Comparative Cervical Test Research Articles

Assessing the feasibility of test-and-cull and test-and-segregation approaches for the control of high-prevalence bovine tuberculosis in Ethiopian intensive dairy farms

Bovine tuberculosis (bTB) is endemic and has a substantial impact on the livestock sector in Ethiopia and other low and middle-income countries (LMICs). With a national emphasis on dairy farm intensification to boost milk production and spur economic growth, the incidence of bTB is anticipated to rise. However, Ethiopia, like other LMICs, lacks a comprehensive national bTB control strategy due to the economic and social infeasibility of traditional test-and-cull (TC) approaches. To inform the development of such a strategy, we evaluated the effectiveness and feasibility of TC and test-and-segregation (TSg) strategies for bTB control on Ethiopian dairy farms. A TC approach was used at Farm A [N = 62; comparative cervical test (CCT) > 4 mm, starting prevalence 11.3%] while TSg was implemented at Farm B (N = 45; CCT > 4 mm, prevalence 22.2%), with testing intervals of 2–4 months. Both strategies achieved a reduction in bTB prevalence to 0%, requiring seven rounds of TC over 18 months at Farm A, and five rounds of TSg over 12 months at Farm B’s negative herd. The results show that adopting more sensitive thresholds [CCT > 0 mm or single cervical test (SCT) > 2 mm] during later rounds was pivotal in identifying and managing previously undetected infections, emphasizing the critical need for optimized diagnostic thresholds. Cost analysis revealed that TC was approximately twice as expensive as TSg, primarily due to testing, labor, and cow losses in TC, versus construction of new facilities and additional labor for TSg. This underscores the economic and logistical challenges of bTB management in resource-limited settings. Taken together, our study highlights an urgent need for the exploration of alternative approaches including TSg and or vaccination to mitigate within herd transmission and enable implementation of bTB control in regions where TC is not feasible.

Comparative analysis of tuberculin and defined antigen skin tests for detection of bovine tuberculosis in buffaloes (Bubalus bubalis) in Haryana state, India

BackgroundBovine tuberculosis (bTB) is a chronic disease that results from infection with any member of the Mycobacterium tuberculosis complex. Infected animals are typically diagnosed with tuberculin-based intradermal skin tests according to World Organization of Animal Health which are presently in use. However, tuberculin is not suitable for use in BCG-vaccinated animals due to a high rate of false-positive reactions. Peptide-based defined skin test (DST) antigens have been identified using antigens (ESAT-6, CFP-10 and Rv3615c) which are absent from BCG, but their performance in buffaloes remains unknown. To assess the comparative performance of DST with the tuberculin-based single intradermal test (SIT) and the single intradermal comparative cervical test (SICCT), we screened 543 female buffaloes from 49 organized dairy farms in two districts of Haryana state in India.ResultsWe found that 37 (7%), 4 (1%) and 18 (3%) buffaloes were reactors with the SIT, SICCT and DST tests, respectively. Of the 37 SIT reactors, four were positive with SICCT and 12 were positive with the DST. The results show that none of the animals tested positive with all three tests, and 6 DST positive animals were SIT negative. Together, a total of 43 animals were reactors with SIT, DST, or both, and the two assays showed moderate agreement (Cohen’s Kappa 0.41; 95% Confidence Interval (CI): 0.23, 0.59). In contrast, only slight agreement (Cohen’s Kappa 0.18; 95% CI: 0.02, 0.34) was observed between SIT and SICCT. Using a Bayesian latent class model, we estimated test specificities of 96.5% (95% CI, 92–99%), 99.7% (95% CI: 98–100%) and 99.0% (95% CI: 97–100%) for SIT, SICCT and DST, respectively, but considerably lower sensitivities of 58% (95% CI: 35–87%), 9% (95% CI: 3–21%), and 34% (95% CI: 18–55%) albeit with broad and overlapping credible intervals.ConclusionTaken together, our investigation suggests that DST has a test specificity comparable with SICCT, and sensitivity intermediate between SIT and SICCT for the identification of buffaloes suspected of tuberculosis. Our study highlights an urgent need for future well-powered trials with detailed necropsy, with immunological and microbiological profiling of reactor and non-reactor animals to better define the underlying factors for the large observed discrepancies in assay performance, particularly between SIT and SICCT.

