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Development and evaluation of a Mycobacterium bovis interferon-γ enzyme-linked immunospot (ELISpot) assay for detection of bovine tuberculosis
Abstract
Bovine tuberculosis (bTB) caused by Mycobacterium bovis is an important zoonotic disease. This infection is difficult to control because of the limited ability of the tuberculin skin test (TST) and ancillary IFN-γ release assay to detect all infected animals. In this study, we aimed to develop an efficient assay based on the enzyme-linked immunospot (ELISpot) technique for the diagnosis of bTB, with IFN-γ monoclonal antibodies 3E9 and Bio-labeled 6F8 used as capture and detection antibodies, respectively. As expected, there were significantly more M. bovis-specific spot-forming units (SFU) in bTB-infected cattle than in healthy cattle when an M. bovis-specific antigen, CFP-10-ESAT-6 fusion protein (CE protein), was used. The M. bovis IFN-γ ELISpot assay demonstrated a high level of agreement (90.83%) with the BOVIGAM ELISA test (Thermo Fisher Scientific) for detecting bTB. Furthermore, 3 of 109 cattle tested negative by both the TST and the BOVIGAM ELISA tests, but positive by the ELISpot assay (TST⁻ ELISA⁻ ELISpot⁺). During subsequent long-term monitoring, these 3 cattle became TST⁺ ELISA⁺ ELISpot⁺. These results suggest that the M. bovis IFN-γ ELISpot assay we established could detect infected cattle earlier than the BOVIGAM ELISA test.
... The development of monoclonal antibodies against bovine IFN-γ made it easier and simpler to measure IFN-γ produced from stimulated lymphocytes using a sandwich ELISA. Application of ELISA for measuring Bo-IFN-γ showed sensitivity and specificity comparable and even better than the standard tuberculin test (Bass et al. 2013;Neeraja et al. 2014;Genç et al. 2015;Risalde et al. 2017;Keck et al. 2018;Li et al. 2022). IFN-γ assay use was also extended for the diagnosis of human tuberculosis using monoclonal antibodies produced against human IFN-γ (McCallan et al. 2021) and for the diagnosis of Johne's disease in cattle (Fan et al. 2012). ...
... We did not employ the hybridoma clones that had low growth to make ascetic fluid. Lilenbaum et al. 1999;Wood and Jones 2001;Gormley et al. 2006;Schiller et al. 2009;Ferrara et al. 2009;Faye et al. 2011;Bass et al. 2013;Genç et al. 2015;Risalde et al. 2017;Li et al. 2022) as follows: ...
ABSTRACT
Bovine tuberculosis is an infectious and chronic disease affecting cattle, caused by Mycobacterium bovis (M. bovis). The zoonotic nature of the disease has a serious worldwide impact on human health. Also, the significant economic costs caused by such disease in addition to the deficiency of precise estimates of the actual disease prevalence necessitate more efficient detection and control measures, particularly in developing countries. The main target of the present work was to develop a local, less expensive bovine tuberculosis interferon-gamma ELISA (Bo-IFN-γ ELISA) kit for the rapid and accurate diagnosis of bovine tuberculosis. In the current study, three murine hybridomas producing monoclonal antibodies (mAbs) against bovine interferon-gamma (Bo-IFN-γ) were developed and their monoclonal antibodies were characterized. The produced mAbs were of IgM isotype and their specificity was proved using the western blot technique. The prepared mAbs were used for the development of the bovine IFN-γ ELISA test that was evaluated for laboratory diagnosis of bovine tuberculosis. The sensitivity and specificity of the developed ELISA kit as compared with the standard tuberculin skin test were determined. This method is based on the measurement of IFN-γ released from sensitized bovine lymphocytes upon exposure to the mycobacterial antigens. Using checkerboard titration, the optimal coating concentration of anti-Bo-IFN-γ was 20μg/well. Blood samples from apparently healthy cattle, which proved negative in the tuberculin test, were examined with the developed kit and the cutoff value (COV) was equal to 0.30 optical density (OD). In a preliminary study to evaluate the sensitivity and specificity of the developed ELISA, 23 cattle were examined with the Bo-IFN-γ ELISA and the standard tuberculin skin test. The developed Bo-IFN-γ ELISA showed high sensitivity (98%) and specificity (71.4%) in the diagnosis of bovine tuberculosis as compared to the standard tuberculin skin test.
... IGRA is a new immunodetection method for Mycobacterium tuberculosis infection, and the accuracy of this method is more than 95% (13). However, its high experimental technical requirements also limit its wide promotion and application (14). The two detection methods are prone to misdiagnosis of infected cattle with low CMI response levels in the early stage of infection or with low immunity in the late stage of infection (15). ...
Bovine tuberculosis (bTB) is a chronic zoonotic disease caused by Mycobacterium bovis. A large number of cattle are infected with bTB every year, resulting in huge economic losses. How to control bTB is an important issue in the current global livestock economy. In this study, the original transcriptome sequences related to this study were obtained from the dataset GSE192537 by searching the Gene Expression Omnibus (GEO) database. Our differential gene analysis showed that there were obvious biological activities related to immune activation and immune regulation in the early stage of bTB. Immune-related biological processes were more active in the early stage of bTB than in the late. There were obvious immune activation and immune cell recruitment in the early stage of bTB. Regulations in immune receptors are associated with pathophysiological processes of the early stage of bTB. A gene module consisting of 236 genes significantly related to the early stage of bTB was obtained by weighted gene co-expression network analysis, and 18 hub genes were further identified as potential biomarkers or therapeutic targets. Finally, by random forest algorithm and logistic regression modeling, FCRL1 was identified as a representative mRNA marker in early bTB blood. FCRL1 has the potential to be a diagnostic biomarker in early bTB.
... Bovine tuberculosis (bovine TB) is a chronic granulomatous disease caused by bacteria of the Mycobacterium tuberculosis complex (MTC), in particular Mycobacterium bovis (Li et al., 2022). Although M. bovis is mainly a pathogen of cattle, a range of other domestic and wild animal hosts are also affected (Sichewo et al., 2020;. ...
... Despite enhanced sensitivity of around 81.8% and specificity of up to 94%, this method is still limited in detecting infection at different stages, as well as in postmortem tests [15] . On the other hand, molecular-based tools such as mycobacterial interspersed repeat units-variable number tandem repeats (MIRU-VNTR), spoligotyping, and restriction fragment length polymorphism (RFLP) are currently proposed for mycobacterial DNA detection. ...
Bovine tuberculosis is a prevalent zoonotic disease that causes high risks for production animals, dairy producers and consumers, together with significant economic losses. Thus, methods for easy, fast and specific detection of Mycobacterium bovis in small and medium-sized livestock under field conditions are very required. In this work, a Loop-Mediated Isothermal Amplification LAMP-PCR targeting the Region of Difference 12 (RD12) of M. bovis genome was designed for the purpose of identification. A set of six primers designed for the isothermal amplification of five different genomic fragments led to the specific identification of M. bovis from other mycobacterial species. A basic colorimetric reaction was clearly observed at first sight under natural light, indicating positive identification of M. bovis in a maximum of 30 min of isothermal amplification at 65 °C. The limit of detection was near 50 fg of M. bovis genomic DNA, corresponding approximately to 10 copies of the genome.
•The proposed LAMP-PCR amplification of M. bovis genomic DNA might be performed by untrained laboratory personnel.
