Epidemiological studies of Johne’s Disease in cattle from Scottish farms, with a focus on slaughterhouse investigations

The PARABAN project has been a Scotland-wide initiative to develop and deliver farm-specific ‘best practice’ for the control of Mycobacterium avium ssp. paratuberculosis (MAP) in cattle using ‘Knowledge Exchange’. A range of partners have been involved, including nine ‘Champion Farms’. With input from the farmer, his/her vet and PARABAN advisors, a tailored monitoring and control programme was devised for each ‘Champion Farm’, taking into account the history of the disease on the farm, the physical facilities available and farmer objectives. Culling decisions based on live animal test results were incorporated into each farm-specific programme to complement the management programme already in place to maintain each herd. Results were analysed and discussed with all the partners throughout the project and then offered for wider scrutiny at farm open days. Feedback and questions from these open days have been used to complete the ‘Knowledge Exchange’ cycle. As a major component of the PARABAN project the author collected samples from all adult animals culled from ‘Champion Farms’ at slaughter or as fallen stock, irrespective of in-life MAP test status. These were then subjected to histopathological examination by experienced veterinary pathologists and the results compared with the results from in-life MAP testing. This was intended to evaluate the contribution slaughterhouse sampling could make towards decision making for disease control on farm and formed the main aim of this thesis. In total, samples of terminal ileum and draining lymph node were collected from three-hundred and fifty-two animals. A positive result on histopathology was defined as the presence of lesions typical of MAP and also the presence of acid-fast bacteria within the sections. There was found to be fair agreement between the overall results from histopathology and serum ELISA (Kappa = 0.33), though there appeared to be some variation in agreement between the tests on the individual ‘Champion Farms’. The presence of MAP was confirmed in seven of the eight farms which contributed animals to this study, despite sometimes prolonged efforts at controlling the disease. A separate study was undertaken to make use of the archives of the Scottish Centre for Production Animal Health and Food Safety at the Veterinary School, University of Glasgow. The archive contained records of cases from across southern Scotland and northern England. Analysis of the data generated from examination of these records suggested that MAP is widespread within the Scottish cattle herd and may well be increasing

Evaluación del desempeño de pruebas diagnósticas de tuberculosis en pacientes VIH positivo en dos hospitales públicos de Bogotá

La tuberculosis TB es una de las principales causas de muerte en el mundo en individuos con infección por VIH. En Colombia esta coinfección soporta una carga importante en la población general convirtiéndose en un problema de salud pública. En estos pacientes las pruebas diagnósticas tienen sensibilidad inferior y la enfermedad evoluciona con mayor frecuencia hacia formas diseminadas y rápidamente progresivas y su diagnóstico oportuno representa un reto en Salud. El objetivo de este proyecto es evaluar el desempeño de las pruebas diagnósticas convencionales y moleculares, para la detección de TB latente y activa pacientes con VIH, en dos hospitales públicos de Bogotá. Para TB latente se evaluó la concordancia entre las pruebas QuantiFERON-TB (QTF) y Tuberculina (PPD), sugiriendo superioridad del QTF sobre la PPD. Se evaluaron tres pruebas diagnósticas por su sensibilidad y especificidad, baciloscopia (BK), GenoType®MTBDR plus (Genotype) y PCR IS6110 teniendo como estándar de oro el cultivo. Los resultados de sensibilidad (S) y especificidad (E) de cada prueba con una prevalencia del 19,4 % de TB pulmonar y extrapulmonar en los pacientes que participaron del estudio fue: BK S: 64% E: 99,1%; Genotype S: 77,8% E: 94,5%; PCRIS6110 S: 73% E: 95,5%, de la misma forma se determinaron los valores predictivos positivos y negativos (VPP y VPN) BK: 88,9% y 94,8%, Genotype S: 77,8% E: 94,5%; PCRIS6110 S: 90% y 95,7%. Se concluyó bajo análisis de curva ROC que las pruebas muestran un rendimiento diagnóstico similar por separado en el diagnóstico de TB en pacientes con VIH, aumentando su rendimiento diagnostico cuando se combinan

