Opportunities for Improved Serodiagnosis of Human Tuberculosis, Bovine Tuberculosis, and Paratuberculosis

Mycobacterial infections—tuberculosis (TB), bovine tuberculosis (bTB), and Johne's disease (JD)—are major infectious diseases of both human and animals. Methods presently in use for diagnosis of mycobacterial infections include bacterial culture, nucleic acid amplification, tuberculin skin test, interferon-γ assay, and serology. Serological tests have several advantages over other methods, including short turn-around time, relatively simple procedures, and low cost. However, current serodiagnostic methods for TB, bTB and JD exhibit low sensitivity and/or specificity. Recent studies that have aimed to develop improved serodiagnostic tests have mostly focused on identifying useful species-specific protein antigens. A review of recent attempts to improve diagnostic test performance indicates that the use of multiple antigens can improve the accuracy of serodiagnosis of these mycobacterial diseases. Mycobacteria also produce a variety of species-specific nonprotein molecules; however, only a few such molecules (e.g., cord factor and lipoarabinomannan) have so far been evaluated for their effectiveness as diagnostic antigens. For TB and bTB, there has been recent progress in developing laboratory-free diagnostic methods. New technologies such as microfluidics and “Lab-on-Chip” are examples of promising new technologies that can underpin development of laboratory-free diagnostic devices for these mycobacterial infections

Characterization of Ethanol Extracted Cell Wall Components of Mycobacterium avium Subsp. paratuberculosis

Antigens extracted using ethanol (EtOH) and incorporated in the EtOH vortex ELISA (EVELISA) test have previously shown high specificity and sensitivity for detecting Mycobacterium avium subspecies paratuberculosis (Map) and M. bovis infections in cattle. The objective of this study is to define the components present in the EtOH extract. We show that this extract is composed of lipid, carbohydrate, and proteins on the surface of the bacilli, and that EtOH removes the outer layer structure of Map which comprise these elements. To identify proteins, polyclonal antibodies to the EtOH prep were produced and used to screen a Map genomic expression library. Seven overlapping clones were identified with a single open reading frame, MAP_0585, common to all. MAP_0585, which encodes a hypothetical protein, was recombinantly produced and used to demonstrate strong reactivity in sera from hyperimmunized rabbits, but this protein is not strongly immunogenic in cattle with Johne’s disease. A panel of monoclonal antibodies was used to determine the presence of additional proteins in the EtOH extract. These antibodies demonstrated that a well-known antigen, termed MPB83, is present in M. bovis EtOH extracts and a fatty acid desaturase (MAP_2698c) is present in Map EtOH extracts, while lipoarabinomannan was common to both. The lipid and carbohydrate components of the extract were analyzed using thin layer chromatography and lectin binding, respectively. Lectin biding and protease treatment of the EtOH extract suggest the antigenic component is carbohydrate and not protein. These results give further insight into this important antigen prep for detecting mycobacterial diseases of cattle

Evaluation of ethanol vortex ELISA for detection of bovine tuberculosis in cattle and deer

Background: The use of serological assays for diagnosis of bovine tuberculosis (TB) has been intensively studied and use of specific antigens have aided in improving the diagnostic accuracy of the assays. In the present study, we report an in-house enzyme linked immunosorbent assay (ELISA), developed by using ethanol extract of Mycobacterium bovis (M. bovis). The assay, named (ethanol vortex ELISA [EVELISA]), was evaluated for detection of anti- M. bovis antibodies in the sera of cattle and white-tailed deer. Methods: By using the EVELISA, we tested sera obtained from two species of animals; cattle (n = 62 [uninfected, n = 40; naturally infected, n = 22]) and white-tailed deer (n = 41 [uninfected, n = 25; naturally infected, n = 7; experimentally infected, n = 9]). To detect species specific molecules, components in the ethanol extract were analyzed by thin layer chromatography and western blotting. Results: Among the tested animals, 77.2% of infected cattle and 87.5% of infected deer tested positive for anti- M. bovis antibody. There were only minor false positive reactions (7.5% in cattle and 0% in deer) in uninfected animals. M. bovis -specific lipids and protein (MPB83) in the ethanol extract were detected by thin layer chromatography and western blotting, respectively. Conclusion: The results warrant further evaluation and validation of EVELISA for bovine TB diagnosis of traditional and alternative livestock as well as for free-ranging animal species