•Specific identification of M. bovis LAMP is possible in 30 min at 65.. C using a simple water bath.
•The basic colorimetric reaction for M. bovis identification could be observed with the naked eye under natural light.
... At present, the quantitative detection method of IFN-γ is conducted through immunological methods. ELISA[16] [17][23] and Enzyme-Linked Immunospot Assay (ELISPOT)[24] [25] are the current clinically existing methods to detect IFN-γ. The reagentX. ...
Zoonotic tuberculosis (ZTB) is a worldwide disease caused by Mycobacterium tuberculosis complex (MTC) members that can infect humans and a wide range of domestic and wild mammals. The majority of zoonoses occur when humans come into contact with relatively abundant animal species, and they continue to pose a genuine threat to public health. Bovine tuberculosis (BTB), caused by Mycobacterium bovis (M. bovis), is one of the world’s most economically significant zoonotic diseases. The common transmission routes from animals to humans are airborne transmission, unpasteurized milk consumption, and direct contact with untreated animal products or infected animals. The diagnosis of M. bovis infection in animal species is critical for limiting disease propagation and management. The detection of M. bovis-infected individuals is difficult because only severely diseased animals show clinical manifestations, limiting its early-stage controls. The utility of conventional and immunological diagnostic tools in detecting infection at an early stage is limited. However, developing novel reagents and technologies for detecting M. bovis infection in domestic species is helping advance bovine TB diagnosis.
The interferon-γ assay has been used worldwide as an ancillary test for the diagnosis of bovine tuberculosis (bTB). This study aimed to describe, based on the bTB-free status in Switzerland, the difference of applying a more stringent cutoff point of 0.05 compared with 0.1 for bTB surveillance. Moreover, the effect of time between blood collection and stimulation, culture results, optical density values, and the influence of testing different breeds were evaluated. Blood samples from a total of 118 healthy cows older than 6 months were tested with three commercial interferon-gamma assays. To confirm the bTB-free status of the tested animals and to investigate potential cross-reactions with nontuberculous mycobacteria, pulmonary and abdominal lymph nodes in addition to ileal mucosa from each cattle were used for the detection of viable Mycobacteria spp. by specific culture. Significant differences regarding the proportion of false-positive results between the two Bovigam tests and between Bovigam 2G and ID Screen were found. Samples analyzed with Bovigam 2G were 2.5 [95% confidence interval (CI) 1.6–3.9] times more likely to yield a false-positive test result than samples analyzed with Bovigam TB. Similarly, the odds ratio (OR) for testing samples false-positive with ID Screen compared with Bovigam TB was 1.9 (95% CI 1.21–2.9). The OR for testing false-positive with ID Screen compared with Bovigam 2G was less to equally likely with an OR of 0.75 (95% CI 0.5–1.1). When using a cutoff of 0.05 instead of 0.1, the OR for a false-positive test result was 2.2 (95% CI 1.6–3.1). Samples tested after 6 h compared with a delayed stimulation time of 22–24 h were more likely to yield a false-positive test result with an OR of 3.9 (95% CI 2.7–5.6). In conclusion, applying a more stringent cutoff of 0.05 with the Bovigam 2G kit generates a questionable high number of false-positive results of one of three tested animals. Furthermore, specific breeds might show an increased risk to result false-positive in the Bovigam 2G and the ID Screen assays.
Bovine tuberculosis (bTB) is one of the major zoonotic concerns of the world, as milk and meat from cattle are major products for human consumption. Bovine tuberculosis not only affects the health of cattle and poses an imminent zoonotic threat, but also causes significant economic loss in both developed and developing countries. This systematic review reports the prevalence of Mycobacterium tuberculosis complex (MTBC) organisms in slaughtered cattle showing tuberculosis like lesion (TBL) with available literature worldwide. Appropriate keywords were used to search various databases to collect articles pertaining to slaughterhouse studies. Bovine TB prevalence, based on the prevalence of MTBC organisms in slaughtered cattle showing TBL by culture, microscopy, PCR and spoligotyping was assessed in each study using a random-effects model and standardized mean with 95% confidence interval (CI). Heterogeneity was assessed by the I2 statistic. Publication bias was evaluated using funnel plots. Out of 72 hits, 37 studies were selected based on title and abstract. Ten articles were excluded due to lack of desired data, and 27 studies were included in the final analysis. From the selected articles, it was found that 426 [95% CI: 302 - 560] per 1,000 slaughtered cattle with TBL were positive for the presence of MTBC organisms. The sensitivity analysis showed that no individual study alone influenced the estimation of pooled prevalence. The prevalence of MTBC organisms in slaughtered cattle showing TBL by culture, microscopy, PCR and spoligotyping was 474[95% CI: 342- 610], 385 [95% CI: 269- 515], 218 [95% CI: 132- 338], 326 [95% CI: 229-442] respectively, per 1,000 slaughtered cattle. Most of the slaughtered cattle were from the same locality as the slaughterhouse. The results obtained in this study suggest that abattoir monitoring can give an estimate of the prevalence of bTB in that locality. This study also emphasizes the need to test cattle and animal handlers who were in contact with bTB positive cattle.
The study and characterization of biomolecules involved in the interaction between mycobacteria and their hosts are crucial to determine their roles in the invasion process and provide basic knowledge about the biology and pathogenesis of disease. Promising new biomarkers for diagnosis and immunotherapy have emerged recently. My-cobacterium is an ancient pathogen that has developed complex strategies for its persistence in the host and environment , likely based on the complexity of the network of interactions between the molecules involved in infection. Several biomarkers have received recent attention in the process of developing rapid and reliable detection techniques for tuberculosis. Among the most widely investigated antigens are CFP-10 (10-kDa culture filtrate protein), ESAT-6 (6-kDa early secretory antigenic target), Ag85A, Ag85B, CFP-7, and PPE18. Some of these antigens have been proposed as biomarkers to assess the key elements of the response to infection of both the pathogen and host. The design of novel and accurate diagnostic methods is essential for the control of tuberculosis worldwide. Presently, the diagnostic methods are based on the identification of molecules in the humoral response in infected individuals. Therefore, these tests depend on the capacity of the host to develop an immune response, which usually is heterogeneous. In the last 20 years, special attention has been given to the design of multiantigenic diagnostic methods to improve the levels of sensitivity and specificity. In this review, we summarize the state of the art in the study and use of mycobacterium biomolecules with the potential to support novel tuberculosis control strategies.
The Ag85 complex functions as the main secretory protein of Mycobacterium tuberculosis (M. tuberculosis) and BCG. This complex is composed of the proteins, Ag85A, Ag85B, and Ag85C, with Ag85A thought to play the largest role within the complex. However, the lack of commercially available monoclonal antibodies (mAbs) against Ag85A still hinders the biological and applicative research on this protein. In this study, we developed and identified anti-Ag85A mAbs, and five hybridoma cells were established. Using the indirect immunofluorescence test, we found that two anti-Ag85A mAbs did not cross-react with Ag85B and/or Ag85C. In addition, we showed that all of the mAbs tested in this study are able to react with endogenous Ag85A protein in BCG and rBCG:Ag85A using indirect ELISA and Western blot analyses. A competitive ELISA (cELISA) based on mAb 3B8 was developed, the analyses of clinic serum samples from cattle with bovine tuberculosis (TB) and healthy cattle demonstrated that the sensitivity of the cELISA was 54.2% (26/48) and the specificity was 83.5% (167/200). This study demonstrated that the mAbs against Ag85A will provide useful reagents for further investigation into the function of the Ag85 complex and can be used for serodiagnosis of bovine TB.
