigenic target-6 kDa), as a potent immunogenic protein, fused to C-terminus of MTB HSP70 (HSP70_359–610), as an appropriate carrier and adjuvant.The constructed gene was inserted into a prokaryotic expression vector (pQE30); consequently, the recombinant fusion protein with a 6xHis-tag was successfully over expressed in Escherichia coli M15. Inclusion bodies from bacterial cell lysates were solubilized and the recombinant fusion protein was easily purified by Ni-NTA affinity chromatography under denaturing conditions followed by urea gradient dialysis. The purified and refolded protein was then applied for immunization of mice that resulted in the detection of high titers of specific antibodies, high level of IFN-γ and cell proliferation.The results of our study could confirm the capability of E6H70C fusion protein, as a potential tuberculosis vaccine candidate, for the efficient induction of specific immune responses in a mouse model. However, further investigation need to evaluate the protectivity of this recombinant protein in host model.     

Mycobacterium Tuberculosis Infection in Rhesus Monkeys (Macaca mulatta) and Evaluation of ESAT-6 and CFP10 as Immunodiagnostic Antigens#

Tuberculosis (TB) in nonhuman primates is a serious menace to the welfare of the animals and human who come into contact with them, while the rapid, accurate, and robust diagnosis is challenging. In this study, we first sought to establish an appropriate primate TB model resembling natural TB in nonhuman primates. Four rhesus monkeys (Macaca mulatta) of Chinese origin were infected intratracheally with two low doses of M. tuberculosis H37Rv. Regardless of the infectious doses, all monkeys were demonstrated to be successfully infected by clinical assessments, tuberculin skin test conversions, peripheral immune responses, gross observations, histopathology analysis, and M. tuberculosis burdens. Furthermore, we extended the usefulness of this model for assessing the following immunodiagnostic antigens: CFP10, ESAT-6, CFP10-ESAT-6, and an antigen cocktail of CFP10 and ESAT-6. The data showed that CFP10 was an M. tuberculosis-specific, “early” antigen used for serodiagnosis of TB in nonhuman primates. In conclusion, we established a useful primate TB model depending on low doses of M .tuberculosis and affording new opportunities for studies of M. tuberculosis disease and diagnostics.     

Optimization of conditions for expression of recombinant interferon-γ in E.coli

Interferon gamma (IFN-γ) is an important immunoregulatory cytokine that has a central role against viral and bacterial infections. In this study, the cDNA encoding 141 amino acids of mature IFN-γ from mice splenocytes was cloned in a prokaryotic expression vector pQE 30. Optimization of expression conditions resulted in high IFN-γ protein. Western blot showed that recombinant IFN-γ was specifically recognized by its counterpart anti-mouse IFN-γ antibodies. In vitro dose-dependent studies, with A549 and HeLa cell lines, showed that cloned IFN-γ was safe and had no effect on cell proliferation. The protein prediction and analysis using SOPMA program, revealed that IFN-γ had 80 α-helices, 8 β-turns jointed by 9 extended strands and 44 random coils. A total of four major clusters were observed with murine IFN-γ sharing 39 % homology with human IFN-γ. Pair-wise alignment studies with human revealed 26 % identity and 43.3 % similarity. The recovery of bioactive proteins from inclusion bodies (IBs) is a complex process and various protocols have been developed. We report here a simple, robust and inexpensive purification approach for obtaining recombinant IFN-γ protein expressed as IBs in E.coli.     

Differential Immune Responses and Protective Effects in Avirulent Mycobacterial Strains Vaccinated BALB/c Mice

Screening live mycobacterial vaccine candidates is the important strategy to develop new vaccines against adult tuberculosis (TB). In this study, the immunogenicity and protective efficacy of several avirulent mycobacterial strains including Mycobacterium smegmatis, M. vaccae, M. terrae, M. phlei, M. trivial, and M. tuberculosis H37Ra were compared with M. bovis BCG in BALB/c mice. Our results demonstrated that differential immune responses were induced in different mycobacterial species vaccinated mice. As BCG-vaccinated mice did, M. terrae immunization resulted in Th1-type responses in the lung, as well as splenocytes secreting IFN-γ against a highly conserved mycobacterial antigen Ag85A. M. smegmatis also induced the same splenocytes secreting IFN-γ as BCG and M. terrae did. In addition, M. terrae and M. smegmatis-immunized mice predominantly increased expression of IL-10 and TGF-β in the lung. Most importantly, mice vaccinated with H37Ra and M. vaccae could provide the same protection in the lung against virulent M. tuberculosis challenge as BCG. The result may have important implications in developing adult TB vaccine.     

