ABSTRACT
BACKGROUND: Multiple techniques exist for detecting Mycobacteria, each having its own advantages and drawbacks. Among them, automated culture-based systems like the BACTEC-MGIT™ are popular because they are inexpensive, reliable and highly accurate. However, they have a relatively long "time-to-detection" (TTD). Hence, a method that retains the reliability and low-cost of the MGIT system, while reducing TTD would be highly desirable. METHODS: Living bacterial cells possess a membrane potential, on account of which they store charge when subjected to an AC-field. This charge storage (bulk capacitance) can be estimated using impedance measurements at multiple frequencies. An increase in the number of living cells during culture is reflected in an increase in bulk capacitance, and this forms the basis of our detection. M. bovis BCG and M. smegmatis suspensions with differing initial loads are cultured in MGIT media supplemented with OADC and Middlebrook 7H9 media respectively, electrical "scans" taken at regular intervals and the bulk capacitance estimated from the scans. Bulk capacitance estimates at later time-points are statistically compared to the suspension's baseline value. A statistically significant increase is assumed to indicate the presence of proliferating mycobacteria. RESULTS: Our TTDs were 60 and 36 h for M. bovis BCG and 20 and 9 h for M. smegmatis with initial loads of 1000 CFU/ml and 100,000 CFU/ml respectively. The corresponding TTDs for the commercial BACTEC MGIT 960 system were 131 and 84.6 h for M. bovis BCG and 41.7 and 12 h for M smegmatis, respectively. CONCLUSION: Our culture-based detection method using multi-frequency impedance measurements is capable of detecting mycobacteria faster than current commercial systems.
Subject(s)
Humans , Bacteriological Techniques/methods , Dielectric Spectroscopy , Mycobacterium/isolation & purification , Mycobacterium/growth & development , Time Factors , Reproducibility of Results , Culture Media , Mycobacterium/classificationABSTRACT
The objective of this report is to provide information on Mycobacterium tuberculosis complex infections in animals and in humans. Included is information on the susceptibility of different species as well as information on etiology, epidemiology, pathogenesis, diagnosis, prevention and control of this disease. The term One Health has been adopted to describe the unified human medical and veterinary interdisciplinary/multidisciplinary collaborative approach to zoonoses and will be critical for future endeavors in the control of the global TB epidemic. This unified paradigm is ideally suited for control of bovine TB and many other international public health and clinical health issues. Sharing resources and increasing interaction between public health and veterinary medical scientists can raise awareness of ‘shared risk' of bovine TB between humans and animals and, in resource-limited situations, can maximize use of existing infrastructure and reduce unnecessary duplication of effort in disease control programs.
El objetivo de este artículo es proporcionar información sobre las infecciones por el Complejo Mycobacterium tuberculosis en animales y en humanos. Se incluye información sobre la susceptibilidad de diferentes especies, así como sobre la etiología, epidemiología, patogenia, diagnóstico, prevención y control de esta enfermedad. La expresión UNA SALUD ha sido adoptada para describir el enfoque unificado de la medicina humana y la veterinaria, de colaboración interdisciplinaria/multidisciplinaria en las zoonosis, que puede resultar fundamental para el control de la endemia mundial de tuberculosis. Este paradigma unificado es especialmente relevante para el control de la tuberculosis bovina. Compartir recursos y lograr una mayor interacción entre la investigación en salud pública y en medicina veterinaria puede elevar la conciencia de “riesgo compartido” de la tuberculosis bovina en humanos y animales y, en situaciones de recursos limitados, puede maximizar el uso de la infraestructura existente y reducir la duplicación innecesaria de esfuerzos en los programas de control de la infección y enfermedad.
Subject(s)
Humans , Animals , Tuberculosis/prevention & control , Tuberculosis/veterinary , Zoonoses/prevention & control , One Health , Tuberculosis/diagnosis , United States/epidemiology , Cattle , Zoonoses/microbiology , Zoonoses/epidemiology , Public Health , Mycobacterium bovis/pathogenicity , Mycobacterium tuberculosis/pathogenicityABSTRACT
The presence of intestinal helminths can down-regulate the immune response required to control mycobacterial infection. BALB/c mice infected with Mycobacterium bovis following an infection with the intestinal helminth Strongyloides venezuelensis showed reduced interleukin-17A production by lung cells and increased bacterial burden. Also, small granulomas and a high accumulation of cells expressing the inhibitory molecule CTLA-4 were observed in the lung. These data suggest that intestinal helminth infection could have a detrimental effect on the control of tuberculosis (TB) and render coinfected individuals more susceptible to the development of TB.
