Intradermal Tuberculin Test Research Articles

Evaluating the ability of non-tuberculous mycobacteria to induce non-specific reactions in bovine tuberculosis diagnostic tests in guinea pigs and cattle

Limitations in diagnostic test performance are one of the major challenges hampering the eradication of bovine tuberculosis (bTB). Non-tuberculous mycobacteria (NTM) are considered one of the main causes of non-specific reactions in the intradermal tuberculin test, the most widely used bTB diagnostic test. To determine the role of NTMs in bTB misdiagnosis in Spain, an experimental study including the NTM species most commonly found in bTB-positive animals from bTB-free farms in the country (M. avium subsp. avium (Maa), “Mycobacterium avium subsp. hominissuis” (Mah), M. bourgelatii, M. intermedium, M. kansasii and M. nonchromogenicum) was carried out on guinea pigs and cattle. First, guinea pigs were sensitized with the selected NTMs, and six weeks post-sensitization four antigen mixtures (bovine-PPD, avian-PPD, P22 and ESAT6-CFP10) were inoculated intradermally and their effect was measured 24- and 48-h post-inoculation. Larger erythematous reactions were observed in guinea pigs sensitized with Mah, M. kansasii, and Maa, with significant differences in the reactions measured at the bovine-PPD inoculation site for the two first bacteria compared with other NTMs. The sensitization process was repeated in cattle, and five months post-sensitization the same antigen mixtures were inoculated in the cervical region and responses were measured at 48- and 72-h post-inoculation. A significantly higher increase in the skinfold thickness measured at the bovine-PPD inoculation site was observed in calves sensitized with Mah, Maa, M. intermedium and M. kansasii. These results demonstrate that certain NTM species may play a more significant role in bTB diagnostic interferences and show that results obtained in guinea pig and bovine models do not always coincide.

Extensive differential DNA methylation between tuberculosis skin test positive and skin test negative cattle

Bovine tuberculosis (bTB), caused by Mycobacterium bovis (M. bovis), represents a significant problem for the agriculture industry as well as posing a risk for human health. Current diagnostic tests for bTB target the cell-mediated immune (CMI) response to infection with M. bovis, primarily through screening of animals with the tuberculin skin test. Epigenetic modifications have been shown to alter the course of the immune response and differentially methylated regions (DMRs) might also influence the outcome of the skin test in cattle. Whole Genome Bisulphite Sequencing (WGBS) was used to profile DNA methylation levels from peripheral blood of a group of cattle identified as test positive for M. bovis (positive for the single intradermal comparative tuberculin test (SICTT) and/or the interferon-γ release assay compared to a test negative control group [n = 8/group, total of 16 WGBS libraries]. Although global methylation profiles were similar for both groups across the genome, 223 DMRs and 159 Differentially Promoter Methylated Genes (DPMGs) were identified between groups with an excess of hypermethylated sites in SICTT positive cattle (threshold > 15% differential methylation). Genes located within these DMRs included the Interleukin 1 receptor (IL1R1) and MHC related genes (BOLA and BOLA-DQB). KEGG pathway analysis identified enrichment of genes involved in Calcium and MAPK signalling, as well as metabolism pathways. Analysis of DMRs in a subset of SICTT negative cattle that were IFN-γ positive showed differential methylation of genes including Interleukin 10 Receptor, alpha (IL10RA), Interleukin 17 F (IL17F) and host defence peptides (DEFB and BDEF109). This study has identified a number of immune gene loci at which differential methylation is associated with SICTT test results and the degree of methylation could influence effective host immune responses.

Differentiation of Bovine Tuberculosis and Paratuberculosis Infections with Antemortem Diagnostic Methods

