Comparative Cervical Tuberculin Test Research Articles

Bovine Tuberculosis Testing in Colombia: Comparative Histopathological, Microbiological, and Molecular Biology Findings

Introduction: Bovine tuberculosis (bTB) is a zoonotic infectious disease present in Colombia, caused by Mycobacterium bovis, and causes tuberculosis in water buffalo (Bubalus bubalis). Diagnosis of bovine tuberculosis through the intradermal test is difficult; evaluating and understanding the behavior of other diagnostic tests is necessary.
 Objective: To describe the behavior and results of different diagnostic methods for bovine tuberculosis in water buffalo positive for the Purifed Proteic Derivate (DPP) intradermal test.
 Methodology: In water buffaloes positive for comparative cervical tuberculin test, different diagnostic methods were applied, described, and compared: Ziehl-Neelsen staining, microbiological culture, histopathological analysis, and PCR-HRM.
 Results: Histopathological tests showed that 26 water buffalo positive for DPP (52%) had histological lesions compatible with bovine tuberculosis. 37% of the evaluated samples from tuberculin-positive Buffalo's lungs and secondary lymph nodes showed acid-alcohol-resistant bacillus with Ziehl-Neelsen staining. Four samples of Mycobacterium bovis from tuberculin-positive buffalo were isolated and identified, with two of these isolates confirmed from tissues with PCR-HRM, and three buffalo with microbiological isolates presented granulomatous lesions through histological analysis. Seventeen tuberculin-positive buffalo (34%) tested positive for real-time PCR HRM, and nine of these buffalo did not have histological lesions compatible with bTB and were confirmed with the molecular test.
 Conclusion: Our results provide positive evidence of histological findings, microbiological isolation, and molecular diagnosis of tuberculin-positive water buffalo in the lowlands of Colombia. None of the complementary tests performed showed 100% concordance with the comparative cervical tuberculin test results for bTB.

Prevalence of bovine tuberculosis and analysis of risk factors among the dairy farms in and around Guwahati metropolitan city, India

Bovine tuberculosis (bTB), a neglected zoonotic disease caused by Mycobacterium bovis is being reported worldwide. The present work was carried out from December 2020 to November 2021 to assess the prevalence and risk factors of bTB in peri-urban and urban dairy farms of Guwahati, Assam, India. A questionnaire was used to collect data on knowledge about bTB on 36 farms, and ten animals per farm were screened by single intradermal comparative cervical tuberculin test (SICCT) to determine the prevalence of bTB, giving a total of 360 animals. The demographic data of the farmers revealed that 61.1% respondents were illiterate, 66.7% had no awareness about bovine tuberculosis and 41.7% consumed unpasteurised milk and milk products. SICCT showed that 38 cattle from 18 of the farms were positive reactors for bTB, yielding an overall animal level prevalence of 10.55% (95% confidence interval (CI = 7.58–14.2%) and a 50% herd prevalence (95% CI 32.9–67.1%). Animals 5 years and above were found to be more likely to be positive for bTB (17.18%). The study highlighted the widespread prevalence of bovine tuberculosis in peri-urban and urban dairy farms of Guwahati which gives a picture also about other major cities of India. Hence, it is of utmost importance to undertake a comprehensive epidemiological study in such cities for effective control and prevention of bTB in a one health approach.

Estimation of the specificity of an antibody ELISA for paratuberculosis generated from a sector of the UK cattle population using results from a paratuberculosis control programme

In the United Kingdom (UK) a voluntary programme to control paratuberculosis in cattle based on herd management and serological screening has been operating since 1998. The programme assigns a risk level to each participating herd according to the within herd seroprevalence and the confirmation of the presence of infection with Mycobacterium avium subspecies paratuberculosis (MAP) by faecal culture or polymerase chain reaction (PCR). From the outset a general concern over the specificity of the paratuberculosis antibody enzyme-linked immunosorbent assay (ELISA) resulted in the use of a faecal screen for the causal organism to negate or confirm infection in individual seropositive animals. Progress in improving the diagnostic tests has been gradual throughout the life of the programme and the under-pinning approach to using tests to determine the risk of paratuberculosis for a herd required to be re-examined. This study used a large data set of more than 143,000 test results over five years from the lowest paratuberculosis risk level category of herds to estimate the specificity of a commercially available paratuberculosis antibody ELISA for cattle. In each year of the study the estimated specificity reached or exceeded 0.998. We also examined the apparent impact that annual or more frequent application of the single intradermal comparative cervical tuberculin (SICCT) test for tuberculosis (TB), using purified protein derivatives of Mycobacterium bovis and Mycobacterium avium subspecies avium, had on specificity of the antibody ELISA for paratuberculosis. We found a statistically significant difference in three of the five years with herds that were officially tuberculosis free and not subject to frequent SICCT testing. This difference was small and considered to be of little practical importance for the paratuberculosis assurance programme. We concluded that, in the UK the mandatory TB surveillance programme of cattle herds is not a limiting factor in the use of serological testing to support herd-level assurance schemes for paratuberculosis. Furthermore, in paratuberculosis, where shedding of MAP is intermittent and the sensitivity of the commercially available PCR tests for detection MAP is highly variable, faecal screening of seropositive animals is an unreliable method for negating infection in seropositive cattle.