There are two different contexts in the Irish bTB eradication programme in which the interferon-gamma assay (IFN-γ) is applied. Firstly, the IFN-γ assay is applied routinely to high risk cohorts in herds with four or more reactors to the SICTT. The IFN-γ test is then carried out on blood samples submitted to the laboratory within 8 h of collection (diagnostic testing). Secondly, the use of the IFN-γ assay has recently been extended to test SICTT reactors as part of a general quality assurance (QA) scheme to monitor the performance of the SICTT. Blood samples from reactors are tested one day after blood collection (QA testing). In this study, we analysed the relative performance of the SICTT and IFN-γ when used in parallel as an 8 hour diagnostic test and as a 24 hour QA test on SICTT reactors. A total of 17,725 IFN-γ tests were included in the analysis (11,658 diagnostic tests and 6,067 QA tests). Of the samples submitted for diagnostic testing, the proportion positive to IFN-γ decreased with the severity of interpretation of the SICTT result. Of the standard reactors that were tested with IFN-γ in the QA programme, 92.2% were positive to the IFN-γ test. Among animals that were SICTT −ve/IFN-γ +ve, 18.9% were positive at post-mortem compared to 11.8% of those that were SICTT +ve (standard reactor)/IFN-γ −ve. These results highlight the risk associated with retaining SICTT −ve/IFN-γ +ve animals, and suggest that prompt removal of these animals is necessary to reduce the potential for future transmission.
Background
Bovine tuberculosis (bTB) is a disease of significant economic importance and is a persistent animal health problem with implications for public health worldwide. Control of bTB in the UK has relied on diagnosis through the single intradermal comparative cervical test (SICCT). However, limitations in the sensitivity of this test hinder successful eradication and the control of bTB remains a major challenge. Genetic selection for cattle that are more resistant to bTB infection can assist in bTB control. The aim of this study was to conduct a quantitative genetic analysis of SICCT measurements collected during bTB herd testing. Genetic selection for bTB resistance will be partially informed by SICCT-based diagnosis; therefore it is important to know whether, in addition to increasing bTB resistance, this might also alter genetically the epidemiological characteristics of SICCT. ResultsOur main findings are that: (1) the SICCT test is robust at the genetic level, since its hierarchy and comparative nature provide substantial protection against random genetic changes that arise from genetic drift and from correlated responses among its components due to either natural or artificial selection; (2) the comparative nature of SICCT provides effective control for initial skin thickness and age-dependent differences; and (3) continuous variation in SICCT is only lowly heritable and has a weak correlation with SICCT positivity among healthy animals which was not significantly different from zero (P > 0.05). These emerging results demonstrate that genetic selection for bTB resistance is unlikely to change the probability of correctly identifying non-infected animals, i.e. the test’s specificity, while reducing the overall number of cases. Conclusions
This study cannot exclude all theoretical risks from selection on resistance to bTB infection but the role of SICCT in disease control is unlikely to be rapidly undermined, with any adverse correlated responses expected to be weak and slow, which allow them to be monitored and managed.
Bovine tuberculosis (bTB), caused by Mycobacterium bovis (M. bovis), is a serious re-emerging disease in both animals and humans. The evolution of the Multi- and Extensively drug-resistant M. bovis strains (MDR-TB and XDR-TB) represents a global threat to public health. Worldwide, the disease is responsible for great economic losses in the veterinary field, serious threat to the ecosystem, and about 3.1% of human TB cases, up to 16% in Tanzania. Only thorough investigation to understand the pathogen's epidemiology can help in controlling the disease and minimizing its threat. For this purpose, various tools have been developed for use in advanced molecular epidemiological studies of bTB, either alone or in combination with standard conventional epidemiological approaches. These techniques enable the analysis of the intra- and inter-species transmission dynamics of bTB. The delivered data can reveal detailed insights into the source of infection, correlations among human and bovine isolates, strain diversity and evolution, spread, geographical localization, host preference, tracing of certain virulence factors such as antibiotic resistance genes, and finally the risk factors for the maintenance and spread of M. bovis. They also allow for the determination of epidemic and endemic strains. This, in turn, has a significant diagnostic impact and helps in vaccine development for bTB eradication programs. The present review discusses many topics including the aetiology, epidemiology and importance of M. bovis, the prevalence of bTB in humans and animals in various countries, the molecular epidemiology of M. bovis, and finally applied molecular epidemiological techniques.
Bovine tuberculosis (bTB) remains a globally significant veterinary health problem. Defining correlates of protection can accelerate the development of novel vaccines against TB. As the cultured IFNγ ELISPOT (cELISPOT) assay has been shown to predict protection and duration of immunity in vaccinated cattle, we sought to characterize the phenotype of the responding T-cells. Using expression of CD45RO and CD62L we purified by cytometric cell sorting four distinct CD4(+) populations: CD45RO(+)CD62L(hi), CD45RO(+)CD62L(lo), CD45RO(-)CD62L(hi) and CD45RO(-)CD62L(lo) (although due to low and inconsistent cell recovery, this population was not considered further in this study), in BCG vaccinated and Mycobacterium bovis infected cattle. These populations were then tested in the cELISPOT assay. The main populations contributing to production of IFNγ in the cELISPOT were of the CD45RO(+)CD62L(hi) and CD45RO(+)CD62L(lo) phenotypes. These cell populations have been described in other species as central and effector memory cells, respectively. Following in vitro culture and flow cytometry we observed plasticity within the bovine CD4(+) T-cell phenotype. Populations switched phenotype, increasing or decreasing expression of CD45RO and CD62L within 24h of in vitro stimulation. After 14 days all IFNγ producing CD4(+) T cells expressed CD45RO regardless of the original phenotype of the sorted population. No differences were detected in behavior of cells derived from BCG-vaccinated animals compared to cells derived from naturally infected animals. In conclusion, although multiple populations of CD4(+) T memory cells from both BCG vaccinated and M. bovis infected animals contributed to cELISPOT responses, the dominant contributing population consists of central-memory-like T cells (CD45RO(+)CD62L(hi)).
Description of the technology Elispot is a workhorse for functional analysis of single immune cells. Originally established for the enumeration of specific antibody-secreting B cells in 1983 [1], the technology has been expanded to the analysis of T cells, NK cells and monocytes. The assay is typically carried out in 96 well plates with a nitrocellulose or PVDF membrane, which is coated with an antibody that is directed against the analyte of interest. Cells and stimulants are added to the plate, and secreted analyte is captured in the immediate surroundings of the cell. After cell removal, bound analyte is made visible via the addition of a biotinylated secondary antibody, avidinenzyme complex and substrate that precipitates onto the membrane and forms spots which can be automatically enumerated. Each spot represents one cell that secreted the analyte. The most common analytes investigated today are cytokines (IFNɣ ,I L-2, IL-5, IL-10, IL-17, Granzyme B, TNF, GM-CSF and many more) and immunoglobulins. Others can also be evaluated, such as chemokines (e.g., CXCL8) or apolipoproteins. The hallmark of the assay is its sensitivity allowing the detection of antigen-specific immune cells in very low frequencies. In contrast to other assays, Elispot measures cells that actually secrete analytes without impairment by receptor binding or protease activity. The assay is easy to perform and transferable, and can be adapted to high throughput. While it was historically limited to the assessment of one parameter per assay, it has now been expanded to the simultaneous assessment of 2 or even 3 parameters in one well by the introduction of fluorescent dyes in the detection cascade (Fluorospot), allowing the identification of up to 7 subsets of immune cells in one well with similar ease of performance as for the enzymatic Elispot evaluating just one parameter (Fig. 1). Because of its substantial penetration of translational research, Elispot underwent rigorous harmonization efforts led by the cancer immunotherapy field [2], and it is the only functional immune monitoring assay for which a proficiency panel program has been made available for any laboratory independent of affiliation or scientific background (www.proficiencypanel.com). Lastly, the Elispot assay performance can readily be standardized, qualified, and, if required, validated according to the International Congress of Harmonization (ICH) guidelines [3].