Direct Detection of Mycobacterium bovis in Bovine Lymph Nodes by PCR

P>Thirty-five lymph node samples were taken from animals with macroscopic lesions consistent with Mycobacterium bovis infection. The animals were identified by postmortem examination in an abattoir in the northwestern region of state of Parana, Brazil. Twenty-two of the animals had previously been found to be tuberculin skin test positive. Tissue samples were decontaminated by Petroff`s method and processed for acid-fast bacilli staining, culture in Stonebrink and Lowenstein-Jensen media and DNA extraction. Lymph node DNA samples were amplified by PCR in the absence and presence (inhibitor controls) of DNA extracted from M. bovis culture. Mycobacterium bovis was identified in 14 (42.4%) lymph node samples by both PCR and by culture. The frequency of PCR-positive results (54.5%) was similar to that of culture-positive results (51.5%, P > 0.05). The percentage of PCR-positive lymph nodes increased from 39.4% (13/33) to 54.5% (18/33) when samples that were initially PCR-negative were reanalysed using 2.5 mu l DNA (two samples) and 1 : 2 diluted DNA (three samples). PCR sensitivity was affected by inhibitors and by the amount of DNA in the clinical samples. Our results indicate that direct detection of M. bovis in lymph nodes by PCR may be a fast and useful tool for bovine tuberculosis epidemic management in the region.

Evidence for the expression of native Mycobacterium tuberculosis phospholipase C: recognition by immune sera and detection of promoter activity

The genome of Mycobacterium tuberculosis H37Rv contains three contiguous genes (plc-a, plc-b and plc-c) which are similar to the Pseudomonas aeruginosa phospholipase C (PLC) genes. Expression of mycobacterial PLC-a and PLC-b in E. coli and M. smegmatis has been reported, whereas expression of the native proteins in M. tuberculosis H37Rv has not been demonstrated. The objective of the present study was to demonstrate that native PLC-a is expressed in M. tuberculosis H37Rv. Sera from mice immunized with recombinant PLC-a expressed in E. coli were used in immunoblots to evaluate PLC-a expression. The immune serum recognized a 49-kDa protein in immunoblots against M. tuberculosis extracts. No bands were visible in M. tuberculosis culture supernatants or extracts from M. avium, M. bovis and M. smegmatis. A 550-bp DNA fragment upstream of plc-a was cloned in the pJEM12 vector and the existence of a functional promoter was evaluated by detection of ß-galactosidase activity. ß-Galactosidase activity was detected in M. smegmatis transformed with recombinant pJEM12 grown in vitro and inside macrophages. The putative promoter was active both in vitro and in vivo, suggesting that expression is constitutive. In conclusion, expression of non-secreted native PLC-a was demonstrated in M. tuberculosis.

Identificação e diferenciação do Mycobacterium tuberculosis pela amplificação do DNA das regiões intergênicas dos operons inhA e pIC