Mycobacterium tuberculosis complex detection in rural goat herds in South Africa using Bayesian latent class analysis

Animal tuberculosis affects a wide range of domestic and wild animal species, including goats (Capra hircus). In South Africa, Mycobacterium tuberculosis complex (MTBC) testing and surveillance in domestic goats is not widely applied, potentially leading to under recognition of goats as a potential source of M. bovis spread to cattle as well as humans and wildlife. The aim of this study was to estimate diagnostic test performance for four assays and determine whether M. bovis infection was present in goats sharing communal pastures with M. bovis positive cattle in the Umkhanyakude district of Northern Zululand, KwaZulu Natal. In 2019, 137 M. bovis-exposed goats were screened for MTBC infection with four diagnostic tests: the in vivo single intradermal comparative cervical tuberculin test (SICCT), in vitro QuantiFERON®-TB Gold (QFT) bovine interferon-gamma release assay (IGRA), QFT bovine interferon gamma induced protein 10 (IP-10) release assay (IPRA), and nasal swabs tested with the Cepheid GeneXpert® MTB/RIF Ultra (GXU) assay for detection of MTBC DNA. A Bayesian latent class analysis was used to estimate MTBC prevalence and diagnostic test sensitivity and specificity. Among the 137 M. bovis-exposed goats, positive test results were identified in 15/136 (11.0%) goats by the SICCT; 4/128 (3.1%) goats by the IPRA; 2/128 (1.6%) goats by the IGRA; and 26/134 (19.4%) nasal swabs by the GXU. True prevalence was estimated by our model to be 1.1%, suggesting that goats in these communal herds are infected with MTBC at a low level. Estimated posterior means across the four evaluated assays ranged from 62.7% to 80.9% for diagnostic sensitivity and from 82.9% to 97.9% for diagnostic specificity, albeit estimates of the former (diagnostic sensitivity) were dependent on model assumptions. The application of a Bayesian latent class analysis and multiple ante-mortem test results may improve detection of MTBC, especially when prevalence is low. Our results provide a foundation for further investigation to confirm infection in communal goat herds and identify previously unrecognized sources of intra- and inter-species transmission of MTBC.

Apparent prevalence and risk factors for bovine tuberculosis in the state of Paraná, Brazil: an assessment after 18 years since the beginning of the Brazilian program

Bovine tuberculosis (bTB) impacts considerably animal production and one health worldwide. To describe the prevalence, risk factors, and spatial pattern of the disease in the state of Paraná, Brazil, a cross-sectional study was conducted from September 2018 to February 2019. The area was divided into seven regions. Within each region, farms were randomly selected, and a predetermined number of cows was selected and tested by a comparative cervical tuberculin test. 17,210 animals were tested across 1757 farms. Herd prevalence of bTB-infected herds in Paraná was 2.5% [1.87–3.00%]. It has varied from 0.8 to 3.98% among seven regions, with clustering being detected in the west, central, and northeast areas. Animal prevalence was 0.35% [0.21–0.59%] and has varied from 0.08 to 0.6% among the pre-set regions. No major shifts in the prevalence of bTB were detected since 2007. Large-sized herds, dairy production, and feeding with whey were detected to be correlated with the presence of bTB. Exclusively among dairy herds, veterinary assistance from cooperatives, possession of self-owned equipment to cool milk, and feeding with whey were correlated with the disease. Considering these results, it is recommended that the state of Paraná seek to implement a surveillance system for the detection of bTB-infected herds transforming them into free ones, if possible, incorporating elements of risk-based surveillance. Health education is also recommended to inform farmers about the risks of introducing animals without testing and of feeding raw whey to calves.

