Diagnosis of goat tuberculosis using tuberculinization and molecular techniques

Tuberculosis is an infectious, chronic, and worldwide disease. It has been known since the beginning of humanity and still negatively influences public health and livestock, especially, in Brazil, in the northeast. Etiologic agents are the mycobacteria of the Mycobacterium tuberculosis complex, which is the most important in mammals' involvement. The state of Bahia has 68.7% of its territory located in the semiarid region and holds the largest goat herd in the country. Goat breeding is a social and economic activity that adds value to this region. Up to the present, data on goat tuberculosis is unknown in this state. Thus, this study seeks data on tuberculosis prevalence in goats in a semiarid region of Bahia by using the comparative tuberculin test and multiplex polymerase chain reaction (PCR). A total of 600 adult animals of both sexes were evaluated. A prevalence of 0.33% (2/600) and 33.33% (1/3) properties were found for positive animals. Each assessed property had a questionnaire to analyze the epidemiological data management and relevant aspects for the disease occurrence. To confirm the positive tuberculin test results, PCR was used to detect and identify the pathogenic mycobacteria involved in the infection. It is concluded that most of the properties performing goat breeding in the region show low technification levels and promote farming between different species. Low prevalence of the disease alerts preventive measures to avoid major proportion situations that could influence the goat breeding in the state.

Bovine tuberculosis: diagnosis in dairy cattle through the association of analyzes / Tuberculose bovina: diagnóstico em bovinos leiteiros através da associação de análises

Tuberculosis is a chronic anthropozoonosis of worldwide occurrence, caused by the bacterium Mycobacterium tuberculosis and its variants. In Brazil, the National Program for the Control and Eradication of Brucellosis and Tuberculosis in cattle, is responsible for diagnosing and the correctly allocate positive animals, but there is still a lack of definitive diagnosis of the disease. This study described the use of five diagnostic tools that can be used, preferably together, for the confirmation of suspected cases. These tools included the clinical examination comparative cervical tuberculin test, macroscopic findings during the slaughtering and histopathology of the damaged tissues followed by histochemistry. We evaluated a total of 211 dairy cattle, where 15.1% (32/211) had classic clinical signs of bovine tuberculosis, 74 (35%) showed reactivity in the comparative cervical tuberculin test. Of the total number of animals, 141 (66.8%) were referred for sanitary slaughter due to legal and control issues in the outbreaks of the disease. In the follow-up of slaughtering and inspection of viscera and carcasses, 74 (52.5%) had macroscopic lesions compatible with bovine tuberculosis, while 67 (47.5%) showed no visible changes. During the inspection, fragments of lymph nodes and liver and lung parenchyma were collected from five cattle with macroscopic lesions and five with no lesions. The histopathological analysis showed numerous areas of caseous necrosis with or without central calcification and granulomatous inflammatory infiltrate. In the special staining of Ziehl-Neelsen, numerous acid-fast bacilli were evidenced in all cases.(AU)

A tuberculose é uma antropozoonose crônica de ocorrência mundial, causada pela bactéria Mycobacterium tuberculosis e suas variantes. No Brasil existe o Programa Nacional de Controle e Erradicação da Brucelose e Tuberculose em bovinos que viabiliza o diagnóstico e a destinação correta dos animais positivos, porém ainda há carência quanto ao diagnóstico da doença. Assim, este trabalho descreve a utilização de cinco ferramentas diagnósticas para a confirmação de casos suspeitos de tuberculose. As ferramentas utilizadas compreenderam o exame clínico, teste tuberculínico cervical comparativo, os achados macroscópicos durante o abate sanitário e a histopatologia dos tecidos lesados seguido de histoquímica. O estudo avaliou um total de 211 bovinos leiteiros, dos quais 15,1% (32/211) apresentaram sinais clínicos clássicos de tuberculose bovina, 35,1% (74/211) apresentaram reatividade no teste tuberculínico cervical comparativo, e 143 animais (67,8%) foram encaminhados para abate sanitário devido a questões legais e de controle nos focos da doença. No acompanhamento do abate e inspeção sanitária de vísceras e carcaças verificou-se que 51,8% (74/143) dos bovinos abatidos apresentavam lesões macroscópicas compatíveis com tuberculose bovina, enquanto 48,2% (69/143) não apresentavam alterações visíveis. Durante a inspeção foram coletados fragmentos de linfonodos e parênquima de fígado e pulmão de cinco bovinos com lesões macroscópicas e de cinco sem lesões, que na análise histopatológica apresentaram numerosas áreas de necrose caseosa com ou sem calcificação central e infiltrado inflamatório granulomatoso. Na coloração de Ziehl-Neelsen foram evidenciados numerosos bacilos álcool-ácido resistentes em todos os casos. Assim, diante dos resultados obtidos verifica-se que as análises empregadas no presente estudo foram de extrema importância para o diagnóstico acurado de tuberculose em bovinos.(AU)

Enzyme-linked immunosorbent assay as complement of intradermal skin test for the detection of mycobacterium bovis infection in cattle.