Cultured IFN-γ ELISPOT assays are primarily a measure of central memory T cell (Tcm) responses with humans; however, this important subset of lymphocytes is poorly characterized in cattle. Vaccine-elicited cultured IFN-γ ELISPOT responses correlate with protection against bovine tuberculosis in cattle. However, whether this assay measures cattle Tcm responses or not is uncertain. The objective of the present study was to characterize the relative contribution of Tcm (CCR7+, CD62Lhi, CD45RO+), T effector memory (Tem, defined as: CCR7-, CD62Llow/int, CD45RO+), and T effector cells (CCR7-, CD62L-/low, CD45RO-), in the immune response to Mycobacterium bovis. Peripheral blood mononuclear cells (PBMC) from infected cattle were stimulated with a cocktail of M. bovis purified protein derivative, rTb10.4 and rAg85A for 13 days with periodic addition of fresh media and rIL-2. On day 13, cultured PBMC were re-stimulated with medium alone, rESAT-6:CFP10 or PPDb with fresh autologous adherent cells for antigen presentation. Cultured cells (13 days) or fresh PBMCs (ex vivo response) from the same calves were analyzed for IFN-γ production, proliferation, and CD4, CD45RO, CD62L, CD44, and CCR7 expression via flow cytometry after overnight stimulation. In response to mycobacterial antigens, ~75% of CD4+ IFN-γ+ cells in long-term cultures expressed a Tcm phenotype while less than 10% of the ex vivo response consisted of Tcm cells. Upon re-exposure to antigen, long-term cultured cells were highly proliferative, a distinctive characteristic of Tcm, and the predominant phenotype within the long-term cultures switched from Tcm to Tem. These findings suggest that proliferative responses of Tcm cells to some extent occurs simultaneously with reversion to effector phenotypes (mostly Tem). The present study characterizes Tcm cells of cattle and their participation in the response to M. bovis infection.
The Delayed type hypersensitivity skin test (DTH) and interferon-gamma assay are used for the diagnosis of bovine tuberculosis (TBB). The specificity of these diagnoses, however, is compromised because both are based on the response against purified protein derivative of Mycobacterium bovis (PPD-B). In this study, we assessed the potential of two cocktails containing M. bovis recombinant proteins: cocktail 1 (C1): ESAT-6, CFP-10 and MPB83 and cocktail 2 (C2): ESAT-6, CFP-10, MPB83, HspX, TB10.3, and MPB70. C1, C2, and PPD-B showed similar response by DTH in M. bovis-sensitized guinea pigs. Importantly, C1 induced a lower response than PPD-B in M. avium-sensitized guinea pigs. In cattle, C1 displayed better performance than PPD-B and C2; indeed, C1 showed the least detection of animals either vaccinated or Map-infected. To optimize the composition of the cocktails, we obtained protein fractions from PPD-B and tested their immunogenicity in experimentally M. bovis-infected cattle. In one highly reactive fraction, seven proteins were identified. The inclusion of FixB in C1 enhanced the recognition of M. bovis-infected cattle without compromising specificity. Our data provide a promising basis for the future development of a cocktail for TBB detection without interference by the presence of sensitized or infected animals with other mycobacteria.
We aimed to estimate the global occurrence of zoonotic tuberculosis (TB) caused by Mycobacterium bovis or M. caprae infections in humans by performing a multilingual, systematic review and analysis of relevant scientific literature of the last 2 decades. Although information from many parts of the world was not available, data from 61 countries suggested a low global disease incidence. In regions outside Africa included in this study, overall median proportions of zoonotic TB of ≤1.4% in connection with overall TB incidence rates ≤71/100,000 population/year suggested low incidence rates. For countries of Africa included in the study, we multiplied the observed median proportion of zoonotic TB cases of 2.8% with the continental average overall TB incidence rate of 264/100,000 population/year, which resulted in a crude estimate of 7 zoonotic TB cases/100,000 population/year. These generally low incidence rates notwithstanding, available data indicated substantial consequences of this disease for some population groups and settings.
Bovine tuberculosis (BTB) is a chronic bacterial disease, and a major animal health problem with zoonotic implications. Screening of mycobacterial infections in bovines is traditionally done using the single intradermal tuberculin test. Though the test is widely used, it has its own disadvantages and they include its inability to distinguish between pathogenic and non-pathogenic mycobacterial infections owing to its low specificity. Furthermore, the associated operative difficulties of this test have driven the quest for discovery of new antigens and diagnostic assays leading to the development of the interferon (IFN)- test. Presently, combinatorial testing using the skin test and the interferon gamma assays are being used in the diagnosis of BTB in various control and surveillance programs. In this study, we report the cloning, expression and purification of ESAT-6-CFP-10 fusion protein and its further use in the development of the IFN- gamma ELISPOT assay for accurate diagnosis of BTB in cattle. The BTB diagnosis employing the ELISPOT assay was evaluated using peripheral blood mononuclear cells from culture positive and culture negative cattle. The ELISPOT assay showed higher specificity and sensitivity in detecting BTB when a recombinant ESAT-6-CFP-10 fusion protein was used. The present study indicated that the usefulness of the fusion protein can replace the ESAT-6, CFP-10 or combination of both proteins for detecting BTB in IFN-gamma ELISPOT assay.
Switzerland has been officially free of bovine tuberculosis (OTF) since 1960. Since 1980 the control of bovine tuberculosis (bTB) has been reduced to passive abattoir surveillance. Isolated cases of bTB, partly due to reactivation of human Mycobacterium bovis infections with subsequent transmission to cattle, have been noticed in the last years. In Europe, the overall prevalence of bTB is slightly increasing. Both OTF and non-OTF countries report increases in the proportion of bTB positive cattle herds.
In vitro production of bovine interferon gamma (BoIFN-γ) cytokine from bovine peripheral blood mononuclear cells (PBMCs) can be detected using the most sensitive enzyme-linked immunosorbent spot (ELISPOT) assay. ELISPOT assays are dependent on the quantity and quality of PBMC preparations and hence contribute significantly to the performance of this assay. In order to standardise the methods for isolation of PBMCs, we compared two methods for the processing of bovine blood which included aliquots of blood that were stored in a horizontal position without dilution or agitation and aliquots of blood that were immediately diluted 1:1 with complete Rosewell Park Memorial Institute (RPMI) 1640 medium and stored in a horizontal position with gentle agitation. PBMCs were isolated at 2, 4, 6, 8 and 24 h and at 4°C and at 22°C ± 2°C. They were stimulated using tuberculosis-specific antigens, after which the ELISPOT assay was performed. Quantities of spot-forming cells (SFC) created by the release of BoIFN-γ in ELISPOT assays were significantly greater in the samples stored at 22°C ± 2°C than those held +4°C and the intensity of the signals dropped following processing after 6 h. A further drop in SFC was observed in those samples that had been stored undiluted and without agitation. These findings demonstrated that optimisation of PBMC isolation procedures can lead to increased sensitivity in the detection of BoIFN-γ using the ELISPOT assay, thus contributing to an enhanced diagnosis of bovine tuberculosis.