Use of ethanol extract of Mycobacterium bovis for detection of specific antibodies in sera of farmed red deer (Cervus elaphus) with bovine tuberculosis

Background: Bovine tuberculosis (bTB) in wildlife species poses a threat to domestic livestock in many situations. Control programs for bTB in livestock depend on testing and slaughtering the positive animals; however, the currently available diagnostic tests often have poor specificity. In our previous study, we developed a specific and sensitive enzyme linked immunosorbent assay (ELISA) for another mycobacterial disease – Johne’s disease, using surface antigens of Mycobacterium avium ssp. paratuberculosis (MAP) extracted by briefly agitating the bacilli in 80% ethanol solution. The ELISA test was named ethanol vortex ELISA (EVELISA). The objective of this study is to examine whether EVELISA technique could be used to specifically detect anti-Mycobacterium bovis (M. bovis) antibodies in the serum of M. bovis-infected farmed red deer (Cervus elaphus). We tested a total of 45 red deer serum samples, divided in 3 groups – uninfected animals (n = 15), experimentally infected with M. bovis (n = 15) and experimentally infected with MAP (n = 15). Results: The presence of anti-M. bovis antibodies was tested using an ethanol extract of M. bovis. Without absorption of anti-MAP cross reactive antibodies, it was found that 13 out of the 15 MAP-infected animals showed high antibody binding. Using heat killed MAP as an absorbent of cross reactive antibodies, anti-M. bovis antibodies were detected in 86.7% of M. bovis-infected animals with minor false positive results caused by MAP infection. Conclusions: The results from this study suggest that EVELISA may form a basis for a sensitive and specific test for the diagnosis of bTB in farmed red deer

Use of ethanol extract of Mycobacterium bovis for detection of specific antibodies in sera of farmed red deer (Cervus elaphus) with bovine tuberculosis

Background: Bovine tuberculosis (bTB) in wildlife species poses a threat to domestic livestock in many situations. Control programs for bTB in livestock depend on testing and slaughtering the positive animals; however, the currently available diagnostic tests often have poor specificity. In our previous study, we developed a specific and sensitive enzyme linked immunosorbent assay (ELISA) for another mycobacterial disease – Johne’s disease, using surface antigens of Mycobacterium avium ssp. paratuberculosis (MAP) extracted by briefly agitating the bacilli in 80% ethanol solution. The ELISA test was named ethanol vortex ELISA (EVELISA). The objective of this study is to examine whether EVELISA technique could be used to specifically detect anti-Mycobacterium bovis (M. bovis) antibodies in the serum of M. bovis-infected farmed red deer (Cervus elaphus). We tested a total of 45 red deer serum samples, divided in 3 groups – uninfected animals (n = 15), experimentally infected with M. bovis (n = 15) and experimentally infected with MAP (n = 15). Results: The presence of anti-M. bovis antibodies was tested using an ethanol extract of M. bovis. Without absorption of anti-MAP cross reactive antibodies, it was found that 13 out of the 15 MAP-infected animals showed high antibody binding. Using heat killed MAP as an absorbent of cross reactive antibodies, anti-M. bovis antibodies were detected in 86.7% of M. bovis-infected animals with minor false positive results caused by MAP infection. Conclusions: The results from this study suggest that EVELISA may form a basis for a sensitive and specific test for the diagnosis of bTB in farmed red deer

Evaluation of low-cost phage-based Microbial Source Tracking tools for elucidating human fecal contamination pathways in Kolkata, India

