Interferon-gamma Assay Research Articles

Diagnosis of Bovine Tuberculosis by Comparative Intradermal Tuberculin Test, Interferon Gamma Assay and <i>esxB</i> (CFP-10) PCR in Blood and Lymph Node Aspirates

Bovine tuberculosis (TB) is a chronic debilitating disease of huge economic importance due to loss in production, morbidity and mortality, and has a potential zoonotic threat. TB is endemic in India and has a worldwide prevalence, therefore, needing early diagnostic technique for the eradication of TB globally. Currently, compared to the eradication programme of TB in Medical sector, Veterinary sector is lagging behind though TB is one of the major zoonotic diseases prevalent in dairy animals and wildlife in India. With the “End TB” strategy by WHO in human, parallel measures for early diagnosis and culling has to be followed in case of animals for an overall successful eradication programme. The objective of this study is diagnosis of TB in cattle and buffaloes by using the cell-mediated immune response tests, i.e. Comparative Intradermal Tuberculin Test (CITT) and Interferon gamma (IFN-γ) assay, and Polymerase Chain Reaction (PCR) targeting esxB gene (CFP-10 protein) and to compare their diagnostic capabilities. This study was carried out in 202 dairy cattle and buffaloes from an organized dairy farm, where almost all of the animals appeared clinically healthy. We found that, the combined use of both CITT and IFN-γ assay lead to more accurate diagnosis of TB, although IFN-γ assay was more specific than CITT. However, esxB PCR showed almost similar sensitivity to IFN-γ assay and may be used as a fast alternative method for the diagnosis of bovine TB from blood samples.

Modeling tuberculosis dynamics with the presence of hyper-susceptible individuals for Ho Chi Minh City from 1996 to 2015

BackgroundThe depletion of CD4 cell is the underlying reason for TB hyper-susceptibility among people with HIV. Consequently, the trend of TB dynamics is usually hidden by the HIV outbreak.MethodsHere, we aim to evaluate the trend of TB dynamics quantitatively by a simple mathematical model using the known prevalence of hyper-susceptible individuals in the population. In order to estimate the parameters governing transmission we fit this model in a maximum likelihood framework to both reported TB cases and data from samples tested with Interferon Gamma Assay from Ho Chi Minh City - a city with high TB transmission and strong synchronization between HIV/AIDS and TB dynamics.ResultsOur results show that TB transmission in HCMC has been declining among people without HIV; we estimate a 18% (95% CI: 9–25%) decline in the transmission parameter between 1996 and 2015. Furthermore, we show that co-infected patients have limited contribution to the transmission process. For hyper-susceptible individuals, our model suggests that the risk of a new active TB infection occurring is significantly higher than the risk of relapsed active TB, while this is not the case for people without hyper-susceptibility.ConclusionsThe increase of TB notifications in Ho Chi Minh City from 1996 to 2008 is evitable when, as occurred, the number of hyper-susceptible individuals increased faster than the decrease of TB transmission rate. The sharp decrease in TB notifications observed in this city from 2008 to 2015 is the combined result of the decrease of TB transmission rate and the decrease of hyper-susceptible individuals in the population. For hyper-susceptible individuals, we propose that the reason for the reduced relapsed active TB risk is HIV treatment delay. According to HIV treatment guidelines issued by Vietnam’s Ministry of Health, hyper-susceptible individuals usually have to wait until their CD4 cell count falls under 350 cells/μl to start ART. Once patients begin ART, they will remain on ART for the rest of their life and thus have greater protection against relapses of TB. We therefore hypothesize that the delay in using ART imposes considerable TB burden on HCMC despite the declining transmission process.