Existing strategies for long-term bovine tuberculosis (bTB) control/eradication campaigns are being reconsidered in many countries because of the development of new testing technologies, increased global trade, continued struggle with wildlife reservoirs of bTB, redistribution of international trading partners/agreements, and emerging financial and animal welfare constraints on herd depopulation. Changes under consideration or newly implemented include additional control measures to limit risks with imported animals, enhanced programs to mitigate wildlife reservoir risks, re-evaluation of options to manage bTB-affected herds/regions, modernization of regulatory framework(s) to re-focus control efforts, and consideration of emerging testing technologies (i.e. improved or new tests) for use in bTB control/eradication programs. Traditional slaughter surveillance and test/removal strategies will likely be augmented by incorporation of new technologies and more targeted control efforts. The present review provides an overview of current and emerging bTB testing strategies/tools and a vision for incorporation of emerging technologies into the current control/eradication programs.
The prevalence of bovine tuberculosis (BTB) in the UK remains a significant economic burden and problem for the agri-food industry. Much effort has been directed towards improving diagnostics, finding vaccine candidates and assessing the usefulness of badger culling. The contribution that host genotype makes to disease outcome has, until recently, been overlooked; yet, it is biologically untenable that genetic variation does not play a role. In this review, we highlight the evidence, past and present, for a role of host genetics in determining susceptibility to BTB in livestock. We then address some of the major issues surrounding the design of future studies tasked with finding the exact causative genetic variation underpinning the TB susceptibility phenotype. Finally, we discuss some of the potential future benefits, and problems, that a knowledge of the genetic component to BTB resistance/susceptibility may bring to the agricultural industries and the wider scientific community.
Bovine tuberculosis (bTB) is a disease of zoonotic and economic importance. In many countries, control is based on test and slaughter policies and/or abattoir surveillance. For testing, cell mediated immune- (CMI-) based assays (i.e., tuberculin skin test (TST) supplemented by the interferon gamma (IFN-gamma) assay) are the primary surveillance and disease control tests for bTB. The combined use of the in vivo and in vitro CMI assays to increase overall sensitivity has raised the question of whether the IFN-gamma response is influenced by injection of purified protein derivatives (PPDs) for TST. Published data on the influence of the TST, applied as the caudal fold test (CFT) or the comparative cervical test (CCT), on the IFN-gamma assay are contradictory. Reviewing published data and including additional data, the following conclusions can be drawn: (1) in naturally infected cattle, PPD administration for the single or repeated short-interval CCT neither boosts nor depresses PPD-specific IFN-gamma production. Disparate results have been concluded from some studies using experimental infections, emphasizing the importance of confirming initial experimental-based findings with studies using cattle naturally infected with Mycobacterium bovis. (2) In cattle experimentally infected with M. bovis, PPD administration for CFT boosts PPD-specific IFN-gamma production for up to 7 days without any effect on test interpretation. Importantly, in naturally infected cattle, CFT-related boosting selectively increases the in vitroM. bovis PPD (PPD-B) response 3 days after CFT, resulting in an increased PPD-B response relative to the response to Mycobacterium avium PPD (PPD-A). In non-infected cattle, it cannot be excluded that the CFT induces a mild boost of the PPD-specific response, particularly in animals sensitized to environmental, non-tuberculous mycobacteria, thus decreasing the specificity of the IFN-gamma assay. (3) In general, there is a lack of data clearly characterizing the effect of TSTs on the IFN-gamma assay. Further studies are required to clearly describe the effects of both CFT and CCT in non-infected animals and in naturally infected cattle, especially in low reacting infected cattle.
Overlapping peptides of Mycobacterium tuberculosis antigens ESAT-6 and CFP-10 offer increased specificity over the purified protein derivative skin test when they were used
in an ex vivo enzyme-linked immunospot (ELISPOT) assay for gamma interferon detection for the diagnosis of M. tuberculosis infection from recent exposure. We assessed whether equivalent results could be obtained for a fusion protein of the two
antigens and whether a combined readout would offer increased sensitivity in The Gambia. We studied the ELISPOT assay results
for 488 household contacts of 88 sputum smear-positive tuberculosis (TB) cases. The proportions of subjects positive by each
test and by the tests combined were assessed across an exposure gradient, defined according to sleeping proximity to a TB
case. Eighty-eight (18%) subjects were positive for CFP-10 peptides, 148 (30%) were positive for ESAT-6 peptides, 161 (33%)
were positive for both peptides, and 168 (34%) were positive for the fusion protein; 188 (39%) subjects had either a positive
result for a peptide or a positive result for the fusion protein. There was reasonable agreement between the peptide and the
protein results (kappa statistic = 0.78) and no significant discordance (P = 0.38). There was a strong correlation between the fusion protein and combined peptide spot counts (r = 0.9), and responses to the peptide and the proteins all increased significantly according to M. tuberculosis exposure. The proportion of subjects positive for either the pool of peptides or the fusion protein offered maximum sensitivity,
being significantly higher than the proportion of subjects positive for ESAT-6 peptides alone (P = 0.007). A fusion protein of ESAT-6 and CFP-10 is equivalent to overlapping peptides for the diagnosis of latent M. tuberculosis infection. Use of a combination of peptides and fusion protein offers improved sensitivity.
The early, preclinical stages of bovine TB can be detected in live animals by the use of tests of cellular immunity (the skin, γ-interferon and lymphocyte transformation tests). Tests of humoral (antibody) immunity, Mycobacterium bovis PCR probes on early tissue cultures or live cattle specimens, and tests based on “electronic nose” technology have been developed more recently. The key measure of diagnostic test accuracy is the relationship between sensitivity and specificity, which determines the false-positive and false-negative proportions. None of the tests currently available for the diagnosis of bovine TB allow a perfectly accurate determination of the M. bovis infection status of cattle. Although various factors can reduce the sensitivity and specificity of the skin tests, these remain the primary ante mortem diagnostic tools for TB in cattle, providing a cost-effective and reliable means of screening entire cattle populations. Despite the inescapable limitations of existing diagnostic tests, bovine TB has been effectively eradicated from many developed countries and regions with the implementation of sound programmes of regular tuberculin skin testing and removal of reactors, coupled with slaughterhouse surveillance for undetected infections, repeat testing and culling of infected herds, cattle movement restrictions to prevent introduction of infected animals and occasional slaughter of entire herds with intractable breakdowns. This is likely to remain the mainstay of bovine TB control programmes for the foreseeable future. Additionally, newer ancillary in vitro diagnostic assays are now available to TB control programme managers to supplement the skin tests in defined circumstances according to the specific disease situation in each country or region. The strategic deployment of ancillary in vitro tests alongside the primary skin tests has enhanced the detection of M. bovis-infected cattle and reduced the number of animals slaughtered as false positives.
