Single Intradermal Tuberculin Test Research Articles

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.

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.

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.

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.

Differences in skin test reactions to official and defined antigens in guinea pigs exposed to non-tuberculous and tuberculous bacteria

The single and comparative intradermal tuberculin tests (SITT and CITT) are official in vivo tests for bovine tuberculosis (TB) diagnosis using bovine and avian purified protein derivatives (PPD-B and PPD-A). Infection with bacteria other than Mycobacterium tuberculosis complex (MTC) can result in nonspecific reactions to these tests. We evaluated the performance of the skin test with PPDs and new defined antigens in the guinea pig model. A standard dose (SD) of Rhodococcus equi, Nocardia sp., M. nonchromogenicum, M. monacense, M. intracellulare, M. avium subsp. paratuberculosis, M. avium subsp. avium, M. avium subsp. hominissuis, M. scrofulaceum, M. persicum, M. microti, M. caprae and M. bovis, and a higher dose (HD) of M. nonchromogenicum, M. monacense, M. intracellulare, M. avium subsp. paratuberculosis were tested using PPD-B, PPD-A, P22, ESAT-6-CFP-10-Rv3615c peptide cocktail long (PCL) and fusion protein (FP). The SD of R. equi, Nocardia sp., M. nonchromogenicum, M. monacense, M. intracellulare and M. avium subsp. paratuberculosis did not cause any reactions. The HD of M. nonchromogenicum, M. monacense, M. intracellulare, and M. avium subsp. paratuberculosis and the SD of M. avium subsp. hominissuis, M. scrofulaceum and M. persicum, caused nonspecific reactions (SIT). A CITT interpretation would have considered M. avium complex and M. scrofulaceum groups negative, but not all individuals from M. nonchromogenicum HD, M. monacense HD and M. persicum SD groups. Only animals exposed to M. bovis and M. caprae reacted to PCL and FP. These results support the advantage of complementing or replacing PPD-B to improve specificity without losing sensitivity.

PREVALENCE OF BOVINE TUBERCULOSIS IN DAIRY CATTLE OF JAIPUR, RAJASTHAN, INDIA

Tuberculosis in cattle is a chronic debilitating infectious disease caused by Mycobacterium bovis, an intracellular acid-fast bacterium. The diseases have become prevalent infection in most of the developing countries and causes considerable economic losses to producers of livestock, particularly in dairy cows. This study was conducted to evaluate the prevalence of tuberculosis in cattle in Jaipur. A total of 200 serum samples were collected from cattle of a gaushala in Jaipur and subjected to ELISA and single intradermal tuberculin test. overall prevalence obtained was 1.5% in cattle. Individual test wise, 6 (3%) and 9 (4.5%) animals were found positive to SITT and ELISA, respectively. Higher prevalence was observed in age group above 5 years as 3.65% and 5.11% by SITT and ELISA, respectively. Also, the prevalence was found higher in crossbred cattle as 3.45 and 6.89 by SITT and ELISA, respectively. Comparison of results for SITT and ELISA tests was found statistically non-significant (p&gt; 0.05) with respect to age and breed. The kappa value to know the agreement between SITT and ELISA test was 0.38. It shows a poor agreement between both the tests for tuberculosis in cattle. The prevalence of bovine tuberculosis in milch animals signifies potential health risk for human and animals. The findings are required further confirmation with large scale surveillance of all susceptible animals

High-Specificity Test Algorithm for Bovine Tuberculosis Diagnosis in African Buffalo (Syncerus caffer) Herds.

