Bovine Tuberculosis Control Programs Research Articles

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.

Fascioliasis may promote tuberculous infectivity in small ruminants

Fascioliasis and bovine tuberculosis (TB) are global impediments to livestock development. We investigated the co-infectivity of fascioliasis and TB in small ruminants at slaughter. A total of 84 goats and 16 sheep were investigated from different slaughter houses in Mymensingh city, Bangladesh from June 2019 to February 2020. Grossly, acute fascioliasis was characterized by hemorrhagic tracts in the liver and chronic fascioliasis with biliary cirrhosis and pipe-stem liver. Grossly, seven goats and two sheep were associated with the acute and sixty goats and seven sheep were associated with the chronic phase of fascioliasis. Five goats’ livers showed both the acute and chronic phases of fascioliasis. In TB, granulomas with central core of caseous necrosis were seen in the lymph nodes (21), livers (10) and lungs (01) of goats or in the lymph nodes (03) and liver (01) of sheep. Histopathologically, biliary cirrhosis was seen in fascioliasis and granuloma, caseous necrosis and calcification in TB. In co-infection revealed granuloma (TB) with acid-fast bacilli and widespread biliary cirrhosis in the livers of goats (14) and sheep (02). The fragments of the 16S rRNA gene (372 bp, M. tuberculosis complex) and MPB83 gene (600 bp, M. bovis) were detected in the lymph nodes, livers and lungs using polymerase chain reaction. This study showed the existence of co-infectivity of Fasciola and M. bovis in goats and sheep in Bangladesh. Chronic fascioliasis may be associated with establishing tuberculous infection in small ruminants. Therefore, extremely zoonotic bovine TB control programs require active management of fascioliasis.

Spatial distribution of Mycobacterium bovis spoligotypes in cattle from the state of Minas Gerais, Brazil.

Genotyping methods have led to a better understanding of the epidemiology of Mycobacterium bovis (M. bovis) infection, and its transmission dynamics, as well as the possible phylogenetic relationships between Mycobacterium strains, thus making bovine tuberculosis control programs more efficient. The goal of this study was to characterize the main spoligotypes of M. bovis isolated from cattle in the State of Minas Gerais, Brazil. It was carried out in 28 municipalities of "Triângulo Mineiro" and "Alto Paranaíba" regions of the state. Viscera samples were obtained from 58 bovines positive for tuberculosis according to comparative cervical tests, and from another 100 bovines with lesions suggestive of tuberculosis, which were donated by the National Agricultural Laboratory of Pedro Leopoldo, Minas Gerais. Microbiological isolation was performed in Stonebrink medium, and molecular identification of mycobacteria was performed by PCR. Genotyping was performed using the spoligotyping method at the Agrobiotechnology and Molecular Biology Institute of National Agricultural Technology Institute-National Scientific and Technical Research Council, Buenos Aires, Argentina. Among the 158 viscera samples, we obtained 40 (25%) isolates of M. bovis, and detected 11 spoligotype patterns, with a predominance of SB1142 (37.5%), SB0121 (25.0%), and SB1145 (10.0%). Other standards, SB0295, SB1050, SB0881, SB1144, SB1802, SB0140, SB0120, and SB0849, varied from 2.5 to 7.5%, heterogeneously distributed among the municipalities. The presence of spoligotypes shared with other Brazilian states and different countries indicates their possible exchange through epidemiological relationships, such as the transit of live animals and/or genetic similarity between strains that share a common ancestor.

Bayesian Estimation of Diagnostic Accuracy of Three Diagnostic Tests for Bovine Tuberculosis in Egyptian Dairy Cattle Using Latent Class Models.