Diagnostic tests based on cell-mediated immunity are used in programs for the control and eradication of bovine tuberculosis (bTB), which is mainly caused by Mycobacterium bovis. Additional serological assays could be performed as an ancillary method to detect an infected animal that fails to produce an immune response against the intradermal reaction (IDR), the official bTB test. In this study, we evaluated the effectiveness of an enzyme-linked immunosorbent assay (ELISA) that uses bovine PPD as a capture antigen as a complement to the IDR in herds with confirmed cases of bTB. The study was conducted in two stages. First, a panel of 200 serum samples was analyzed by ELISA. The sensitivity and specificity obtained were 60% and 99%, respectively. The subsequent stage consisted of evaluating 7,494 bovines from 14 selected dairy farms. The number of animals yielding a IDR negative/ELISA positive result were 200. A necropsy analysis of 33 of these IDR negative/ELISA positive animals revealed that 30 (91%) presented granulomatous lesions and positive M. bovis isolation. This finding confirmed bTB in most cases. Altogether, the results obtained in the present study suggest that the combined use of IDR and ELISA is an effective strategy to improve the control of bTB in endemic herds.

Evaluation of tuberculin skin test (TST) in vervet monkeys (Chlorocebus pygerythrus) using experimentally tuberculin sensitized animals.

BACKGROUND: Nowadays, several test methods with different useful values are available for diagnosis of the tuberculosis (TB) in non-human primates (NHPs). Despite some limitations of tuberculin skin test (TST), it is still the most commonly used method for TB testing of NHPs. METHODS: During this investigation, TST was performed upon three groups of experimentally tuberculin sensitized and one group of non-sensitized vervet monkeys (Chlorocebus pygerythrus) by means of two types of old tuberculin (OT) and two types of purified protein derivative (PPD) tuberculin. RESULTS: The data obtained from this study revealed that PPD tuberculin prepared from both Mycobacterium tuberculosis and Mycobacterium bovis has more advantages over OT in tuberculin testing of the vervet monkeys. The potency of the PPD tuberculin prepared from M bovis was estimated almost twice as much of the M tuberculosis. CONCLUSIONS: Intrapalpebral injection of 0.1 mL of a concentration of ≥1 mg/mL of PPD tuberculin prepared from M bovis is the preferred method for TST of vervet monkeys.

Prevalence of bovine tuberculosis in the Aksu Region of Xinjiang, China, between 1985 and 2016 / Prevalência de tuberculose bovina na região Aksu de Xinjiang, China, entre 1985 e 2016

Prevalence of bovine tuberculosis infection in cattle in Aksu Prefecture determined by intradermal tuberculin skin test (TST), between 1985 and 2016. Cattle were analyzed according to region, feeding pattern, herds and age. A total of 890,009 cattle were tested, with overall bovine tuberculosis prevalence of 0.13% (1172/890009). Statistically significant difference was found in feeding pattern and herds. Prevalence in cows (0.19%, 615/327022) was higher than that in beeves (P< 0.01, OR= 1.903, 95% CI = 1.696 to 2.134). Significant difference (P< 0.01; OR= 2.238, 95%; CI= 1.937 to 2.585) was evident for rates for bovine tuberculosis in the peasant household (0.12%, 942/802343) and farm groups (0.26%, 230/87666). The overall prevalence of bTB was decreased in the Aksu Prefecture, especially the positive rate was under 0.1% in 2010s. We concluded that the control measures forbovine tuberculosis in the Aksu region cattle herds are effective.(AU)

Prevalência de infecção por tuberculose bovina em gado na prefeitura de Aksu determinada por teste cutâneo tuberculínico (TST) entre 1985 e 2016 foi avaliada. O gado foi analisado de acordo com região, padrão alimentar, rebanho e idade. Um total de 890009 animais foram testados, com prevalência de 0,13% de tuberculose bovina (1172/890009). Diferença estatisticamente significativa foi encontrada em padrão alimentar e rebanhos. Prevalência em vacas (0,19%, 615/327022) foi mais alta que em bois (P< 0,01, OR= 1,903, 95% CI = 1,696 a 2,134). Diferenças significativas (P< 0,01; OR= 2,238, 95%; CI= 1,937 a 2,585) foram evidentes em taxas para tuberculose bovina em casas de camponeses (0,12%, 942/802343) e grupos de fazendeiros (0,26%, 230/87666). A prevalência de bTB caiu na prefeitura Aksu, a taxa positiva se encontrava abaixo de 0.1% a partir de 2010. Conclui-se que as medidas de controle para tuberculose bovina na região de Aksu foram eficazes.(AU)

Tuberculosis (TB) outbreak in a closed Aotus monkey breeding colony: Epidemiology, diagnosis and TB screening using antibody and interferon-gamma release testing.