PECTUS EXCAVATUM AND ATYPICAL MYCOBACTERIAL PULMONARY DISEASE

SESSION TITLE: Imaging SESSION TYPE: Med Student/Res Case Rep Postr PRESENTED ON: 10/09/2018 01:15 pm - 02:15 pm INTRODUCTION: NTM ( nontuberculous mycobacterial disease ) has been well reported in immunocompetent hosts. MAC (Mycobacterium Avium Complex ) is the most widely encountered form of NTM disease. The clinical presentation and radiographic imaging differs based on whether the patient has pre existing lung disease. In immunocompetentpatients without pre exisiting lung disease MAC is typically encountered in non smoking females greater than 50 years of age. Additional patient specific risk factors are poorly understood. The association between chest wall deformities and MAC lung disease has been previously described but not commonly appreciated by clinicians and radiologists. CASE PRESENTATION: 44 y/o f with a history of Vitamin D deficiency, Thallasemia minor and GERD presented for outpatient pulmonary evaluation of abnormal CT imaging associated with a + PPD. The patient is employed in a hospital setting as a nurse and has had multiple prior PPDs which were + with negative CXR imaging. She had received BCG vaccination as a child in Albania and was never previously treated for latent tuberculosis. On a routine physical evaluation for a new position she was required to have a new CXR performed which revealed subtle airspace and reticular densities in the bilateral mid lung fields. She had a subsequent gamma interferon assay (Qunatiferon GOLD) performed which was negative and was referred for CT imaging and additional pulmonary evaluation to rule out tuberculosis. The patient reported intermittent productive cough with discolored sputum for several years. She denied any weight loss, night sweats, hemoptysis or any known TB exposure. CT chest revealed evidence of bronchiectasis in the RML and lingula associated with centrilobular tree in bud opacities. Additionally on CT imaging and physical exam the pt was noted to have a pectusexcavatum deformity of the chest wall. 3 sets of sputum AFB were + MAC. Treatment was started with ethambutol, rifampin, clarithromycin on a 3 time weekly basis with significant improvement in symptoms. DISCUSSION: Pectus Excavatum, also known as sunken or funnel chest, is a congenital chest wall deformity in which several ribs and the sternum grow abnormally, producing a concave, or caved-in, appearance in the anterior chest wall. Chest wall deformities including pectusexcavatum have been associated with MAC lung disease more so than tuberculous lung disease, but the precise mechanism is poorly understood and not commonly appreciated by clinicians and radiologists. CONCLUSIONS: The combination of a pectus deformity with RML and lungula bronchiectasis in an immunocompetenthost should immediately raise the concern of MAC lung disease. Additional research is warranted to better elucidate risk factors for pulmonary NTM disease. Reference #1: 1- Cherra Pumphrey, MD , Abigail Chandler, MD and et al.MAC Infection in a Man With Pectus Excavatum. November 01, 2007. Pediatr Surg Int. 2001;17:344-350. 4. Reference #2: 2- Honda JR, Knight V, Chan ED. Pathogenesis and risk factors for nontuberculous mycobacterial lung disease. Clin Chest Med. 2015;36:1-11. DISCLOSURES: No relevant relationships by Mona Alipour, source=Web Response No relevant relationships by Farhad Arjomand, source=Web Response No relevant relationships by Louis Gerolemou, source=Web Response No relevant relationships by Nabil Mesiha, source=Web Response No relevant relationships by Viswanath Vasudevan, source=Web Response

Evaluation of the Performance of the IDvet IFN-Gamma Test for Diagnosis of Bovine Tuberculosis in Spain.