In this study, based on the results of tuberculin skin tests (Bovine and Avian PPD) used in the antemortem diagnosis and differentiation of Bovine Tuberculosis, the animals in the farms with suspected Tuberculosis were serologically examined to diagnose Paratuberculosis infection and fecal bacterioscopy was performed. In addition, it was aimed to obtain data that will contribute to the eradication studies of Bovine Tuberculosis disease by comparing the antemortem diagnostic methods of Bovine Tuberculosis disease, which is endemic in Türkiye and by determining the sensitivity and specificity values of the interferon gamma (IFN-γ) test. In this context, intradermal tuberculin test was applied to 423 cattle with suspected Tuberculosis in a total of 5 dairy cattle farms, one each from Çankırı, Çorum, Ankara, Eskişehir and Konya regions, and this test was determined as the gold standard method and the sensitivity and specificity of the IFN-γ test were determined as 86% and 97%, respectively. For the diagnosis of Paratuberculosis infection, antibody ELISA, fecal bacterioscopy and IFN-γ ELISA were performed on these animals and the prevalence of these tests were 10.4%, 5.44% and 4.96% respectively and 4 (0.95%) of the cattle were positive for each of the diagnostic methods for Map infection. As a result, it was concluded that IFN-γ test, which gives similar results to intradermal tuberculin test results, can also be used in the antemortem diagnosis of Bovine Tuberculosis. Also, in the comparative intradermal tuberculin test for the diagnosis of Tuberculosis infection, avian PPD positive animals were found to play a decisive role in the detection of nonspecific reactions or Paratuberculosis infected animals, supported by other tests used for the diagnosis of Paratuberculosis.

Expression and field evaluation of new Mycobacterium bovis antigens

Bovine tuberculosis (bTB) represents a threat to livestock production. Mycobacterium bovis is the main causative agent of bTB and a pathogen capable of infecting wildlife and humans. Eradication programs based on surveillance in slaughterhouses with mandatory testing and culling of reactive cattle have failed to eradicate bTB in many regions worldwide. Therefore, developing effective tools to control this disease is crucial. Using a computational tool, we identified proteins in the M. bovis proteome that carry predictive binding peptides to BoLADRB3.2 and selected Mb0309, Mb1090, Mb1810 and Mb3810 from all the identified proteins. The expression of these proteins in a baculovirus-insect cell expression system was successful only for Mb0309 and Mb3810. In parallel, we expressed the ESAT-6 family proteins EsxG and EsxH in this system. Among the recombinant proteins, Mb0309 and EsxG exhibited moderate performance in distinguishing between cattle that test positive and negative to bTB using the official test, the intradermal tuberculin test (IDT), when used to stimulate interferon-gamma production in blood samples from cattle. However, when combined as a protein cocktail, Mb0309 and EsxG were reactive in 50 % of positive cattle. Further assessments in cattle that evade the IDT (false negative) and cattle infected with Mycobacterium avium paratuberculosis are necessary to determine the potential utility of this cocktail as an additional tool to assist the accurate diagnosis of bTB.

Prevalence of pulmonary tuberculosis and HIV infections and risk factors associated to tuberculosis in detained persons in Antananarivo, Madagascar

The incidence rate of tuberculosis in prisons is estimated to be 8 times greater than that in the general population in Madagascar. Our objectives were to estimate the prevalence of pulmonary tuberculosis and HIV infection among prisoners and to identify risk factors associated with tuberculosis. We conducted a cross-sectional study at the central prison of Antananarivo from March to July 2021. Individual male and female inmates aged ≥ 13 years who had lived in the prison for at least three months prior to the study period were included as participants. Acid-fast bacilli detection by microscopy and/or culture, an intradermal tuberculin test, a chest X-ray, and a rapid diagnostic orientation test for HIV were performed. Among 748 participants, 4 (0.5%) were confirmed to have pulmonary tuberculosis. Overall, 14 (1.9%) patients had “confirmed” or “probable” tuberculosis [0.90–2.84, 95% CI]. The proportion of participants with latent tuberculosis infection was 69.6% (517/743) based on a positive tuberculin test without clinical symptoms or radiography images indicating tuberculosis. Out of 745 HIV screening tests, three showed reactive results (0.4%). Age (OR = 4.4, 95% CI [1.4–14.0]) and prior tuberculosis treatment (or episodes) were found to be associated with confirmed and probable tuberculosis.

Evaluation of the Effect of a Recent Comparative Intradermal Tuberculin Test on the Humoral Diagnosis of Paratuberculosis Using Serum and Milk Samples from Goats.

Paratuberculosis (PTB) and tuberculosis (TB) are two mycobacterial diseases with a severe economic and health impact on domestic ruminants. The ante mortem diagnosis of PTB is hampered, among other factors, by the limited sensitivity of all the available diagnostic techniques. Since TB-infected goats subjected to the comparative intradermal tuberculin test (CITT) may experience a booster effect on their antibody titer and a potential enhancement to the sensitivity of humoral techniques for tuberculosis, in the present study we aimed to evaluate this diagnostic strategy on the humoral diagnosis of PTB in serum and milk samples collected from a caprine herd that was TB free and PTB infected. The results from 120 goats indicated a significant increase (p < 0.001) in the quantitative response detected using an ELISA technique, conducted using serum and milk samples taken 15 and 30 days after performing a CITT (day 0 of the study); although, it did not translate into a significant increase in the number of reactors during any of the testing events (0, 3,15, 30 and 60 days post-CITT). Additionally, the number of ELISA-positive animals was higher for the serum versus the milk samples at both 15 and 30 days post-CITT. The increase in the quantitative ELISA result suggested a diagnostic strategy that maximizes ELISA sensitivity, mainly using serum samples, in PTB-infected herds; although, it may depend on individual differences and the interpretation criteria.