Tuberculosis (TB) caused by Mycobacterium tuberculosis (MTB) is a devastating and terminal disease in non-human primates (NHPs). Regular TB screenings using the intradermal tuberculin test (TST) have been the mainstay of TB surveillance and control in NHPs. Historically, Aotus monkeys have been considered less susceptible to TB than other NHPs. Here we present the diagnosis and epidemiology of a TB outbreak at The Gorgas Memorial Institute Aotus colony in Panama, and the results of two cross-sectional randomized TB screening studies, using antibody (Ab) and IFN-gamma release assay testing. RESULTS: Epidemiological and spatial analysis confirmed that the outbreak was the result of a continuing intermittent exposure, with human to monkey transmission as the most likely source. During the outbreak that lasted five months (January-June 2015), Mycobacterium kansassi and MTB were isolated from lung caseous granulomas in 1/7 and 3/7 TB suspicious animals respectively. Furthermore, MTB was detected by qRT-PCR in formalin fixed lung and liver granulomas in 2/7 and 1/6 monkeys respectively, suggesting an aerosol route of infection. Likewise, a random sample that included 63 / 313 adult (>2 year-old) monkeys, screened for latent TB with the Primagam® IFN-gamma release assay, between March-May, 2016, were all non-reactors; indicating that the outbreak was self-limiting and the colony was likely free or latent TB infection. Control measures included, quarantine, disinfection and TST screening of all personnel. In conclusion, this study demonstrates that Aotus are highly susceptible to TB, therefore, TB prevention measures should be strictly enforced in Aotus monkey colonies.

Investigation of bovine tuberculosis outbreaks by using a trace-back system and molecular typing in Korean Hanwoo beef cattle.

Bovine tuberculosis is a chronic contagious disease responsible for major agricultural economic losses. Abattoir monitoring and trace-back systems are an appropriate method to control bovine tuberculosis, particularly in beef cattle. In the present study, a trace-back system was applied to bovine tuberculosis cases in Korean native Hanwoo beef cattle. Bovine tuberculosis was detected in three index beef cattle during abattoir monitoring in Jeonbuk Province, Korea, and the original herds were traced back from each index cow. All cattle in each original herd were subjected to tuberculin skin test. The positive rates in the tuberculin skin test were 64.6% (62 of 96), 4.8% (2 of 42), and 8.1% (3 of 37) at farms A, B, and C, respectively. On post-mortem examination of 56 tuberculin-positive cattle, 62% had granulomatous lesions, and Mycobacterium bovis was cultured from 40 (71.4%) of the cattle. Molecular typing by spoligotyping and the mycobacterial interspersed repetitive unit-variable-number tandem repeat assay revealed the genotype of the M. bovis strains from the index cattle were same as the M. bovis genotype in each original herd. The results suggest that tracing back from index cattle to the original herd is an effective method to control bovine tuberculosis in beef cattle.

Nasal swab real-time PCR is not suitable for in vivo diagnosis of bovine tuberculosis / A PCR em tempo real de swab nasal não é adequada para o diagnóstico in vivo de tuberculose bovina

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.(AU)

A tuberculose bovina (bTB) é uma zoonose que causa perdas econômicas e riscos à saúde pública em muitos países. O diagnóstico da doença em animais vivos é realizado pelo teste intradérmico da tuberculina, que é baseado em reações de hipersensibilidade tardia. Como a tuberculose tem resposta imunológica complexa, este teste tem limitações em termos de sensibilidade e especificidade. Este estudo procurou desenvolver uma abordagem alternativa para o diagnóstico in vivo da tuberculose bovina, com base na reação em cadeia da polimerase (PCR) em tempo real. As amostras de DNA, extraídas de suabes nasais de vacas vivas, foram usadas para PCR em tempo real com SYBR® Green, capaz de diferenciar os complexos Mycobacterium tuberculosis e Mycobacterium avium. A análise estatística foi realizada para comparar os resultados de teste de tuberculina, padrão ouro para o diagnóstico in vivo da bTB, com PCR em tempo real, determinando-se assim a especificidade e sensibilidade do método molecular. O teste cervical comparativo (TCC) foi realizado em 238 animais, dos quais 193 tiveram DNA dos suabes nasais adequados para análise molecular, como indicado pela amplificação do gene gliceraldeído-3-fosfato-desidrogenase (GAPDH), e foram incluídos no estudo. No total, 25 (10,5%) animais foram reativos no TCC, dos quais nenhum foi positivo no teste molecular. Dos 168 animais negativos no TCC, quatro foram positivos para o complexo M. tuberculosis na PCR em tempo real a partir dos suabes nasais. A comparação destes resultados gerou valores de sensibilidade e especificidade de 0% e 97,6%, respectivamente; além disso, baixos coeficientes de concordância e correlação (-0,029 e -0,049, respectivamente) entre os resultados obtidos com ambos os testes também foram observados. Este estudo mostrou que a PCR em tempo real a partir de suabes nasais não é adequada para o diagnóstico in vivo da tuberculose bovina; portanto, o teste da tuberculina ainda é a melhor opção para este fim.(AU)