Marked Microevolution of a Unique Mycobacterium tuberculosis Strain in 17 Years of Ongoing Transmission in a High Risk Population

The transmission and persistence of Mycobacterium tuberculosis within high risk populations is a threat to tuberculosis (TB) control. In the current study, we used whole genome sequencing (WGS) to decipher the transmission dynamics and microevolution of M. tuberculosis ON-A, an endemic strain responsible for an ongoing outbreak of TB in an urban homeless/under-housed population. Sixty-one M. tuberculosis isolates representing 57 TB cases from 1997 to 2013 were subjected to WGS. Sequencing data was integrated with available epidemiological information and analyzed to determine how the M. tuberculosis ON-A strain has evolved during almost two decades of active transmission. WGS offers higher discriminatory power than traditional genotyping techniques, dividing the M. tuberculosis ON-A strain into 6 sub-clusters, each defined by unique single nucleotide polymorphism profiles. One sub-cluster, designated ON-A^NM (Natural Mutant; 26 isolates from 24 cases) was also defined by a large, 15 kb genomic deletion. WGS analysis reveals the existence of multiple transmission chains within the same population/setting. Our results help validate the utility of WGS as a powerful tool for identifying genomic changes and adaptation of M. tuberculosis.     

Exploration of Novel Cellular and Serological Antigen Biomarkers in the ORFeome of Mycobacterium tuberculosis