In Spain, the national bovine tuberculosis (bTB) eradication program is based on yearly skin testing of every ≥6 weeks old animal using the single or comparative tuberculin test and parallel use of the interferon-gamma (IFN-γ) assay as an ancillary diagnostic test in infected herds. There are several versions of the latter. Recently, a new commercial IDvet IFN-γ assay has been authorized for use in the program, but there is limited scientific evidence about its performance in different epidemiological settings. Therefore, two studies to evaluate the performance of the IDvet assay were conducted. In study 1, a concordance analysis between the new IDvet and the Bovigam IFN-γ assay in use in Spain for over 10 years was conducted. In study 2, results from the IDvet assay when applied in tandem with a single intradermal tuberculin (SIT) test were used to evaluate the concordance between both tests and to estimate their sensitivity (Se) and specificity (Sp) using a Bayesian latent-class model. Field data from cattle herds located in Madrid and Castilla y Leon (Spain) were collected. For study 1, herd selection was based on a high expected prevalence of reactors to the IFN-γ assay, while herds were selected at random to estimate Se and Sp of the new IDvet assay in study 2. Agreement between the results obtained with both kits for IFN-γ assay was poor (Kappa = 0.20), and a receiver operating characteristic (ROC) analysis indicated a low Se of the new IDvet relative to the Bovigam in a heavily bTB infected population. The Bayesian latent-class analysis estimated the Se of the IDvet assay to be 36.7% [95% probability posterior interval (PPI) 14.7–78.8%] with estimated Sp close to 100% when the cut-off recommended by the manufacturer (35) was applied. At the alternative cut-off values of 16 and 4, the estimated Se of the IDvet assay increased to 49.0% (PPI: 24.8–94.1%) and 56.0% (PPI: 30.8–96.3%), respectively, while maintaining a high specificity. The results suggest that the new IDvet assay may have lower sensitivity than the Bovigam for diagnosis of bTB in cattle herds in Spain, and that adjusting its cut-off might be considered.

Development and evaluation of two multi-antigen serological assays for the diagnosis of bovine tuberculosis in cattle

There is currently an increased interest in the use of serological approaches in combination with traditional cell-mediated immunity-based techniques to improve the detection of tuberculosis (TB)-infected animals. In the present study, we developed and validated two different serological TB-detection assays using four antigens, MPB70, MPB83, ESAT6 and CFP10, and the tuberculin PPDb. A conventional multi-antigen TB-ELISA method and a novel TB multiplex test, based on Luminex technology, were developed to detect antibodies to multiple antigen targets. The performance levels of the two tests were evaluated and compared using selected panels of samples having known TB states. The TB-ELISA test (containing five antigens, including PPDb) had a sensitivity (Se) of 74.2% and a specificity (Sp) of 94.9%, while the TB-Luminex test had higher Se (79.0%) and Sp (99.1%) rates even when only one reactive antigen was used to classify the test as positive. If a more restrictive criterion, requiring two positive antigens to classify the test as positive, was used, then the TB-ELISA's Sp rate increased to 99.8% but the Se decreased to 61.3%, while the TB-Luminex test's Sp rate increased to 100% but the Se decreased to 51.2%. TB-ELISA and TB-Luminex were applied to a panel of 257 sera collected from bTB-positive herds, as determined by a post-mortem inspection. They showed good performance levels, identifying 49 (80.3%) and 48 (78.7%), respectively, of 61 samples that had tested positive by the intradermal tuberculin (IDT) test and/or interferon-gamma assay. In addition, TB-ELISA and TB-Luminex were able to identify 60 and 42 samples as positive, respectively, out of the 196 samples that tested negative to IDT and interferon-gamma at the time of serum collection. Subsequent IDT tests performed after 1–2 months, confirmed the positivity of 18 samples, indicating the strategic value of having two serological assays to detect TB-infected herds that were not reactive to initial IDT testing, thereby allowing for the rapid control of outbreaks and eradication of the disease.

Evaluation of therapeutic potency of human papillomavirus-16 E7 DNA vaccine alone and with interleukin-18 as a genetic adjuvant