Effect of a recent intradermal test on the specificity of P22 ELISA for the diagnosis of caprine tuberculosis.

Caprine tuberculosis (TB) is a zoonotic disease caused by members of the Mycobacterium tuberculosis complex. TB eradication programs in goats are based on the single and comparative intradermal tuberculin tests (SITT and CITT, respectively). Antibody-based diagnostic techniques have emerged as potential diagnostic tools for TB. P22 ELISA has been previously evaluated using samples collected after the intradermal tuberculin tests to maximize the sensitivity, a phenomenon known as booster effect. However, there is no information available on whether the use of this diagnostic strategy could lead to a decrease of its specificity (Sp). The aim of the present study was to elucidate the interference effect of a recent CITT on the Sp of the P22 ELISA in serum and milk samples collected at different times after the CITT from a TB-free herd (n = 113). The number of reactors to P22 ELISA was significantly higher (p < 0.01) on serum samples collected 15 days post-CITT compared to day 0, showing a decrease in Sp from 99.1% (95% CI; 95.2-99.8%) to 88.5% (95% CI; 81.3-93.2%). The number of reactors and the quantitative values of P22 ELISA were significantly higher (p < 0.01) in serum samples compared to milk. No significant (p > 0.05) changes in the Sp of the P22 ELISA were observed throughout the different time samplings using milk No significant (p > 0.05) changes were observed on days 30 and 60 post-CITT. In conclusion, the booster effect strategy may significantly decrease the Sp of P22 ELISA in TB-free herds when serum samples are used but not when milk is tested.

Prevalence and risk factors associated with bovine tuberculosis in the southern zone of Nasarawa State, north-central Nigeria

A cross-sectional study was conducted on 500 grazing cattle under a pastoral production system in the southern zone of Nasarawa state, North-Central Nigeria to determine the prevalence and risk factors associated with bovine tuberculosis using a Single intradermal Comparative Tuberculin Test (SCITT) from April 2019 to February 2020. The total number of animals with bTB was 46 (9.2%) and the results were significantly higher in older animals between 5-6years (OR: 7.4; CI: 1.54-35.4; P=0.000) and in those animals that were ≥ 7 years (OR: 5.7; CI: 1.24-25-9; P=0.00) old and likely at risk of having more bTB. Additionally, the prevalence was significantly higher in female animals (OR:3.7; CI:1.5-8.8; p=0.003), also a significant prevalence was observed as the herd size increased from 31-80 (OR:1.95; CI: 0.75-5.0; p=0.000) as well as from 81-171 (OR:5.5; CI: 2.14-14.4; p=0.000). Moreso, the prevalence was significant with the location of the animals, as areas with grazing reserves appeared to have more odds and more likely to have bTB: Awe (OR:9.6; C I:.2-4.4); Doma (OR:6.9; I:1.5-30.1; p= 0.01); Keana (OR:4.5; CI:0.96-21.6; p=0.01); Obi (OR:3.5; CI:0.7-17; p=0.01). The major risk factors for bovine tuberculosis in this study were sex, age, herd size, and location, contributing largely to the spread of bTB in the study area.

Non-tuberculous mycobacteria: occurrence in skin test cattle reactors from official tuberculosis-free herds.

Non-tuberculous mycobacteria (NTM) are considered a relevant cause of non-specific reactions to the most widely applied bovine tuberculosis (bTB) test, the intradermal tuberculin test. In order to establish which NTM species might act as a potential source of such diagnostic interference, a collection of 373 isolates obtained from skin test positive cows from 359 officially tuberculosis-free (OTF) herds, culled in the framework of the bTB eradication campaign in Spain, were identified at the species level through PCR and Sanger sequencing of the 16S rDNA, hsp65 and rpoB genes. Of the 308 isolates for which a reliable identification was achieved, 32 different mycobacterial species were identified, with certain species being most represented: among M. avium complex members (n = 142, 46.1%), M. avium subsp. hominissuis (98; 69.0%) was the most abundant followed by M. avium subsp. avium (33, 23.2%), and M. intracellulare (7, 4.9%). Among non-MAC members (n = 166, 53.9%), M. nonchromogenicum (85; 27.6%) and M. bourgelatii (11; 5.6%) were the predominant species. In addition, mixed results were obtained in 53 isolates presenting up to 30 different genotypes, which could be indicative of new mycobacterial species. Our results represent a first step toward characterizing the diversity of NTM species that could interfere with official diagnostic tests for bTB eradication in Spain.