Phages, such as those infecting Bacteroides spp., have been proven to be reliable indicators of human fecal contamination in microbial source tracking (MST) studies, and the efficacy of these MST markers found to vary geographically. This study reports the application and evaluation of candidate MST methods (phages infecting previously isolated B. fragilis strain GB-124, newly isolated Bacteroides strains (K10, K29, and K33) and recently isolated Kluyvera intermedia strain ASH-08), along with non-source specific somatic coliphages (SOMCPH infecting strain WG-5) and indicator bacteria (Escherichia coli) for identifying fecal contamination pathways in Kolkata, India. Source specificity of the phage-based methods was first tested using 60 known non-human fecal samples from common animals, before being evaluated with 56 known human samples (municipal sewage) collected during both the rainy and dry season. SOMCPH were present in 40-90% of samples from different animal species and in 100% of sewage samples. Phages infecting Bacteroides strain GB-124 were not detected from the majority (95%) of animal samples (except in three porcine samples) and were present in 93 and 71% of the sewage samples in the rainy and dry season (Mean = 1.42 and 1.83 log(10)PFU/100mL, respectively), though at lower levels than SOMCPH (Mean = 3.27 and 3.02 log(10)PFU/100mL, respectively). Phages infecting strain ASH-08 were detected in 89 and 96% of the sewage samples in the rainy and dry season, respectively, but were also present in all animal samples tested (except goats). Strains K10, K29, and K30 were not found to be useful MST markers due to low levels of phages and/or co-presence in non-human sources. GB-124 and SOMCPH were subsequently deployed within two low-income neighborhoods to determine the levels and origin of fecal contamination in 110 environmental samples. E. coli, SOMCPH, and phages of GB-124 were detected in 68, 42, and 28% of the samples, respectively. Analyses of 166 wastewater samples from shared community toilets and 21 samples from sewage pumping stations from the same districts showed that SOMCPH were present in 100% and GB-124 phages in 31% of shared toilet samples (Median = 5.59 and <1 log(10) PFU/100 mL, respectively), and both SOMCPH and GB-124 phages were detected in 95% of pumping station samples (Median = 5.82 and 4.04 log(10) PFU/100 mL, respectively). Our findings suggest that GB-124 and SOMCPH have utility as low-cost fecal indicator tools which can facilitate environmental surveillance of enteric organisms, elucidate human and non-human fecal exposure pathways, and inform interventions to mitigate exposure to fecal contamination in the residential environment of Kolkata, India

Co-Creation of Breast Cancer Risk Communication Tools and an Assessment of Risk Factor Awareness: A Qualitative Study of Patients and the Public in India

The incidence of breast cancer (BC) is increasing worldwide, and India reported 179,790 cases in 2020. It is important to inform people of risk factors and methods for risk management through interactive risk communication techniques. Affordable, easy-to-understand transmedia tools for the communication of BC risk were co-created by a multidisciplinary team of doctors, clinical researchers, epidemiologists, health economists, digital designers, and public representatives. Qualitative in-depth interviews were conducted in regional languages to assess the views of patients, relatives, the public, and health professionals in India of these prototypes. There was low awareness of BC, with some understanding of age and hereditary risk factors but limited knowledge of reproductive factors amongst the general public, patients, and relatives. Participants favored storytelling techniques (animation and comic strips/infographics) to explain complex issues such as genetic risk and testing. Co-created BC risk communication transmedia tools should be used to support informed decision making

Comparing the genetic typing methods for effective surveillance and rabies control in Georgia

A full nucleoprotein gene sequencing of 68 isolates collected from passive rabies surveillance system in Georgia between 2015 and 2016 identified two distinct dog rabies phylogroups, GEO_V1 and GEO_V2, which both belonged to the cosmopolitan dog clade. GEO_V1 was found throughout the country and was further divided into four sub-phylogroups that overlapped geographically; GEO_V2 was found in the southeast region and was closely related to dog rabies in Azerbaijan. A sequence analysis of the full N gene, partial nucleoprotein gene of N-terminal and C-terminal, and the amplicon sequences of pan-lyssavirus RT-qPCR LN34 showed that all four sequencing approaches provided clear genetic typing results of canine rabies and could further differentiate GEO_V1 and GEO_V2. The phylogenetic analysis results vary and were affected by the length of the sequences used. Amplicon sequencing of the LN34 assay positive samples provided a rapid and cost-effective method for rabies genetic typing, which is important for improving rabies surveillance and canine rabies eradication globally