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.

Real-time PCR using atpE, conventional PCR targeting different regions of difference, and flow cytometry for confirmation of Mycobacterium bovis in buffaloes and cattle from the Delta area of Egypt

BackgroundMycobacterium bovis notoriously causes detrimental infections in bovines and humans. In this study, 1500 buffaloes and 2200 cattle were tested by single intradermal comparative cervical tuberculin test and compared with the detection rates of M. bovis isolation, real-time and simplex PCR, and flow Cytometry.ResultsThe tuberculin test is the reference test in Egypt, the positive rate was 54/3700 (1.5%) composed of 18/1500 (1.2%) buffaloes and 36/2200 (1.6%) cattle which were mandatorily slaughtered under the Egyptian legislation, after postmortem examination the non-visible-lesion proportion was 39/54 (72.2%) which surpassed the visible-lesion rate 15/54 (27.8%) with (p < 0.0001). The samples from each case were pooled into one sample representing the case, and the isolation rate of M. bovis was 25/54 (46.3%). Real-time PCR using atpE was positive for mycobacteria on the genus level in 18/18 (100%) and 5/5 (100%) of tissue samples and isolates, respectively; simplex PCR detected M. bovis in 44/54 (81.5%) and 25/25 (100%) of tissue samples and isolates, respectively. Flow Cytometry evaluation of the CD4+, CD8+, WC1+δγ, and CD2+ cell phenotypes showed increased counts in the tuberculin-positive cases compared with negative cases (p < 0.0001), and these phenotypes in the tuberculin-positive cases increased after antigen stimulation than in the negative cases (p < 0.0001). Detection rates of PCR techniques and flow Cytometry exceeded that of bacterial isolation (p < 0.0001) and exhibited a strong correlation.ConclusionsThe skin test suffers from interference from non-tuberculous mycobacteria able to cause false-positive reactions in cattle and other species. Real-time PCR using atpE, conventional PCR targeting RDs, and flow Cytometry are rapid and accurate methods that correlate with the isolation and can be promising for detection and confirmation of infected live and slaughtered cases.

Prevalence of Bovine Tuberculosis among Cattle and Buffaloes in the Central Province of Sri Lanka

The Bovine tuberculosis (bTB) is a chronic disease condition in dairy cattle and a proven global zoonosis. This study was designed to identify the prevalence of bTB in dairy cattle and buffaloes in the Central Province (CP) of Sri Lanka. Single Intradermal Comparative Cervical Tuberculin (SICCT) test was performed in 20 farms (n=616 cattle and buffaloes) in three districts (NuwaraEliya; NE, Kandy; KN, and Matale; MT) in the CP. Out of the SICCT positive samples, randomly selected serum samples (n=33) of eight farms were subjected to the rapid antibody (Ab) test for further confirmation. Results were evaluated for different risk factors; age, sex, parity, body condition score (BCS), breed, herd origin, reproductive status, herd size, type of management, and duration of farm establishment. The prevalence of bTB among individual cattle and buffaloes was 22% with a 50% herd-level prevalence. In NE and KN, 34% and 19% of individuals showed positive reactions for SICCT, respectively, while all the individuals in MT were negative. There were significant statistical associations (P&lt;0.05) were observed with the prevalence of bTB and BCS, breed, herd origin, and reproductive status; however, age, sex, parity, herd size, type of management, and duration of farm establishment were not statistically significant (P&gt;0.05) with the prevalence of bTB.The conclusion is that, based on the SICCT test, the estimated prevalence of bTB in cattle and buffaloes in the central province of Sri Lanka is relatively high (&gt;20%). The SICCT test could be recommended for the screening of the bTB in cattle and buffaloes in all regions of Sri Lanka to assess the island-wide prevalence of bTB, as this disease carries the risk of transmitting to humans and other susceptible animal species.

Utility of the Intradermal Skin Test in a Test-and-Cull Approach to Control Bovine Tuberculosis: A Pilot Study in Ethiopia.