Experimental infection of goats with Mycobacterium microti induces subclinical pulmonary tuberculosis and mild responses to tuberculin skin tests

Mycobacterium microti is a member of the Mycobacterium tuberculosis complex that seldom causes disease in livestock and humans. This study evaluated the effects on immunodiagnosis and the pathological findings in goats after experimental exposure by different routes and doses to M. microti. In a first experiment goats were challenged orally (PO, n = 7) or intranasally (IN, n = 7) with 104 CFU. In a second experiment, the endobronchial route was assessed, with a low dose of 102 CFU (EB-LD, n = 7) and a high dose of 105 CFU (EB-HD, n = 7) as well as the subcutaneous route (SC, n = 5). Temperature, body weight, clinical signs and immunological responses were monitored. Pathological evaluation was carried out and samples were processed for mycobacterial detection. Resultsdemonstrated the induction of a subclinical pulmonary infection in all the EB-HD challenged animals. Infection was also confirmed in one animal of the SC group, but not in the EB-LD, PO or IN groups. Two animals belonging to the EB-HD and SC groups, respectively, showed positive results to the single intradermal tuberculin test, and another two animals of the EB-HD and EB-LD groups showed doubtful (inconclusive) results, indicating that M. microti can induce mild responses to tuberculin skin testing. No positive results were observed when defined antigens absent in M. microti (ESAT-6 and CPF-10) were used.Our results indicate that animals exposed to M. microti can yield positive results to the skin tests currently performed in livestock tuberculosis eradication campaigns and reinforce the need to use specific antigens in antemortem tests to avoid interference with M. bovis/M. caprae diagnosis.

A humoral diagnostic test outperforms cellular tests in a farm with a latent tuberculosis outbreak caused by a new Mycobacterium tuberculosis complex spoligotype that affected sheep but not goats.

Tuberculosis (TB) is a disease caused by members of the M. tuberculosis complex (MTC) that affects numerous species. M. caprae, a member of the complex which is close to M. bovis, is emerging and affects several different hosts that include goats, cattle, sheep, pigs, rabbits, wild boar, red deer, foxes and also humans. A new M. caprae spoligotype (SB2737) was isolated from an outbreak of sheep tuberculosis affecting a mixed sheep (323)-goat (29) farm in 2021. The index case was detected by the La Rioja slaughterhouse veterinary inspection. Tracing back to the farm of origin, both species were submitted to Comparative Intradermal Tuberculin Test (CITT) and M. bovis-specific antibody ELISA tests. A subsample was also examined by IFN-γ release assay (IGRA) and all positives were slaughtered and pathologically and microbiologically investigated. Only 1.2% of sheep and no goat were positive in the CITT, and 11.4% in the IGRA sheep subsample, while up to 36.8% were positive in two consecutive M. bovis-specific antibody ELISA tests. Goats had always tested negative in annual intradermal follow-up since 2013. Upon confirmation of the immunologically positive sheep at slaughter, all the remaining negative animals were killed and 29.2% of sheep were still found infected. This raised the final overall prevalence to 37.5%. Antibody ELISA was the most sensitive (81.4%) in vivo detection method still showing a 85.0% specificity relative to pathological and microbiological tuberculosis status. It was nearly 10 times more sensitive than skin test and had an 86.8% positive predictive value. Notwithstanding a possible singular pathogenesis of the new spoligotype, this outbreak adds up to previous reports suggesting that sheep tuberculosis could be huge reservoir of infection worldwide overlooked by skin test low sensitivity or simply lack of investigation. This makes it urgent to extend the use antibody tests to address the Trojan horse of hidden M. tuberculosis complex infections on bovine TB control programs.