Ante-mortem bovine tuberculosis (bTB) tests for buffaloes include the single comparative intradermal tuberculin test (SCITT), interferon-gamma (IFN-γ) release assay (IGRA) and IFN-γ-inducible protein 10 release assay (IPRA). Although parallel test interpretation increases the detection of Mycobacterium bovis (M. bovis)-infected buffaloes, these algorithms may not be suitable for screening buffaloes in historically bTB-free herds. In this study, the specificities of three assays were determined using M. bovis-unexposed herds, historically negative, and a high-specificity diagnostic algorithm was developed. Serial test interpretation (positive on both) using the IGRA and IPRA showed significantly greater specificity (98.3%) than individual (90.4% and 80.9%, respectively) tests or parallel testing (73%). When the SCITT was added, the algorithm had 100% specificity. Since the cytokine assays had imperfect specificity, potential cross-reactivity with nontuberculous mycobacteria (NTM) was investigated. No association was found between NTM presence (in oronasal swab cultures) and positive cytokine assay results. As a proof-of-principle, serial testing was applied to buffaloes (n = 153) in a historically bTB-free herd. Buffaloes positive on a single test (n = 28) were regarded as test-negative. Four buffaloes were positive on IGRA and IPRA, and M. bovis infection was confirmed by culture. These results demonstrate the value of using IGRA and IPRA in series to screen buffalo herds with no previous history of M. bovis infection.

Effect of a recent parenteral dexamethasone and ketoprofen administration on the immunological diagnosis of tuberculosis in goats.

Caprine tuberculosis (TB) is a zoonosis caused by members of the Mycobacterium tuberculosis complex (MTBC). Caprine TB eradication programmes are based mainly on intradermal tuberculin tests and slaughterhouse surveillance. Different factors may affect the performance of the TB diagnostic tests used in caprine herds and, therefore, their ability to detect infected animals. The present study evaluates the effect of the fraudulent administration of two anti-inflammatory substances, dexamethasone and ketoprofen, on the performance of the TB diagnostic techniques used in goats, as well as the suitability of high performance liquid chromatography (HPLC) for their detection in hair samples. The animals (n = 90) were distributed in three groups: (1) a group treated with dexamethasone (n = 30); a second group treated with ketoprofen (n = 30); and a third non-treated control group (n = 30). Both dexamethasone and ketoprofen groups were subjected to intramuscular inoculation with the substances 48 h after the administration of bovine and avian purified protein derivatives (PPDs), that is, 24 h before the tests were interpreted. All the animals were subjected to the single and comparative intradermal tuberculin (SIT and CIT, respectively) tests, interferon-gamma release assay (IGRA) and P22 ELISA. The number of SIT test reactors was significantly lower in the dexamethasone (p = 0.001) and ketoprofen (p < 0.001) groups 72 h after the bovine PPD inoculation compared with the control group. A significantly higher number of positive reactors to IGRA was detected within the dexamethasone group (p = 0.016) 72 h after PPD administration compared to the control group. Dexamethasone and ketoprofen detection in either hair or serum samples was challenging when using HPLC since these substances were not detected in animals whose skin fold thickness (SFT) was reduced, what could be an issue if they are used for fraudulent purposes. In conclusion, the parenteral administration of dexamethasone or ketoprofen 48 h after the PPDs administration can significantly reduce the increase in SFT (mm) and subsequently the number of positive reactors to SIT test.

Field evaluation of the tuberculin skin test for the detection of Mycobacterium tuberculosis complex infection in communal goats (Capra hircus) in KwaZulu-Natal, South Africa

In South Africa, animal tuberculosis (TB) control programs predominantly focus on domestic cattle and African buffaloes (Syncerus caffer) despite increasing global reports of tuberculosis in goats (Capra hircus). Left undetected, Mycobacterium tuberculosis complex (MTBC) infected goats may hinder TB eradication efforts in cattle and increase zoonotic risk to humans. Since the publication of animal TB testing guidelines in 2018, prescribing the use of the tuberculin skin test (TST) for goats in South Africa by the Department of Agriculture, Land Reform, and Rural Development (DALRRD), there have been no published reports of any field application of the prescribed test criteria in goat herds. Therefore, this study aimed to evaluate the performance of these DALRRD guidelines using the single intradermal cervical tuberculin test (SICT) and the single intradermal comparative cervical tuberculin test (SICCT). Between October and December 2020, 495 goats from communal pastures of Kwa-Zulu Natal (KZN), where M. bovis infection has been identified in cattle and where cattle and goats cohabitate, were tested using the SICT and SICCT (M. bovis-exposed group). Additionally, 277 goats from a commercial Saanen dairy herd, with no history of M. bovis, were also tested (M. bovis-unexposed group). Estimated apparent prevalence of TST positive goats was determined based on published test interpretation criteria as described by DALRRD. When proportions of test-positive goats were compared between different DALRRD criteria, the ≥ 4 mm cut-off criterion for the SICCT resulted in the lowest proportion of positive results in the presumably uninfected group (1/277 positive in the unexposed group). The apparent prevalence of TB in the exposed group was estimated at 3.0% (95% CI: 1.7–4.9%), which is similar to previous reports of M. bovis prevalence in cattle from this area (6%). The detection of a significantly greater proportion of SICCT positive goats in the M. bovis-exposed group compared to the unexposed group suggests that MTBC infection is present in this population. Further investigations should be undertaken, in conjunction with confirmatory molecular tests, mycobacterial culture, and advanced pathogen sequencing to establish whether MTBC infection in domestic goats is a true under-recognized threat to the eradication of animal TB in South Africa.