Evidence of Mycobacterium tuberculosis complex bacteraemia in intradermal skin test positive cattle detected using phage-RPA.

Bovine tuberculosis is a zoonotic infectious disease caused by Mycobacterium bovis that affects cattle and can cause tuberculosis in a range of wildlife animals. A bacteriophage-based method combined with PCR (phage-PCR) has been recently used to detect and identify viable pathogenic mycobacteria in the peripheral blood mononuclear cells (PBMCs) of animals suffering from paratuberculosis. To adapt this method for the detection of M. bovis in blood, a new isothermal DNA amplification protocol using Recombinase Polymerase Amplification (RPA) was developed and was found to be able to detect M. bovis BCG within 48 h, with a limit of detection of approximately 10 cells per ml of blood for artificially inoculated blood samples. When blood samples (2 ml) from a Single Comparative Cervical Intradermal Tuberculin (SCCIT)- negative beef herd were tested, Mycobacterium tuberculosis complex (MTC) cells were not detected from any (45) of the blood samples. However when blood samples from SCCIT-positive animals were tested, viable MTC bacteria were detected in 66 % (27/41) of samples. Of these 41 animals sampled, 32 % (13) had visible lesions. In the visible lesion (VL) group, 85 % (11/13) had detectable levels of MTC whereas only 57 % (16/28) of animals which had no visible lesions (NVL) were found to have detectable mycobacteraemia. These results indicated that this simple, rapid method can be applied for the study of M. bovis infections. The frequency with which viable mycobacteria were detected in the peripheral blood of SCCIT-positive animals changes the paradigm of this disease.

An outbreak of bovine tuberculosis in a fallow deer herd (Dama dama) in Sicily.

Wild ruminants have an important role in the epidemiology of bovine tuberculosis (bTB). This study describes an outbreak of bovine tuberculosis occurring in a fallow deer herd in Sicily. In 2012 a Sicilian herd of 47 animals was referred for cachexia. Pathological examination of 2 dead animals revealed disseminated granulomas predominantly involving the skin and subcutaneous tissues. Tissue samples were submitted for histological analysis, bacteriological culture, and biomolecular assay. PCR analysis identified Mycobacterium strains. Genotyping by spoligotyping and MIRU-VNTR profiles identified Mycobacterium bovis spoligotype SB0120 in both animals. In 2014, bTB skin testing of 28 fallow deer from the same group was positive in 4 and inconclusive in another 4. All 8 positive/inconclusive reactors were euthanized. Disseminated granulomatous lesions were noted in 6 of these animals, 3 of which (2 positive and 1 negative to skin tests) also presented cutaneous lesions. M. bovis spoligotype SB0120 was identified from all animals in which tuberculous-like lesions were observed, including 2 negative reactors. Many of the animals involved in this outbreak presented diffuse skin lesions, a potential route of transmission of M. bovis infection. Given the epidemiological role wildlife play in the maintenance of bTB infection and its potential risk for humans, a comprehensive monitoring plan for this zoonosis in wildlife species in Sicily is needed.

Field application of immunoassays for the detection of Mycobacterium bovis infection in the African buffalo (Syncerus caffer).