Assessment of the diagnostic performance of intradermal tuberculin test and post-mortem inspection for the diagnosis of bovine tuberculosis according to WOAH guidelines

Bovine tuberculosis (bTB) constitutes a global challenge for public and animal health with still some deficiencies regarding its diagnosis. This study aimed to estimate the accuracy of the single intradermal tuberculin test (SIT) and post-mortem inspection for different diagnostic objectives following WOAH guidelines. Tissue samples from 59 microbiological culture/PCR-positive and 58 microbiological culture/PCR-negative cattle were evaluated. The diagnostic sensitivity and specificity, the positive and negative probability indices as well as the positive and negative predictive values (PPV and NPV) of each technique were estimated for different pretest probabilities. The SIT with strict interpretation demonstrated moderate precision in confirming the absence of infection in populations historically free of bTB, with a 12.1% rate of false positives, but also detecting positive animals in the early stage of the eradication programs, with a 13.6% rate of false negatives. The diagnostic performance for ruling out bTB was notably high (NPV > 90%) in animals with a pre-test probability (PTP) below 42%. Post-mortem inspection constituted an interesting alternative tool to confirm suspected and positive cases for SIT, particularly in areas with bTB prevalence exceeding 19%, where implementing SIT and eradication measures may be impractical. In these areas, the likelihood that animals with tuberculosis-like lesions are affected by the disease surpasses 90%. Similarly, in herds with a PTP below 25%, the absence of bTB could be confidently ruled out with over 90% certainty. These findings highlight the effectiveness of SIT and post-mortem inspection as valuable techniques for current eradication programs and controlling bTB in high-prevalence areas where molecular techniques may not be feasible.

The impact of changing the cut-off threshold of the interferon-gamma (IFN-γ) assay for diagnosing bovine tuberculosis in Ireland

In Ireland, the interferon-gamma (IFN-γ) assay is routinely used as an ancillary test interpreted in parallel with the single intradermal comparative tuberculin test (SICTT) to maximize the detection of bovine tuberculosis (bTB) infected animals. Up until 2018, a positive test result was recorded in the IFN-γ ELISA assay following whole blood stimulation with purified protein derivative (PPD)-bovine (B), PPD-avian (A) and nil sample (N), using the interpretation criteria, B-N > 50 optical density units (OD), B > 100 and B-A > 0. Following a review of available data, the threshold of the B-A component changed to B-A > 80. As predicting the impact of changing the cut-off thresholds for the IFN-γ test de novo is challenging, the aims of this study were to follow animals that initially tested negative using the new IFN-γ assay interpretation criteria and investigate their future risk of disclosure with bTB, with a focus on animals that otherwise would have been removed when using the older interpretation criteria (0 < B-A ≤ 80). Enrolled animals (n = 28,669 cattle from 527 herds) were followed up for two years (2019–2021), or to point of bTB detection or death. At the end of follow-up, 1151 (4.0%) of enrolled animals were bTB cases. The majority of these cases were diagnosed using SICTT (80.5%). The cumulative number of positive animals that would have been removed if the old cut-off (0 < B-A ≤ 80) was used amounted to 1680 cattle (5.9% of the enrolled cohort). Of these, 127 (7.5%) were diagnosed with bTB during follow-up. In contrast, 1024 of the 1151 cattle which subsequently tested positive during the study period following a negative IFN-γ test would not have been identified with the old or new IFN-γ cut-off criteria. Survival analysis showed that animals that would have been removed under the old interpretation criteria were at increased risk of a positive diagnosis with bTB during follow-up compared to other test negative animals. A newly developed risk prediction model (using a Cox proportional hazard model) showed that age, animal number of SICTT tests, number of inconclusive SICTT tests, B-A (IFN-γ assay), B-N (IFN-γ assay), animals from store herds and the percentage of the rest of the herd that were positive during the breakdown were statistically significantly associated with bTB detection. However, inclusion of the IFN-γ OD variables did not show added value in terms of prediction performance of the model.

Prevalence and zoonotic risk factors of Mycobacterium bovis tuberculosis in cattle at the cattle-wildlife-human interface in South and East Cameroon.