Zoonotic Importance of Bovine Tuberculosis in Ethiopia: An Overview

Bovine tuberculosis (BTB) is an important zoonotic disease that affects both domestic animals as well as humans in many countries of the world. The disease is caused by Mycobacterium bovis, which is responsible for 10–15% of human tuberculosis cases. BTB poses an occupational health hazard to the cattle owners, butchers, animal attendants, and veterinarians. The source of infection is exogenous and the respiratory tract is considered the chief portal of entry of the organism. The mode of spread of bacteria is aerogenous. BTB is endemic in Ethiopia, and it is mostly transmitted to people through the consumption of unpasteurized contaminated milk. Miliary tubercular lesions, chronic cough, blockages of the airways, alimentary system, or blood vessels, and lymph node enlargement are all symptoms of tuberculosis. Single intradermal or comparative intradermal tuberculin tests are useful delayed-type hypersensitivity tools for diagnosing tuberculosis in live animals. It is also employed in tuberculosis eradication programs and international trade activity. Vaccination of calves with an attenuated BTB strain known as Bacillus of Calmette and Guerin, as well as testing and culling, is significant measures in the prevention and control of BTB in endemic areas like Ethiopia. The proper pasteurization of milk before human consumption is a significant measure to reduce the public health risk posed by BTB.

The Role of Histopathology as a Complementary Diagnostic Tool in the Monitoring of Bovine Tuberculosis.

The diagnosis of bovine tuberculosis (bTB) is based on the single intradermal tuberculin test (SIT), interferon gamma, and compulsory slaughter of reactor animals. Culture and PCR from fresh tissue are regarded as gold standard techniques for post-mortem confirmation, with the former being time-consuming and presenting moderate to low sensitivity and the latter presenting promising results. Histopathology has the advantage to identify and categorize lesions in both reactor and non-reactor animals. Therefore, this study aims to highlight the role of histopathology in the systematic diagnosis of bTB to shorten the time to disclose positive animals. Blood (212) and lymph node (681) samples were collected for serological, bacteriological, and histopathological analyses from a total of 230 cattle subjected to the Spanish bTB eradication program. Seventy-one lymph nodes and 59 cattle yielded a positive result to bacteriology, with 59 lymph nodes and 48 cattle presenting a positive result in real-time PCR from fresh tissue. Roughly 19% (40/212) of sera samples gave a positive result to ELISA. Tuberculosis-like lesions (TBLs) were observed in 11.9% (81/681) of the lymph nodes and 30.9% (71/230) of cattle. Noteworthy, TBLs were evidenced in 18 out of 83 SIT− and real-time PCR and bacteriology negative animals, with 11/18 disclosing a positive result to Ziehl-Neelsen technique and two of them to ddPCR from paraffin blocks targeting IS6110. Six out of these 11 ZN+ corresponded with mesenteric LN and were confirmed positive to paratuberculosis. Histopathology yielded a sensitivity of 91.3% (CI95 83.2–99.4%) and a specificity of 84.4% (CI95 78.6–89.3%) with good agreement (κ = 0.626) when compared with real-time PCR. Our results confirm that histopathology allows a rapid confirmation of real-time PCR and bacteriology, emphasizing its contribution to bTB control and monitoring.