The African buffalo (Syncerus caffer) is considered the most important maintenance host of bovine tuberculosis (BTB) in wildlife in Southern Africa. The diagnosis of Mycobacterium bovis infection in this species mostly relies on the single intradermal comparative tuberculin test (SICTT). As an alternative, the BOVIGAM® 1G, an interferon-gamma (IFN-γ) release assay, is frequently used. The test performance of cell-mediated immunity (CMI-) and humoral immunity (HI-) based assays for the detection of M. bovis infections in buffaloes was compared to identify the test or test combination that provided the highest sensitivity in the study. Buffaloes were sampled during the annual BTB SICTT testing in the Hluhluwe-iMfolozi-Park (KwaZulu-Natal, South Africa) during June 2013. A total of 35 animals were subjected to the SICTT, 13 of these tested positive and one showed an inconclusive reaction. CMI-based assays (BOVIGAM® 1G (B1G) and BOVIGAM® 2G (B2G)) as well as a serological assay (IDEXX TB ELISA) were used to further investigate and compare immune responsiveness. Thirteen SICTT positive buffaloes and one inconclusive reactor were slaughtered and a post-mortem (PM) examination was conducted to confirm BTB. Lesions characteristic of BTB were found in 8/14 animals (57.1%). Test results of individual assays were compared with serial and parallel test interpretation and the sensitivity was calculated as a percentage of test positives out of the number of SICTT positive animals with granulomatous lesions (relative sensitivity). The B1G assay showed the highest individual sensitivity (100%; 8/8) followed by the B2G assay (75%; 6/8) and the IDEXX TB ELISA (37.5%; 3/8). Therefore, using in parallel interpretation, any combination with the B1G showed a sensitivity of 100% (8/8), whereas combinations with the B2G showed a 75% sensitivity (6/8). Out of the 21 SICTT negative animals, 7 animals showed responsiveness in the B2G or IDEXX TB ELISA. In conclusion, this study has shown that the BOVIGAM® IFN-γ assay had the highest test performance.

Pathology and diagnosis of Mycobacterium bovis in naturally infected dromedary camels (Camelus dromedarius) in India.

The present study investigated the pathological features of tuberculosis (TB) caused by Mycobacterium bovis and its diagnosis in naturally infected dromedary camels from an organized farm in India. During the period of the 5-year study, a total of 18 (19.56 %) camels out of 92 examined showed gross lesions compatible with TB at post-mortem. The clinical signs and pathological lesions in these camels were studied, and the efficacy of different diagnostic tests was also assessed. On the basis of occurrence and distribution of gross TB lesions, the infected camels revealed two different lesional patterns as pulmonary (n = 15) and disseminated (n = 3) form. The histopathology of affected organs revealed typical granulomatous lesions wherein the giant cells and acid-fast bacilli were occasionally observed in pulmonary form whereas they frequently observed in disseminated form. The single intradermal tuberculin test (SIDT) detected TB in 10 (55.55 %) whereas the Ziehl-Neelsen (ZN) stain and IS6110 PCR from tissue lesions detected 13 (72.22 %) and 18 (100 %) of the infected camels, respectively. The study suggests that pulmonary form of the TB is more common in camels indicating respiratory route as the major source of exposure in camel herds. Moreover, very low sensitivity of SIDT was observed which highlights the difficulty for confirmation of TB in live camels.

Padronização do teste imunoalérgico aplicado ao diagnóstico da tuberculose e micobacterioses em suínos (Sus scrofa) experimentalmente sensibilizados com suspensões oleosas de Mycobacterium bovis ou M. avium inativados / Standardization of the immunoallergic skin test applied for the diagnosis of tuberculosis and mycobacterioses in swine (Sus scrofa) experimentally sensitized with oil suspensions of inactivated Mycobacterium bovis or M. avium

Foi investigado o valor diagnóstico da resposta alérgica cutânea em leitões experimentalmente sensibilizados, pela via intramuscular, com suspensões oleosas de Mycobacterium bovis ou M. avium inativados pelo calor.Foram utilizados 91 animais, divididos em quatro grupos: grupos A e B, cada um com 25 indivíduos, grupos C e D com 21 e 20 indivíduos respectivamente, balanceando-se as características de raça, linhagem, faixa etária e sexo. Aos 30 dias de idade, todos os animais foram submetidos a uma triagem com a aplicação de tuberculina PPD bovina, pela via intradérmica na base da orelha e não houve qualquer tipo de reação. Decorridos 60 dias do teste tuberculínico de triagem, o grupo A recebeu injeção intramuscular de 0,5 mL de uma suspensão oleosa de M. avium estirpe D4; o grupo B recebeu 0,5 mL de uma suspensão oleosa de M. bovis estirpe AN5; o grupo C (controle I), recebeu 0,5 mL do adjuvante oleoso; e o grupo D (controle II), recebeu 0,5 mL de solução fisiológica. Após 30 dias da sensibilização foi realizada a prova de tuberculinização comparativa com reação medida pela variação da espessura da pele com cutímetro de mola às 0h, 24h, 48h e 72h, após a aplicação das tuberculinas. No teste comparativo, lido às 48 ou 72 horas, a reação foi considerada negativa quando a diferença das reações entre o PPD bovino e o PPD aviário foi menor que 6,7 mm; suspeito ou inconclusivo quando a diferença se situou na faixa de 6,7 a 7,5 mm; e positiva de acordo com o tipo de PPD, considerando-se tuberculose para PPD M. bovis e micobacteriose para PPD M. avium, quando a diferença da reação foi superior a 7,5 mm.

