Development of an enzyme-linked immunosorbent assay for the diagnosis of feline tuberculosis.

Cases of feline tuberculosis (TB) can be challenging to diagnose. Currently, this is achieved through a combination of mycobacterial culture, polymerase chain reaction (PCR), or interferon-gamma release assay (IGRA); however, these each have limitations. There is limited data regarding the use of humoral immunodiagnostics for TB in cats. Therefore, we sought to develop an enzyme-linked immunosorbent assay (ELISA) to further facilitate the diagnosis of feline TB. A comparative PPD (purified protein derivative) antibody ELISA was optimised for use on serum and plasma, and was tested against samples from 14 cats with culture-confirmed TB and 24 uninfected controls. Selection of an appropriate positive cut-off value based on receiver-operator characteristic curve analysis gave test sensitivity of 64.3 % and specificity of 100 %. When tested on further samples from cats with strongly suspected mycobacteriosis, 32.9 % (23/70) were antibody positive. Notably, positive results were recorded in cats that failed to respond to the IGRA, and in one PCR and IGRA negative cat. No positive responses were identified in cats with non-tuberculous mycobacterial infections, or with non-mycobacterial diseases (n = 12). Therefore, antibody-based diagnostics may be useful adjunctive tests for cases of TB missed by the IGRA, helping protect both feline and, in turn, human health.

Diagnostic accuracy of the interferon-gamma release assay (IGRA) for cases of feline mycobacteriosis.

The aim of this study was to evaluate the sensitivity and specificity of the interferon-gamma release assay (IGRA) for diagnosing infections with members of the Mycobacterium (M.) tuberculosis-complex (MTBC) and non-tuberculous mycobacteria (NTM) in domestic cats, and to generate defined feline-specific cut-off values using receiver operating characteristic (ROC) curve analysis to improve test performance. Records of 594 cats that had been tested by IGRA were explored to identify individuals that had a culture and/or polymerase chain reaction (PCR)-confirmed case of mycobacterial disease, and those that had a final diagnosis of non-mycobacterial disease. A total of 117 cats - 80 with mycobacterial disease and 37 diagnosed with a condition other than mycobacteriosis - were identified for further detailed analysis. This population was used to estimate test sensitivity and specificity, as well as likelihood ratios for the IGRA to correctly identify a cat with or without mycobacterial disease. Agreement between IGRA results and culture/PCR using current and proposed new cut-off values was also determined. ROC analysis of defined confirmed infected and non-mycobacterial disease control cats allowed an adjustment of current test cut-offs that increased the overall test sensitivity for MTBC infections from 83.1 % (95 % confidence interval [CI]: 71.5-90.5 %) to 90.2 % (95 % CI: 80.2-95.4%), and M. bovis infection from 43 % (95 % CI: 28.2-60.7%) to 68 % (95 % CI: 51.4-82.1%) while maintaining high test specificity (100 % in both cases). Overall agreement between IGRA results and culture/PCR, while recognising that neither culture nor PCR tests have perfect sensitivity, improved from weak (κ = 0.57) to moderate (κ = 0.71) using new proposed IGRA test cut-off values. Application of these results, based upon the statistical analysis of accumulated test data, can improve the diagnostic performance of the feline IGRA, particularly for identifying infections with M. bovis, without compromising specificity.

Interactions between helminths and tuberculosis infections: Implications for tuberculosis diagnosis and vaccination in Africa.

Africa is the second most populous continent and has perennial health challenges. Of the estimated 181 million school aged children in sub-Saharan Africa (SSA), nearly half suffer from ascariasis, trichuriasis, or a combination of these infections. Coupled with these is the problem of tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb) infection, which is a leading cause of death in the region. Compared to the effect of the human immunodeficiency virus on the development of TB, the effect of chronic helminth infections is a neglected area of research, yet helminth infections are as ubiquitous as they are varied and may potentially have profound effects upon host immunity, particularly as it relates to TB infection, diagnosis, and vaccination. Protection against active TB is known to require a clearly delineated T-helper type 1 (Th1) response, while helminths induce a strong opposing Th2 and immune-regulatory host response. This Review highlights the potential challenges of helminth-TB co-infection in Africa and the need for further research.

Use of antigen-specific interleukin-2 to differentiate between cattle vaccinated with Mycobacterium bovis BCG and cattle infected with M. bovis.