Increasing evidence demonstrates that antigen-specific cellular and humoral immunity plays an indispensable role in protection against Mycobacterium tuberculosis infection. Antigen is a key element in the development of a successful diagnostic method and vaccine. However, few antigens are available, and a systemic study on M. tuberculosis ORFeome-based antigen screening is still lacking. In the current study, a genome-wide examination was conducted on high-throughput M. tuberculosis encoding proteins and novel antigens were identified via a comprehensive investigation of serological and antigen-specific cellular responses. The serological immunoglobulin G level of each protein was detected in pooled sera from 200 pulmonary tuberculosis patients by means of semi-quantitative Western blot. Of the 1,250 detected proteins, 29 were present at a higher level relative to the commercialized 38-kDa protein. Furthermore, the top 12 of the 29 proteins had not been previously reported, and their antigenicity was validated in serum from each individual patient. Results confirmed that the 12 proteins displayed nearly identical immunoglobulin G antibody levels in patients with pulmonary and extrapulmonary tuberculosis. Antigen-specific cellular interferon-γ secretion was also evaluated using a cell-based ELISPOT assay. Thirty-four of the proteins were able to induce positive interferon-γ production by peripheral blood mononuclear cells from pulmonary tuberculosis patients as judged by positive (commercial ESAT-6 antigen) and negative controls. The top 4 candidates out of the 34 proteins displayed good accuracy ranging from 50% to 80% compared with the commercial ESAT-6 antigen. Subsequent epitope examination confirmed that a pool of peptides, including a 25aa peptide from Rv1198, demonstrated significant tuberculosis-specific cellular interferon-γ production. Overall, the current study draws significant attention to novel M. tuberculosis antigens, many of which have not been previously reported. This discovery provides a large amount of useful information for the diagnosis of tuberculosis and the development of vaccines to provide protection against tuberculosis.Despite great efforts to improve its diagnosis, prevention, and treatment, tuberculosis (TB)¹ remains a serious burden on global health, especially in developing countries (1). Diagnostic inaccuracy and delays, vaccine failure, and drug resistance are the main contributors to the current TB epidemic (2). The World Health Organization (WHO) reports that only 60% of the estimated total TB cases have been detected; nearly half of active TB cases remain undetectable, and the pathogen continues to be transmitted (3). A critical step in the control of TB is the early and sensitive diagnosis of infection and disease; however, antigen biomarkers in Mycobacterium tuberculosis that are able to induce antigen-specific immune responses in diverse patients are not yet comprehensively understood. Scientists have been working to identify novel antigens in order to develop new, efficacious, and long-lasting vaccines; to improve the accuracy and specificity of diagnostic tools; and to fill gaps in knowledge regarding TB (4–7). It is well known that cell-mediated immunity plays a central role in controlling the proliferation of bacteria and that IFN-γ is the dominant cytokine that indicates infection. T-cell-based IFN-γ release assays were developed several decades ago to assess IFN-γ production after in vitro stimulation with M. tuberculosis antigens and are among the methods used to identify M. tuberculosis infection, although their reliability is still controversial (8). Among the over 4,000 predicted open reading frames (ORFs) in the M. tuberculosis genome, only three M. tuberculosis antigens, ESAT-6, CFP-10, and Ag85, have been commonly studied and are used in practice. Furthermore, the diagnostic methods based on ESAT-6 and CFP-10 are still in dispute and have at least two disadvantages: (i) neither is significantly superior to tuberculin skin tests or able to differentiate latent M. tuberculosis infection from active TB (9), and (ii) both miss diagnoses, as only 60% to 80% of active pulmonary TB is diagnosed as positive by the present established methods (10–13). Thus, the discovery of novel M. tuberculosis antigens is vital in order for antigenic epitopes to be identified that have better accuracy and specificity for the diagnosis of active TB. In addition to cell-mediated immune response, emerging evidence indicates that the B-cell-mediated humoral immune response plays a critical role in defending against M. tuberculosis (14, 15). Although the current commercial serodiagnostic kits are used in developing countries, antibody-based diagnosis is controversial, and current test performance is poor. WHO recommends against using these inaccurate TB serological tests because most lack sensitivity and specificity (16, 17). But the identification of new serum biomarkers is still highly recommended by WHO, and the study of novel M. tuberculosis serum biomarkers is still important. Moreover, the demand for serodiagnostic methods for TB diagnosis is still huge in developing and undeveloped countries because they are cost-effective and easy to use. Thus, it is worthwhile to examine the effectiveness of providing more antigen choices and a better panel, rather than using a single antigen, when diagnosing TB using these methods.High-throughput multi-omics technologies have made it feasible to do genome-wide studies of certain pathogens. Though this necessitates translating a large amount data from biological studies into a form that can be used clinically, it is necessary to meet urgent clinical requirements. A few proteome-scale studies have been performed, and several antigenic proteins of M. tuberculosis have been identified using serum antibody-based screening. However, certain limitations still exist. Inconsistent results have been obtained from different groups using various methods, and systemic, functional antigen screening, based on the cellular immune response, is missing because of the experimental complexity and difficulty of recruiting enough patients to obtain fresh blood specimens that contain viable white blood cells.In the current study, we screened and identified M. tuberculosis antigens that were specific to serological and cellular responses in one study of active pulmonary TB patients. After genome-based high-throughput cloning and expression, 1,250 purified candidate proteins were obtained for the identification of M. tuberculosis T and B cell antigens. The serological IgG level of each protein was determined by means of semi-quantitative Western blot. In addition, antigen-specific IFN-γ secretion was evaluated in 1,250 proteins using a cell-based ELISPOT assay. To our knowledge, this is the first time that cellular antigenic response has been examined on a large scale in TB patients, and the results provide an overview of a broad range of novel antigen biomarkers that could be beneficial for improving TB diagnosis and vaccine development.     