Bovine tuberculosis (bTB) is one of the top three, high-priority, livestock diseases in Ethiopia and hence, the need for evaluation of potential control strategies is critical. Here, we applied the test-and-segregate followed by cull strategy for the control of bTB in the intensive Alage dairy farm in Ethiopia. All cattle reared on this farm were repeatedly skin tested using the Comparative Cervical Tuberculin (CCT) test for a total of five times between 2015 and 2021. During the first (October 2015) and second (March 2017) rounds of testing, all reactor animals (>4 mm) were culled, while those that were deemed as inconclusive (1–4 mm) were segregated and retested. At retest, animals with CCT >2 mm were removed from the herd. In the third (December 2017) and fourth (June 2018) rounds of tuberculin testing, a more stringent approach was taken wherein all reactors per the severe mode of CCT test interpretation (>2 mm) were culled. A final herd status check was performed in May 2021. In summary, the number of CCT positives (>4 mm) in the farm dropped from 23.1% (31/134) in October 2015 to 0% in December 2017 and remained 0% until May 2021. In contrast, the number of Single Cervical Tuberculin (SCT) test positives (≥4 mm) increased from 1.8 to 9.5% (from 2017 to 2021), indicating that CCT test might not be sufficient to effectively clear the herd of bTB. However, a more stringent approach would result in a drastic increase in the number of false positives. The total cost of the bTB control effort in this farm holding 134–200 cattle at any given time was conservatively estimated to be ~US$48,000. This, together with the need for culling an unacceptably high number of animals based on skin test status, makes the test-and-cull strategy impractical for nationwide implementation in Ethiopia and other low- and middle-income countries (LMICs) where the infection is endemic. Hence, there is an increased emphasis on the need to explore alternate, affordable measures such as vaccination alongside accurate diagnostics to help control bTB in endemic settings.

ELISA indreto como método complementar de diagnóstico da tuberculose bovina

ABSTRACT: Bovine tuberculosis is an economic and health problem, requiring precise diagnostic methods for its control and eradication. The aim of this study was to evaluate the performance of a commercial enzyme-linked immunosorbent assay (ELISA) test for the diagnosis of bovine tuberculosis. A total of 1,644 cattle from eight dairy herds were evaluated using the comparative cervical tuberculin test (CCTT). Three of the herds had no recent tuberculosis infection, and the other five had shown positive results in a previous tuberculin test. For the serological diagnosis of tuberculosis, a commercial ELISA antibody test kit for Mycobacterium bovis was used. Serum samples from 846 cattle from the eight herds were evaluated using ELISA for M. bovis. Animals that were positive based on either CCTT or ELISA for M. bovis or both were sent to slaughter. Samples of their lungs, livers, and lymph nodes were collected and stored under refrigeration for microbiological culture and subsequent confirmation by polymerase chain reaction. Samples from the same tissues were also fixed with 10% formaldehyde in bottles for histopathological examination and stained with hematoxylin and eosin (HE). Of the 1,644 cattle, 61 were considered positive and 65 inconclusive based on CCTT. Retesting of the inconclusive samples identified an additional 19 positive cases, totaling 80 (4.8%) CCTT-positive animals from five herds. ELISA for M. bovis identified 4.2% (36/846) positive cattle, of which 35 were considered negative and one inconclusive based on CCTT. Of the 36 positive cases identified by ELISA for M. bovis, 27 were euthanized, 11% (3/27) showed suggestive lesions of tuberculosis on macroscopic examination, and two were confirmed by histological, microbiological, and PCR methods. The weak association of ELISA for M. bovis with the results obtained by macroscopic, histological, and microbiological isolation indicates the fragility of ELISA performance in field conditions. Therefore, it is suggested that its use as a complementary method for herd sanitation be based on the local epidemiological situation.

Machine learning classification methods informing the management of inconclusive reactors at bovine tuberculosis surveillance tests in England