We describe here the application of a novel bovine interleukin-2 (IL-2) enzyme-linked immunosorbent assay (ELISA) for the measurement of antigen-specific IL-2 in cattle naturally infected with Mycobacterium bovis and in cattle vaccinated with Mycobacterium bovis BCG and then experimentally challenged with pathogenic M. bovis. Supernatants from whole-blood cultures stimulated with mycobacterial antigen (bovine purified protein derivative [PPDB] or the peptide cocktail ESAT6-CFP10) were assessed using a sandwich ELISA consisting of a new recombinant monoclonal fragment capture antibody and a commercially available polyclonal anti-bovine-IL-2. The production of IL-2 was compared to the production of gamma interferon (IFN-γ) in the same antigen-stimulated whole-blood supernatants. The data show that cattle infected with M. bovis produced quantifiable levels of antigen-specific IL-2, while IL-2 levels in cattle vaccinated with M. bovis BCG did not. Furthermore, cattle vaccinated with M. bovis BCG and then challenged with pathogenic M. bovis displayed a more rapid induction of IL-2 but ultimately had lower levels of infection-induced IL-2 than did unvaccinated challenge control cattle. These data suggest that IL-2 responses are not detectable post-BCG vaccination and that these responses may require infection with virulent M. bovis to develop. This may be useful to differentiate infected cattle from uninfected or BCG-vaccinated cattle, although the overall sensitivity is relatively low, particularly in single intradermal comparative cervical tuberculin (SICCT)-negative infected animals. Furthermore, the strength of the IL-2 response may correlate with pathology, which poses interesting questions on the immunobiology of bovine tuberculosis in contrast to human tuberculosis, which is discussed.

Comparative study of IFNγ and antibody tests for feline tuberculosis.

This study describes the comparison of the cell-based interferon-gamma (IFNγ) test with serological rapid antibody tests (STAT-PAK and DPP VetTB) for the ante mortem testing of tuberculosis in domestic cats. The antibody specificities of rapid antibody test-positive cats were further discerned using multi-antigen print immunoassay. A total of 62 cats with culture-confirmed Mycobacterium bovis, Mycobacterium microti, Mycobacterium avium and Mycobacterium malmoense, as well as negative controls and dangerous-contact cats were tested. Tests were also applied longitudinally to one further cat undergoing TB chemotherapy for suspected M. bovis infection. Our data from this small study show excellent test specificity (100% for all tests) and encouraging levels of test sensitivity for M. bovis and TB Complex infections (IFNγ 70-100% depending upon test interpretation criteria; rapid tests both 90% for M. bovis infection and up to 46.2% for M. microti infection). The differential diagnosis of very pathogenic TB Complex (M. bovis, Mycobacterium tuberculosis), as opposed to less-pathogenic TB Complex (M. microti) was possible where positive responses to the protein cocktail ESAT6/CFP10 were observed (80% of M. bovis-infected cats in this study showed positive IFNγ responses to ESAT6/CFP10, while 20% had antibody responses to ESAT6/CFP10 using MAPIA). Finally, preliminary data from a longitudinal study of one M. bovis-exposed cat with a positive IFNγ test pre-treatment suggest that a decrease in bacterial burden may be reflected in the IFNγ response, and thus the IFNγ test may provide a monitor for TB chemotherapy.

Repeat tuberculin skin testing leads to desensitisation in naturally infected tuberculous cattle which is associated with elevated interleukin-10 and decreased interleukin-1 beta responses.

The principal surveillance tool used to control bovine tuberculosis in cattle is the removal of animals that provide a positive response to the tuberculin skin-test. In this study we performed a longitudinal investigation of the immunological and diagnostic consequences of repeated short-interval skin-tests in cattle naturally infected with Mycobacterium bovis. Tuberculin skin-test positive cattle were subjected to up to four further intradermal comparative cervical skin-tests at approximately 60-day intervals. A significant progressive reduction in the strength of the skin-test was observed after successive tests. In contrast, the magnitude of interferon-gamma (IFN-gamma) responses was not influenced by repeat skin-testing either transiently around the time of each skin-test or longitudinally following repeated tests. A significant boost in blood interleukin-10 (IL-10) production was observed within 3 days following each skin-test although the magnitude of this boosted response returned to lower levels by day 10 post-test. The application of a novel multiplex assay to simultaneously measure seven cytokines and chemokines also identified that skin-testing resulted in a significant and progressive reduction in antigen specific interleukin-1beta (IL-1beta) whilst confirming stable IFN-gamma and elevated IL-10 responses in the blood. Therefore, we have demonstrated that in cattle naturally infected with M. bovis, repeat short-interval skin-testing can lead to a progressive reduction in skin-test responsiveness which has potential negative consequences for the detection of infected animals with marginal or inconclusive skin-test responses. The desensitising effect is associated with decreased IL-1beta and elevated IL-10 responses, but importantly, does not influence antigen specific IFN-gamma responses.