The aim of the present study was to calculate the sensitivity (Se) and specificity (Sp) of the single cervical tuberculin test (SCT), rapid lateral flow test (RLFT), and real-time polymerase chain reaction (RT-PCR) for the diagnosis of Mycobacterium bovis (M. bovis) infection in Egyptian dairy cattle herds within a Bayesian framework. The true M. bovis infection within-herd prevalence was assessed as a secondary objective. Data on the test results of SCT, RLFT, and RT-PCR for the detection of M. bovis were available from 245 cows in eleven herds in six major governorates in Egypt. A Bayesian latent class model was built for the estimation of the characteristics of the three tests. Our findings showed that Se of SCT (0.93 (95% Posterior credible interval (PCI): 0.89–0.93)) was higher than that of RT-PCR (0.83 (95% PCI: 0.28–0.93)) but was similar to the Se of RLFT (0.93 (95% PCI: 0.31–0.99)). On the contrary, SCT showed the lowest Sp estimate (0.60 (95% PCI: 0.59–0.65)), whereas Sp estimates of RT-PCR (0.99 (95% PCI: 0.95–1.00)) and RLFT (0.99 (95% PCI: 0.95–1.00)) were comparable. The true prevalence of M. bovis ranged between 0.07 and 0.71. In conclusion, overall, RT-PCR and RLFT registered superior performance to SCT, making them good candidates for routine use in the Egyptian bovine tuberculosis control program.

Bovine Tuberculosis: A Review of Molecular Diagnostic Methods and Impact on Public Health

Bovine tuberculosis (BTB) is a zoonotic infectious disease of cattle, other domesticated animals, and certain wildlife populations. It has been widely distributed throughout the world, and it has been a cause for great economic loss in animal production. In developed countries, the eradication programs have reduced or eliminated TB in cattle. Many factors contribute to the persistence of the disease, such as the limitations of diagnostic tests, larger herd sizes, increase in animal movements and trade, and limited options for control, such as restrictions on whole herd depopulation. The available advanced TB diagnostic techniques can detect and differentiate the causative mycobacterial species, and help an early confirmation of the diagnosis, which is useful to design appropriate control measures in the national BTB control programs. Molecular diagnostics, such as polymerase chain reaction, spoligotyping, restriction fragment length polymorphism (RFLP), variable number tandem repeats typing (VNTR), and polymorphism GC-rich repeat sequence (PGRS) are the techniques used for concurrent detection and typing of Mycobacterium species at the strain level. The molecular epidemiology is also being used to identify the source of contamination, to determine the risk factors of BTB transmissions, to investigate the drug resistance pattern, and to track the geographic distribution and spread of clones of Mycobacteria species. The molecular diagnosis is the tool to check whether active transmission or reactivation of BTB, hence, it is better to adopt these methods for the epidemiological survey of BTB. Since BTB is a major public health problem, at least a single reference laboratory should be available for molecular based diagnosis as part of its control.

Evaluation of the Control Options of Bovine Tuberculosis in Ethiopia Using a Multi-Criteria Decision Analysis.

Bovine tuberculosis (BTB) is a zoonotic bacterial infection caused by Mycobacterium bovis and is characterized by the development of granulomatous lesions in the lymph nodes, lungs and other tissues. It poses serious public health impacts and food security challenges to the agricultural sector in terms of dairy and meat productions. In Ethiopia, BTB has been considered as a priority disease because of its high prevalence in urban and peri-urban dairy farms. However, there has not been any national control program in the country. Thus, in order to initiate BTB control program in the country, information on control options is needed to tailor the best option for the Ethiopian situation. The objective of this study was to identify, evaluate and rank various BTB control options in Ethiopia using a multi-criteria decision analysis based on preference ranking organization method for enrichment evaluations (PROMETHEE) approach while accounting for the stakeholders' preferences. Control options were evaluated under two scenarios: with (scenario 1) and without (scenario 2) bacillus Calmette–Guérin (BCG) vaccination. Nine potential control options were identified that include combinations of three control options (1) test and slaughter with or without government support, (2) test and segregation, and (3) BCG vaccination. Under scenario 1, BCG vaccination, BCG vaccination and test and slaughter with partial compensation by government, and BCG vaccination and test and slaughter with full compensation by government were the top three ranked control options. Under scenario 2, test and slaughter with full compensation by government was the preferred control option, followed by test and segregation supported by test and slaughter with full government compensation, and test and slaughter with half compensation by government. Irrespective of the variability in the weighting by the stakeholders, the sensitivity analysis showed the robustness of the ranking method. In conclusion, the study demonstrated that BCG vaccination, and test and slaughter with full compensation by government were the two most preferred control options under scenarios 1 and 2, respectively. National level discussions were strongly recommended for further concretization and implementation of these control measures.