AIMS: Despite the existence of effective preventive vaccines for human papillomavirus (HPV), therapeutic vaccines that trigger cell-mediated immune responses are required to treat established infections and malignancies. The aim of this study was to evaluate the therapeutic potency of HPV-16 E7 deoxyribonucleic acid (DNA) vaccine alone and with interleukin (IL)-18. METHODS: In vitro expressions of IL-18 were performed on human embryonic kidney 293 cells and confirmed it by Western blotting methods. DNA vaccine was available from a previous study. A total of 45 female C57BL/6 mice divided into five groups (DNA vaccine, DNA vaccine adjuvanted with IL-18, pcDNA3.1, and phosphate buffer saline) were inoculated with murine tissue culture-1 cell line of HPV related carcinoma, expressing HPV-16 E6/E7 antigens. They were then immunized subcutaneously twice at a seven-day interval. The antitumor and antigen specific-cellular immunity were assessed by lymphocyte proliferation (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide: MTT) assay, lactate dehydrogenase release assay, IL-4 assay and interferon-gamma (IFN-γ) assay. Tumor size was followed for 62 days.RESULTS: MTT assay, which measures the lymphocyte proliferation in response to the specific antigen, increased in the co-administration and the DNA vaccine groups as compared to control and genetic adjuvant groups (p<0.001). The mice immunized with the co-administration generated significantly more IFN-γ and IL-4 than other immunized mice (p<0.001). Reduction of the tumor size in the co-administration and the DNA vaccine groups was significantly more pronounced than in the control and genetic adjuvant groups (p<0.001), but no statistically significant difference between DNA vaccine and co-administration groups (p=0.15) occurred.CONCLUSIONS: IL-18 as a genetic adjuvant and E7 DNA vaccine alone enhanced immune responses in mouse model systems against cervical cancer. However, using of IL-18 as a genetic adjuvant with E7 DNA vaccine had no significant synergistic effect on the immune responses in vivo.

Development of a novel immunoproteasome digestion assay for synthetic long peptide vaccine design.

Recently, many autologous tumor antigens have been examined for their potential use in cancer immunotherapy. However, the success of cancer vaccines in clinical trials has been limited, partly because of the limitations of using single, short peptides in most attempts. With this in mind, we aimed to develop multivalent synthetic long peptide (SLP) vaccines containing multiple cytotoxic T-lymphocyte (CTL) epitopes. However, to confirm whether a multivalent vaccine can induce an individual epitope-specific CTL, the only viable screening strategies currently available are interferon-gamma (IFN-μ enzyme-linked immunospot (ELISPOT) assays using human peripheral blood mononuclear cells, or expensive human leukocyte antigen (HLA)-expressing mice. In this report, we evaluated the use of our developed murine-20S immunoproteasome (i20S) digestion assay, and found that it could predict the results of IFN-μ ELISPOT assays. Importantly, the murine-i20S digestion assay not only predicted CTL induction, but also antitumor activity in an HLA-expressing mouse model. We conclude that the murine-i20S digestion assay is an extremely useful tool for the development of “all functional” multivalent SLP vaccines.

The use of serological tests in combination with the intradermal tuberculin test maximizes the detection of tuberculosis infected goats

The diagnosis of tuberculosis (TB) in goats is based mainly on the single and comparative intradermal tuberculin (SIT and CIT) tests and, exceptionally, on the interferon-gamma (IFN-γ) assay, however they are not perfect in terms of sensitivity and specificity. Nevertheless, various serological assays that provide a potential cost-effective approach for the control of TB are also available or under development, and a variety of results have been reported regarding the ability of these tests to detect infected animals, particularly in the early stages of infection. In the present study, SIT/CIT and IFN-γ tests and three different serological assays were evaluated during two consecutive herd testing events in a recently infected caprine herd (n = 447) with a high prevalence of infection in order to evaluate their performance and provide field data with which to improve the TB control programs in this species. The proportion of infected animals that tested positive among all the infected goats (T+/I+ value) in the last herd testing event ranged from 26.2% (IC95%; 19.3–34.5) to 85.7% (IC95%; 78.5–90.7) using cell-based diagnostic tests. The SIT/SCIT tests detected more infected goats than the IFN-γ test, regardless of the interpretation criteria. The T+/I+ value of serology was 83.2 (IC95%; 75.2–89), although it increased significantly (P < 0.05) when using samples collected 15 days after the intradermal test (100%, IC95%; 97–100). In general, a parallel interpretation of intradermal tests with serology maximized the detection of infected goats. These results demonstrate that serological tests are valuable diagnostic tools to maximize the detection of TB infected goats, even in recent outbreaks, accelerating the eradication process.