Tuberculose bovina no estado do Rio Grande do Norte com base em dados secundários

ABSTRACT: This study determined the frequency of positive tests for bovine tuberculosis in animals and breeders/establishments in Rio Grande do Norte, Brazil. Data were provided by the Instituto de Defesa e Inspeção Agropecuária do Rio Grande do Norte (IDIARN), from its Unidade Local de Sanidade Animal e Vegetal (ULSAV’S), acquired in monthly reports issued by qualified Veterinarians under the National Program for the Control and Eradication of Brucellosis and Animal Tuberculosis (PNCEBT), from June 2012 to December 2021. For diagnosis, the comparative cervical test was used as a routine and confirmatory test. A total of 45,804 cattle were tested, 53 (0.1%) of which tested positive. Secondary data are essential in the evaluation of sanitary measures, allowing rapid generation of hypotheses about diseases and providing support for decision-making. Measures such as raising awareness among producers, sanitary control in the acquisition and sale of cows for reproduction, inspection of sanitary barriers, and conducting epidemiological surveys to understand the actual situation of this disease in the state of Rio Grande do Norte are crucial.

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.

A Defined Antigen Skin Test for Diagnosis of Bovine Tuberculosis in Domestic Water Buffaloes (Bubalus bubalis)

Bovine tuberculosis (bTB) remains endemic in domestic water buffaloes (Bubalus bubalis) in India and elsewhere, with limited options for control other than testing and slaughter. The prescribed tuberculin skin tests with purified protein derivative (PPD) for diagnosis of bTB preclude the use of Bacille Calmette-Guérin (BCG)-based vaccination because of the antigenic cross-reactivity of vaccine strains with Mycobacterium bovis and related pathogenic members of the M. tuberculosis complex (MTBC). For the diagnosis of bTB in domestic water buffaloes, we here assessed a recently described defined-antigen skin test (DST) that comprises overlapping peptides representing the ESAT-6, CFP-10 and Rv3615c antigens, present in disease-causing members of the MTBC but missing in BCG strains. The performance characteristics of three doses (5, 10 or 20 μg/peptide) of the DST were assessed in natural tuberculin skin test reactor (n = 11) and non-reactor (n = 35) water buffaloes at an organized dairy farm in Hisar, India, and results were compared with the single intradermal skin test (SIT) using standard bovine tuberculin (PPD-B). The results showed a dose-dependent response of DST in natural reactor water buffaloes, although the SIT induced a significantly greater (P < 0.001) skin test response than the highest dose of DST used. However, using a cut-off of 2 mm or greater, the 5, 10, and 20 μg DST cocktail correctly classified eight, 10 and all 11 of the SIT-positive reactors, respectively, suggesting that the 20 μg DST cocktail has a diagnostic sensitivity (Se) of 1.0 (95% CI: 0.72–1.0) identical to that of the SIT. Importantly, none of the tested DST doses induced any measurable skin induration responses in the 35 SIT-negative animals, suggesting a specificity point estimate of 1.0 (95% CI: 0.9–1.0), also identical to that of the SIT and compares favorably with that of the comparative cervical test (Se = 0.85; 95% CI: 0.55–0.98). Overall, the results suggest that similar to tuberculin, the DST enables sensitive and specific diagnosis of bTB in water buffaloes. Future field trials to explore the utility of DST as a defined antigen replacement for tuberculin in routine surveillance programs and to enable BCG vaccination of water buffaloes are warranted.