Introduction and Application of the Interferon-γ Assay in the National Bovine Tuberculosis Control Program in South Korea.

Bovine tuberculosis is a chronic disease impacting both public health and the livestock industry. The interferon (IFN)-γ assay has been introduced as an ancillary test for diagnosing bovine tuberculosis to overcome limitations of the skin test. The objective of this study was to assess the IFN-γ assay in terms of diagnostics and as a nationwide surveillance program in South Korea. From 2012 to 2013, cattle (n = 120) with bovine tuberculosis and cattle (n = 426) from bovine tuberculosis free herds were subjected to the IFN-γ assay to evaluate the sensitivity and specificity of the assay, respectively, depending on various cut-offs (0–3.5). When optical density of the cut-off was 0.1, the sensitivity and specificity were found to be 81.7% (74.7–88.6) and 99.5% (98.9–100.0), respectively. After introducing the IFN-γ assay as part of the national control program, the IFN-γ assay and single caudal fold skin test data were collected from 47 regional veterinary services to compare the results of these two tests. Overall, the agreement between the IFN-γ assay and the single caudal fold skin test (n = 492,068) was 98.2%, and Cohen's kappa value for the two methods was 0.47. Serial and parallel use of the IFN-γ assay and skin test for the bovine tuberculosis control program were compared using samples (n = 91) from cattle confirmed as bovine tuberculosis positive in laboratories from 2014 to 2016. Parallel screening for bTB showed much higher sensitivity (86/91, 94.5%) than the following screening approaches: serial (47.2%, 43/91), single screening using CFT (63.7%, 58/91), or the IFN-γ assay (78.0%, 71/91). These results indicate that the IFN-γ assay and single caudal fold skin test are complementary to each other; therefore, parallel use of these two tests is considered a useful approach to reduce the prevalence of bovine tuberculosis in South Korea.

Intercurrence of Paratuberculosis in Intradermal Tuberculin Test Reactive Cattle

Background: Bovine tuberculosis control programs are based on a standard diagnostic method, the intradermal test with purified protein derivatives, which is used to identify and eliminate diseased animals. Currently none of the tests available allow complete differentiation between infected and uninfected animals. The main limitations of the tests available are related to diagnostic sensitivity and specificity, which results in false positive reactions due to the existence of cross infections, and also false negative, inherent to the state of energy of some animals. The aim of this work was to study the intercurrence of paratuberculosis in tuberculosis reactive cattle by the comparative cervical test.Materials, Methods & Results: Three hundred and thirty four cattle were evaluated using the comparative cervical test (CCT) and serology for tuberculosis (TB) and paratuberculosis (PTB) ELISA IDEXX®. All of the animals testing positive by CCT were euthanized and necropsied. Fragments of lymph node, lung and intestine were collected and analyzed using histopathological techniques, with staining by Hematoxylin-Eosin (HE). Samples of lung and lymph nodes (retropharyngeal, submandibular, cervical and mediastinal) of the animals testing positive by CCT were evaluated using qPRC for M. bovis, and intestinal and mesenteric lymph nodes using PCR for PTB. Of the 334 cattle evaluated using the comparative cervical test, 16 were considered positive. No lesions suggestive of tuberculosis were found in the macroscopic inspection of the carcasses. The most evident anatomical and pathological finding was a thickening of intestinal mucosa, found in 12 of the 16 cattle submitted to necropsy. No microscopic lesions suggestive of TB were identified nor was the presence of M. bovis detected by qPCR. The main histopathological findings were observed in the small intestine and mesenteric lymph nodes and identified as enteritis, lymphangitis, lymphangiectasia and granulomatous lymphadenitis. In the intestine the changes are characterized by dilated and inflamed lymphatic vessels and intense inflammatory infiltrate on the mucosa and submucosa. Of the 334 serum samples evaluated, the M. bovis ELISA Antibody Test (IDEXX®) identified 17 positive animals. All the cattle considered positive by M. bovis ELISA were considered negative by CCT. In the samples from nine animals (9/16), DNA from M. avium subsp. paratuberculosis (MAP) was identified and in twelve carcasses (12/16) lesions characteristic of PTB were found, which were subsequently confirmed by histopathological techniques. In another nine animals of the herd anti-MAP antibodies were detected. None of those that tested positive by PTB ELISA were reactive by CCT.Discussion: Animals considered positive by TB ELISA that were not positive in the intradermal test does not mischaracterize the clinical picture of the disease. Considering the inverse relationship between cell-mediated and humoral responses to M. bovis, the intradermal test and the serological tests are designed to measure different immunological responses, which develop during different stages of infection. The progress of the cellular immunological response to humoral immunity occurs in the most advanced stages of tuberculosis. Of the 16 cattle considered positive by CCT, 12 animals presented macroscopic and histological lesions suggestive of PTB and DNA from MAP was detected in nine. Although it is the official test for the control of TB in different countries, the intradermal test with PPD has presented limitations, primarily related to specificity. M. avium subsp. Paratuberculosis is considered the main cause of false positive reactions in the intradermal test. The PPD bacterial extract is a complex mixture of proteins, lipids, sugars and nucleic acids, and many of these components are also shared by numerous species of mycobacteria (tuberculous or not).