Entre as doenças causadas por bactérias do gênero Mycobacterium, a tuberculose por M. tuberculosis é a mais conhecida. O diagnóstico da doença é feito utilizando-se um conjunto de exames que possibilitam a identificação da mesma (WATT, 2000). Contudo, sabe-se que o diagnóstico combinado de microscopia direta e com o posterior isolamento em meio de cultivo é o “padrão-ouro”. A principal desvantagem desse método é que tal bactéria possui um crescimento lento (cerca de 8 semanas). Recentemente, a detecção de doenças através da técnica de reação em cadeia da polimerase (PCR) tem proporcionado avanços significativos no diagnóstico. O uso da amplificação específica de genes, para identificar a M. tuberculosis, tais como rDNA 16S, IS6110 ou a região intergênica senX3-regX3, tem apresentado algumas restrições, ao nível de confiabilidade e sensibilidade, para a aplicação da técnica de PCR. O presente estudo mostra a construção e a aplicação de um novo alvo para a aplicação da PCR no diagnóstico da tuberculose, baseado no ensaio da diferença de organização gênica do operon plcA, B e C diferenciando a M. tuberculosis das demais micobactérias. Neste trabalho, foram examinadas 273 amostras de pacientes com suspeita de tuberculose, sendo estas submetidas ao estudo comparativo da técnica de PCR versus cultivo (padrão ouro). A PCR amplificou fragmentos de 439pb. Os resultados mostram 93,7% de acurácia para PCR/Cultivo (p<000,1), 93,1% de sensibilidade com intervalo de confiança de 88,7-96,0 e especificidade de 96,4% com intervalo de confiança de 96,4-99,4. O valor da estatística Kappa (k) foi de 0,82 com erro padrão de 0,041, demonstrando um alinhamento quase perfeito para a verificação do grau de concordância entre os testes. Desta forma, o uso desta nova região para a amplificação da PCR se mostra uma importante e confiável ferramenta no diagnóstico específico da tuberculose. Outra região que compreende parte dos genes mbaA e inhA foi utilizada para diferenciar o Complexo tuberculosis do Complexo avium. Porém, novos experimentos serão necessários para o emprego desta região como uma ferramenta de diagnóstico.

Isolation of Mycobacterium spp. in milk from cows suspected or positive to tuberculosis

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

INCIDENCE OF MYCOBACTERIUM-TUBERCULOSIS AND OTHER MYCOBACTERIA ON PULMONARY INFECTIONS IN ARARAQUARA - SP, 1993

The incidence of tuberculosis and other infections by mycobacteria was analyzed in 559 patients admitted to the Tisiology Section of the Special Health Care Unit of Araraquara (SESA). Mycobacteria were isolated from 78 individuals out of this total. Among these patients, 15 were also HIV positive. The occurrence of isolated species was: M. tuberculosis: 69 patients; M. avium-intracellulare: 5 patients; M. fortuitum: 2 patients; M. chelonae: 1 patient; and M. simiae 1 patient. The latter was for the first time isolated from humans in Brazil. In most cases, non tubercular mycobacteria (NTM) were found in the HIV positive patients.

Ocorrência e identificação molecular de espécies do gênero Mycobacterium e marcadores de virulência em linhagens de Rhodococcus equi isoladas de linfonodos e das fezes de suínos de abatedeouro

Pós-graduação em Medicina Veterinária - FMVZ

Detecção de DNA de micobactérias dos complexos M. tuberculosis e M. avium em amostras parafinadas de pacientes transplantados renais

Introduction: Tuberculosis (TB) is a granulomatous disease caused by Mycobacterium tuberculosis. The genus Mycobacteriumhas two different complexes: M. tuberculosis Complex and M. avium Complex. This is a global health epidemic and remains a major global health problem, besides, the clinical severity of TB is significantly higher in transplanted patients. The detection of these mycobacteria complexes in transplanted patients, by molecular methods, is fundamental for quick treatment of patients and can contribute for rapid and accuracy of diagnosis. Objective: To detect mycobacteria DNA of M. tuberculosis and M. avium Complexes in formalin fixed paraffin-embedded samples (FFPE) of two patients groups: non transplanted and transplanted. Materials and Methods: The study includes 40 FFPE biopsies separated in four groups: NTP – presence of epithelioid granuloma and positive ZN, non-transplanted patients – 9 samples; NTN - presence of epithelioid granuloma and negative ZN, non-transplanted patients – 10 samples; TP – positive ZN, transplanted patients – 9 samples; TN – negative ZN, transplanted patients – 7 samples. Sections were cut for DNA extraction. Samples were submitted to PCR for amplification of: a) β-actin, b) IS6110 insertion and c) IS1245 insertion. DNA evaluation was made by spectrophotometry and efficiency and PCR analysis was made by agarose gels under UV light. Results: In all samples processed, 97.1% were positive for human β-actin gene. In22.2% of NTP group were found the IS6110 insertion sequencebut the IS1245 wasn´t. In the NTN group was not found any sequence. In theTP group, 11.1% of the samples were positive for IS6110 and also 11,1% werepositive for IS1245. In the TN group, 14.3% of the samples were positive forIS6110 and for IS1245, 14.3% was also positive. Conclusion: Although factors such as DNA degradation after formalin fixation and paraffin embedding, were possible to detect DNA from the human gene ...