Development of a tree shrew-specific interferon-gamma assay

ABSTRACTTree shrews (Tupaia belangeri) are small squirrel-like mammals closely related to primates. Due to their susceptibility to several human viruses, tree shrews have been proposed as potential animal models for the study of human viral infections. However, there are no standardized assays currently available for the detection of tree shrew-specific interferon (IFN)-γ, a major cytokine secreted during the antiviral immune response. Herein, we developed a novel enzyme-linked immunosorbent assay (ELISA) for the quantification of IFN-γ in tree shrew serum samples. Tree shrew-specific IFN-γ was expressed in Escherichia coli via fusion with glutathione S-transferase (GST-TS-IFN-γ) to obtain recombinant IFN-γ. To generate anti-IFN-γ monoclonal antibodies, mice were immunized with the GST-TS-IFN-γ recombinant fusion protein, and hybridoma cell lines were established. Similarly, anti-IFN-γ polyclonal antibodies were obtained from immunized rabbits, purified, and conjugated to horseradish peroxidase (HRP). Based on the results obtained from the antibody matching test, we optimized the monoclonal antibody (1:2000) and the HRP-conjugated polyclonal antibody (1:8000) as coating and detection antibodies, respectively. Titration curves were generated with recombinant IFN-γ to develop a sensitive sandwich ELISA; the lowest detection limit of the assay was 20 ng/mL. We also tested mitogen-stimulated tree shrew blood samples in this ELISA, and found significantly higher levels of IFN-γ in the stimulated versus the unstimulated samples. Most importantly, our ELISA system detected native IFN-γ in serum samples from 50 healthy tree shrews. We have thus developed a novel ELISA, and have demonstrated the first ELISA-based measurement of IFN-γ in tree shrew serum samples.

Successful Application of the Gamma-Interferon Assay in a Bovine Tuberculosis Eradication Program: The French Bullfighting Herd Experience.

In the French Camargue region, where bovine tuberculosis had been enzootic for several years in bullfighting cattle herds, the gamma-interferon (IFN) assay was used since 2003 in parallel with the intradermal test in order to increase overall disease detection sensitivity in infected herds. This study presents the results of a field-evaluation of the assay during a 10-year period (2004–2014) of disease control and surveillance program and explores the particular pattern of IFN assay results in bullfight herds in comparison to cattle from other regions of France. The low sensitivity [59.2% (50.6; 67.3)] of IFN assay using the tuberculin stimulation could be related to the poor gamma-IFN production from bullfight cattle blood cells which is significantly lower than in animals of conventional breeds. The characteristics of the assay were progressively adapted to the epidemiological situation and the desired strategic applications. Data analysis with a receiver operating characteristic curve based on a simple S/P value algorithm allowed for the determination of a new cutoff adapted for a global screening, giving a high specificity of 99.9% results and a high accuracy of the assay. Having regularly risen to above 5% since 2005, with a peak around 10% in 2010, the annual incidence dropped to under 1% in 2014. The positive predictive value relative to the bacteriological confirmation evolved during the years, from 33% in 2009 to 12% during the last screening period, a normal trend in a context of decreasing prevalence. The estimated rate of false-positive reactions during screening campaigns was 0.67%, confirming the high specificity of the test, measured in bTB negative herds, in this epidemiological context. The proportion of false-positive reactions decreased with the age and was higher in males than in females. Although these results indicate that the IFN assay is accurate in the field, it also emphasizes great differences between interferon quantities produced by bullfight cattle blood samples compared to those of classical bovine breeds, which underlines the necessity to adapt the algorithms and combinations of the assay according to local epidemiological contexts.