Simultaneous measurement of antigen-induced CXCL10 and IFN-γ enhances test sensitivity for bovine TB detection in cattle

Bovine tuberculosis (BTB) is a disease of economic and zoonotic importance caused mainly by Mycobacterium bovis. In addition to the tuberculin skin test, an interferon-gamma (IFN-γ) release assay (IGRA) blood test has been incorporated in the BTB control programs of numerous countries as an ancillary test to the skin test. A potential disadvantage of the IGRA assay is that it relies solely on the measurement of a single readout (i.e. IFN-γ) for the detection of BTB. In this study we have assessed the practical use of CXCL10 as an additional biomarker for the diagnosis of BTB in the setting of the current testing approach alongside IGRA. To do so, we have assessed both IFN-γ and CXCL10 readouts in blood cultures from a variety of different BTB cattle groups stimulated with standard tuberculin reagents and also with more specific defined antigens (ESAT-6, CFP-10 and Rv3615c). When using a tuberculin based whole blood assay, CXCL10 alone could not substitute for IFN-γ as the analyte measured in the test without reducing the sensitivity of detecting BTB animals. However, when used as an additional test readout, CXCL10 identified BTB animals that failed to induce IFN-γ responses. When tested in non-infected animals, the use of the dual biomarker system had the potential to lower overall test specificity, however this could be overcome by raising the cut-off values for CXCL10 test positivity. Taken together, the results demonstrate that in particular settings, measurement of CXCL10 has the potential to complement the current use of IFN-γ in blood assays to maximise the detection of BTB.

Prevalence and risk factors of bovine tuberculosis in dairy cattle in Eritrea.

BackgroundThe prevalence of bovine tuberculosis (BTB) in dairy cattle in the three major milk producing regions of Eritrea was assessed by subjecting 15,354 dairy cattle, 50 % of Eritrea’s dairy cattle population, to the single intradermal comparative tuberculin test (SICTT). Skin test results were interpreted according to guidelines of the World Organization for Animal Health (OIE) with >4 mm as cutoff in skin thickness increase. In addition, we studied the relation between ‘physiological’ variables related to pregnancy and lactation, and the variable ‘region’ on the probability to be skin test positive.ResultsThe BTB prevalences at animal and herd levels were: 21.5 % and 40.9 % in Maekel, 7.3 % and 10 % in Debub, and 0.2 % and 1.6 % in the Anseba region, respectively. Overall, in the regions included, prevalence was 11.3 % (confidence interval (CI) 95 % CI, 11.29 – 11.31 %) and 17.3 % (95 % CI, 17.27–17.33 %), at animal and herd level, respectively. Considering positive herds only, the animal BTB prevalence was 36.8 %, 30.1 %, and 1.8 %, in Maekel, Debub and Anseba, respectively, and the overall animal prevalence within these herds was 32 %. In adult dairy cattle the probability of positive reactivity in the SICTT test was highest in pregnant animals as compared to the other categories.ConclusionThis study reports persistent prevalence of BTB as defined by positive SICTT in the dairy sector of Eritrea, especially in the regions of Maekel and Debub that are located in the central highlands of the country. To our understanding this is the first report that has encompassed all the major dairy farms in Eritrea and it will be instrumental in advocating future BTB control programs in the dairy sector.Electronic supplementary materialThe online version of this article (doi:10.1186/s12917-016-0705-9) contains supplementary material, which is available to authorized users.