Animal-side serologic assay for rapid detection of Mycobacterium Bovis infection in multiple species of free-ranging wildlife

Numerous species of mammals are susceptible to Mycobacterium bovis, the causative agent of bovine tuberculosis (TB). Several wildlife hosts have emerged as reservoirs of M. bovis infection for domestic livestock in different countries. In the present study, blood samples were collected from Eurasian badgers (n = 1532), white-tailed deer (n = 463), brushtail possums (n = 129), and wild boar (n = 177) for evaluation of antibody responses to M. bovis infection by a lateral-flow rapid test (RT) and multiantigen print immunoassay (MAPIA). Magnitude of the antibody responses and antigen recognition patterns varied among the animals as determined by MAPIA; however, MPB83 was the most commonly recognized antigen for each host studied. Other seroreactive antigens included ESAT-6, CFP10, and MPB70. The agreement of the RT with culture results varied from 74% for possums to 81% for badgers to 90% for wild boar to 97% for white-tailed deer. Small numbers of wild boar and deer exposed to M. avium infection or paratuberculosis, respectively, did not cross-react in the RT, supporting the high specificity of the assay. In deer, whole blood samples reacted similarly to corresponding serum specimens (97% concordance), demonstrating the potential for field application. As previously demonstrated for badgers and deer, antibody responses to M. bovis infection in wild boar were positively associated with advanced disease. Together, these findings suggest that a rapid TB assay such as the RT may provide a useful screening tool for certain wildlife species that may be implicated in the maintenance and transmission of M. bovis infection to domestic livestock.

Mycobacterium bovis–Infected White-Tailed Deer (Odocoileus Virginianus): Detection of Immunoglobulin Specific to Crude Mycobacterial Antigens by ELISA

White-tailed deer (Odocoileus virginianus) have recently emerged as a source of Mycobacterium bovis infection for cattle within North America. The objective of this study was to evaluate the antibody response of M. bovis–infected deer to crude mycobacterial antigens. Deer were experimentally inoculated with M. bovis strain 1315 either by intratonsilar instillation or by exposure to M. bovis–infected (i.e., in contact) deer. To determine the time course of the response, including the effects of antigen administration for comparative cervical skin testing, serum was collected periodically and evaluated by enzyme-linked immunosorbent assay (ELISA) for immunoglobulin (i.e., IgG heavy and light chains) reactivity to mycobacterial antigens. The reactivity to M. bovis purified protein derivative (PPDb) exceeded (P < 0.05) the reactivity to M. avium PPD (PPDa) only after in vivo administration of PPDa and PPDb for comparative cervical testing of the infected deer. The mean immunoglobulin response, as measured by ELISA, of intratonsilar-inoculated deer to a proteinase K–digested whole-cell sonicate (WCS-PK) of M. bovis strain 1315 exceeded (P < 0.05) the mean of the prechallenge responses to this antigen at approximately 1 month after inoculation and throughout the remainder of the study (i.e., ~11 months). This response also exceeded (P < 0.05) that of the uninfected deer. Although this is encouraging, further studies are necessary to validate the use of the proteinase K–digested M. bovis antigens in the antibody-based assays of tuberculosis.