The regression of the bovine tuberculin reaction: Results from the Reactor Quality Assurance study in Northern Ireland

Tuberculin skin tests remain widely used in the control of bovine tuberculosis (bTB) in cattle. Little is known about the rate of regression of tuberculin reactions after the comparative intradermal cervical test (CICT) in cattle. This study aimed to collect data to describe tuberculin regression in reactors following the CICT at 72 ± 4 h post injection. Reactors were also tested using the interferon gamma (IFN-γ) assay to establish if any pattern existed between these results and the CICT reaction regression. The data were derived from 108 herds, 112 herd-level CICTs and 1008 animals. A multivariable linear mixed model was built to explore the regression of the bovine tuberculin reaction over time and the influence of potential predictors.The results confirmed a proportional decline in the bovine tuberculin reaction occurred over time. The predictors in the final model demonstrated that regression of the tuberculin reaction differed between reactors according to their IFN-γ test results and whether visible lesions were present at slaughter. Follow-up measurement of tuberculin reactions and the serial use of the IFN-γ assay in large breakdowns has the potential to provide both a mechanism for quality assurance of the current CICT bTB surveillance and the identification of atypical breakdowns or reactors requiring further investigation.

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 &amp; 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).

Northern Ireland farm-level management factors for prolonged bovine tuberculosis herd breakdowns.

This study determined farm management factors associated with long-duration bovine tuberculosis (bTB) breakdowns disclosed in the period 23 May 2016 to 21 May 2018; a study area not previously subject to investigation in Northern Ireland. A farm-level epidemiological investigation (n = 2935) was completed when one or more Single Intradermal Comparative Cervical Test (SICCT) reactors or when one or more confirmed (positive histological and/or bacteriological result) lesion at routine slaughter were disclosed. A case-control study design was used to construct an explanatory set of management factors associated with long-duration bTB herd breakdowns; with a case (n = 191) defined as an investigation into a breakdown of 365 days or longer. Purchase of infected animal(s) had the strongest association as the most likely source of infection for long-duration bTB herd breakdowns followed by badgers and then cattle-to-cattle contiguous herd spread. However, 73.5% (95% CI 61.1-85.9%) of the herd type contributing to the purchase of infection source were defined as beef fattening herds. This result demonstrates two subpopulations of prolonged bTB breakdowns, the first being beef fattening herds with main source continuous purchase of infected animals and a second group of primary production herds (dairy, beef cows and mixed) with risk from multiple sources.

Modeling the Accuracy of Two in-vitro Bovine Tuberculosis Tests Using a Bayesian Approach.

Accuracy of new or alternative diagnostic tests is typically estimated in relation to a well-standardized reference test referred to as a gold standard. However, for bovine tuberculosis (bTB), a chronic disease of cattle, affecting animal and public health, no reliable gold standard is available. In this context, latent-class models implemented using a Bayesian approach can help to assess the accuracy of diagnostic tests incorporating previous knowledge on test performance and disease prevalence. In Uruguay, bTB-prevalence has increased in the past decades partially because of the limited accuracy of the diagnostic strategy in place, based on intradermal testing (caudal fold test, CFT, for screening and comparative cervical test, CCT, for confirmation) and slaughter of reactors. Here, we evaluated the performance of two alternative bTB-diagnostic tools, the interferon-gamma assay, IGRA, and the enzyme-linked immunosorbent assay (ELISA), which had never been used in Uruguay in the absence of a gold standard. In order to do so animals from two heavily infected dairy herds and tested with CFT-CCT were also analyzed with the IGRA using two antigens (study 1) and the ELISA (study 2). The accuracy of the IGRA and ELISA was assessed fitting two latent-class models: a two test-one population model (LCA-a) based on the analysis of CFT/CFT-CCT test results and one in-vitro test (IGRA/ELISA), and a one test-one population model (LCA-b) using the IGRA or ELISA information in which the prevalence was modeled using information from the skin tests. Posterior estimates for model LCA-a suggested that IGRA was as sensitive (75–78%) as the CFT and more sensitive than the serial use of CFT-CCT. Its specificity (90–96%) was superior to the one for the CFT and equivalent to the use of CFT-CCT. Estimates from LCA-b models consistently yielded lower posterior Se estimates for the IGRA but similar results for its Sp. Estimates for the Se (52% 95%PPI:44.41-71.28) and the Sp (92% 95%PPI:78.63–98.76) of the ELISA were however similar regardless of the model used. These results suggest that the incorporation of IGRA for detection of bTB in highly infected herds could be a useful tool to improve the sensitivity of the bTB-control in Uruguay.