Cost-benefit analysis of vaccination against Mycobacterium avium ssp. paratuberculosis in dairy cattle, given its cross-reactivity with tuberculosis tests

Johne’s disease (JD), or paratuberculosis, is a chronic enteric disease of ruminants, caused by infection with Mycobacterium avium ssp. paratuberculosis (MAP). Johne’s disease causes considerable economic losses to the US dairy industry, estimated to be over $200 million annually. Available control strategies include management measures to improve calf hygiene, test-and-cull strategies, and vaccination. Although the first 2 strategies have shown to reduce the prevalence of MAP, they require dedicated and long-term efforts from dairy producers, with often relatively slow progress. As a result, uptake of both strategies has not been as wide as expected given the economic benefits especially of improved hygiene. Vaccination has also been found to reduce the prevalence and economic losses of JD, but most economic estimates have been based on simulation of hypothetical vaccines. In addition, if an animal is vaccinated, cross-reactivity between MAP antibodies and bovine tuberculosis (BTB) antigens may occur, decreasing the specificity of BTB tests. Therefore, MAP vaccination would cause additional indirect costs to the BTB surveillance and control program. The objective of the present study was to use data from a MAP vaccine trial together with an epidemiologic and economic model to estimate the direct on-farm benefits of MAP vaccination and to estimate the indirect costs of MAP vaccination due to the cross-reactivity with BTB tests. Direct economic benefits of MAP vaccination were estimated at $8.03 (90% predictive interval: −$25.97 to $41.36) per adult animal per year, all accruing to the dairy producers. This estimate is likely an underestimation of the true direct benefits of MAP vaccination. In addition, indirect economic costs due to cross-reactivity were $2.14 per adult animal per year, making MAP vaccination economically attractive. Only in regions or states with a high frequency of BTB testing (because of, for example, Mycobacterium bovis outbreaks in a wild deer population) and areas where typically small groups of animals are BTB tested would MAP vaccination not be economically attractive.

Extent of Mycobacterium bovis transmission among animals of dairy and beef cattle and deer farms in South Korea determined by variable-number tandem repeats typing

Identifying sources of Mycobacterium bovis transmission would be essential for establishing effective control programs of bovine tuberculosis (TB), a major zoonosis threatening human health worldwide. As an effort to determine the extent of M. bovis transmission among dairy and beef cattle and deer populations, a mycobacterial interspersed repetitive units (MIRU)-variable-number tandem repeats (VNTR) typing method was employed for analysis of 131 M. bovis isolates from 59 Holstein dairy cattle, 39 Korean beef cattle, and 33 deer. Of 31 MIRU-VNTR markers, 15 showed allelic diversity. The most discriminatory locus for M. bovis isolates was VNTR 3336 (h=0.59) followed by QUB 26, MIRU 31, VNTR 2401, and VNTR 3171 which showed high discriminatory power (h=0.43). The combined VNTR loci had an allelic diversity of 0.83. On the basis of the VNTR profiles of 30 VNTR loci, 24 genotypes were identified, and two genotypes were highly prevalent among all M. bovis isolates (33.6% and 19.1%, respectively), thus indicating that more than 50% of the isolates shared common molecular characteristics. Six additional genotypes were common in 2 of the 3 animal species, suggesting a wide interspecies transmission of M. bovis. This study thus demonstrates that MIRU-VNTR typing is useful in differentiation of M. bovis isolates and that M. bovis transmission occurs frequently among farmed animal species, highlighting the importance of bovine TB control programs in different animal species which are often raised in the same villages.