Viral booster vaccines improve Mycobacterium bovis BCG-induced protection against bovine tuberculosis.

Previous work with small-animal laboratory models of tuberculosis has shown that vaccination strategies based on heterologous prime-boost protocols using Mycobacterium bovis bacillus Calmette-Guérin (BCG) to prime and modified vaccinia virus Ankara strain (MVA85A) or recombinant attenuated adenoviruses (Ad85A) expressing the mycobacterial antigen Ag85A to boost may increase the protective efficacy of BCG. Here we report the first efficacy data on using these vaccines in cattle, a natural target species of tuberculous infection. Protection was determined by measuring development of disease as an end point after M. bovis challenge. Either Ad85A or MVA85A boosting resulted in protection superior to that given by BCG alone: boosting BCG with MVA85A or Ad85A induced significant reduction in pathology in four/eight parameters assessed, while BCG vaccination alone did so in only one parameter studied. Protection was particularly evident in the lungs of vaccinated animals (median lung scores for naïve and BCG-, BCG/MVA85A-, and BCG/Ad85A-vaccinated animals were 10.5, 5, 2.5, and 0, respectively). The bacterial loads in lymph node tissues were also reduced after viral boosting of BCG-vaccinated calves compared to those in BCG-only-vaccinated animals. Analysis of vaccine-induced immunity identified memory responses measured by cultured enzyme-linked immunospot assay as well as in vitro interleukin-17 production as predictors of vaccination success, as both responses, measured before challenge, correlated positively with the degree of protection. Therefore, this study provides evidence of improved protection against tuberculosis by viral booster vaccination in a natural target species and has prioritized potential correlates of vaccine efficacy for further evaluation. These findings also have implications for human tuberculosis vaccine development.

Adaptation of IFN-gamma ELISA and ELISPOT tests for feline tuberculosis.

There are currently no reliable immunodiagnostic tests for feline tuberculosis. Infection of domestic cats in the UK is thought to occur via their contact with the relevant reservoir of infection, e.g. cattle and badgers for Mycobacterium bovis, and rodents for M. microti. In the African National Parks, where M. bovis infection of Bovidae is an increasing problem, transmission to big cats is occurring via their ingestion of infected carcasses. We have adapted feline ELISA and ELISPOT assays to potentially provide the first cell-based diagnostic test for the detection of tuberculosis in cats. We tested peripheral blood mononuclear cell antigen-specific IFN-gamma responses of 18 cats suspected of mycobacterial infection for which biopsy material was co-submitted to the Veterinary Laboratories Agency for mycobacterial culture and identification. Seventeen cats were tested by ELISA while seven cats were tested by ELISPOT (six cats were tested by both ELISA and ELISPOT). Six healthy control cats provided baseline data for these tests. Responses to bovine and avian tuberculins (PPDB and PPDA) and a protein cocktail of ESAT6 and CFP10 were measured, together with positive mitogen (PMA and calcium ionophore) and negative (medium) controls. Overall, both ELISPOT and ELISA tests were found to be suitable for generating rapid results (2 and 4 days, respectively), which provided good predictive information for M. bovis and M. microti infections, but were unable to reliably discern M. avium infection.

Is interleukin-4delta3 splice variant expression in bovine tuberculosis a marker of protective immunity?