The diagnostic value of the cutaneous allergic response to tuberculin in piglets experimentally sensitized intramuscularly with the oily suspensions of heat inactivated M. bovis or M. avium was investigated. Ninety-one animals were used and divided into four groups: groups A and B were formed each with 25 individuals, and groups C and D, with 21 and 20 individuals, respectively, balancing the characteristics of race, ancestry, age and sex. At the age of 30 days, all the animals were submitted to the screening test with the use of M. bovis PPD, by the intradermal route at the base of the ear and no reaction was detected. Sixty days after the screening tuberculin test, animals of the group A were injected intramuscularly with 0.5 mL of oily suspension of M. avium D4 strain; animals of the group B received 0.5 mL of an oily suspension of M. bovis, AN5 strain; group C (control I) received 0.5 mL of an oily adjuvant; and the individuals of the group D (control II) received 0.5 mL of saline solution. Following 30 days of sensitization, comparative skin reactions were measured by the variation in skin thickness with a caliper at 0h, 24h, 48h an 72h after applications of tuberculins. In the comparative test measured at 48 or 72h, the reaction was considered negative when the difference of the reactions between bovine PPD and avian PPD was less than 6.7 mm; suspected or inconclusive, when the difference stood in the range of 6.7 to 7.5 mm; and positive according to the type of PPD, considering tuberculosis the M. bovis PPD and mycobacteriosis the M. avium PPD, when the difference of the reaction was greater than 7.5 mm.

Tuberculin screening of some selected Fulani lactating cows in North-Central Nigeria.

The prevalence of mycobacterial infection among lactating Fulani cows was investigated in the Federal Capital Territory, Abuja and Kaduna State of Nigeria. Tuberculin testing using single comparative intradermal tuberculin test showed a 14.6 % positive, 4 % doubtful, and 81.4 % negative reactors. Mycobacterial infection was found to be present in the nomadic (constantly moving) and seminomadic (limited movement) management systems studied but management showed no significant effect on the prevalence of the disease. However, the prevalence was significantly higher in older age groups than the younger ones (P < 0.05).

A multidisciplinary approach to diagnose naturally occurring bovine tuberculosis in Brazil / Uma abordagem multidisciplinar para o diagnóstico de tuberculose bovina em um rebanho naturalmente infectado no Brasil

A herd infected naturally with tuberculosis was investigated by different diagnostic methods. Ninety days after a screening test that identified 21 cows as skin test positive, a Comparative Intradermal Tuberculin Test (CITT) was performed in those 21 cows and in 29 other randomly selected skin test negative cows. Milk samples and nasal swabs were collected prior to the CITT for bacteriological culture and PCR, while blood samples were collected for IFN release and antibody responses to MPB70 and MPB83, at three time points post tuberculin injection. Animals positive by CITT were slaughtered and disease confirmation undertaken. Based on the Kappa test, IFN was comparable to the standard tests (culture, PCR and CITT) at all three sampling points. Results from both antibody ELISAs were similar but were not comparable to the standard tests. T-test analysis of the CITT, IFN and ELISAs demonstrated that their performances were not correlated. There is increasing recognition that individually, available diagnostic tests do not detect all infected cattle. Therefore, a comprehensive strategy for the diagnosis of bovine TB should include test results for the detection of both cellular and humoral immune responses where there may be animals at different stages of infection.

Um rebanho bovino naturalmente infectado por tuberculose foi analisado através de diferentes métodos diagnósticos. Um teste intradérmico simples (TIC) identificou 21 animais como positivos. Após 90 dias deste resultado, um teste intradérmico comparativo (TIC) foi aplicado nos 21 animais positivos ao TIS, além de outros 29 animais com resultados prévios negativos escolhidos aleatoriamente. De todos estes animais (50), foram coletadas amostras de leite e secreção nasal para isolamento e identificação de microrganismos por cultura e PCR; amostras de sangue de cada um dos animais foram coletadas para exames de ELISA: produção de Interferon-gama (IFN) e pesquisa de anticorpos frente aos antígenos MPB70 e MPB83. Tais amostras sanguíneas foram coletadas em três diferentes momentos: no dia da execução do TIC e nos dias dia 7 e dia 21 após a execução do TIC. Os animais que foram positivos a este teste foram abatidos; exames de identificação do agente, tais como cultivo e PCR foram realizados post-mortem para confirmação da doença. Baseado na análise Kappa, IFN apresentou resultados estatisticamente comparáveis aos resultados de isolamento e identificação bacteriana por cultura e PCR, além do TIC ao longo de todo o experimento. No entanto, TIC, ELISA e IFN não foram estatisticamente comparáveis. Tais resultados sugeriram que nenhum dos atuais métodos de diagnóstico para tuberculose possibilitou a identificação de todos os animais infectados. Por este motivo, uma estratégia mais abrangente deveria incluir métodos de diagnóstico que pudessem identificar a resposta imune celular e humoral, uma vez que animais de um mesmo rebanho poderiam se encontrar em diferentes estágios da infecção.