Subject(s)
Animals , Mice , /biosynthesis , Intestinal Diseases, Parasitic/immunology , Mycobacterium Infections/immunology , Mycobacterium bovis/immunology , Strongyloides/immunology , Strongyloidiasis/immunology , Bacterial Load/methods , Coinfection , Coinfection/immunology , Coinfection/pathology , Disease Susceptibility , Intestinal Diseases, Parasitic , Intestinal Diseases, Parasitic/pathology , Lung , Lung , Mice, Inbred BALB C , Mycobacterium Infections , Mycobacterium Infections/pathology , Strongyloidiasis , Strongyloidiasis/pathologyABSTRACT
Las pruebas de tuberculina son las de uso generalizado para el diagnóstico y el control de la tuberculosis (TBC) en el hombre y en los animales. Se caracteriza por una compleja mezcla de antígenos de mycobacterias capaces de inducir reacciones de hipersensibilidad en animales infectados, incluso con mycobacterias diferentes al Mycobacterium bovis, por efectos de reactividad cruzada. La preparación del derivado protéico purificado (PPD), es similar a la de la tuberculina, a diferencia de la concentración de proteínas, las cuales se separan por precipitación con agentes químicos y no por calor, aumentando su especificidad. Los primeros resultados obtenidos con las pruebas serológicas para el diagnóstico de tuberculosis bovina muestran que existe una gran reactividad antigénica cruzada entre las especies de mycobacterias, por lo que se requiere de antígenos más específicos. Se implementó un ELISA-TBC para la detección de anticuerpos anti M. bovis. El ensayo inmunoenzimático para el IFN-y bovino cuando se utilizó conjuntamente con el sistema de cultivo de sangre completa resultó en un ensayo in vitro rápido y sensible para detectar la reactividad de la inmunidad mediada por células al M. bovis en el ganado infectado. A partir de estas pruebas se compararon los resultados obtenidos para establecer la sensibilidad y especificidad utilizando como prueba oro, los datos obtenidos en el cultivo bacteriológico y la reacción en cadena de la polimerasa (PCR). Los animales reaccionantes a la tuberculina incluyeron animales positivos a PPD-B y PPD-A, así como animales negativos a cultivo bacteriológico y PCR. Los PPD-B positivos, no son en su totalidad, los mismos reaccionantes al IFN-y o al ELISA-TBC. Aún cuando su sensibilidad es baja, muestra mayor especificidad y concordancia que el resto de las pruebas utilizadas.
The tuberculin tests are widely used for diagnosis and control of tuberculosis (TB) in humans and animals. It is characterized by a complex mixture of mycobacteria antigens able to induce hypersensitivity reactions even in animals infected with mycobacteria other than M. bovis, for purposes of cross-reactivity. The preparation of purified protein derivative (PPD) is similar to the tuberculin, unlike the concentration of proteins which are separated by precipitation with chemical agents and not by increasing its specific heat. The first results obtained with the serological tests for diagnosis of bovine tuberculosis show that there is a great antigenic cross-reactivity between mycobacterias species so it requires more specific antigens. It implemented a cattle IFN-g test and ELISA-TBC to detect anti M. bovis activity. The immunoassay test for IFN-g used in conjunction with the cropping system of whole blood resulted in an essay in vitro rapid and sensitive to detect the reactivity of the cell-mediated immunity to M. bovis in livestock infected. Comparative test of the tuberculina, test of Gamma Interferon (INF-y) and a test ELISA-TBC, soon was taken to slaughter house to take linfoides weave samples and nodules, to which the test of chain reaction of Polimerasa was applied to them, to bacteriological culture and (PCR), for the identification from the pathogen. From these tests the patterns of immune response settled down and the obtained results of the different tests were compared to establish sensitivity and specificity using with t gold standard, the data collected in culture and PCR. The results were analyzed using the statistical method of analysis of variance for nonparametric tests. The PPD B-positive, are not the same reacting to IFN-y or at ELISA-TBC. Although its sensitivity is low, it shows greater specificity and consistency as the rest of the tests used.