Bovine tuberculosis is a major problem in many countries; hence, new and better diagnostic tools are urgently needed. In this
work, we have tested ESAT6, CFP10, PE13, PE5, MPB70, TB10.4, and TB27.4 for their potentials as diagnostic markers in field
animals from Northern Ireland, Mexico, and Argentina, regions with low, medium, and high prevalences of bovine tuberculosis,
respectively. At all three sites, ESAT6 and CFP10 were superior diagnostic antigens, while their combination performed even
better at the two sites where the combination was tested, providing the best coverage for the detection of diseased populations.
The high sensitivity in the skin test reactor groups, combined with the high specificity in the tuberculosis-free groups,
indicated that a diagnosis could correctly be made for 85% of the infected animals, based on their responses to these two
antigens. Furthermore, TB10.4, PE13, and PE5 have the potential to supplement ESAT6 and CFP10 in a future five-component diagnostic
cocktail.
Mycobacterium bovis, the causative agent of bovine tuberculosis (bTB), poses a risk of infection for livestock, humans, and wildlife. An interferon (IFN)-γ release assay has been used with tuberculin skin tests to detect bTB; however, infected animals may still be missed. Previous studies have suggested that bovine interleukin-2 (BoIL-2) may act as a potential biological marker for the diagnosis of bovine infectious diseases. However, a detailed evaluation of IL-2 as a diagnostic target for bTB is lacking. Therefore, we established hybridoma cell lines that produced monoclonal antibodies (mAbs) recognizing the native BoIL-2 and developed a flow cytometry assay, based on the BoIL-2 mAbs, for detecting M. bovis-specific IL-2. Subsequently, the method was utilized for a preliminary investigation of bTB in cattle; significantly (P < 0.0001) more CD4⁺IL-2⁺ T cells were detected in infected cattle than in healthy animals when a specific mycobacterial antigen CFP-10–ESAT-6 fusion protein was used. Moreover, our method demonstrated high coincidence rates with the BOVIGAM® test and an IFN-γ flow cytometry assay for the diagnosis of bTB. These findings show that the present method may be useful for detecting bTB.
Programs for the eradication of bovine tuberculosis (bTB) focus on the tuberculin skin test (TST) and slaughter of reactor cattle. However, the disease remains an animal health concern in several countries and improving the efficiency of the TST has become a critical issue. The detection of Mycobacterium bovis antibodies in serum, within weeks after the TST, may be a rapid and inexpensive way to improve bTB control. This study reports the validation of an enzyme-linked immunosorbent assay (ELISA) to detect bovine tuberculosis as an ancillary test to TST in dairy farms in Argentina. The estimated validation parameters were within the established requirements of the World Organization for Animal Health (OIE). The test demonstrated high repeatability, with coefficients of variation <25%. High test reproducibility through interlaboratory testing was also found, with an estimated Pearson coefficient of 0.9648 (95% confidence intervals 0.9315-0.9820). The ELISA detected tuberculous cattle unidentified by the TST. Of 43 animals sent to slaughterhouses that were ELISA positive 15-17 days after a negative TST, 36 were confirmed as infected with M. bovis by histopathology and IS6110 PCR. According to ROC curve analysis of results of 145 cattle from M. bovis-free herds and the 36 M. bovis-infected cattle, at a corrected optical density cut-off point of 0.3853, specificity was 95.95% and the positive predictive value at this cut-off was 83.72%. The ELISA detection test validated in this study could be readily applied in dairy farms, to complement a prior TST and improve livestock health.
Objectives:
Currently-available IFN-γ-release assays (IGRA) cannot discriminate active tuberculosis (TB) from latent TB infection (LTBI), and so have limited clinical utility for diagnosing active TB. Since numbers of TNF-α-producing T cells are highly correlated with active TB, we hypothesized that detecting IFN-γ- and/or TNF-α-producing T cells would overcome this limitation of IGRA. This study evaluated the diagnostic performances of the IFN-γ and TNF-α dual release fluorospot assay for active TB.
Methods:
Adult patients with suspected TB including recent TB exposers were prospectively enrolled over a 28-month period. In addition to the conventional IGRA test (i.e., QuantiFERON-In-Tube), a fluorospot assay for detecting IFN-γ- and TNF-α-producing T cells was performed. The final diagnoses were classified by clinical category. Patients with confirmed or probable TB were regarded as active TB, and patients with not active TB were further classified as having not active TB with and without LTBI, based on the QuantiFERON-In-Tube results.
Results:
A total of 153 patients including 45 with active TB and 108 with not active TB (38 LTBI vs 70 not LTBI) were finally analyzed. The sensitivity and specificity of the QuantiFERON-In-Tube assay for active TB were 84% (95% CI, 70-93) and 70% (95% CI 61-79), respectively. The IFN-γ/TNF-α-dual release assay by fluorospot had substantially higher diagnostic specificity (94%) for diagnosing active TB than the IFN-γ-single release assay (72%, p < 0.001), without compromising sensitivity (84% vs. 89%, p = 0.79).
Conclusions:
The fluorospot-based IFN-γ/TNF-α-dual release assay appears to be a simple and useful test for diagnosing active TB.
Human studies have identified the potential of measuring Mycobacterium tuberculosis specific IFN-γ and/or IL-2 secreting T cell subsets to distinguish different clinical stages of human tuberculosis (TB). To assess these functional T cell subsets in different states of bovine TB we have established a bovine dual IFN-γ/IL-2 fluorescence-immunospot (FluoroSpot) assay and analysed the frequencies of Mycobacterium bovis (M. bovis) specific IL-2 and/or IFN-γ producing cells in PBMC from 30 cattle naturally infected with M. bovis. Depending on their post mortem results the animals were grouped in 22 cattle with visible lesions (VL) and 8 cattle without visible lesions (NVL). In response to bovine tuberculin purified protein derivative (PPD-B) the frequencies of cytokine producing cells and proportions of IL-2 single producers were significantly higher in VL compared to NVL while PWM-induced cytokine responses were similar between the two groups. Dual IL-2+IFN-γ+ T cells could be identified as the largest PPD-B responsive T cell subset in both cattle groups. In conclusion, our FluoroSpot is a valid method to enumerate individual antigen-specific IFN-γ+ and IL-2+ T cell subsets ex vivo. The greater levels of single IL-2 producing T cells associated with the presence of pathology could be a potential biomarker for active TB in cattle.
Bovine tuberculosis (bTB) is an important animal and zoonotic disease, which causes severe economic losses. The main focus of this study was to assess the predictive power of previously identified biomarkers of bTB in infected animals that were negative to the tuberculin skin test (TST). We studied 16 animals with bTB, in which the disease was confirmed by necropsy, and 16 healthy animals. The level of expression of ten biomarkers (CXCL9, THBS1, MMP9, IL-22, CXCL10, IFNγ, IL-17, FYVE, CD14, IL-1R) was evaluated by RT-qPCR upon stimulation or not of peripheral blood mononuclear cells with PPDb (purified protein derivative of bovine tuberculin). In this assay, CXCL9, THBS1, MMP9, IL-22 and IFNγ changed their expression level depending on the bTB status. In addition, we evaluated different biomarker candidates simultaneously to infer the animal condition. By performing an analysis with classification trees, we found that the sturdiest combination was IL-22, IFNγ and IL-1R. On the other hand, CXCL10, IFNγ and IL-22's expression distinguished between bTB positive animals that were negative to TST (TST false negative animals) and the bTB negative groups. Thus, these biomarkers are promising candidates to be tested as an ancillary diagnostic assay. In addition, the expression of CXCL10 and IL-22 exhibited also significant differences between the bTB positive animals that were undetectable by IFNγ release assay (IGRA) and TST tests (TST and IGRA false negative animals) and the bTB negative groups. Therefore, CXCL10 and IL-22 constitute candidate biomarkers that could complement the two most widely used diagnostic tests.