Cross reactive immune responses in cattle arising from exposure to Mycobacterium bovis and non-tuberculous mycobacteria

Accurate diagnosis of tuberculosis in cattle may be compromised in areas where there are high rates of exposure to environmental/non-tuberculous mycobacteria (NTM). This cross reaction of immune responses to Mycobacterium bovis antigens shared with NTMs can result in reduced specificity of commonly used diagnostic tests including tuberculin skin tests and the interferon gamma assay (IFN-ɣ). In this study we assessed the cross-reactive immune responses of M. bovis (infected) and NTM exposed animals to M. bovis and M. avium tuberculin, the ESAT6/CFP10 cocktail antigen, tuberculin derived from cultures of selected NTMs, and a panel of recombinant mycobacterium tuberculosis complex (MTBC) antigens sharing homology with orthologues in NTM. Gamma interferon (IFN-ɣ) responses were measured in whole blood cultures using the IFN-ɣ assay and the IFN-ɣ elispot assay on purified peripheral blood mononuclear cells (PBMC).We observed the expected strong IFN-ɣ response to PPD-B in the M. bovis infected animals that distinguished this group from non-infected NTM exposed cattle. The IFN-ɣ responses to PPD-N (M. nonchromogenicum), were relatively high in both infected and non-infected NTM exposed cattle, but were not significantly different to classify the true infection status of each group. The results indicated that the cross-reactive responses to PPD-B and/or PPD-A with PPD-N, likely arose from prior exposure to environmental non-tuberculous mycobacteria. The IFN-ɣ immune responses to the 10 R-Mag measured by the IFN-ɣ elispot assay revealed that three of the selected antigens, Rv3615 (ESpC), Rv0287 (esxG) and the ESAT6/CFP10, were immunogenic in the infected cattle, and distinguished the infected cattle from the non-infected NTM exposed animals. The combined data of PPDs and R-Mags derived from NTM mycobacteria may prove useful in future development of novel bTB diagnostic tests.

Seroprevalence and risk factors for selected respiratory and reproductive tract pathogen exposure in European bison (Bison bonasus) in Poland

After the complete extinction from the wild of European bison (Bison bonasus) at the beginning of the twentieth century, the worldwide species population was restored to approximately 5500 individuals, with the species however remaining endangered. Despite numerous studies on the ecology and genetics of European bison, the threats of infectious diseases have been largely unexamined. The aim of this study was to screen the exposure of the world’s largest population of European bison to the pathogens, which may influence the condition and development of the endangered species. A total of 240 free-ranging and captive European bison from eight main Polish populations sampled were tested for the presence of specific antibodies against ten different viruses, bacteria or protozoan. The samples were collected from chemically immobilized, selectively culled or found dead animals. Based on serology, the exposure to bovine viral diarrhea virus (BVDV), bovine herpesvirus type 1 (BoHV-1), Mycoplasma and Brucella spp. was determined as rather accidental. Using gamma-interferon assay followed by Mycobacterium tuberculosis subs. caprae detection in tissues, diagnosis of bovine tuberculosis was made for 6 out of 78 (7.7%) bison from one captive herd. The highest seroprevalence was found for bovine adenovirus type 3 (BAdV-3) −60.2% and bovine parainfluenza type 3 (PIV-3) −34.0%, while the antibodies against bovine respiratory syncytial virus (BRSV), Toxoplasma gondii and Leptospira spp. were found in 10.4%, 10.4% and 8.7% of samples, respectively. In the multivariable statistical analysis using generalized linear mixed models (GLMMS), the risk factors for PIV-3 seropositivity included population type (free-living/captive), age and health status (apparently healthy/eliminated due to the poor condition). Higher risk of BAdV-3 seropositive result was observed in free-living female European bison. The high BAdV-3 and PIV-3 seroprevalences may suggest involvement of these pathogens in the most frequently observed respiratory disorders in European bison. Moreover, this is the first study demonstrating BAdV-3 exposure in the species.