Performance of Loop-Mediated Isothermal Amplification Technique in Milk Samples for the Diagnosis of Bovine Tuberculosis in Dairy Cattle Using a Bayesian Approach.

This study aimed to estimate the sensitivity (Se) and specificity (Sp) of loop-mediated isothermal amplification (LAMP) and single intradermal tuberculin (SIT) tests for the diagnosis of bovine tuberculosis (bTB) in dairy cattle in Thailand using a Bayesian approach. The SIT test was performed in 203 lactating dairy cattle from nine dairy farms located in Chiang Mai province, Thailand. Milk samples were collected for the LAMP test. Kappa analysis was performed to determine the agreement between the two tests. A one-population conditional independence Bayesian model was applied to estimate the Se and Sp of the two tests. Of 203 dairy cattle, 2 were positive for the SIT test using standard interpretation, whereas 38 were positive for the LAMP test. A poor agreement (kappa = 0) was observed between the two tests. The median Se and Sp of the SIT test using standard interpretation were 63.5% and 99.1%, respectively. The median Se and Sp of the LAMP test were 67.2% and 82.0%, respectively. The estimated true prevalence of bTB was 3.7%. The LAMP test with milk samples can potentially be used as a non-invasive screening test for the diagnosis of bTB in dairy cattle.

Tuberculin test errors and its effect on detection of bovine tuberculosis

Bovine tuberculosis is an endemic disease in Egypt, a notable gap exists between limited cases identified by single intradermal tuberculin test performed through the national control program and higher detected cases at abattoirs. Therefore, this study aimed to investigate epidemiological situation and causes of the previously mentioned gap in Middle-Delta Region. A total of 25 emergency-slaughtered animals of unknown tuberculosis-status were investigated by cocktail-antigens ELISA and post-mortem examination. Five visible tuberculous-lesion cases were detected and confirmed by PCR, ELISA was sensitive and predictive of the existence of tuberculous-lesions; 4(80%) out of 5 visible-lesion cases were seropositive. True prevalence among the slaughtered animals was 27%. In addition, tuberculin-testing of 400 animals during the national control program was evaluated, many technical and procedural errors were detected, and all animals were negative.Out of them, 55 animals were tested by ELISA before the application of tuberculin test, 30 (54.5%) animals were seropositive. To confirm the effect of the reported errors on reliability of tuberculin test, reference serum of 20 tuberculosis-positive animals that were tested by standard-procedures tuberculin test and their status were confirmed by PCR after slaughtering, were tested by ELISA. A complete matching was evident, the 20 standard-tuberculin positive animals were all seropositive. In conclusion, bovine tuberculosis is endemic at high levels in the study area, reported errors of tuberculin test during national control program may be the cause for missing tuberculosis cases and not tuberculin test itself and finally, a further wide-scale funded study is required to discover the situation throughout Egypt.

Epidemiology of Avian Tuberculosis in Selected Districts of Oromia Region, Ethiopia.

Avian tuberculosis is a growing public health concern and a significant impediment to socioeconomic development, especially in developing countries, where the risk of infection is high. The disease is predominantly caused by Mycobacterium avium belonging to serotypes 1, 2, 3, and 6 (genotypes IS901 and IS1245) and Mycobacterium genavense. It mostly occurs in older birds and immunocompromised individuals due to the greater opportunity for infection with age and host immunity. A cross-sectional study anticipated to generate epidemiological information on avian tuberculosis was carried out from November 2016 to June 2017 at highland areas of Gerar Jarso, Ada'a (midland), and Boset (lowland) districts of Oromia region, Ethiopia. Two hundred seventy-three village chickens comprising local breeds, exotic breeds, and crossbreeds of both sexes were used in the study. Single intradermal avian tuberculin test, postmortem inspection of positive reactors, mycobacteriological culturing, and histopathological examination were used to assess information on the epidemiology of the disease. Subsequently, avian tuberculin test revealed an overall apparent prevalence of 11.4% (31/273) and a specific prevalence of 6.8% (6/88) in the highland, 13.4% (13/97) in the midland, and 13.6% (12/88) in the lowland study districts. Besides, it signified a higher odd of exposure in crossbreeds and females as compared to locals and males. In addition, greater odd of exposure was observed in chickens at mid- and low altitudes as compared to those at the highland. Moreover, 40.9% (9/22) of positive reactor chickens sacrificed for necropsy showed gross pathological lesions. Similarly, histopathological examination revealed a granuloma characterized by central necrosis and peripheral mononuclear lymphocytes. Nevertheless, only 0.02% (2/120) of the cultured tissues had shown colonial growth up to 12 weeks of incubation, and both were seen on sodium pyruvate-enriched Lowenstein–Jensen medium slants. Generally, the study revealed an overall increment of the apparent prevalence of avian tuberculosis with decreasing altitude. Besides, it signified a relative breed and sex variation in the risk of acquiring the disease, with crossbreeds and female chickens having higher odds of exposure.