ESAT-6, CFP-10 and EspC are virulence factors that have been extensively assayed for bovine and human tuberculosis diagnosis due their potent T-cell inducing activities. While polymorphisms of ESAT-6 and CFP-10 were analyzed, with the description of CFP-10 variants in M. tuberculosis, this fact has not been explored in M. bovis field isolates. The coding sequences of esxA (ESAT-6), esxB (CFP-10) and mb3645c (EspC) from 58 M. bovis strains exhibiting genomic variability (spoligotyping) were analyzed. Two genes –esxA and esxB – remained invariant while mb3645c exhibited one synonymous polymorphism (G to A mutation, position 66bp) in one isolate, compared to M. bovis AF2122/97 reference strain. All isolates exhibited a synonymous nucleotide polymorphism simultaneously (G to A mutation, position 255bp), compared to M. tuberculosis H37Rv reference strain. This study confirms the high conservation for ESAT-6, CFP-10 and EspC in local M. bovis field isolates and reinforce the use of these three antigens in the diagnosis of bovine tuberculosis. Further studies should be performed to globally confirm these findings.
Bovine tuberculosis remains a major economic and animal welfare concern worldwide. Cattle vaccination is being considered as part of control strategies. This approach, used alongside conventional control policies, also requires the development of vaccine-compatible diagnostic assays to distinguish vaccinated from infected animals (DIVA). We discuss progress made on optimizing the only potentially available vaccine, bacille Calmette Gueacuterin (BCG), and on strategies to improve BCG efficacy. We also describe recent advances in DIVA development based on the detection of host cellular immune responses by blood-testing or skin-testing approaches. Finally, to accelerate vaccine development, definition of host biomarkers that provide meaningful stage-gating criteria to select vaccine candidates for further testing is highly desirable. Some progress has also been made in this area of research, and we summarize studies that defined either markers predicting vaccine success or markers that correlate with disease stage or severity.
The quantifi cation of single cell interferon-gamma (IFN-γ) release for assessing cellular immune responses using the Enzyme-linked immunospot (ELISPOT) assay is an invaluable technique in immunology. Peripheral blood mononuclear cells (PBMC) are stimulated in vitro with recombinant proteins, peptides and recently whole malaria organisms. Stimulation may be short term (20–36 h) or long term (cultured ELISpot, up to 7 days). ELISpot is also able to quantify other cytokines secreted by antigen-specifi c T-cells, such as interleukin-2, interleukin-5, and other interleukins. ELISpot is playing an important role especially in vaccine research studies.
The ELISPOT is a powerful functional assay used to detect biological activity and immunological secretions from immune cells. In this chapter, we specifically discuss T cell ELISPOT methods for the detection of secreted cytokines. A detailed protocol is given enabling the detection of interferon gamma-secreting CD8(+) T cells and/or peripheral blood mononuclear cells following their isolation and polyclonal activation. Included is a brief discussion on choosing the activation method for your T cell ELISPOT assay, as well as additional instructions for the adaptation of this protocol for the study of memory and antigen-specific T cell responses.
The enzyme-linked immunospot (ELISPOT) assay is a widely used method for enumerating antigen-specific cytokine-producing or antibody-secreting immune cells. It is one of the most effective immunological and diagnostic approaches to detect and quantify low-frequency cytokine- or antibody-producing cells in human and animal tissues, such as peripheral blood, lymph nodes, and spleen. Detection and quantification of specific cytokine-producing cells by the ELISPOT assay is based on the formation of visible spots at the site of cytokine release by the cells under investigation (e.g., T cells) using pairs of different capture and detection antibodies under optimized conditions.
Here we focus mainly on practical, optimized protocols for cytokine ELISPOT assays for detection of mouse and human cytokine-producing immune cells (e.g., peripheral blood mononuclear cells, PBMC), including suggestions for trouble-shooting and optimizing steps for problematic tissue samples.
The enzyme-linked immuno-spot (ELISPOT) technique is a sensitive method used for measurement of elusive immune markers in limited-volume samples. By virtue of the exquisite sensitivity of the ELISPOT assay, frequency analysis of rare cell populations (e.g., antigen-specific responses), which was not possible before, is now relatively easy. However, development of a method sensitive enough to pinpoint elusive immune markers at the single-cell level is a challenge since there are a number of demands that have to be fulfilled and traps to avoid, achieving a valuable outcome.
To optimize the environment for in vitro culture and analysis of immune spots by ELISPOT, a number of criteria have to be fulfilled: processing of sample and perhaps also cryopreservation of cells before analysis and, for the ELISPOT assay, optimal cell culture, positive and negative controls, antigen concentration, and, finally, development and readout of spots.
If these criteria are fulfilled for your ELISPOT assay, you will likely have the opportunity to pinpoint elusive immune markers at the single-cell level. This chapter describes the ELISPOT assay for detection of cytokines (e.g., IFN-γ and IL-4), with focus on the main criteria that affect the assay. However, this method could be easily adapted to measure other immune markers in small volumes of biological samples.
Mycobacterium bovis is both the causative agent of bovine tuberculosis (TB) and a zoonotic pathogen. In humans, considerably fewer cases of TB are caused by M. bovis than M. tuberculosis; nevertheless, diagnostic limitations mean that currently available data on prevalence grossly underestimate the true dimension of the problem. The routes of transmission from animals to humans are well known and include direct exposure to infected animals or consumption of contaminated animal products. Application of fingerprinting tools facilitates analysis of the molecular epidemiology of M. bovis in animal-to-human and human-to-human transmission. Apart from cattle and M. bovis, other animal species and members within the M. tuberculosis complex can contribute to the zoonosis. Improvements in diagnostic techniques, application of more advanced discriminatory genotyping tools, and collaboration between veterinary and human health care researchers are key to our understanding of this zoonosis.
The infection status of cattle for bovine tuberculosis (bTB) was determined by real-time PCR, comparing the levels of IFN-γ mRNA in blood cultures stimulated with either bovine or avian tuberculin with non-stimulated control (phosphate buffer saline, PBS) blood culture. Totally, 137 cattle were tested to validate the assay, in which 54 were IFN-γ real-time quantitative PCR (RT-qPCR) positive, while the remaining 83 were found negative. Meanwhile, the IFN-γ ELISA test was carried out using the Bovigam IFN-γ detection ELISA kit and these results were used as a standard. The results of the single intradermal tuberculin tests (SIDT) and IFN-γ RT-qPCR tests were compared and revealed that the RT-qPCR correlated better with the ELISA and its accuracy was higher than SIDT. This indicates the RT-qPCR is a useful diagnostic method for bTB in cattle. However, several limitations remain for our approach, such as lack of a TB lesions or postmortem test results as a gold standard. Further improvements should be made in the future to increase accuracy of diagnosis of bTB in cattle.