A cross-sectional study on prevalence of bovine tuberculosis in Indian and crossbred cattle in Gangetic delta region of West Bengal, India

Aim : The aim of this study was to investigate the incidence of bovine tuberculosis (BTB), an old chronic disease having zoonotic potential, covering four districts in Gangetic delta region of West Bengal, India, and to find the prevalence in organized as well as backyard herds and variation in relation to their age, sex, and breeds. Methods : The incidence of BTB in exotic and indigenous breeds of cattle (n=173) of various age groups was investigated employing tuberculin (single intradermal tuberculin and comparative cervical tuberculin) tests and gamma interferon assay. Further, milk samples (n=96) from milching animals and antemortem (n=519) samples (nasal swab, buccal swab, and aspirates from pre-scapular lymph nodes) were also screened employing bacteriological and molecular techniques. Results : In total, 36 (25.4%) animals from organized and one (3.2%) from backyard farming sector were found positive to BTB. Polymerase chain reaction (PCR) of milk samples based on 16S rRNA amplified the 1030 bp band in four samples indicating them belonging to genus Mycobacterium. Species-specific primers used to differentiate between Mycobacterium bovis and M. tuberculosis confirmed the presence of M. bovis. Prevalence of BTB in exotic crossbred animals (34.6%) was significantly higher (p<0.001) compared to indigenous cattle (10.5%). Further, gender-wise analysis of data with respect to BTB revealed higher positivity (p<0.05) among cows/heifers (25.8%) compared to bulls/bullocks (7.3%). Although BTBpositive cattle were detected in all the age groups, no statistical difference (p=0.779) was found among them. Conclusion : The findings indicate a higher prevalence of BTB in exotic crossbred animals in Gangetic delta and variation in breed susceptibility, thereby suggesting an urgent review of the present policy on adopting national crossbreeding program and implementation of “One Health” approach.

Development and evaluation of an interferon gamma assay for the diagnosis of tuberculosis in red deer experimentally infected with Mycobacterium bovis

BackgroundRed deer (Cervus elaphus) is regarded as an epidemiologically relevant host for Mycobacterium bovis (M. bovis) and closely related members of the Mycobacterium tuberculosis complex that cause animal tuberculosis (TB). The standard antemortem screening test for the detection of TB in deer is the intradermal tuberculin skin test, but the detection of interferon-gamma (IFNγ) produced by white blood cells exposed to M. bovis antigens can be used as an alternative or supplemental assay in most TB eradication/control programs. This study aims to develop an in-house sandwich ELISA for deer IFNγ, based on the cross-reactivity of the antibodies to both cervid and bovine IFNγ, and to evaluate the potential of this assay to detect M. bovis-infected red deer in response to the in vitro stimulation of whole-blood cells with bovine purified protein derivative (bPPD), p22 protein complex derived from bPPD or using the specific tuberculous mycobacterial proteins ESAT-6/CFP-10, Rv3615c and Rv3020c. The positive control stimulant used in this study was pokeweed mitogen, which resulted in a consistent induction of IFNγ in samples from red deer, thus allowing the interpretation of the assay.ResultsThe percentage of animals correctly classified by this technique as M. bovis non-infected was 100%. The detection of infected animals as positive was high and ranged widely depending upon the antigen and the cut-off value applied, as well as the time after infection. Our findings indicate that this protocol may serve as a reliable assay for the antemortem diagnosis of TB from the initial stage of M. bovis-infection, and may also be adequately sensitive.ConclusionsThe suggested optimal antigens and cut-off are bPPD, p22 and the combination of ESAT-6/CFP-10 and Rv3020c with a 0.05 Δ optical density, which yielded a up to 100% correct classification of TB positive and negatve red deer under our experimental conditions. This technique will aid in TB testing of farmed and translocated deer. Future studies should evaluate the ability of this IFNγ assay to detect specific responses under field conditions.

SCREENING FOR BOVINE TUBERCULOSIS IN AFRICAN BUFFALO (SYNCERUS CAFFER) IN NGORONGORO CONSERVATION AREA, NORTHERN TANZANIA: IMPLICATIONS FOR PUBLIC HEALTH.

