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

Morphology and ploidy level determination of Pteris vittata callus during induction and regeneration

Background: Morphological and ploidy changes of the arsenic hyperaccumulator, Chinese brake fern (Pteris vittata) callus tissue are described here to provide insight into fern life cycle biology and for possible biotechnology applications. Pteris vittata callus was studied using transmission and scanning electron microscopy, and flow cytometry. Results: Callus induction occurred both in light and dark culture conditions from prothallus tissues, whereas rhizoid formation occurred only in dark culture conditions. Callus tissues contained two types of cells: one actively dividing and the other containing a single large vacuole undergoing exocytosis. Sporophytes regenerated from callus asynchronously form clusters of cells in a manner apparently analogous to direct organogenesis. Extracellular matrices were observed in actively-growing callus and at the base of regenerating sporophytes. Callus tissue nuclei were found to be primarily diploid at induction and throughout maintenance of cultures indicating that callus cell fate is determined at induction, which closely follows apogamous sporophyte development. Presence of a dense extracellular matrix in conjunction with sporophyte development suggests a link between the suspensor-like activity of the embryonic foot during normal fern embryo development and the suspected functions of extracellular matrices in angiosperms. Conclusions: Further investigation could lead to a better understanding of genes involved in P. vittata embryo development and apogamous sporophyte development. The methodology could be useful for in vitro propagation of rare and valuable fern germplasm

Inferring biomarkers for Mycobacterium avium subsp. paratuberculosis infection and disease progression in cattle using experimental data

Available diagnostic assays for Mycobacterium avium subsp. paratuberculosis (MAP) have poor sensitivities and cannot detect early stages of infection, therefore, there is need to find new diagnostic markers for early infection detection and disease stages. We analyzed longitudinal IFN-γ, ELISA-antibody and fecal shedding experimental sensitivity scores for MAP infection detection and disease progression. We used both statistical methods and dynamic mathematical models to (i) evaluate the empirical assays (ii) infer and explain biological mechanisms that affect the time evolution of the biomarkers, and (iii) predict disease stages of 57 animals that were naturally infected with MAP. This analysis confirms that the fecal test is the best marker for disease progression and illustrates that Th1/Th2 (IFN-γ/ELISA antibodies) assays are important for infection detection, but cannot reliably predict persistent infections. Our results show that the theoretical simulated macrophage-based assay is a potential good diagnostic marker for MAP persistent infections and predictor of disease specific stages. We therefore recommend specifically designed experiments to test the use of a based assay in the diagnosis of MAP 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

Induction of B Cell Responses upon Experimental Infection of Neonatal Calves with Mycobacterium avium subsp. paratuberculosis

The objective of this study was to determine if experimental infection of neonatal calves with Mycobacterium avium subsp. paratuberculosis would invoke changes in the percentages of total B cells in the peripheral blood mononuclear cell population and of subpopulations of B cells as determined by CD5, CD25, and CD45RO markers during a 12-month period. Experimental infection groups included control (noninfected), oral (infected with M. avium subsp. paratuberculosis strain K-10), oral/DXM (pretreatment with dexamethasone before oral inoculation), i.p. (intraperitoneal inoculation), and oral/M (oral inoculation with mucosal scrapings from a cow with clinical disease) groups. Over the course of the study, the percentages of total B cells in nonstimulated and antigen-stimulated cell cultures increased for oral and i.p. group calves, with the highest percentages noted at 3 and 6 months. Oral/M group calves had increased percentages of activated B cells, as determined by CD5dim and CD5bright markers, at 9 and 12 months. Experimental infection by all methods resulted in increased expression of CD25 and CD45RO B cells early in the study, but the most significant results were observed at 12 months for oral/DXM and oral/M group calves. Immunoblot analyses with a whole-cell sonicate of M. avium subsp. paratuberculosis demonstrated the most reactivity with sera from i.p. group calves and the least reactivity with sera from oral group calves. Further evidence of M. avium subsp. paratuberculosis-specific antibody responses in the i.p. group calves was demonstrated using the ethanol vortex enzyme-linked immunosorbent assay (EvELISA) method. In summary, an induction of B cell responses was noted after experimental infection with M. avium subsp. paratuberculosis, with differences in responses noted according to the method of experimental inoculation