Assessment of the specificity of a gamma-interferon test performed with specific antigens to detect bovine tuberculosis, after non-negative results to intradermal tuberculin testing

In cattle herds in France, cervical skin tests (STs) using simple intradermal tuberculin (SIT) are performed to detect bovine tuberculosis (bTB). When positive results are found on ST screening, the...

The prevalence of brucellosis and bovine tuberculosis in ruminants in Sidi Kacem Province, Morocco.

Bovine tuberculosis (BTB) and brucellosis are major endemic zoonoses in ruminants in Morocco that impact on both animal and human health. This study presents an assessment of the epidemiological and socioeconomic burden of bacterial zoonoses in Sidi Kacem Province in Northern Morocco from a cross-sectional survey of 125 cattle and/or small ruminant-owning households. In total, 1082 sheep and goats were examined from 81 households. The single intradermal comparative cervical test to screen for bovine tuberculosis was undertaken on 1194 cattle from 123 households and all cattle were blood sampled. Cattle and small ruminant sera were tested for brucellosis using the standard Rose Bengal Test (sRBT) and the modified Rose Bengal Test (mRBT). Bacteriology was performed on 21 milk samples obtained from cattle that were seropositive for brucellosis for isolation and phenotyping of circulating Brucella strains. Individual and herd prevalence for BTB in cattle of 20.4% (95% CI 18%-23%) and 57.7% (95% CI 48%-66%), respectively, were observed in this study. The prevalence of brucellosis in cattle at individual and herd level was 1.9% (95% CI 1.2%-2.8%) and 9% (95% CI 4.5%-1.5%), respectively. Brucella pathogens were isolated from three cattle milk samples and were identified as B. abortus using Bruceladder® multiplex PCR and B. abortus biovar 1 by classical phenotyping. All small ruminants were seronegative to sRBT, two were positive to mRBT. A higher risk of BTB and brucellosis was observed in cattle in intensive livestock systems, in imported and crossed breeds and in animals from larger herds (>15). The three risk factors were usually present in the same herds, leading to higher transmission risk and persistence of both zoonoses. These results highlight the importance of implementing control strategies for both BTB and brucellosis to reduce productivity losses and the risk of transmission to humans. Prioritising control for BTB and brucellosis in intensive livestock production systems is essential for human and animal health.

Comparison of the cutaneous response of the comparative cervical tuberculin (CCT) test with different reading times in buffaloes

Tuberculosis is an infectious disease that affects ruminants, causing losses in milk and meat production. Thus, the aim of this research was to compare the cutaneous response of the comparative cervical tuberculin test in buffaloes. Thus, the Comparative Cervical Test (CCT) examined 473 buffaloes, with 440 females and 33 males of different ages and races. Measurement of the allergic skin response was performed at 24, 48, 72 and 96 hours after tuberculinization. For avian tuberculin, the mean skin increases ranged from 12.24mm (0 hour) to 14.82mm (96 hours). As for bovine tuberculin ranged from 11.99mm (0 hour) to 13.68mm (96 hours). The maximum point of measurement of the allergic skin reaction was observed at 72 hours after the application of avian tuberculin, the same was observed for bovine tuberculin. However, there was no statistically significant difference in relation to the amplitude of the reactions observed at 48, 72 and 96 hours. Therefore, it was concluded that the skin allergic reaction occurred in a gradual manner and that the maximum amplitude value of the cutaneous allergic response in buffalo in the diagnosis of tuberculosis was at 72 hours.