The assessment of host and bacterial proteins in sputum from active pulmonary tuberculosis

Pulmonary tuberculosis (TB) is caused by Mycobacterium tuberculosis. The protein composition of sputum may reflect the immune status of the lung. This study aimed to evaluate the protein profiles in spontaneous sputum samples from patients with active pulmonary TB. Sputum samples were collected from patients with pulmonary TB and healthy controls. Western blotting was used to analyze the amount of interleukin 10 (IL-10), interferon-gamma (IFN-γ), IL-25, IL-17, perforin-1, urease, albumin, transferrin, lactoferrin, adenosine deaminase (also known as adenosine aminohydrolase, or ADA), ADA-2, granzyme B, granulysin, and caspase-1 in sputum. Results of detection of IL-10, IFN-γ, perforin-1, urease, ADA2, and caspase-1, showed relatively high specificity in distinguishing patients with TB from healthy controls, although sensitivities varied from 13.3% to 66.1%. By defining a positive result as the detection of any two proteins in sputum samples, combined use of transferrin and urease as markers increased sensitivity to 73.2% and specificity to 71.1%. Furthermore, we observed that the concentration of transferrin was proportional to the number of acid-fast bacilli detected in sputum specimens. Detection of sputum transferrin and urease was highly associated with pulmonary TB infection. In addition, a high concentration of transferrin detected in sputum might correlate with active TB infection. This data on sputum proteins in patients with TB may aid in the development of biomarkers to assess the severity of pulmonary TB.     

Effect of attenuation of Treg during BCG immunization on anti-mycobacterial Th1 responses and protection against Mycobacterium tuberculosis

Background

The functional equilibrium between natural regulatory T cells (Treg) and effector T cells can affect the issue of numerous infections. In unvaccinated mice, the influence of Treg in the control of primary infection with mycobacteria remains controversial.

Methodology

Here, we evaluated the role of Treg during prophylactic vaccination with Mycobacterium bovis BCG (Bacillus Calmette-Guérin) on the induction of T cell responses and on the protective effect against subsequent M. tuberculosis challenge in mice.

Principal Findings

We demonstrated that, subsequent to BCG injection, Treg were recruited to the draining lymph nodes and negatively control anti-mycobacterial CD4⁺ — but not CD8⁺ — T-cell responses. Treatment of BCG-immunized mice with an anti-CD25 mAb (PC61) induced an increase IFN-γ response against both subdominant and immunodominant regions of the protective immunogen TB10.4. In Treg-attenuated, BCG-immunized mice, which were then infected with M. tuberculosis, the lung mycobacterial load was significantly, albeit moderately, reduced compared to the control mice.

Conclusions

Our results provide the first demonstration that attenuation of Treg subset concomitant to BCG vaccination has a positive, yet limited, impact on the protective capacity of this vaccine against infection with M. tuberculosis. Thus, for rational design of improved BCG, it should be considered that, although the action of Treg does not represent the major cause of the limited efficiency of BCG, the impact of this cell population on the subsequent control of M. tuberculosis growth is significant and measurable.     

Bacterial Thymidine Kinase as a Non-Invasive Imaging Reporter for Mycobacterium tuberculosis in Live Animals

Background

Bacteria can be selectively imaged in experimentally-infected animals using exogenously administered 1-(2′deoxy-2′-fluoro-β-D-arabinofuranosyl)-5-[¹²⁵I]-iodouracil ([¹²⁵I]-FIAU), a nucleoside analog substrate for bacterial thymidine kinase (TK). Our goal was to use this reporter and develop non-invasive methods to detect and localize Mycobacterium tuberculosis.

Methodology/Principal Findings

We engineered a M. tuberculosis strain with chromosomally integrated bacterial TK under the control of hsp60 - a strong constitutive mycobacterial promoter. [¹²⁵I]FIAU uptake, antimicrobial susceptibilities and in vivo growth characteristics were evaluated for this strain. Using single photon emission computed tomography (SPECT), M. tuberculosis P_hsp60 TK strain was evaluated in experimentally-infected BALB/c and C3HeB/FeJ mice using the thigh inoculation or low-dose aerosol infection models. M. tuberculosis P_hsp60 TK strain actively accumulated [¹²⁵I]FIAU in vitro. Growth characteristics of the TK strain and susceptibility to common anti-tuberculous drugs were similar to the wild-type parent strain. M. tuberculosis P_hsp60 TK strain was stable in vivo and SPECT imaging could detect and localize this strain in both animal models tested.