Parameter estimation and use of gamma interferon assay for the diagnosis of bovine tuberculosis in Brazil / Estimação dos parâmetros e uso do teste do interferon gama para o diagnóstico da tuberculose bovina no Brasil

This study aimed to evaluate the interference of tuberculin test on the gamma-interferon (INFg) assay, to estimate the sensitivity and specificity of the INFg assay in Brazilian conditions, and to simulate multiple testing using the comparative tuberculin test and the INFg assay. Three hundred-fifty cattle from two TB-free and two TB-infected herds were submitted to the comparative tuberculin test and the INFg assay. The comparative tuberculin test was performed using avian and bovine PPD. The INFg assay was performed by the BovigamTM kit (CSL Veterinary, Australia), according to the manufacturer's specifications. Sensitivity and specificity of the INFg assay were assessed by a Bayesian latent class model. These diagnostic parameters were also estimate for multiple testing. The results of INFg assay on D0 and D3 after the comparative tuberculin test were compared by the McNemar's test and kappa statistics. Results of mean optical density from INFg assay on both days were similar. Sensitivity and specificity of the INFg assay showed results varying (95% confidence intervals) from 72 to 100% and 74 to 100% respectively. Sensitivity of parallel testing was over 97.5%, while specificity of serial testing was over 99.7%. The INFg assay proved to be a very useful diagnostic method.(AU)

O presente estudo avalia a interferência do teste de tuberculinização no teste do interferon gama (INFg), estima a sensibilidade e a especificidade do INFg em condições brasileiras e simula a utilização dos testes múltiplos usando a tuberculinização comparada e o teste do INFg. Trezentos e cinquenta animais oriundos de dois rebanhos livres e dois rebanhos positivos foram submetidos à tuberculinização comparada e ao teste de INFg. A tuberculinização comparada foi realizada utilizando PPD aviária e bovina. O teste de INFg foi realizado utilizando o kit Bovigam® (CSL Veterinary, Austrália) de acordo com as especificações do fabricante. A sensibilidade e especificidade do teste de INFg foram calculadas pelo Modelo Bayesiano de Classe Latente. Esses parâmetros foram também estimados para os testes múltiplos. Os resultados do teste de INFg no D0 e D3 após o teste de tuberculinização foram comparados pelos testes estatísticos de McNemar e kappa. Os resultados das médias de densidade ótica do teste de INFg em ambos os dias foram similares. A sensibilidade e a especificidade do teste de INFg apresentaram resultados variando (95% intervalo de confiança) de 72 a 100% e 74 a 100%, respectivamente. A sensibilidade do teste em paralelo foi acima de 97,5% enquanto a especificidade do teste em série foi acima de 99,7%. O teste do INFg provou ser um método de diagnóstico muito útil.(AU)

Tuberculosis in camelids: a review.

Tuberculosis is a chronic, contagious, granulomatous disease caused by mycobacterial species belonging to the Mycobacterium tuberculosis complex. Camelids were not considered highly susceptible to tuberculosis, but in recent years increased numbers of cases have been experienced in some countries. In most of the cases, transmission probably occurs through contact with infected cattle or wildlife. None of the ante-mortem tests currently available can consistently provide accurate diagnosis of the infection in live camelids. Recently developed serological assays have the potential for rapid and accurate diagnosis of tuberculosis but still need to be validated.

Colonization with nontuberculous mycobacteria is associated with positive tuberculin skin test reactions in the common marmoset (Callithrix jacchus).

Mycobacterium tuberculosis infections can result in significant morbidity and mortality in nonhuman primate colonies. Preventative health programs designed to detect infection routinely include tuberculin skin testing (TST). Because Mammalian Old Tuberculin used for TST contains antigens common to a variety of mycobacterial species, false-positive results can occur in animals sensitized to nontuberculous mycobacteria (NTM). Over 11 mo, a large colony of common marmosets (Callithrix jacchus) demonstrated a 3.6% prevalence of equivocal or positive TST reactions (termed 'suspect reactions'). Culture of gastric aspirates, bronchoalveolar lavage fluid, and feces revealed a single animal with a positive fecal culture for Mycobacterium gordonae. PCR amplification of M. gordonae DNA in feces collected from animals with suspect TST reactions (demonstrating a 66.7% colonization rate) and colony controls (demonstrating a 14.3% colonization rate) revealed a significant association between suspect TST reactions and intestinal colonization. Gross and histopathologic evaluation revealed a multifocal lymphadenopathy and granulomatous lymphadenitis in 2 of 4 TST-positive marmosets examined. Counter to expectations, granulomatous lymphoid tissue was culture-positive for M. kansasii rather than M. gordonae. Detection of M. gordonae in the feces of TST-suspect animals likely represents an apathogenic intestinal colonization that may serve as an indicator of NTM exposure, whereas evidence of histopathologic disease is associated with the more pathogenic M. kansasii. Although a high index of suspicion for M. tuberculosis should always be maintained, colonization with NTM organisms represents a cause of suspect TST reactions in common marmosets.