Subject(s)
Cattle , Animals , Clonal Anergy , Mycobacterium bovis , Enzyme-Linked Immunosorbent Assay/methods , Enzyme-Linked Immunosorbent Assay/veterinary , Tuberculin Test/methods , Tuberculin Test/veterinary , Tuberculosis, Bovine/diagnosis , Veterinary MedicineABSTRACT
With the purpose of identifying management factors that may be influencing the prevalence of bovine tuberculosis under tropicalconditions, namely in Rio de Janeiro, Brazil, 1632 cows were tested through the single cervical tuberculin test. A questionnairewas completed for each herd. A total of 207 positive reactions were observed, corresponding to 12.7% of the studied cattle. Themain factors observed that may be influencing the prevalence of bovine tuberculosis on those farms were the absence orreduced veterinary assistance and the herd size. The presence of adequate cattle houses and the highly intensive managementare also considered to be likely to influence the prevalence of the disease. Under tropical conditions, a tuberculosis controlprogram, in addition to the test-and-slaughter control method, should include an investigation of herd management practices totry to identify factors that are likely to influence the prevalence of the disease.
ABSTRACT
Mycobacterium tuberculosis es el agente causal de tuberculosis en humanos, sin embargo, la transmisión zoonótica de Mycobacterium bovis de animales a humanos, principalmente a individuos inmunocomprometidos, es de gran impacto en salud pública. La necesidad de diseñar métodos rápidos y precisos que permitan diferenciar entre M. tuberculosis y M. bovis ha conducido al desarrollo de estrategias de identificación genotípica. El objetivo de este estudio fue evaluar el poder discriminatorio de un ensayo de reacción en cadena de la polimerasa en formato múltiple para diferenciar entre aislados clínicos de M. tuberculosis y M. bovis. Se estandarizaron condiciones para amplificación simultánea con oligonucleótidos dirigidos a secuencias de genes Rv0577 y Rv1510. Rv0577 es específico de micobacterias del Complejo M. tuberculosis y Rv1510 se localiza en regiones polimórficas (RD4) del genoma de cepas del complejo M. tuberculosis, ausentes en M. bovis y M. bovis BCG, permitiendo diferenciar entre estas dos especies del complejo M. tuberculosis. Para evaluar la especificidad del ensayo se analizaron 46 aislados clínicos. El amplicón correspondiente a Rv0577 se detectó en todos los aislados clínicos del complejo M. tuberculosis, mientras que el producto de 1033 pb Rv1510 (RD4), se observó exclusivamente en M. tuberculosis, pero no en M. bovis, ni en M. bovis BCG, demostrando la especificidad de este ensayo de RCP para identificar micobacterias del complejo M.tuberculosis y diferenciar entre M. tuberculosis y M. bovis en una única reacción de amplificación. Dada la especificidad del ensayo, éste puede ser aplicado para la identificación y diferenciación de micobacterias del complejo M. tuberculosis en muestras clínicas, para hacer control de calidad de productos derivados de animales con sospechas de infección y desarrollar estudios a gran escala de transmisión zoonótica de M. bovis en poblaciones vulnerables.
Mycobacterium tuberculosis is the causal agent of tuberculosis in humans, however, the zoonotic transmission of Mycobacterium bovis from animals to humans, mainly immunocompromised individuals, is of great impact in public health. The necessity to design rapid and precise methods to differentiate between M. tuberculosis and M. bovis has lead to the development of strategies of genotypic identification. The aim of this study was to evaluate the discriminatory power of a multiplex PCR assay to differentiate between M. tuberculosis and M. bovis clinical isolates. Conditions for simultaneous amplification with oligonucleótidos targeted Rv0577 and Rv1510 genes sequences were standardized. Rv0577 is specific of M. tuberculosis complex mycobacteria and Rv1510 is located in polymorphic regions (RD4) of the mycobacterial genome of M. tuberculosis complex, but is absent in M. bovis and M. bovis BCG, allowing to differentiate between M. tuberculosis complex strains. To evaluate the specificity of the PCR assay, 46 clinical isolates were analyzed. The amplicon from Rv0577 gene was detected in all of the clinical isolates from M. tuberculosis complex, whereas the 1033 pb product from Rv1510 (RD4), was exclusively observed in M. tuberculosis and was absent in M. bovis and M. bovis BCG, showing the specificity of this PCR assay to identify mycobacteria of the M. tuberculosis complex and to differentiate between M. tuberculosis and M. bovis in a single-step assay. This multiplex PCR assay can be applied to the identification and differentiation of mycobacteria of M. tuberculosis complex in clinical samples, to the quality control of products derived from animals with infection suspicions and to develop studies on great scale of zoonotic transmission of M. bovis in susceptible populations.