Nasal swab real-time PCR is not suitable for in vivo diagnosis of bovine tuberculosis

ABSTRACT: Bovine tuberculosis (bTB) is a zoonosis causing economic losses and public health risks in many countries. The disease diagnosis in live animals is performed by intradermal tuberculin test, which is based on delayed hypersensitivity reactions. As tuberculosis has complex immune response, this test has limitations in sensitivity and specificity. This study sought to test an alternative approach for in vivo diagnosis of bovine tuberculosis, based on real-time polymerase chain reaction (PCR). DNA samples, extracted from nasal swabs of live cows, were used for SYBR® Green real-time PCR, which is able to differentiate between Mycobacterium tuberculosis and Mycobacterium avium complexes. Statistical analysis was performed to compare the results of tuberculin test, the in vivo gold standard bTB diagnosis method, with real-time PCR, thereby determining the specificity and sensitivity of molecular method. Cervical comparative test (CCT) was performed in 238 animals, of which 193 had suitable DNA from nasal swabs for molecular analysis, as indicated by amplification of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene, and were included in the study. In total, 25 (10.5%) of the animals were CCT reactive, of which none was positive in the molecular test. Of the 168 CCT negative animals, four were positive for M. tuberculosis complex at real time PCR from nasal swabs. The comparison of these results generated values of sensitivity and specificity of 0% and 97.6%, respectively; moreover, low coefficients of agreement and correlation (-0.029 and -0.049, respectively) between the results obtained with both tests were also observed. This study showed that real-time PCR from nasal swabs is not suitable for in vivo diagnosis of bovine tuberculosis; thus tuberculin skin test is still the best option for this purpose.

Situação da tuberculose em ovinos consorciados com bovinos leiteiros na região sudoeste maranhense

RESUMO: Objetivando conhecer a situação da tuberculose ovina no Maranhão, avaliaram-se 343 ovinos da região sudoeste do estado, 247 fêmeas e 96 machos, com idade acima de 6 meses, por meio do teste cervical comparativo (TCC), com inoculação de tuberculinas na região cervical direita. Cranialmente, injetou-se derivado proteico purificado (PPD) aviário; e caudalmente, PPD bovino, em doses individuais de 0,1 mL. Todos os animais apresentaram resultado negativo para tuberculose, entretanto, 65 ovinos apresentaram reação à tuberculina bovina superior à aviária. Para investigar a possibilidade de baixa sensibilização ou anergia em animais com tuberculose avançada, foi necropsiado um ovino caquético, idade acima de quatro anos, com resultado negativo ao TCC, porém, com reação à tuberculina aviária e linfonodos aumentados com consistência endurecida. Os resultados indicaram infecções por C. pseudotuberculosis, Actinomyces pyogenes, Staphylococcus aureus e Mycobacterium sp. A cultura e o isolamento resultaram em Pseudomonas aeruginosas e Bacillus sp. Não foram visualizadas estruturas bacterianas compatíveis com bacilo álcool ácido resistente (BAAR). Os resultados confirmam ausência de animais reagentes ao M. bovis, pressupondo inexistência da tuberculose em ovinos na região estudada.

Análise de custo-efetividade de protocolos no diagnóstico da tuberculose bovina em um rebanho naturalmente infectado

Resumo: Em diversos países, a tuberculose bovina, causada por Mycobacterium bovis, é tanto um problema econômico quanto de saúde pública. Mundialmente, são implementados programas de erradicação da enfermidade com políticas baseadas em testes tuberculínicos e abate dos animais reativos, porém pouco se sabe sobre os custos e a efetividade das políticas de erradicação. O presente estudo avaliou a relação de custo-efetividade dos protocolos de diagnóstico da tuberculose bovina, em uma abordagem multidisciplinar, empregados em um rebanho naturalmente infectado. Após realização da análise de custo-efetividade (C/Ef) dos protocolos diagnósticos ante-mortem observou-se que o Teste Cervical Comparativo (C/Ef=4,68), quando utilizado isoladamente, é a escolha diagnóstica mais custo-efetiva para um rebanho naturalmente infectado. Para os protocolos confirmatórios de diagnóstico post-mortem a histopatologia (C/Ef=17,47) associada ao Teste Cervical Simples e Teste Cervical Comparativo foi a escolha mais custo-efetiva para os animais do rebanho estudado. Entretanto, o único protocolo eficaz em diagnosticar 100% dos animais infectados foi o uso em conjunto do teste humoral ELISA associado ao teste celular IFN.