Effect of the topical administration of corticosteroids and tuberculin pre-sensitisation on the diagnosis of tuberculosis in goats

BackgroundCaprine tuberculosis (TB) is a zoonosis caused by members of the Mycobacterium tuberculosis complex (MTBC). Caprine TB control and eradication programmes have traditionally been based on intradermal tuberculin tests and slaughterhouse surveillance. However, this strategy has limitations in terms of sensitivity and specificity. Different factors may affect the performance of the TB diagnostic tests used in goats and, subsequently, the detection of TB-infected animals. In the present study, the effect of two of the factors that may affect the performance of the techniques used to diagnose TB in goats, the topical administration of corticosteroids and a recent pre-sensitisation with tuberculin, was analysed.MethodsThe animals (n = 151) were distributed into three groups: (1) a group topically treated with corticosteroids 48 h after intradermal tuberculin tests (n = 53); (2) a group pre-sensitised with bovine and avian purified protein derivatives (PPDs) 3 days before the intradermal tuberculin test used for TB diagnosis (n = 48); and (3) a control group (n = 50). All the animals were tested using single and comparative intradermal tuberculin (SIT and CIT, respectively) tests, an interferon-gamma release assay (IGRA) and a P22 ELISA.ResultsThe number of SIT test reactors was significantly lower in the group treated with corticosteroids when compared to the pre-sensitised (p < 0.001) and control (p = 0.036) groups. In contrast, pre-sensitisation with bovine and avian PPDs did not cause a significant reduction in the number of SIT and CIT test reactors compared with the control group. In fact, a higher number of reactors was observed after the prior tuberculin injection in the pre-sensitised group (p > 0.05). No significant effect was observed on IGRA and P22 ELISA due to corticosteroids administration. Nevertheless, a previous PPD injection affected the IGRA performance in some groups.ConclusionsThe application of topical corticosteroid 24 h before reading the SIT and CIT tests can reduce the increase in skin fold thickness and subsequently significantly decrease the number of positive reactors. Corticosteroids used can be detected in hair samples. A previous pre-sensitisation with bovine and avian PPDs does not lead to a significant reduction in the number of intradermal tests reactors. These results are valuable in order to improve diagnosis of caprine TB and detect fraudulent activities in the context of eradication programs.

Host Serum Proteins as Potential Biomarkers of Bovine Tuberculosis Resistance Phenotype.