Bovine tuberculosis (bTB) remains one of the most complex, challenging, and costly animal health problems in England. Identifying and promptly removing all infected cattle from affected herds is key to its eradication strategy; the imperfect sensitivity of the diagnostic testing regime remaining a serious obstacle.The main diagnostic test for bTB in cattle in England, the Single Intradermal Comparative Cervical Tuberculin Test (SICCT: also known as the skin test), can produce inconclusive results below the reactor threshold. The immediate isolation of inconclusive reactor (IR) animals followed by a 60-day retest may not prevent lateral spread within the herd (if it is substandard, allowing transmission) or transmission to wildlife. Over half of IR-only herds that went on to have a positive skin test result (a bTB herd ‘incident’) in 2020, had it triggered by at least one IR not clearing their 60-day retest, instead of by another test within the previous 15 months.Machine learning classification algorithms (classification tree analysis and random forest), applied to England’s 2012–2020 IR-only surveillance herd tests, identified at-risk tests for an incident at the IRs’ 60-day retest. In this period, 4 739 out of 22 946 (21 %) IR-only surveillance tests disclosing 6 296 out of 42 685 total IRs, had an incident at retest (2 716 IRs became reactors and 3 580 IRs became two-time IRs). Both models showed an AUC above 80 % in the 2012–2019 dataset. Classification tree analysis was preferred due to its easy-to-interpret outputs, 70 % sensitivity, and 93 % specificity in the 20 % of 2019–2020 testing dataset.The paper aimed to identify IR-only surveillance tests at-risk of an incident at the 60-day retest to target them with appropriate measures to mitigate the IRs’ risk. Sixteen percent (341 out of 2 177) of IR-only herd tests were identified as high-risk in the 2020 dataset, with 265 (78 %) of these having at least one reactor or IR at retest. Severe-level reinterpretation of the high-risk IR-only disclosing tests identified in this dataset would turn 68 out of the 590 (12 %) IRs into reactors, generating 23 incidents, the majority (19 or 83 %) part of the 265 incidents that would have been declared at the retest.Classification tree analysis used to identify IR-only high-risk tests in herds eligible for severe interpretation would enhance the sensitivity of the test-and-slaughter regime, cornerstone of the bTB eradication programme in England, further mitigating the risk of disease spread posed by IRs.

Cytokine Expression in Peripheral Blood Mononuclear Cell Cultures Obtained from Cattle with Different Stages of Natural Mycobacterium bovis Infection

In bovine tuberculosis (bTB), cellular, humoral, or both types of immune responses have been observed. The purpose of this study was to examine the immune status of tuberculous cows based on the differential cytokine gene expression associated with Th1 (IFN-γ, IL-2), or Th2 (IL-4, IL-10) responses. Twenty-three (23) cows belonging to a dairy herd located in a rural region of the State of Hidalgo, México, were selected for the study. Single Intradermal Comparative Cervical Tuberculin (SICCT) Test, Interferon-Gamma (IFN-γ) Release Assay (BOVIGAM), and Enzyme-Linked Immunosorbent Assay (ELISA) were used for detection of cattle infected by M. bovis. Thirteen cows were positive to all the tests (Group 1); ten cows were positive only to ELISA (Group 2), and the remaining Group (Group 3, control) included cows negative to all the tests. Peripheral blood mononuclear cells (PBMC) from animals were in vitro stimulated by bovin purified protein derivative (PPD), avian PPD, and Concanavalin A (Con A) mitogen for 72h. Changes in the levels of expression of mRNA of the respective cytokines was measured by Reverse Transcription-Polymerase Chain Reaction (RT-PCR) using β-actin gene as internal control. In group 1, PPD bovis and Con A-stimulated cells exhibited high production of IFN-γ, IL-2 and IL-4, but not IL-10. In contrast, PPD avium-stimulated cells displayed a low production of cytokine transcripts. In group 2, cells showed a significant production of IL-10 in response to bovine PPD (P&lt; 0.001). In the control group, a high production of IFN-γ and IL-2 was observed only in Con A-stimulated cells. Post-mortem examinations in animals of group 1 showed slight and medium lesions in lymph nodes, whereas in group 2, the lesions were more extensive. Results indicate differences on gene expression levels of cytokines considered to determine balance in Th1/Th2 response among the evaluated groups. In addition, high levels of antibodies against M. bovis and high IL-10 expression in PBMC together are indicators of progressive bTB when both tuberculin test and IFN-γ assay are negative in tuberculous anergic cattle. Inclusion of serology and IL-10 cytokine expression in in the diagnosis checklist improves detection of infected cattle to help control bovine tuberculosis.

Intradermal comparative cervical tuberculin test in the diagnosis of caprine tuberculosis.