Nationwide Cross-Sectional Study on Bovine Tuberculosis by Intra Vitam Testing in Germany, 2013-2014

Germany was declared officially free from bovine tuberculosis (bTB) effective from 1 July 1996. After the occurrence of several Mycobacterium (M.)bovis outbreaks in north-western Germany in recent years with high intraherd prevalence at the time of detection, the reliability of abattoir surveillance as the principal component of the national bTB control programme was debated by veterinary public health officials. Rising numbers of wildlife-associated outbreaks caused by M.caprae in southern Germany eventually prompted a nationwide cross-sectional study on bTB. A total of 51999 cattle, that is 0.41% of the national herd kept on 1.73% of German cattle farms, were tested. Despite 4 positive and 152 inconclusive single intradermal comparative cervical test results, none of the animals was confirmed as bTB-positive by a subsequent interferon-release assay or by post-mortem PCR testing. The estimated prevalence of bTB in Germany was thus calculated as 0.0% (CI 0.0000-0.0064%) affirming that Germany still qualifies as an officially tuberculosis-free (OTF) country. Occasional randomized nationwide testing can be an appropriate tool to reassure the OTF status and may also help to maintain an appropriate training level for the diagnostic procedures and for supporting sustained disease awareness among stakeholders.

Risk factors for visible lesions or positive laboratory tests in bovine tuberculosis reactor cattle in Northern Ireland

An observational case–control study was conducted to investigate risk factors for confirmed bovine tuberculosis (bTB) infection in cattle reacting positively to the single intradermal comparative cervical test (SICCT) in Northern Ireland in the years 1998, 2002 and 2006.Macroscopic lesions were detected at slaughter (positive visible lesion (VL) status) in 43.0% of reactor cattle, whilst 45.3% of those sampled were confirmed as bTB positive due to the presence of lesions or positive histopathology/mycobacterial culture (positive bTB status). In 97.5% of the reactors, the VL status and bTB status were either both negative or both positive. Generalized linear mixed model analyses were conducted on data of 24,923 reactor cattle with the variables herd identifier, local veterinary office (DVO) and abattoir being used as random effects within all the models generated at univariable and multivariable level. The other variables within the dataset were used as fixed effects. Significant risk factors associated with VL status and bTB status at multivariable level (p<0.05) included age at death, breed, sex, test year, net increase in skin thickness at bovine tuberculin injection site, epidemiological status of skin test, total number of reactors at the disclosure test, mean herd size and prior response to the skin test.These risk factors are likely related to the time since infection, the strength of the challenge of infection and the susceptibility of the animal. These findings are important as the detection of visible lesions and the confirmation of bTB are an integral part of the overall bTB control programme in Northern Ireland and the veterinary meat inspection and hygiene programme. The visible lesion status and bTB status of an animal can affect the way in which bTB breakdowns are managed, since failure to detect visible lesions and recovery of Mycobacterium bovis can lead to a less stringent follow-up after other risk factors have been taken into account.

Effects of Serial Skin Testing with Purified Protein Derivative on the Level and Quality of Antibodies to Complex and Defined Antigens in Mycobacterium bovis-Infected Cattle.

Several serological tests designed to detect antibodies to immunodominant Mycobacterium bovis antigens have recently emerged as ancillary tests for the detection of bovine tuberculosis in cattle, particularly when used after the injection of purified protein derivative (PPD) for skin testing, which significantly boosts M. bovis-specific antibody responses. The present findings demonstrate the onset and duration of boosted antibody responses after the injection of M. bovis PPD for the caudal fold test (CFT) and Mycobacterium avium and M. bovis PPDs for the comparative cervical test (CCT), administered in series in cattle experimentally infected with M. bovis. While skin tests boosted the responses to certain antigens (i.e., MPB83 and MPB70), they did not affect the responses to other antigens (e.g., ESAT-6, CFP10, MPB59, and MPB64). Administration of the CCT 105 days after the CFT resulted in an even greater secondary boost in antibody responses to MPB83 and MPB70 and to a proteinase K-digested whole-cell sonicate (WCS-PK) of M. bovis. Both IgM and IgG contributed to the initial boost in the MPB83/MPB70-specific antibody response after the CFT. The secondary boost after the CCT was primarily due to increased IgG levels. Also, the avidity of antibodies to MPB83 and MPB70 increased after the CCT in M. bovis-infected cattle. The avidity of antibodies to the WCS-PK antigens increased in the interval between the CFT and the CCT but did not increase further after the CCT. Together, these findings demonstrate that the administration of PPDs for skin tests results in additive enhancement (i.e., when the CFT and CCT are performed in series), both qualitative and quantitative, of MPB83/MPB70-specific antibody responses.