Intercorrência entre leucose enzoótica e tuberculose em bovinos leiteiros do estado de Pernambuco / Assessment of intercurrence of enzootic leucosis and bovine tuberculosis in dairy cows in the state of Pernambuco, Brazil

Objetivou-se com este estudo avaliar a intercorrência entre leucose enzoótica e tuberculose em rebanhos bovinos leiteiros em oito municípios do Estado de Pernambuco, pelo estabelecimento da prevalência de bovinos reagentes às provas diagnósticas específicas. Foram analisados sorologicamente 662 animais, pela técnica de imunodifusão dupla em gel de agarose e 612 foram avaliados imunoalergicamente, por meio do teste de tuberculinização. Nos 15 rebanhos examinados, as prevalências de bovinos que apresentaram positividade aos testes IDGA e tuberculina foram 32,2% (213/662) e 14,1% (86/612), respectivamente. Os resultados obtidos nesta pesquisa permitiram concluir que as doenças estudadas encontram-se amplamente disseminadas na população avaliada, com crescimento em níveis significativos da leucose, e as infecções pelo vírus da leucose bovina (VLB) e Mycobacterium bovis encontram-se ativas e em expansão, com risco do comprometimento da saúde dos rebanhos bovinos e, pelo caráter zoonótico da tuberculose bovina, das pessoas que lidam com os bovinos.

We carried out this study to evaluate the intercurrence of tuberculosis and enzootic leukosis in dairy cattle herds in 8 districts of the State of Pernambuco, Brazil, by establishing the prevalence of bovines reagent to specific diagnostic tests, and to also verify the correlation between enzootic leucosis, cattle tuberculosis, the leukocytes count and lymphocytes count among the cattle studied. A total of 662 animals were tested serologically and hematologically, by the techniques of double immunodiffusion in agarose gel and the white blood cell count with the total and differential leukocyte count, respectively, and 612 were evaluated immunoallergically by the tuberculin test. In the 15 herds examined, the prevalence of cattle that were positive to the AGID and tuberculin tests were 32.2% (213/662) and 14.1% (86/612), respectively. Analyzing the correlation between the variables total leukocytes and lymphocytes, we found that total leukocyte is highly related to the variable lymphocytes (72%). The results in this study indicated that the diseases studied are widespread in the population studied, with significant levels of growth in leukemia, and infections of bovine leukosis virus (VLB) and Mycobacterium bovis are active and growing, with risk of impairment of the health of cattle herds, and, due to the zoonotic nature of bovine tuberculosis, of the health of people who work with cattle.

Assessment of antemortem tests used in the control of an outbreak of tuberculosis in llamas (Lama glama).

An outbreak of tuberculosis (TB) caused by Mycobacterium bovis in a llama herd is described. Over a 25-month period, a total of 70 llamas were selected for postmortem examination using four distinct criteria: clinical suspicion of disease (15 animals), positive tuberculin skin test result (three animals), antibody positive using a novel serological test (Rapid Test, 54 animals) and elective cull (five animals). Some animals qualified on more than one criterion. Gross lesions of TB were detected in 15 animals, with lung and lymph node lesions consistently observed. Samples were collected from 14 of 15 animals with visible lesions as well as those with no visible lesions, for histopathology and mycobacterial culture. All 14 llamas with visible lesions had caseonecrotic granulomatous lesions associated with acid-fast bacteria and variable mineralisation, and M bovis was isolated from 13. There were no histopathological lesions of TB in llamas with no grossly visible lesions, and M bovis was not isolated from any of these. The predictive value of suspicious gross lesions at postmortem examination was therefore high in the herd. Molecular typing results indicated that the outbreak was caused by a single strain likely to have originated from a local reservoir, probably cattle or wildlife. Antemortem indicators of infection assisted control of the outbreak, but no single test accurately identified all TB cases. Visible lesions were detected in nine of 15 llamas with clinical suspicion of disease, in two of three that had positive tuberculin skin test results and in 10 of 54 that were antibody positive; there was none (zero out of five) in llamas that were electively culled.