Bovine tuberculosis (bTB) is a contagious and notifiable disease, which is prevalent in cattle populations of many countries and in several wildlife species worldwide. However, the role of wildlife in the transmission and/or maintenance of bTB at the human-wild animal-animal interface and the epidemiology of zoonotic disease are poorly understood in Cameroon, where many wildlife species exist. This study aimed to estimate the prevalence and zoonotic risk factors of bTB at the cattle-wildlife-human interface in the South and East regions of Cameroon. We conducted a descriptive cross-sectional study from May to October 2022 in the southern region (Vallée du Ntem and Dja et Lobo) and eastern region (Haut Nyong and Lom et Djérem) of Cameroon to determine risk factors for bTB in Zebu Bororo, Goudali, Ndama, and Simmental cattle breeds. A comparative intradermal tuberculin testing (CIDT) was performed on 160 cattle randomly selected from herds using the threshold recommended by the World Organization for Animal Health. An interviewee-administered questionnaire was used to gather epidemiological data on sociodemographics, interaction between cattle and wildlife, and awareness of zoonotic tuberculosis (TB) from 90 cattle professionals. The prevalence of bTB at the herd level and associated risk factors were estimated using multiple logistic regression models. Based on the comparative intradermal tuberculin test (CIDT), the estimated prevalence of bTB in 160 cattle (Zebu Bororo, Goudali, Ndama, and Simmental) in South and East Cameroon was 6.8% (4.35%-9.41%) and 1.8% (0%-3.6%) for threshold values 3 mm and 4 mm, respectively. The prevalence obtained by simple intradermal tuberculin test (IDT) was 0.6% (0%-1.2%) for a threshold value 4 mm. Univariate analysis revealed three risk factors associated with bTB with significant odds ratios (OR; p = 0.05): herd size (OR = 4.88; 95% confidence interval [CI]: 1.24-32.56); cattle aged>10 years (OR = 0.17; 95% CI: 0.05-0.53); and victims of bTB organ seizure (OR = 0.015; 95% CI: 0.002-0.067). Multivariate analysis showed that being a cattle herder and contact between wildlife and livestock due to forage was significantly associated with bTB exposure (adjusted OR = 0.02; p = 0.001). Bovine TB is prevalent in cattle of the South and East Cameroon. Comparative IDT of cattle reared in the epidemiological and environmental context of the study areas yielded better results at a threshold of 3 mm than at a threshold of 4 mm recommended by the World Health Organization. Factors associated with exposure to/appearance of bTB were high herd size, cattle aged >10 years old, seizures of tuberculous organs, shepherding as a profession, and contact between cattle and wildlife can be due to lack of forage.

Prevalence and risk factors of bovine tuberculosis in cattle in selected districts of Fafan pastoral settings, Eastern Ethiopia

Cross-sectional study was conducted from October 2021 to August 2022 to investigate the prevalence and associated risk factors of bovine tuberculosis in cattle in selected districts of the pastoral settings of Fafan zone, Somali region, eastern Ethiopia. A comparative intradermal tuberculin test was performed using purified protein derivatives. Animal-related characteristics, and the owner's knowledge on the importance of BTB were collected using a structured questionnaire. The prevalence was 11.24 % (95 % CI, 8.61–14.35) and 43.3 % (95 % CI, 33.27–53.75) at the individual and herd levels, respectively. There were statistically significant differences in the proportions of positive reactor animals according to body condition score (P = 0.000), age (P = 0.048), seasonal migration (P = 0.038), parity number (P = 0.005), and reproductive status (P = 0.037). Animals with poor body condition scores had a significantly higher likelihood of testing positive, with their odds being 11.4 times greater (COR = 11.408, CI = 3.43–37.94, P < 0.001). In multivariate logistic regression, poor body condition score remained significantly associated with the odds of a positive reaction to tuberculosis (AOR = 0.137, CI = 0.053–0.356, P < 0.001). Similarly, the analysis showed that seasonal migration (AOR = 2.882, CI = 1.155–7.191, P = 0.023) and parity number (AOR = 11.64, CI = 1.818–74.464, P = 0.010) were significant predictors of bovine tuberculosis infection in cattle. According to the questionnaire, 14.2 % (17 of 120) and 13.3 % (16 of 120) of the respondents were knowledgeable about bovine tuberculosis and its transmission from animals to humans, and vice versa, respectively. The general judgment of herders' understanding of bovine tuberculosis transmission methods to humans was very low. The study findings showed a high prevalence of bovine tuberculosis in the study area, emphasizing the need for an effective control and prevention strategy.

Preferential differential gene expression within the WC1.1+ γδ T cell compartment in cattle naturally infected with Mycobacterium bovis.