Conclusion

We have developed a novel tool for non-invasive assessment of M. tuberculosis in live experimentally-infected animals. This tool will allow real-time pathogenesis studies in animal models of TB and has the potential to simplify preclinical studies and accelerate TB research.     

Cloning and expression of MPT83 gene from Mycobacterium tuberculosis in E. coli BL21 as vaccine candidate of tuberculosis: A preliminary study

The appearance of Mycobacterium tuberculosis strains leading to drug resistance has caused new problems in TB treatment in various parts of the world and forces WHO to declare TB as a global emergency. With the increase of TB drug resistance, it is convinced that a more effective vaccine development will stop the epidemic of TB. Some M. tuberculosis antigens, one of which is MPT83, have been examined as TB vaccine candidate. MPT83 antigen, which is very immunogenic in lipoprotein micro bacteria, is identified as surface cell interrelated to antigen with cytometry circulation. Having TB resistance from BCG vaccine, MPT83 is considered TB vaccine candidate that can protect people against TB at adult age. The purpose of this research is to conduct amplification of MPT83 antigen cloning, and expression of its antigen on E. coli bacteria. From the result of the research, it is expected that raw material to produce TB vaccine as well as a high-quality antigen can be obtained. The band of DNA in PCR product is 660?bp, while the one in pGEMT-Easy-Mpt83 recombinant plasmid is 3678?bp. This is expressed in E. coli BL21 strain and produces 48?kDa protein as well as GST-MPT83 fusion protein.     

表达HBHA和hIL-12融合蛋白的重组耻垢分枝杆菌在小鼠体内诱导的免疫应答及保护力

目的:测定表达肝素结合血凝素(HBHA)和人白细胞介素12(hIL-12)融合蛋白的重组耻垢分枝杆菌在小鼠体内诱导产生的免疫应答及对结核分枝杆菌感染的保护作用。方法:将表达HBHA和hIL-12融合蛋白的重组耻垢分枝杆菌采用同源加强免疫的方法免疫小鼠,检测小鼠外周血中IFN-γ、IL-2和IL-12的表达水平;用结核分枝杆菌感染免疫小鼠,检测小鼠肺部荷菌量和组织病理变化。结果:表达HBHA和hIL-12融合蛋白的重组耻垢分枝杆菌诱导小鼠产生以IFN-γ、IL-2分泌量增加为主的Th1型免疫应答,并能有效减少感染小鼠肺部结核分枝杆菌的荷菌量和病理损伤。结论:表达HBHA和hIL-12融合蛋白的重组耻垢分枝杆菌免疫小鼠可诱导产生与卡介苗相当的保护作用,可能成为控制结核病的有效疫苗。     

Spoligotyping and drug resistance analysis of Mycobacterium tuberculosis strains from national survey in China

Background

Tuberculosis (TB), caused by Mycobacterium tuberculosis complex (MTBC), is one of the major causes of death in the world today. Although China has the second largest global case rate of tuberculosis, a systematic study of TB prevalence in China has not been completed. From 2006 to 2007, the base line surveillance of tuberculosis was carried out by Ministry of Health, and more than 4000 representative strains were selected from 31 provinces in China.

Methodology/Principal Findings

The aim of the present research was to survey the genotypes of representative Mycobacterium tuberculosis (M. tuberculosis) strains from China using spacer oligonucleotide typing (spoligotyping), and to analyze the relationship between genotype and drug resistance for the first time. A total of 4017 clinical isolates were collected from 2007 to 2008 throughout China. Among those M. tuberculosis isolates, 2500 (62.2%) isolates were Beijing genotypes. The percentage of Beijing genotypes in northern China was higher than in southern China (76.5% vs. 53.2%). Additionally, the frequencies of rifampin-resistant, ofloxacin-resistant and multidrug-resistant isolates were significantly higher in Beijing genotype strains than non-Beijing strains. Furthermore, a novel genotype named “China Southern genotype (CS)” was only isolated from Fujian and Guangdong provinces. Hence, it is very practical to uncover the reason for prevalence of the CS type in southern China.