In the Ngorongoro Conservation Area (NCA), Tanzania, where wildlife and livestock interaction is intense, greater potential for intra- and interspecies disease transmission is expected. We assessed the prevalence of bovine tuberculosis in African buffalo (Syncerus caffer) residing on the valley floor of the crater in the NCA. Apparently healthy animals were randomly selected from herds in nine sites of the Ngorongoro Crater. Syncerus caffer buffalo herds were located using very high-frequency radio-aided rangers positioned in various observation points around the crater in the NCA. A total of 102 African buffalo from 16 herds were immobilized from the ground using a cocktail of 4-10 mg etorphine hydrochloride (M99) and 60-150 mg azaperone tartrate. The M99 was reversed using 10-25 mg diprenorphine hydrochloride depending on age of animals. An interferon gamma assay was performed on harvested plasma samples using sandwich enzyme linked immunosorbent assay. Of the 102 animals sampled, two (2%) African buffalo tested positive for bovine tuberculosis. These results corroborate those of the skin test done recently in cattle in the NCA. The presence of bovine tuberculosis in livestock and wildlife suggested the possibility of cross-species transmission of the disease, indicating the need for appropriate intervention measures.

Evaluation of gamma interferon assay for detection of bovine tuberculosis in inactive foot and mouth disease vaccine administered cattle

Evaluation of gamma interferon assay for detection of bovine tuberculosis in inactive foot and mouth disease vaccine administered cattle

Antibody detection tests improve the sensitivity of tuberculosis diagnosis in cattle

We evaluated the sensitivity (Se) of the single cervical intradermal tuberculin (SIT) test, two interferon-gamma (IFN-γ) assays and three different antibody detection techniques for bovine tuberculosis (bTB) diagnosis in 131 mixed beef breed cattle. The results of the diagnostic techniques performed over the whole herd, and over the animals confirmed as infected based on the presence of lesions compatible with the disease and/or M. bovis isolation were compared to determine apparent prevalence (AP) and Se.The Se of the SIT test (severe interpretation) was 63.7% (95% CI, 54.54–72.00), while the Se of the IFN-γ assays ranged between 60.2% and 92%. The proportion of infected cattle detected by the different antibody detection techniques ranged from 65.5% to 87.6%. Three of the antibody detection techniques yielded a significant higher (p<0.05) Se than that achieved with the official diagnostic techniques. In addition, the interpretation in parallel of cellular and antibody detection techniques reached the highest Se: 98.2% (95% CI, 93.78–99.51) suggesting that the use of diagnostic techniques detecting both cellular and humoral responses could be considered as an alternative in the control of bTB outbreaks in high prevalence settings.

Diagnosis of Mycobacterium bovis infection in livestock using gamma interferon assay and single intradermal comparative tuberculin test in Assam and Meghalaya

The present study was carried out to investigate the diagnostic potential of gamma interferon (IFN-ã) assay and single intradermal comparative tuberculin test (SICTT), including species specification of bovine tuberculosis infection in different livestock farms of Assam and Meghalaya. A total of 199 animals (cattle and buffalo) were examined for bovine tuberculosis symptoms and swab samples were cultured. Biochemical tests and PCR were used for species specification of bovine tuberculosis. Out of 199 cases examined, 33 (16.58%) showed positive for SICTT, 39 (19.59%) for IFN-ã and 35(17.59%) for PCR. Based on PCR targeting pncA region, the confirmation was done for M. Bovis. IFN-ã thus ensures a sensitive and specific detection of early bovine tuberculosis infection together with SICTT and hence may be considered as a screening method of choice.

The performance of the interferon gamma assay when used as a diagnostic or quality assurance test in Mycobacterium bovis infected herds

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 8h 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 8h diagnostic test and as a 24h QA test on SICTT reactors. A total of 17,725 IFN-γ tests were included in the analysis (11,658 diagnostic tests and 6067 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.

AGREEMENT OF QUANTIFERON TEST AND TUBERCULIN SKIN TEST IN DIAGNOSING LATENT TUBERCULOSIS INFECTION AMONG HIV-INFECTED PEOPLE IN KISUMU COUNTY, KENYA

BackgroundHIV-infected people are at greatest risk of progression from latent tuberculosis infection (LTBI) to development of active tuberculosis (TB) disease. Accurate diagnosis and treatment of LTBI in this group is...