Bovine tuberculosis (bTB), caused by infection with Mycobacterium bovis, continues to cause significant issues for the global agriculture industry as well as for human health. An incomplete understanding of the host immune response contributes to the challenges of control and eradication of this zoonotic disease. In this study, high-throughput bulk RNA sequencing (RNA-seq) was used to characterise differential gene expression in γδ T cells - a subgroup of T cells that bridge innate and adaptive immunity and have known anti-mycobacterial response mechanisms. γδ T cell subsets are classified based on expression of a pathogen-recognition receptor known as Workshop Cluster 1 (WC1) and we hypothesised that bTB disease may alter the phenotype and function of specific γδ T cell subsets. Peripheral blood was collected from naturally M. bovis-infected (positive for single intradermal comparative tuberculin test (SICTT) and IFN-γ ELISA) and age- and sex-matched, non-infected control Holstein-Friesian cattle. γδ T subsets were isolated using fluorescence activated cell sorting (n = 10-12 per group) and high-quality RNA extracted from each purified lymphocyte subset (WC1.1+, WC1.2+, WC1- and γδ-) was used to generate transcriptomes using bulk RNA-seq (n = 6 per group, representing a total of 48 RNA-seq libraries). Relatively low numbers of differentially expressed genes (DEGs) were observed between most cell subsets; however, 189 genes were significantly differentially expressed in the M. bovis-infected compared to the control groups for the WC1.1+ γδ T cell compartment (absolute log2 FC ≥ 1.5 and FDR P adj. ≤ 0.1). The majority of these DEGs (168) were significantly increased in expression in cells from the bTB+ cattle and included genes encoding transcription factors (TBX21 and EOMES), chemokine receptors (CCR5 and CCR7), granzymes (GZMA, GZMM, and GZMH) and multiple killer cell immunoglobulin-like receptor (KIR) proteins indicating cytotoxic functions. Biological pathway overrepresentation analysis revealed enrichment of genes with multiple immune functions including cell activation, proliferation, chemotaxis, and cytotoxicity of lymphocytes. In conclusion, γδ T cells have important inflammatory and regulatory functions in cattle, and we provide evidence for preferential differential activation of the WC1.1+ specific subset in cattle naturally infected with M. bovis.

Enhancing bovine tuberculosis screening at Dhaka city in Bangladesh: Integrating gamma interferon blood test as ancillary testing with tuberculin skin test

Tuberculin skin test (TST) is the standard method for screening of bovine tuberculosis (bTB). However, gamma interferon blood test has been introduced in the bTB control program as an ancillary testing with TST in many countries of the world. The objective of this study was to recommend this screening test as an ancillary testing with TST for field application in Bangladesh. In this study 577 cattle of different age, sex and breeds from twenty nine (29) cattle herds were examined to determine skin response against bTB through single intradermal comparative tuberculin test (SICTT) that comprised of positive (n = 81), inconclusive (n = 44) and negative (n = 452) animals. Of which 74 animals that included positive (n = 63), inconclusive (n = 8) and negative (n = 3) animals were taken under this study. Blood samples were collected in heparinized tube and stimulated overnight with bovine and avian purified protein derivatives (PPDs) for the secretion of gamma interferon, and measured via sandwich ELISA. Cohen’s kappa statistics was performed for the evaluation of agreement between the two tests. The agreement obtained between two tests was fair (Kappa agreement, K = 24.0%, 95% CI = 16.9−30.5%, P = 0.037). Of positive (n = 63), inconclusive (n = 8) and negative (n = 3) status of animals at SICTT, 82.54% (n = 52), 62.50% (n = 5), and 33.33% (n = 1) were found to be bTB positive respectively through this ancillary test. This test notably corroborates to TST result. A considerable number of inconclusive TB status animals were found to be positive through this gamma interferon assay. Therefore, this test could be used as an ancillary test with TST to maximize the proportion of bTB estimation in the infected cattle herd for early detection of zoonotic tuberculosis in Bangladesh before transmission at the animal-human interface.

Divergent proinflammatory immune responses associated with the differential susceptibility of cattle breeds to tuberculosis.