Eradication of bovine tuberculosis (bTB) continues to be a worldwide challenge. The lack of reliable vaccines dampens the control and eradication programs of Mycobacterium bovis infection and spread. Selection and breeding of cattle resistant to M. bovis infection would greatly enhance the effectiveness of bTB eradication programs. Here, we have evaluated the potential of serum proteins as biomarkers of cattle resistance to bTB in Holstein-Friesian cows, 6–8-year-old, born and raised in similar conditions in herds with bTB prevalence >30%. Serum proteins obtained from uninfected cows (bTB-resistant; R) were compared to those from infected cows (bTB-susceptible; S), defined by a negative or positive bTB diagnosis, respectively. bTB diagnosis included: (i) single intradermal (caudal fold) tuberculin test, (ii) whole blood IFN-gamma test, (iii) gross visible lesions in lymph nodes and lungs by inspection at the abattoir, and (iv) a bacteriological culture for M. bovis. Using 2D-GE and LC-ESI-MS/MS, we found higher expression levels of primary amine oxidase (AO), complement component 5 (C5), and serotransferrin (TF) in R cattle than S cattle. In-house developed and standardized ELISAs for these novel biomarkers showed the best sensitivities of 72, 77, 77%, and specificities of 94, 94, 83%, for AO, C5, and TF, respectively. AUC-ROC (95% CI) values of 0.8935 (0.7906–0.9964), 0.9290 (0.8484–1.010), and 0.8580 (0.7291–0.9869) were obtained at cut-off points of 192.0, 176.5 ng/ml, and 2.1 mg/ml for AO, C5, and TF, respectively. These proteins are involved in inflammatory/immunomodulatory responses to infections and may provide a novel avenue of research to determine the mechanisms of protection against bTB. Overall, our results indicate that these proteins could be novel biomarkers to help identify cattle resistant to bTB, which in turn could be used to strengthen the effectiveness of existing eradication programs against bTB.

Effect of the Inoculation Site of Bovine and Avian Purified Protein Derivatives (PPDs) on the Performance of the Intradermal Tuberculin Test in Goats From Tuberculosis-Free and Infected Herds.

The single and comparative intradermal tuberculin (SIT and CIT) tests are used for the ante-mortem diagnosis of caprine tuberculosis (TB). The tuberculin injection site has been associated with a different performance of the test in cattle. In contrast to that required in cattle in Europe (cervical injection), it can be carried out in the scapular region in goats. Nevertheless, there are no previous data concerning the effect of the injection site on the performance of the test in goats. The aim of the present study was to evaluate the effect of two different inoculation sites (cervical and scapular) on the performance of the SIT/CIT tests. This was done by intradermally inoculating 309 goats from two infected herds and one TB-free herd with both avian and bovine PPDs in the mid-cervical and scapular regions. None of the animals from the TB-free herd had positive reactions, and the number of reactors was not significantly higher, regardless of the inoculation site, in the high and low prevalence herds. However, significantly higher increases in skin fold thickness were observed on the cervical site when compared to the scapular site after the avian and bovine PPD inoculations in the TB-free herd (p < 0.001) and after the bovine PPD injection in the high prevalence herd (p = 0.003). The presence of clinical signs was also more evident on the cervical site when using avian and bovine PPDs in the high prevalence herd (p < 0.01). In contrast, increases in higher skin fold thickness were observed on the scapular site when compared to the cervical site after the bovine and avian PPD inoculations were employed in the low prevalence herd (p < 0.01). These results suggest that the cervical injection of PPDs may improve the sensitivity of the intradermal tuberculin test in high TB prevalence caprine herds, mainly owing to the increased presence of local clinical signs and a better performance of the CIT test. Moreover, specificity was not affected when using standard interpretations, although further analyses in a great number of herds are required in order to confirm these findings.

Evaluation of ELISA and PCR in parallel to single intradermal cervical tuberculin test (SICT) for diagnosis of tuberculosis in buffaloes.

Bovine tuberculosis is an economically important disease with very high zoonotic potential. Single intradermal cervical tuberculin test (SICT) is considered a gold standard assay for the diagnosis of bovine tuberculosis. However, bovines especially buffaloes may produce a false negative result when the animal becomes cell-mediated immune (CMI) anergic in the advanced stage of the disease. In the present study, ELISA and PCR assays were successfully demonstrated to be useful in diagnosing tuberculosis especially in the CMI anergic buffaloes infected with Mycobacterium bovis. ELISA and PCR assays are able to detect 8.94% and 8.13%, respectively, more animals as positive in comparison to standard SICT assay in a selected population of 123 buffaloes. The moderate agreement between SICT and ELISA (k: 0.528; 0.249-0.807), a substantial agreement between SICT and PCR (k: 0.648; 0.364-0.931), and high agreement between ELISA and PCR (k: 0.856; 0.697-1.0) highlight that ELISA and PCR, if used in parallel with SICT, will provide better sensitivity over single assay. Reduction of false negative reactors may help in minimizing the zoonotic threat from bovine tuberculosis especially in disease endemic region where human and livestock interface is quite high.