Tuberculosis remains one of the most important infectious diseases with well-known zoonotic nature that affect humans, wildlife, and domestic animals, including goats. Nonetheless, no intradermal tuberculin test has been standardized for caprine diagnosis of tuberculosis. The present study investigated the intradermal comparative cervical tuberculin test (ICCTT) in the diagnosis of tuberculosis among 60 goats from farms with history of tuberculosis. The cutoff applied to goats was based on a study where goats had been experimentally infected with Mycobacterium bovis and Mycobacterium avium. Clinical examination, bacteriological culture, and histopathological staining were assessed to the diagnosis. Isolates compatible with mycobacteria were subjected for molecular diagnosis based on gyrB-restriction fragment length polymorphism (RFLP) analysis and PCR restriction-enzyme analysis (PRA) of hsp65 gene by BstEII and HaeIII, namely PRA-hsp65 assay. From all goats, 60% (n = 36/60), 3.3% (n = 2/60), and 36.7% (n = 22/60) showed positive, inconclusive, and negative reactions, respectively. Out of 36 goats with ICCTT positive, 75% (n = 27/36) had isolation of mycobacteria and were detected M. bovis by gyrB-RFLP. Molecular diagnosis and histopathological findings compatible with tuberculosis showed 86.1% (n = 31/36) concordance with the ICCTT. When compared ICCTT with M. bovis isolation, gyrB-RFLP, and histopathology, the better arithmetic means of sensitivity and specificity were 2.5mm for ICCTT compared with M. bovis isolation and gyrB-RFLP, and 4.55mm when compared with histopathology. Both receiver operating characteristic (ROC) curves presented statistical significance (P < 0.001). The identification of other mycobacteria, e.g., M. kansasii, M. flavescens, M. avium, M. florentinum, M. lentiflavum, M. simiae, and Corynebacterium pseudotuberculosis, not influenced positive results in ICCTT. The concordance between bacteriological, histopathological, and molecular identification with ICCTT findings indicate that the tuberculin test may be used as a valuable tool for diagnosis of caprine tuberculosis and reinforce the importance of association of methods to diagnostic of the disease from animal origin.

Using of Gamma Interferon γ-IFN and Multiplex PCR (m-PCR) for Detection of Bovine Tuberculosis in Dairy Herds in Egypt

Many diagnostic tools are essential for Mycobacterium bovis (M. bovis) eradication program. This study aimed to apply γ-IFN assay to detect bovine tuberculosis and multiplex PCR (m-PCR) for rapid identification of Mycobacterial isolates. A total no. of 150 cattle in 10 small farms at different Governorates in Egypt, were previously gave suspected results with comparative cervical tuberculin test (SICCT), they retested after 60 days later again with SICCT and bovine gamma-interferon (γ-IFN) immunoassay. Eighty-seven (58%) out of total 150 animals were +ve reactors by SICCT test while 80 (53.3%) animals gave +ve γ-IFN assay. The isolated M. bovis by conventional culturing and identification tests were +ve 55 (63.2%) out 87. The γ-IFN assay sensitivity and specificity gave 82.9% and 93.8% respectively. For rapid identification of different mycobacterial isolates using m-PCR two set of primers were used. The first set gave 123bp DNA PCR product expressing IS6110 insertion element for Mycobacterium tuberculosis (MTBC). The other one gave 500bp from RvD1Rv2031c genomic sequence definite to M. bovis. M-PCR findings were in a concordance with results of conventional culturing and identification tests with high sensitivity and specificity (100%). From this study, it is concluded that diagnosis of bovine tuberculosis (BTB) used tuberculin test and γ-IFN assay with m-PCR for rapid identification M. bovis isolates in living herds.

Epidemiological situation of bovine tuberculosis in the state of Tocantins, Brazil

The epidemiological situation of bovine tuberculosis was studied from October 2014 to August 2015 in the state of Tocantins, a traditional beef exporter. The state was divided into five regions and the farms were randomly selected in each region. A predetermined number of animals was randomly selected and diagnosed using the Comparative Cervical Tuberculin Test. A total of 11,926 animals from 757 farms were tested. Animals with inconclusive test results were retested with the same diagnostic procedure at least 60 days later. The prevalence of infected herds in the state was 0.16% [0.02-1.15] and the prevalence of infected animals 0.009% [0.001-0.063]. Only one positive animal was detected in the Araguaína region. The state of Tocantins has a very low prevalence of bovine tuberculosis and, therefore, should evaluate the need of structuring a surveillance system to eradicate the disease.