Tuberculosis (TB) in the bovine is one of the most predominant chronic debilitating infectious diseases primarily caused by Mycobacterium bovis. Besides, the incidence of TB in humans due to M. bovis, and that in bovines (bovine TB, bTB) due to M. tuberculosis- indicates cattle as a major reservoir of zoonotic TB. While India accounts for the highest global burden of both TB and multidrug-resistant TB in humans, systematic evaluation of bTB prevalence in India is largely lacking. Recent reports emphasized markedly greater bTB prevalence in exotic and crossbred cattle compared to indigenous cattle breeds that represent more than one-third of the total cattle population in India, which is the largest globally. This study aimed at elucidating the immune responses underlying the differential bTB incidence in prominent indigenous (Sahiwal), and crossbred (Sahiwal x Holstein Friesian) cattle reared in India. Employing the standard Single Intradermal Tuberculin Test (SITT), and mycobacterial gene-targeting single as well as multiplex-PCR-based screening revealed higher incidences of bovine tuberculin reactors as well as Mycobacterium tuberculosis Complex specific PCR positivity amongst the crossbred cattle. Further, ex vivo mycobacterial infection in cultures of bovine peripheral blood mononuclear cells (PBMC) from SITT, and myco-PCR negative healthy cattle exhibited significantly higher intracellular growth of M. bovis BCG, and M. tuberculosis H37Ra in the crossbred cattle PBMCs compared to native cattle. In addition, native cattle PBMCs induced higher pro-inflammatory cytokines and signaling pathways, such as interferon-gamma (IFN-γ), interleukin-17 (IL-17), tank binding kinase-1 (TBK-1), and nitric oxide (NO) upon exposure to live mycobacterial infection in comparison to PBMCs from crossbred cattle that exhibited higher expression of IL-1β transcripts. Together, these findings highlight that differences in the innate immune responses of these cattle breeds might be contributing to the differential susceptibility to bTB infection, and the resultant disparity in bTB incidence amongst indigenous, and crossbred cattle.

Evaluation of the diagnostic accuracy of the serological test for paratuberculosis in cattle according to tuberculosis status.

Enzyme-linked immunosorbent assays (ELISAs) are the most widely used diagnostic tools in bovine paratuberculosis (bPTB) control. However, their diagnostic accuracy may be compromised by bovine tuberculosis (bTB) infection, as both diseases share diagnostic targets. The bPTB and bTB infection status of 228 animals was determined using microbiological tissue culture as a reference test. The diagnostic performance (sensitivity, specificity, likelihood ratios and predictive values) of the bPTB-ELISA on blood serum samples, taking into account the bPTB animal-level prevalence of the area and the bTB status of the animals, was evaluated. A sensitivity of 40.7% (95% confidence interval [CI]: 27.5%-53.9%) and a specificity of 94.7% (95% CI: 91.4%-98.0%) were obtained for bPTB-ELISA in all animals. A bPTB-ELISA-positive animal would have a post-test probability of 70% or more of being infected in areas with a bPTB prevalence of 23% or more. A negative bPTB-ELISA result, in areas with a bPTB prevalence of 41% or less, would rule out the disease with more than 70% certainty. In bTB-positive animals, sensitivity increased (94.4% [95% CI: 81.4%-100%] vs. 25.1% [95% CI: 11.8%-38.4%]) and specificity decreased (82.6% [95% CI: 71.8%-93.4%] vs. 99.4% [95% CI: 98.0%-99.9%]). The bPTB-ELISA is a good tool to rule out bPTB co-infection in bTB-positive animals, while in bTB-negative animals, it allows confirmation of disease with more than 70% probability if disease prevalence is 6% or more. The observed differences could be enhanced by the effect of frequent application of the intradermal tuberculin test, which was unknown in the animals studied. These results provide useful guidance for the application and interpretation of ELISA as a tool for bPTB disease control.

Investigation of the association between the Enferplex bovine tuberculosis antibody test and the future risk of bovine tuberculosis in irish cattle in infected herds: a pilot field study

The Single Intradermal Comparative Tuberculin Test (SICTT) and the interferon-gamma (IFN-γ) assay are the approved diagnostic tests for bovine tuberculosis (bTB) in Ireland. The aim of this pilot study was to explore if there was any added diagnostic benefit from applying the Enferplex bTB test (an antibody test) in severe bTB herd breakdowns after the removal of cattle that had tested positive to the SICTT and the IFN-γ test. In addition to the normal bTB testing and management protocols, the animals in these herds that tested negative to SICTT and the IFN-γ test were followed forward for a period of two years. All animals were tested by Enferplex at enrolment. The time to subsequent bTB detection (diagnosed with SICTT/IFN-γ tests or detection of visible lesions at routine slaughter) for animals that tested positive or negative to the Enferplex bTB test at the start of the study was compared using Kaplan–Meier survival curves and Cox based survival models. Of the 484 enrolled animals (from 11 herds), 171 (35.3%) and 151 (31.1%) initially tested positive in the Enferplex assay under the high sensitivity and high specificity interpretation settings respectively. The results of the survival analysis showed that there was no difference in the survival time to a positive diagnosis with bTB during the follow-up period between animals initially classified as positive and negative by the Enferplex test. Further research is warranted to explore the potential benefit of using the Enferplex test in other scenarios.