The Single Intradermal Cervical Comparative Test Interferes with Johne's Disease ELISA Diagnostics.

Enzyme-linked immunosorbent assays (ELISA) of milk and serum samples are a routinely used method of screening herds for Mycobacterium avium subspecies paratuberculosis (MAP). Infection with MAP causes granulomatous enteritis of ruminants known as Johne’s disease (JD). The sensitivity (Se) and specificity (Sp) of MAP ELISAs leads to difficulties in the identification of both infected and infectious animals. Interference with MAP ELISA Se and Sp has been reported in MAP seronegative cows following administration of purified protein derivative (PPD) as part of intradermal testing for bovine tuberculosis (bTB). The aim of this study is to examine the impact of the single intradermal cervical comparative test (SICCT) for bTB, on both serum and milk MAP ELISA tests, in a herd containing both seropositive and seronegative cows pre-SICCT. A secondary objective is to provide appropriate timing of JD ELISA tests in relation to the SICCT. A herd of 139 cows were serum and milk sampled pre- and post-SICCT administration. Prior to SICCT, 6% of the herd tested seropositive for MAP using milk ELISA, with 8% positive on serum. ID Screen Paratuberculosis Indirect Screening Test (ID Vet) was used to screen the herd. Within 14 days of PPD administration, a significant increase in the prevalence of seropositive cows was recorded. Identical prevalence’s were recorded with both test matrices (39%). ELISA values remained significantly higher until day 43 post-SICCT in milk (P = 0.850), and day 71 in serum (P = 0.602). If the “new” positives detected post-bTB testing are deemed false positives due to generation of cross-reacting antibodies by administration of PPD, milk would appear a more suitable sample for JD ELISA testing within 2 months of SICCT. In summary, sampling for JD utilizing milk ELISA should be avoided in the 43-day period following PPD administration, with serum ELISA sampling avoided for an additional 28 days.

Assessment of the sensitivity of the gamma-interferon test and the single intradermal comparative cervical test for the diagnosis of bovine tuberculosis under field conditions

In some French départements, the eradication of bovine tuberculosis is incomplete and usual skin tests [single intradermal tuberculin test (SIT) and single intradermal comparative cervical test (SICCT)] have poor specificity due to cross-reactions with non-pathogenic mycobacteria, causing economic losses. In Côte d'Or (Burgundy, France), an experimental serial testing scheme based on the combination of SICCT and gamma-interferon (IFN-γ) tests has been initiated in order to shorten the interval between suspicion and its invalidation in herds with false-positive results to skin tests. Our aim was to assess the scheme's sensitivity and to compare it to the sensitivity of the screening scheme recommended by the European Commission. Our study included 1768 animals from Côte d'Or. The sensitivities of both schemes were estimated using a Bayesian approach. The individual sensitivity of the IFN-γ test [88·1%, 95% credibility interval (CrI) 72·8-97·5] was not significantly different from individual SICCT sensitivity (80·3%, 95% CrI 61·6-98·0) and individual SIT sensitivity (84·2%, 95% CrI 59·0-98·2). The individual specificity of the IFN-γ test was 62·3% (95% CrI 60·2-64·5). No significant difference could be demonstrated between the sensitivities of the serial testing scheme used in Côte d'Or (73·1%, 95% CrI 41·1-100) and the European Union serial testing scheme (70·1%, 95% CrI 31·5-100·0).