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

Quantifying Limits on Replication, Death, and Quiescence of Mycobacterium tuberculosis in Mice

When an individual is exposed to Mycobacterium tuberculosis (Mtb) three outcomes are possible: bacterial clearance, active disease, or latent infection. It is generally believed that most individuals exposed to Mtb become latently infected and carry the mycobacteria for life. How Mtb is maintained during this latent infection remains largely unknown. During an Mtb infection in mice, there is a phase of rapid increase in bacterial numbers in the murine lungs within the first 3 weeks, and then bacterial numbers either stabilize or increase slowly over the period of many months. It has been debated whether the relatively constant numbers of bacteria in the chronic infection result from latent (dormant, quiescent), non-replicating bacteria, or whether the observed Mtb cell numbers are due to balance between rapid replication and death. A recent study of mice, infected with a Mtb strain carrying an unstable plasmid, showed that during the chronic phase, Mtb was replicating at significant rates. Using experimental data from this study and mathematical modeling we investigated the limits of the rates of bacterial replication, death, and quiescence during Mtb infection of mice. First, we found that to explain the data the rates of bacterial replication and death could not be constant and had to decrease with time since infection unless there were large changes in plasmid segregation probability over time. While a decrease in the rate of Mtb replication with time since infection was expected due to depletion of host\u27s resources, a decrease in the Mtb death rate was counterintuitive since Mtb-specific immune response, appearing in the lungs 3–4 weeks after infection, should increase removal of bacteria. Interestingly, we found no significant correlation between estimated rates of Mtb replication and death suggesting the decline in these rates was driven by independent mechanisms. Second, we found that the data could not be explained by assuming that bacteria do not die, suggesting that some removal of bacteria from lungs of these mice had to occur even though the total bacterial counts in these mice always increased over time. Third and finally, we showed that to explain the data the majority of bacterial cells (at least ~60%) must be replicating in the chronic phase of infection further challenging widespread belief of nonreplicating Mtb in latency. Our predictions were robust to some changes in the structure of the model, for example, when the loss of plasmid-bearing cells was mainly due to high fitness cost of the plasmid. Further studies should determine if more mechanistic models for Mtb dynamics are also able to accurately explain these data

\u3cem\u3eMycobacterium avium\u3c/em\u3e subsp. \u3cem\u3eparatuberculosis\u3c/em\u3e lipophilic antigen causes Crohn’s disease-type necrotizing colitis in Mice

Background: A 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced murine colitis model was developed to investigate the pathogenesis and to evaluate a method of treating human Crohn’s disease. This experimental model rapidly induces colitis similar to human Crohn’s disease lesion in a reproducible manner. However, natural exposure of the human digestive tract to TNBS is unrealistic. A novel animal model based on realistic data is eagerly anticipated in future research on pathogenesis of CD. Method: We evaluated the potency of Map antigen molecules in an effort to develop a novel colitis model using a more realistic source than TNBS. We prepared the Map antigen by ethanol extraction and developed a mouse model in a manner similar to that of the well-known TNBS-induced colitis in mice. In the experiment, seven days after subcutaneous (SC) injection of the antigen into normal C57BL/6 mice, the same antigen in 50% ethanol was injected into the colon by the transanal route with a fine cannula. Results: On the fifth day after the transanal injection, histopathological examination revealed full-thickness necrotizing colitis with erosion and ulcers; severe infiltration with neutrophils, lymphocytes, macrophages, and perforation. However, no change was detected with each single Map-antigen injection. Conclusion: The present results provide a novel animal model for research on CD and may be the key to clarifying the relationship between CD and Map. This is the first evidence that mycobacterium antigen induces necrotizing colitis

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