Bovine tuberculosis (BTB) is a major animal health problem with zoonotic implications. Current control programs are based on test and slaughter strategies utilizing skin tests with tuberculins as antigens. The low specificity and associated operative difficulties of these tests have driven the search for new antigens and diagnostic assays. In this multicenter study, using herds from Argentina, Mexico and Northern Ireland, we selected skin test positive and negative animals from herds with different prevalence's of BTB and compared tuberculin (PPDB) and ESAT-6+CFP10 as antigens ex vivo. In low prevalence herds, crossreactivity of PPDB was apparent since up to 60% of the PPDB skin test and ex vivo positive animals did not responded to ESAT-6+CFP10 ex vivo. The superior specificity of ESAT-6+CFP10 was confirmed in a Mycobacterium avium sp. paratuberculosis infected herd where several of the animals had strong crossreactivity to PPDB and PPDA but not to ESAT-6+CFP10. In high prevalence herds 85% of the skin test-positive animals, were confirmed ex vivo using either PPDB or ESAT-6+CFP10 as antigen. However, within this group 60% of the skin test negative animals were PPDB and ESAT-6+CFP10 positive ex vivo indicating that the skin test can in some herds yield a significant number of false negative results. In conclusion, the ex vivo test is recommended as an ancillary test to accelerate BTB eradication. In high prevalence herds, PPDB or ESAT-6+CFP10 can be used as antigen whereas in low and medium prevalence herds ESAT-6+CFP10 is the preferred choice.
The tuberculin skin test is effective in the early detection of pre-clinical cases of Mycobacterium bovis infection in cattle. This allows the rapid removal of infected animals, thus limiting transmission of the disease, and has resulted in the eradication of bovine tuberculosis (Tb) from many countries. This test is very likely to remain the primary screening test for M. bovis infection in cattle as it is a simple, robust and inexpensive test. However, a number of ancillary tests are being used, or are currently being validated. These ancillary tests are likely to provide a more accurate diagnosis following skin-testing. The blood-based BOVIGAM interferon-gamma (IFN-gamma) test is a cellular immune assay which can detect early infection, and has become the main ancillary test in New Zealand. It can be used for re-testing skin test-positive animals, to improve specificity and minimise wastage from slaughtering animals with false-positive tests. Alternatively, it can be used in locations of increased risk of infection in parallel with skin-testing, for examining skin test-negative animals for pre-movement testing or in problem herds to identify M. bovis-infected animals that do not respond to the skin test. Several modifications of the test are now being used to improve specificity by altering the cut-off or using specific antigens present in virulent mycobacteria such as the 6 kDa early secreted antigenic target (ESAT-6) and 10 kDa culture filtrate protein (CFP-10). While antibody based tests generally lack sensitivity, as high levels of antibodies tend to occur late in the disease process, they may have unique desirable properties such as the ability to be used as a cow-side test. The use of these new ancillary tests in association with skin-testing will improve the detection of M. bovis-infected cattle and reduce the unnecessary slaughter of false-positive reactors.
Bovine tuberculosis (BTB), caused by Mycobacterium bovis, continues to pose a threat to livestock worldwide and, as a zoonotic infection, also has serious implications for human health. The implementation of comprehensive surveillance programmes to detect BTB has been successful in reducing the incidence of infection in many countries, yet BTB has remained recalcitrant to eradication in several EU states, particularly in Ireland and the UK. There are well-recognized limitations in the use of the current diagnostics to detect all infected animals and this has led to renewed efforts to uncover novel diagnostic biomarkers that may serve to enhance the performance of the tests. Studies of single immunological parameters have so far been unable to unlock the complexities of the immune response to mycobacterial infection. However, the development of high-throughput methods including pan-genomic gene expression technologies such as DNA microarrays has facilitated the simultaneous identification and analysis of thousands of genes and their interactions during the immune response. In addition, the application of these new genomic technologies to BTB has identified pathogen-associated immune response signatures of host infection. The objective of these investigations is to understand the changing profile of immune responses throughout the course of infection and to identify biomarkers for sensitive diagnosis, particularly during the early stages of infection. Transcriptional profiling via microarray and more recently via next-generation sequencing technologies may lead to the development of specific and sensitive diagnostics for M. bovis infection and will enhance the prospect of eradication of tuberculosis from cattle populations.
Whereas in vitro assays of antibody responses to infectious agents are commonly used for routine diagnostic purposes, diagnostic tests for cellular responsiveness are confined to in vivo intradermal tests. This paper describes a simple and rapid in vitro cellular assay for bovine tuberculosis. The assay system is based on the detection of gamma interferon (gamma IFN), which is released in response to specific antigen. This assay can be carried out with whole blood samples thus avoiding the time-consuming task of isolating lymphocytes. A simple bioassay has been developed to quantitate the amount of gamma IFN produced, but this will eventually be replaced with an enzyme-linked immunoassay using monoclonal antibodies specific for bovine gamma IFN. The application of this system to bovine tuberculosis and other infectious diseases may provide a convenient in vitro cellular assay for routine diagnostic purposes.
Tuberculin tests in general use today rely on the response to intradermal injections of tuberculin with assessment of the injection site for swelling at 72 hours post injection. Estimates of the sensitivity of tuberculin tests range from 68-95% while specificity is estimated to be 96-99%. The sensitivity of the test is affected by the potency and dose of tuberculin administered, the interval post-infection, desensitisation, deliberate interference, post-partum immunosuppression and observer variation. Specificity is influenced by sensitisation as a result of exposure to M. avium, M. paratuberculosis and environmental mycobacteria and by skin tuberculosis.
BOVIGAM which is based on the detection of gamma interferon (IFN- gamma) is a rapid, laboratory assay of a cell mediated immune response that may be used for the detection of tuberculosis (TB) infection in animals. Whole blood is first incubated overnight with bovine PPD, avian PPD or negative control antigens, and IFN- gamma in the supernatant plasma is then measured by EIA. TB infection is indicated by a predominant IFN- gamma response to bovine PPD. Since 1988, BOVIGAM has been extensively trialed on more than 200 000 cattle in Australia, Brazil, Ireland, Northern Ireland, Italy, New Zealand, Romania, Spain and the USA. Sensitivity has varied between 81.8% and 100% for culture-confirmed bovine TB and specificity between 94% and 100%. The IFN- gamma assay detects M. bovis infection earlier than the skin test and in New Zealand is applied to detect skin-test negative cattle with TB, where after slaughter a significant number of IFN- gamma reactors have TB. BOVIGAM is also approved in New Zealand for serial testing skin test positive cattle when non-specificity is suspected. Cattle are tested 7-30 days after a positive caudal fold test. The boosting effect of the skin test on T-cell activity allows blood to be cultured with PPD up to 30 h after collection without effecting accuracy. The BOVIGAM results are not affected by poor nutritional condition and are only mildly and briefly affected by dexamethasone treatment and parturition. IFN- gamma responses of cattle vaccinated with BCG are dose-dependent and short-lived. The BOVIGAM kit is now used routinely in many countries for the detection of M. bovis infected cattle, buffalo and goats.
The strategic use of the gamma-interferon (IFN-gamma) assay (Bovigam) can provide a means for the early identification of Mycobacterium bovis infected cattle, thus ensuring their removal from an infected herd. When used in parallel with the tuberculin test, it is capable of identifying infected cattle, which might otherwise not be detected until later, if at all. The early detection and removal of these animals reduces the risk that they will become a source of infection for other cattle. When targeted in herds of high prevalence the benefits to the herd owner directly concerned can be considerable as the assay provides a means of shortening the period of restriction for such herds. This serves to generate confidence among herd owners and other stakeholders that effective schemes, based on sound scientific principles, can be developed to eradicate tuberculosis from infected cattle populations.