Ashwagandha Derived Withanone Targets TPX2-Aurora A Complex: Computational and Experimental Evidence to its Anticancer Activity

Cancer is largely marked by genetic instability. Specific inhibition of individual proteins or signalling pathways that regulate genetic stability during cell division thus hold a great potential for cancer therapy. The Aurora A kinase is a Ser/Thr kinase that plays a critical role during mitosis and cytokinesis and is found upregulated in several cancer types. It is functionally regulated by its interactions with TPX2, a candidate oncogene. Aurora A inhibitors have been proposed as anticancer drugs that work by blocking its ATP binding site. This site is common to other kinases and hence these inhibitors lack specificity for Aurora A inhibition in particular, thus advocating the need of some alternative inhibition route. Previously, we identified TPX2 as a cellular target for withanone that selectively kill cancer cells. By computational approach, we found here that withanone binds to TPX2-Aurora A complex. In experiment, withanone treatment to cancer cells indeed resulted in dissociation of TPX2-Aurora A complex and disruption of mitotic spindle apparatus proposing this as a mechanism of the anticancer activity of withanone. From docking analysis, non-formation/disruption of the active TPX2-Aurora A association complex could be discerned. Our MD simulation results suggesting the thermodynamic and structural stability of TPX2-Aurora A in complex with withanone further substantiates the binding. We report a computational rationale of the ability of naturally occurring withanone to alter the kinase signalling pathway in an ATP-independent manner and experimental evidence in which withanone cause inactivation of the TPX2-Aurora A complex. The study demonstrated that TPX2-Aurora A complex is a target of withanone, a potential natural anticancer drug

Role of surface antigens of Mycobacterium spp. in Diagnosis

Mycobacterial species are ubiquitous in nature and a worldwide concern for human and animal health. The major mycobacterial infections in animals are Johne’s disease (JD) and bovine tuberculosis (bTB). Controlling these infections is difficult due to the lack of highly sensitive and sensitive diagnostic test. Currently available diagnostic tests have to be carried out in laboratory settings with well experienced and trained examiners. My goal is to develop a sensitive on-site (in-field) device for diagnosis of Johne’s disease and bovine tuberculosis. The specific aims of this thesis were (1) to review currently-used or recently developed diagnostic tests for mycobacterial infections, (2) to optimize a milk-based enzyme linked immunosorbant assay (ELISA) for diagnosis of JD, (3) to evaluate a serum-based ELISA for detection of bTB in red deer and (4) to develop a bead-based microfluidic assay as a prototype of on-site diagnostic device for JD. Previous reports on currently-used or recently developed diagnostic tests for mycobacterial infections were reviewed to summarize challenges and opportunities in development of sensitive on-site diagnostic devices. Most of the current serological tests for mycobacterial infections utilize crude extract of the pathogen or single molecule causing low diagnostic specificity and specificity. Use of multiple antigens was shown to be effective in improving test accuracy. Ethanol extract of Mycobacterium avium subspecies paratuberculosis (MAP) was used to optimize a serum ELISA to test milk samples for JD. Using the optimized conditions, the average of ELISA values in the JD-positive milk samples was found to be significantly higher than that in the JD-negative milk samples. Using ethanol extract from Mycobacterium bovis (MB), an ELISA was developed to detect anti-MB antibodies in serum of farmed red deer. The tentative diagnostic sensitivity and specificity was estimated to be 90% and 93.3%, respectively. Magnetic beads coated with ethanol extract of MAP were used to develop a microfluidic immunoassay for diagnosis of JD. The antigen-coated magnetic beads were tested in the microfluidic system using bovine serum samples and a high level of antibody binding in JD-positive serum was observed