Study on supplemental test to improve the detection of bovine tuberculosis in individual animals and herds

BackgroundBovine tuberculosis (bTB), is a worldwide disease caused by Mycobacterium bovis (M. bovis). The success of bTB eradication and control programs is based on early detection and the removal of reactors from a herd thus routine testing and cull strategy have been applied globally. Since the late nineteenth century, the Tuberculin Skin Test (TST) has been the primary antemortem test available to support bTB eradication campaigns. Due to the TST limitations in terms of Se and Sp, the credibility of the diagnosis is frequently questioned given the occurrence of false-positive and false-negative reactions, therefore, it is necessary to confirm reactive animals using other methods, ensuring the reliability of the diagnosis. The aim of this study was to evaluate the sensitivity and specificity of a multiplex enzyme-linked immunosorbent assay (ELISA) relative to the tuberculin test used for the diagnosis of tuberculosis in cattle in Brazil.ResultsLack of agreement between comparative cervical tuberculin test and ELISA IDEXX TM was observed. The 2 animals positive on the comparative cervical tuberculin test did not react at the ELISA IDEXX TM and 22 negative reactors by comparative cervical tuberculin test were positive by the ELISA IDEXX TM. The ELISA IDEXX TM showed sensitivity that is significantly lower than the official screening test the single cervical tuberculin. ELISA IDEXX TM also detected infected animals and herds undetected by the comparative cervical tuberculin test. The parallel use of comparative cervical tuberculin test and ELISA IDEXX TM increased sensitivity and the feasibility bTB screening.ConclusionsThe results obtained here suggest that the ELISA IDEXX TM may be a supplemental test for the detection of Mycobacterium bovis infection in regions without routine testing and slaughter, where the disease generally progresses to more advanced stages and antibody responses are likely to be more prevalent. Evidence to support the validation of the ELISA IDEXX™ as a supplemental test for bTB eradication programs was provided.

Risk factors and true prevalence of bovine tuberculosis in Bangladesh

Bovine tuberculosis (bTb) is endemic in Bangladesh but the true prevalence has not yet been reported. Our objectives for this study were to determine the true prevalence and identify risk factors for bTb at the animal- and herd-level in Bangladesh. A total of 510 cows were randomly selected during January 2018 to December 2018. Caudal fold (CFT) and comparative cervical tuberculin tests (CCT) were serially interpreted. Animal- and herd-level risk factor data were collected using a pre-tested questionnaire. The hierarchical true prevalence of bTb was estimated within a Bayesian framework. The herd- and animal-level risk factors were identified using mixed effects logistic regression. The apparent prevalence of bTb was 20.6% [95% Confidence Interval (CI): 17.3; 24.3] based on CFT. The animal-level true prevalence of bTb was 21.9 (13.0; 32.4). The herd-level true prevalence in different regions varied from 41.9% to 88.8%. The region-level true prevalence was 49.9 (13.8; 91.2). There is a 100% certainty that herds from Bhaluka and Mymensingh Sadar upazilas are not free from bTb. The odds of bTb were 3.9 times (1.2; 12.6) higher in herds having more than four cows than those with ≤ 4 cows. On the other hand, the risk of bTb was 3.3 times higher (1.0; 10.5) in non-grazing cows than grazing cows. Crossbred cows were 2.9 times (1.5; 5.9) more likely to be infected with bTb than indigenous cows. The risk of bTb in animals with cough was 2.3 times (1.2; 4.3) higher than those without cough. Crossbred, non-grazing cows with cough should be targeted for bTb surveillance. Herds of the Mymensingh, Sadar and Bhaluka regions should be emphasized for bTb control programs. Estimation of Bayesian hierarchical true prevalence facilitates identification of areas with higher prevalence and can be used to indicate regions that where true prevalence exceeds a pre-specified critical threshold.

Epidemiology of Bovine Tuberculosis and Its Zoonotic Implication in Addis Ababa Milkshed, Central Ethiopia