Evaluation of an In Vitro Blood-Based Assay to Detect Production of Interferon-γ By Mycobacterium Bovis–Infected White-Tailed Deer (Odocoileus Virginianus)

Tuberculosis due to Mycobacterium bovis in captive Cervidae was identified as an important disease in the United States in 1990 and prompted the addition of captive Cervidae to the USDA Uniform Methods and Rules for eradication of bovine tuberculosis. As well, M. bovis infection was identified in free-ranging white-tailed deer in northeast Michigan in 1995. Tuberculosis in both captive and free-ranging Cervidae represents a serious challenge to the eradication of M. bovis infection from the United States. Currently, the only approved antemortem tests for tuberculosis in Cervidae are the intradermal tuberculin skin test and the blood tuberculosis test (BTB). At present, the BTB is not available in North America. Tuberculin skin testing of Cervidae is time-consuming and involves repeated animal handling and risk of injury to animals and humans. This study evaluated the potential of a new blood-based assay for tuberculosis in Cervidae that would decrease animal handling, stress, and losses due to injury. In addition, a blood-based assay could provide a more rapid diagnosis. Twenty 6–9-month-old white-tailed deer, male and female, were experimentally inoculated by instillation of 300 colony-forming units of M. bovis in the tonsillar crypts. Seven, age-matched uninfected deer served as controls. Blood was collected on days 90, 126, 158, 180, 210, 238, 263, and 307 after inoculation and was analyzed for the production of interferon-γ (IFN-γ) in response to incubation with M. bovis purified protein derivative (PPDb), M. avium PPDa, pokeweed mitogen (PWM), or media alone. Production of IFN-g in response to PPDb was significantly greater (P < 0.05) at all time points in samples from M. bovis–infected deer as compared with uninfected control deer, whereas IFN-γ production to PWM did not differ significantly between infected and control deer. Measurement of IFN-γ production to PPDb may serve as a useful assay for the antemortem diagnosis of tuberculosis in Cervidae.

EPIDEMIOLOGIC TRENDS OF LUNG DISEASE DUE TO MYCOBACTERIA KANSASII AND AVIUM-INTRACELLULARE IN TEXAS, 1977-1983

This study compared the reported isolations of Mycobacterium kansasii (MK) and Myobacterium avium-intracellulare (MAI) with Mycobacterium tuberculosis (MTB) between 1977-1983 in Texas. A total of 15,395 mycobacterial cases were identified of which 1,352 (8.8%) were MK or MAI. The incidence of MK was higher in urban areas than nonurban areas (p < .005). The incidence of MAI has increased in the Dallas metroplex from 34 cases to 251 for the same time period. Although the number of MK cases previously reported has always exceeded those of MAI, the numbers were equal in the last year (1983) of the study.^ More than 75% of patients with MK or MAI were Caucasians compared to only 18% of patients with MTB. Male to female ratios for MK and MAI are 3:1 and 3:2, respectively. The age distribution of MK patients were an average of 5 years younger than patients with MAI, a finding which concurs with previous studies. MK and MAI pulmonary infections continue to be absent among children and relatively absent among Hispanics.^ MK appears to be associated with occupations in construction, whereas MAI is more often associated with farm work. ^

Tuberculosis determined by Mycobacterium bovis in captive waterbucks (Kobus ellipsiprymnus) in São Paulo, Brazil

Two waterbucks from São Paulo Zoo Foundation exhibited respiratory symptoms in July 2004. After euthanasia, granulommas in lungs and mediastinic lymph nodes were observed. Acid-fast bacilli isolated were identified as Mycobacterium bovis spoligotype SB0121 by PRA and spoligotyping. They were born and kept in the same enclosure with the same group, without any contact to other species housed in the zoo. This is the first detailed description of M. bovis infection in Kobus ellipsiprymnus.

Comparison of three decontamination methods for Mycobacterium bovis isolation

Sixty samples of tissue fragments with lesions suggestive of tuberculosis from bovine abattoirs, kept in saturated solution of sodium borate, were subjected to four treatments: 4% NaOH (Petroff Method), 12 % H2SO4 and 1.5% HPC (1-Hexadecylpyridinium Chloride) decontamination, and physiological saline solution (control). The HPC method showed the lowest contamination rate (3%) when compared to control (88%, p<0.001), NaOH (33%, p<0.001) and H2SO4 (21.7%, p<0.002). Regarding the isolation success, the HPC method was better (40%) than the control (3%, p<0.001), NaOH (13%, p=0.001) and H2SO4 (1.7%, p<0.001) methods. These results indicate that HPC is an alternative to the Petroff method.