Conclusions/Significance

In conclusion, Beijing family genotypes were still the predominant genotype throughout China, which exhibited a greater correlation with rifampin-resistance, ofloxacin-resistance and MDR phenotypes than other TB spoligotypes, and some regions of China showed several unique characters in the distribution of M. tuberculosis genotypes. Our research represents an important contribution for the TB control and research community, which completes broad pictures on drug resistance levels and distribution of M. tuberculosis strain types over China.     

Multiplex analysis of cytokines/chemokines as biomarkers that differentiate healthy contacts from tuberculosis patients in high endemic settings

Differentiation of latent tuberculosis infection (LTBI) from active disease is one of the crucial elements in the control of tuberculosis. Earlier in Indian population which is tuberculosis endemic, we identified that 10 Mycobacterium tuberculosis secreted protein fractions, induced IFN-γ response only in healthy contacts of TB patients (HCs) and not in tuberculosis patients (TB). These fractions were termed as “Contact Specific Fractions” (“CS” fractions) and found useful for differentiating HC from TB. Proteomic analysis revealed that “CS” fractions have 16 different proteins, of which three were novel T cell antigens. Using these “CS” fractions as stimulants, earlier IFN-γ, TNF-α and IL-4 cytokine responses were studied. In the present study, in order to identify the other useful cytokine biomarkers that were differentially expressed between HC and TB, Cytokine/chemokine response to “CS” fractions were analyzed using multiplex cytokine assay system. This preliminary investigation in our tuberculosis endemic population showed six cytokine (G-CSF, IL-6, IL-7, IL-8, IL-9, and PDGF) and one receptor antagonist (IL-1Ra) that were differentially expressed between HC and TB, for the first time. Especially IL-6 and PDGF were more promising biomarkers. IL-6 measurement identified seven as HC out of 10 HC analyzed. The measurement of PDGF identified eight as TB out of 10 TB tested. Studies are underway to further validate these biomarkers for the differentiation of LTBI from active tuberculosis.     

Transmission of Mycobacterium tuberculosis between Farmers and Cattle in Central Ethiopia

Background

Transmission of Mycobacterium tuberculosis (M. tuberculosis) complex could be possible between farmers and their cattle in Ethiopia.

Methodology/Principal Findings

A study was conducted in mixed type multi-purposes cattle raising region of Ethiopia on 287 households (146 households with case of pulmonary tuberculosis (TB) and 141 free of TB) and 287 herds consisting of 2,033 cattle belonging to these households to evaluate transmission of TB between cattle and farmers. Interview, bacteriological examinations and molecular typing were used for human subjects while comparative intradermal tuberculin (CIDT) test, post mortem and bacteriological examinations, and molecular typing were used for animal studies. Herd prevalence of CIDT reactors was 9.4% and was higher (p<0.01) in herds owned by households with TB than in herds owned by TB free households. Animal prevalence was 1.8% and also higher (p<0.01) in cattle owned by households with TB case than in those owned by TB free households. All mycobacteria (141) isolated from farmers were M. tuberculosis, while only five of the 16 isolates from cattle were members of the M. tuberculosis complex (MTC) while the remaining 11 were members of non-tuberculosis mycobacteria (NTM). Further speciation of the five MTC isolates showed that three of the isolates were M. bovis (strain SB1176), while the remaining two were M. tuberculosis strains (SIT149 and SIT53). Pathology scoring method described by “Vordermeier et al. (2002)” was applied and the average severity of pathology in two cattle infected with M. bovis, in 11 infected with NTM and two infected with M. tuberculosis were 5.5, 2.1 and 0.5, respectively.

Conclusions/Significance

The results showed that transmission of TB from farmers to cattle by the airborne route sensitizes the cows but rarely leads to TB. Similarly, low transmission of M. bovis between farmers and their cattle was found, suggesting requirement of ingestion of contaminated milk from cows with tuberculous mastitis.     