Bovine tuberculosis (bTB) continues to be one of the most widely distributed chronic infectious diseases of zoonotic importance, which causes a significant economic loss in animal production. A cross-sectional study was conducted to estimate the prevalence of bTB and its associated risk factors and type the Mycobacterium bovis isolated in central Ethiopia. A total of 65 dairy farms and 654 cattle were tested for bTB using a single intradermal comparative cervical tuberculin (SICCT) test. Data on farm management, animal-related characteristics, and the owner's knowledge of the zoonotic importance of bTB were collected using a structured questionnaire. In addition, a total of 16 animals from different farms were identified for postmortem examination. Lowenstein Jensen (LJ) culture was also conducted, and spoligotyping was used to type the M. bovis strains isolated. Chi-square test and logistic regression models were used to analyze the herd- and animal-level risk factors. Herd- and animal-level prevalence rates of bTB were 58.5% (95% CI: 46.2%−69.2%) and 39.3% (95% CI: 35.5%−43.5%), respectively. At the herd level, poor farm management was the predictor for bTB positivity (p < 0.05). Animal breed, poor BCS, farm type, and poor farm management conditions were significant predictors of bTB positivity (p < 0.05) at an individual animal level. All animals identified for postmortem examination were found to have gross TB-like lesions. A total of 14 M. bovis strains were identified from 12 animals that were positive for LJ culture. The strain with the largest number of clusters (five isolates) was SB1176, followed by SB0134 (three isolates), SB0192 (two isolates), and SB2233 (two isolates), and two new strains, each consisting of only one isolate. The majority (58.5%) of the respondents did not know the zoonotic importance of bTB. The result of this study showed a high prevalence of bTB in the Addis Ababa milkshed and a low level of consciousness of the owners on its transmission to humans. Therefore, the launching of acceptable control measures of bTB and the creation of public awareness about its zoonotic transmission and prevention measures are required.

Dynamics and risk of transmission of bovine tuberculosis in the emerging dairy regions of Ethiopia.

The Ethiopian government has several initiatives to expand and intensify the dairy industry; however, the risk of bovine tuberculosis (bTB) spread is a challenge. To assess the rate of expansion and risk factors for transmission of bTB within-herds, we carried out a repeated cross-sectional survey at two time points, 2016/17 and 2018, in three regional cities, namely, Gondar, Hawassa and Mekelle, representing the emerging dairy belts of Ethiopia. The total number of herds involved was 128, comprising an average of 2303 cattle in each round. The Single Intradermal Comparative Cervical Tuberculin (SICCT) test was used to identify reactor status and data on herd-level risk factors were collected using a structured questionnaire. In the first survey, the apparent prevalence of bTB, as measured by the SICCT test, was 4.5% (95% CI 3.7-5.4%) at the individual animal-level and 24% (95% CI 17.5-32%) at the herd-level. There was no statistically significant change in the overall apparent prevalence or regional distribution at the second survey, consistent with the infection being endemic. The incidence rate was estimated at 3.6 (95% CI 2.8-4.5) and 6.6 (95% CI 3.0-12.6) cases/100 cattle (or herd)-years at the animal- and herd-levels, respectively. Risk factors significantly associated with the within-herd transmission of bTB were age group and within-herd apparent prevalence at the start of the observation period. We noted that farmers voluntarily took steps to remove reactor cattle from their herds as a consequence of the information shared after the first survey. Removal of reactors between surveys was associated with a reduced risk of transmission within these herds. However, with no regulatory barriers to the sale of reactor animals, such actions could potentially lead to further spread between herds. We therefore advocate the importance of setting up regulations and then establishing a systematic bTB surveillance programme to monitor the impact prior to implementing any control measures in Ethiopia.

Intra-vitam Diagnosis of Tuberculosis in Pigs: Concordance Between Interferon-Gamma Release Assay and Comparative Tuberculin Skin Test.

The role of pigs in the maintenance of bovine tuberculosis caused by Mycobacterium bovis has been demonstrated in many settings; however, the current control programs usually do not state any intra-vitam diagnostic procedure in this species, as for the cattle. Carcass inspection has shown to be insufficient to detect infection in swine; thus, the assessment of intradermal tuberculin test and interferon-gamma release assay (IGRA) in this species is mandatory. The current study compares the performances of the single intradermal comparative cervical tuberculin (SICCT) test and IGRA. A total of 628 Nebrodi Black pigs raised in free-roaming farms were subjected to the two tests simultaneously. Besides, 124 animals underwent postmortem examination for the detection of tuberculous lesions and isolation of mycobacteria from target organs. The two tests showed a concordance of 94.42% with a Cohen's k coefficient of 0.786 and McNemar chi-square of 4.83 (P = 0.03). Slightly lower levels of concordance (90.32%) between SICCT and IGRA were obtained in the group of 124 animals, with a Cohen's k = 0.797 and McNemar chi-squared value of 0.69 with a non-significant P = 0.41. Moreover, the results showed how IGRA tends to result positive in higher rates, mostly when non-tuberculous mycobacteria (NTM) were isolated, suggesting a possible impairment of specificity in the event of coinfections in the swine. In conclusion, the results obtained support the possibility of the strategic use of IGRA or SICCT in combination or alternatively one to the other, particularly IGRA which showed lower specificity but has evident advantages over SICCT.