Spatial and Temporal Distribution of Mycobacterium tuberculosis Complex Infection in Eurasian Badger (Meles meles) and Cattle in Asturias, Spain.

Simple SummaryThe aim of the present work was to investigate the prevalence, spatial distribution, and temporal distribution of tuberculosis in 673 free-ranging Eurasian badgers (Meles meles) and cattle from Asturias (Atlantic Spain) during a 13-year follow-up. The study objective was to assess the role of badgers as a reservoir of tuberculosis for cattle and other sympatric wild species in the region. During the follow-up, 27/639 badgers (4.23%) were positive for the Mycobacterium tuberculosis complex based on bacterial isolation, while 160/673 (23.77%) were positive based on P22 ELISA. Badger infection was spatially and temporally associated with cattle herd infection.The present work investigated the prevalence, spatial distribution, and temporal distribution of tuberculosis (TB) in free-ranging Eurasian badgers (Meles meles) and cattle in Asturias (Atlantic Spain) during a 13-year follow-up. The study objective was to assess the role of badgers as a TB reservoir for cattle and other sympatric wild species in the region. Between 2008 and 2020, 673 badgers (98 trapped and 575 killed in road traffic accidents) in Asturias were necropsied, and their tissue samples were cultured for the Mycobacterium tuberculosis complex (MTC) isolation. Serum samples were tested in an in-house indirect P22 ELISA to detect antibodies against the MTC. In parallel, data on MTC isolation and single intradermal tuberculin test results were extracted for cattle that were tested and culled as part of the Spanish National Program for the Eradication of Bovine TB. A total of 27/639 badgers (4.23%) were positive for MTC based on bacterial isolation, while 160/673 badgers (23.77%) were found to be positive with the P22 ELISA. The rate of seropositivity was higher among adult badgers than subadults. Badger TB status was spatially and temporally associated with cattle TB status. Our results cannot determine the direction of possible interspecies transmission, but they are consistent with the idea that the two hosts may exert infection pressure on each other. This study highlights the importance of the wildlife monitoring of infection and disease during epidemiological interventions in order to optimize outcomes.

The effects of intradermal M. bovis and M. avium PPD test on immune-related mRNA and miRNA in dermal oedema exudates of water buffaloes (Bubalus bubalis)

Tuberculosis (TB) is a zoonotic disease primarily caused by pathogens belonging to the genus of Mycobacterium. Programs of control and eradication for bovine TB include a screening using single intradermal tuberculin (SIT) test with Mycobacterium bovis (M. bovis)-purified protein derivatives (PPD-B) single or concurrent with Mycobacterium avium (M. avium)-purified protein derivatives (PPD-A). This study aimed to determine the effects of intradermal PPD-B and PPD-A test on immune-related mRNA and microRNAs in dermal oedema exudates of water buffaloes (Bubalus bubalis). The investigation was carried out on RNA extracted from dermal oedema exudates of 36 animals, of which 24 were M. bovis positive (M. bovis+) and 12 M. avium positive (M. avium+). The lymphocyte polarization toward Th1, Th2, TReg, and Th17 lineages was addressed by measuring the abundance of the respective cytokines and transcription factors, namely TBET, STAT4, IFNγ, and IL1β for Th1; STAT5B, and IL4 for Th2; FOXP3 and IL10 for TReg; and RORC, STAT3, and IL17A for Th17. Due to the very low abundance of Th17-related genes, a digital PCR protocol was also applied. The abundance of microRNAs involved in the immune response against PPDs, including miR-122-5p, miR-148a-3p, miR30a, and miR-455-5p, was equally measured. Results showed that IFNγ (fold change = 2.54; p = 0.037) and miR-148a-3p (fold change = 2.54; p = 0.03) were upregulated in M. bovis+ as compared to M. avium+ samples. Our preliminary results supported the pivotal role of IFNγ in the local immune response related to PPD-B and highlighted the differential expression of miR-148a-3p, which downregulates the proinflammatory cytokines and the TLR4-mediated NF-κB activation, providing an anti-inflammation modulator in responses to mycobacterial infection.