Splice variants of the interleukin-4 (IL-4) cytokine gene have been described for humans, mice, and cattle. IL-4 splice variants have been shown to inhibit IL-4-mediated cellular responses and thus act as IL-4 antagonists. Recent work has highlighted the possibility of a correlation between IL-4 splice variants and protection against clinical tuberculosis. In this study we investigated the potential role of IL-4 splice variants IL-4delta2 and IL-4delta3 in cattle with bovine tuberculosis, using quantitative real-time reverse transcription-PCR. For this analysis we used naturally exposed tuberculin skin test-positive field reactor cattle, uninfected control cattle, and cattle from two experimental models of protective immunity against Mycobacterium bovis: (i) vaccination with M. bovis BCG and challenge with virulent M. bovis and (ii) infection with M. bovis and treatment with isoniazid (INH) prior to rechallenge. The cytokine levels of field reactor cattle were compared to the levels of uninfected controls, while in kinetic studies of BCG vaccination and INH treatment we compared pre-experimental values with sequential samples for each individual animal. The data revealed a significant increase in IL-4delta3 mRNA expression in field reactor cattle, which had no visible pathology compared to cattle with gross pathology typical of bovine tuberculosis. Increased IL-4delta3 expression in both cattle models of protective immunity (BCG vaccination and INH treatment) was transient over time, reaching significance in the INH treatment model. Our results support the hypothesis that IL-4delta3 is involved in protective immunity against M. bovis infection in cattle and are in accordance with clinical studies that have suggested a role for IL-4 splice variants in protective immunity in tuberculosis.

Minimum infective dose of Mycobacterium bovis in cattle.

The aim of this work was to determine the minimum infective dose of Mycobacterium bovis necessary to stimulate specific immune responses and generate pathology in cattle. Four groups of calves (20 animals) were infected by the intratracheal route with 1,000, 100, 10, or 1 CFU of M. bovis. Specific immune responses (gamma interferon [IFN-gamma] and interleukin-4 [IL-4] responses) to mycobacterial antigens were monitored throughout the study, and the responses to the tuberculin skin test were assessed at two times. Rigorous post mortem examinations were performed to determine the presence of pathology, and samples were taken for microbiological and histopathological confirmation of M. bovis infection. One-half of the animals infected with 1 CFU of M. bovis developed pulmonary pathology typical of bovine tuberculosis. No differences in the severity of pathology were observed for the different M. bovis doses. All animals that developed pathology were skin test positive and produced specific IFN-gamma and IL-4 responses. No differences in the sizes of the skin test reactions, the times taken to achieve a positive IFN-gamma result, or the levels of the IFN-gamma and IL-4 responses were observed for the different M. bovis doses, suggesting that diagnostic assays (tuberculin skin test and IFN-gamma test) can detect cattle soon after M. bovis infection regardless of the dose. This information should be useful in modeling the dynamics of bovine tuberculosis in cattle and in assessing the risk of transmission.

Cellular immune responses induced in cattle by heterologous prime-boost vaccination using recombinant viruses and bacille Calmette-Guérin.

The development of novel vaccine strategies to replace or supplement bacille Calmette-Guérin (BCG) is urgently required. Here we study, in cattle, the use of heterologous prime-boost strategies based on vaccination with BCG and the mycobacterial mycolyl transferase Ag85A (Rv3804c) expressed either in recombinant modified vaccinia virus Ankara (MVA85A) or attenuated fowlpox strain FP9 (FP85A). Five different vaccination schedules were tested in the first experiment: MVA85A followed by BCG (group 1); BCG followed by MVA85A (group 2); BCG followed by FP85A and then MVA85A (group 3); MVA85A followed by MVA85A and then FP85A (group 4); and FP85A followed by FP85A and then MVA85A (group 5). Vaccine-induced levels of cellular immunity were assessed by determining interferon-gamma (IFN-gamma) responses in vitro. Prime-boost protocols, using recombinant MVA and BCG in combination (groups 1-3), resulted in significantly higher frequencies of Ag85-specific IFN-gamma-secreting cells than the two viral vectors used in combination (P=0.0055), or BCG used alone (groups 2 and 3, P=0.04). The T-cell repertoires of the calves in all five groups were significantly broader following heterologous booster immunizations than after the primary immunization. In a second experiment, the effects of BCG\MVA85A heterologous prime-boost vaccination were compared with BCG\BCG homologous revaccination. The results suggested a higher Ag85A-specific response with a wider T-cell repertoire in the MVA85A-boosted calves than in the BCG\BCG-vaccinated calves. In conclusion therefore, the present report demonstrates the effectiveness of heterologous prime-boost strategies based on recombinant MVA and BCG to induce strong cellular immune responses in cattle and prioritise such vaccination strategies for rapid assessment of protective efficacy in this natural target species of tuberculosis.