Subunit Vaccine Consisting of Multi-Stage Antigens Has High Protective Efficacy against Mycobacterium tuberculosis Infection in Mice

To search for more effective tuberculosis (TB) subunit vaccines, antigens expressed in different growth stages of Mycobacterium tuberculosis (M. tuberculosis), such as RpfE (Rv2450c) produced in the stage of resuscitation, Mtb10.4 (Rv0288), Mtb8.4 (Rv1174c), ESAT6 (Rv3875), Ag85B (Rv1886c) mainly secreted by replicating bacilli, and HspX (Rv2031c) highly expressed in dormant bacilli, were selected to construct six fusion proteins: ESAT6-Ag85B-MPT64_190-198-Mtb8.4 (EAMM), Mtb10.4-HspX (MH), ESAT6-Mtb8.4, Mtb10.4-Ag85B, ESAT6-Ag85B, and ESAT6-RpfE. The six fusion proteins were separately emulsified in an adjuvant composed of N,N’-dimethyl-N, N’-dioctadecylammonium bromide (DDA), polyribocytidylic acid (poly I:C) and gelatin to construct subunit vaccines, and their protective effects against M. tuberculosis infection were evaluated in C57BL/6 mice. Furthermore, the boosting effects of EAMM and MH in the adjuvant of DDA plus trehalose 6,6''-dimycolate (TDM) on BCG-induced immunity were also evaluated. It was found that the six proteins were stably produced in E. coli and successfully purified by chromatography. Among them, EAMM presented the most effective protection against M. tuberculosis. Interestingly, the mice that received EAMM+MH had significantly lower bacterial counts in the lungs and spleens than the single protein vaccinated groups, and had the same effect as those that received BCG. In addition, EAMM and MH could improve BCG-primed protective efficacy against M. tuberculosis infection in mice. In conclusion, the combination of EAMM and MH containing antigens from both replicating and dormant stages of the bacilli could induce robust immunity against M. tuberculosis infection in mice and may serve as promising subunit vaccine candidate.     

The forest behind the tree: phylogenetic exploration of a dominant Mycobacterium tuberculosis strain lineage from a high tuberculosis burden country

Background

Genotyping of Mycobacterium tuberculosis isolates is a powerful tool for epidemiological control of tuberculosis (TB) and phylogenetic exploration of the pathogen. Standardized PCR-based typing, based on 15 to 24 mycobacterial interspersed repetitive unit-variable number of tandem repeat (MIRU-VNTR) loci combined with spoligotyping, has been shown to have adequate resolution power for tracing TB transmission and to be useful for predicting diverse strain lineages in European settings. Its informative value needs to be tested in high TB-burden countries, where the use of genotyping is often complicated by dominance of geographically specific, genetically homogeneous strain lineages.

Methodology/Principal Findings

We tested this genotyping system for molecular epidemiological analysis of 369 M. tuberculosis isolates from 3 regions of Brazil, a high TB-burden country. Deligotyping, targeting 43 large sequence polymorphisms (LSPs), and the MIRU-VNTRplus identification database were used to assess phylogenetic predictions. High congruence between the different typing results consistently revealed the countrywide supremacy of the Latin-American-Mediterranean (LAM) lineage, comprised of three main branches. In addition to an already known RDRio branch, at least one other branch characterized by a phylogenetically informative LAM3 spoligo-signature seems to be globally distributed beyond Brazil. Nevertheless, by distinguishing 321 genotypes in this strain population, combined MIRU-VNTR typing and spoligotyping demonstrated the presence of multiple distinct clones. The use of 15 to 24 loci discriminated 21 to 25% more strains within the LAM lineage, compared to a restricted lineage-specific locus set suggested to be used after SNP analysis. Noteworthy, 23 of the 28 molecular clusters identified were exclusively composed of patient isolates from a same region, consistent with expected patterns of mostly local TB transmission.

Conclusions/Significance

Standard MIRU-VNTR typing combined with spoligotyping can reveal epidemiologically meaningful clonal diversity behind a dominant M. tuberculosis strain lineage in a high TB-burden country and is useful to explore international phylogenetical ramifications.