Clinical and Diagnostic Developments of a Gamma Interferon Release Assay for Use in Bovine Tuberculosis Control Programs

Currently, the Bovigam assay is used as an official supplemental test within bovine tuberculosis control programs. The objectives of the present study were to evaluate two Mycobacterium bovis-specific peptide cocktails and purified protein derivatives (PPDs) from two sources, liquid and lyophilized antigen preparations. PPDs and peptide cocktails were also used for comparison of a second-generation gamma interferon (IFN-γ) release assay kit with the currently licensed first-generation kit (Bovigam; Prionics AG). Three strains of M. bovis were used for experimental challenge: M. bovis 95-1315, M. bovis Ravenel, and M. bovis 10-7428. Additionally, samples from a tuberculosis-affected herd (i.e., naturally infected) were evaluated. Robust responses to both peptide cocktails, HP (PC-HP) and ESAT-6/CFP10 (PC-EC), and the PPDs were elicited as early as 3 weeks after challenge. Only minor differences in responses to Commonwealth Serum Laboratories (CSL) and Lelystad PPDs were detected with samples from experimentally infected animals. For instance, responses to Lelystad M. avium-derived PPD (PPDa) exceeded the respective responses to the CSL PPDa in M. bovis Ravenel-infected and control animals. However, a 1:4 dilution of stimulated plasma demonstrated greater separation of PPDb from PPDa responses (i.e., PPDb minus PPDa) with the use of Lelystad PPDs, suggesting that Lelystad PPDs provide greater diagnostic sensitivity than CSL PPDs. The responses to lyophilized and liquid antigen preparations did not differ. Responses detected with first- and second-generation IFN-γ release assay kits (Bovigam) did not differ throughout the study. In conclusion, antigens may be stored in a lyophilized state without loss in potency, PC-HP and PC-EC are dependable biomarkers for aiding in the detection of bovine tuberculosis, and second-generation Bovigam kits are comparable to currently used kits.

Situation of bovine tuberculosis in Ecuador

Bovine tuberculosis (BTB) is a chronic and contagious disease that affects domestic animals, wildlife, and humans. Caused by Mycobacterium bovis, BTB causes major economic losses and poses a serious constraint to international livestock trade. Moreover, in developing countries where BTB controls are lacking, M. bovis is a public health concern. In most developing countries, the prevalence of BTB in livestock is unknown because the information is either not reported or not available. In Ecuador, there is no national BTB control program. This article reviews the BTB situation in Ecuador by examining exhaustive data from tuberculin testing surveys and slaughterhouse surveillance studies conducted in 1972-2008 in a variety of the country's geographic areas. In Ecuador, several factors, including the dairy industry's expansion (preempted by the high demand for milk and its by-products), intensified efforts to increase the cattle population, the presence of M. bovis, and a lack of BTB controls, have caused a rise in BTB prevalence, and consequently, a growing push for the implementation of a national BTB control program.

Comparative Intradermal Tuberculin Test in Dairy Cattle in the North of Ecuador and Risk Factors Associated with Bovine Tuberculosis

We studied the prevalence of bovine tuberculosis (BTB) in Mejia canton, the major dairy cattle production area in northern Ecuador. Twenty dairy herds comprising 2,022 cattle were selected. In 2007, each animal was tested using the comparative intradermal tuberculin test (CITT). In 2008, a follow-up test was performed in the same herds. The true annual incidence was 1.70%, and the true prevalence was 7.41% and 7.13% in 2007 and 2008, respectively. The prevalence was 0.27% and 0.57% in medium-sized herds in 2007 and 2008, respectively, compared with 8.63% and 8.43% in large herds (P < 0.01). The number of skin test-positive cases also increased significantly with age (P = 0.03), contacts with other species of animals (P < 0.01), and introduction of new cattle (P = 0.04). Herd prevalence was 55% in 2007 and 65% in 2008. This study shows the lack of knowledge in cattle farmers about this zoonosis and the necessity for a national BTB control program in Ecuador.