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1.
Appl Environ Microbiol ; 79(5): 1612-8, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23275511

RESUMO

Paratuberculosis is an infectious, chronic, and incurable disease that affects ruminants, caused by Mycobacterium avium subsp. paratuberculosis. This bacterium is shed primarily through feces of infected cows but can be also excreted in colostrum and milk and might survive pasteurization. Since an association of genomic sequences of M. avium subsp. paratuberculosis in patients with Crohn's disease has been described; it is of interest to rapidly detect M. avium subsp. paratuberculosis in milk for human consumption. IS900 insertion is used as a target for PCR amplification to identify the presence of M. avium subsp. paratuberculosis in biological samples. Two target sequences were selected: IS1 (155 bp) and IS2 (94 bp). These fragments have a 100% identity among all M. avium subsp. paratuberculosis strains sequenced. M. avium subsp. paratuberculosis was specifically concentrated from milk samples by immunomagnetic separation prior to performing PCR. The amplicons were characterized using DNA methylase Genotyping, i.e., the amplicons were methylated with 6-methyl-adenine and digested with restriction enzymes to confirm their identity. The methylated amplicons from 100 CFU of M. avium subsp. paratuberculosis can be visualized in a Western blot format using an anti-6-methyl-adenine monoclonal antibody. The use of DNA methyltransferase genotyping coupled to a scintillation proximity assay allows for the detection of up to 10 CFU of M. avium subsp. paratuberculosis per ml of milk. This test is rapid and sensitive and allows for automation and thus multiple samples can be tested at the same time.


Assuntos
Metilases de Modificação do DNA/metabolismo , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Leite/microbiologia , Técnicas de Diagnóstico Molecular/métodos , Tipagem Molecular/métodos , Mycobacterium avium subsp. paratuberculosis/isolamento & purificação , Animais , Bovinos , Enzimas de Restrição do DNA/metabolismo , Mycobacterium avium subsp. paratuberculosis/classificação , Mycobacterium avium subsp. paratuberculosis/genética , Paratuberculose/microbiologia , Sensibilidade e Especificidade
2.
Braz J Microbiol ; 47(2): 506-12, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26991290

RESUMO

The aim of this study was to standardize a diagnosis procedure to detect Mycobacterium avium subsp. paratuberculosis (Map) DNA in raw cow milk samples under field conditions. A procedure that combines both immunomagnetic separation and IS900-PCR detection (IMS-IS1 PCR) was employed on milk samples from 265 lactating Holstein cows from Map infected and uninfected herds in Argentina. IMS-IS1 PCR results were analyzed and compared with those obtained from milk and fecal culture and serum ELISA. The extent of agreement between both tests was determined by the Kappa test. IMS-IS1 PCR showed a detection limit of 10(1) CFU of Map/mL of milk, when 50:50 mix of monoclonal and polyclonal antibodies were used to coat magnetic beads. All of the 118 samples from the Map uninfected herds were negative for the set of the tests. In Map infected herds, 80 out of 147 cows tested positive by milk IMS-IS1 PCR (55%), of which 2 (1.4%) were also positive by milk culture, 15 (10%) by fecal culture, and 20 (14%) by serum ELISA. Kappa statistics (95% CI) showed a slight agreement between the different tests (<0.20), and the proportions of agreement were ≤0.55. The IMS-IS1 PCR method detected Map in milk of the cows that were not positive in other techniques. This is the first report dealing with the application of IMS-IS1 PCR in the detection of Map in raw milk samples under field conditions in Argentina.


Assuntos
Doenças dos Bovinos/microbiologia , Separação Imunomagnética/métodos , Leite/microbiologia , Mycobacterium avium subsp. paratuberculosis/isolamento & purificação , Paratuberculose/microbiologia , Reação em Cadeia da Polimerase/métodos , Animais , Argentina , Bovinos , Doenças dos Bovinos/diagnóstico , Doenças dos Bovinos/fisiopatologia , Fezes/microbiologia , Feminino , Lactação , Leite/química , Mycobacterium avium subsp. paratuberculosis/química , Mycobacterium avium subsp. paratuberculosis/genética , Paratuberculose/diagnóstico , Paratuberculose/fisiopatologia
3.
Vet Med Int ; 2012: 145318, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22792511

RESUMO

Johne's Disease or Paratuberculosis is a chronic granulomatous enteritis disease affecting ruminants. Detection of subclinically infected animals is difficult, hampering the control of this disease. The aim of this work was to evaluate the performance of detection of IgG isotypes in a PPA-ELISA to improve the recognition of cattle naturally infected with Map in different stages. A total of 108 animals from Tuberculosis-free herds were grouped as follows: exposed (n = 30), subclinically infected (n = 26), clinically infected (n = 14), and healthy controls (n = 38). Receiver-operating characteristic (ROC) curves of isotypes/PPA-ELISAs were constructed and areas under the curves were compared to evaluate the performance of each test. Our study demonstrated that the conventional PPA-ELISA (detecting IgG) is the best to identify clinically infected animals with high sensitivity (92.9%) and specificity (100%). Meanwhile, IgG2/PPA-ELISA improved the number of subclinically infected cattle detected as compared with conventional IgG/PPA-ELISA (53.8 versus 23.1%). In addition, it had the maximum sensitivity (65.0%, taking into account all Map-infected cattle). In conclusion, the combination of IgG and IgG2/PPA-ELISAs may improve the identification of Map-infected cattle in different stages of disease. The usefulness of IgG2 detection in serological tests for Johne's Disease diagnosis should be further evaluated.

4.
Braz. j. microbiol ; 47(2): 506-512, Apr.-June 2016. tab, graf
Artigo em Inglês | LILACS | ID: lil-780831

RESUMO

Abstract The aim of this study was to standardize a diagnosis procedure to detect Mycobacterium avium subsp. paratuberculosis (Map) DNA in raw cow milk samples under field conditions. A procedure that combines both immunomagnetic separation and IS900 -PCR detection (IMS-IS1 PCR) was employed on milk samples from 265 lactating Holstein cows from Map infected and uninfected herds in Argentina. IMS-IS1 PCR results were analyzed and compared with those obtained from milk and fecal culture and serum ELISA. The extent of agreement between both tests was determined by the Kappa test. IMS-IS1 PCR showed a detection limit of 101 CFU of Map/mL of milk, when 50:50 mix of monoclonal and polyclonal antibodies were used to coat magnetic beads. All of the 118 samples from the Map uninfected herds were negative for the set of the tests. In Map infected herds, 80 out of 147 cows tested positive by milk IMS-IS1 PCR (55%), of which 2 (1.4%) were also positive by milk culture, 15 (10%) by fecal culture, and 20 (14%) by serum ELISA. Kappa statistics (95% CI) showed a slight agreement between the different tests (<0.20), and the proportions of agreement were ≤0.55. The IMS-IS1 PCR method detected Map in milk of the cows that were not positive in other techniques. This is the first report dealing with the application of IMS-IS1 PCR in the detection of Map in raw milk samples under field conditions in Argentina.


Assuntos
Paratuberculose/microbiologia , Doenças dos Bovinos/microbiologia , Reação em Cadeia da Polimerase/métodos , Mycobacterium avium subsp. paratuberculosis/isolamento & purificação , Separação Imunomagnética/métodos , Leite/microbiologia , Paratuberculose/diagnóstico , Paratuberculose/fisiopatologia , Argentina , Lactação , Bovinos , Doenças dos Bovinos/diagnóstico , Doenças dos Bovinos/fisiopatologia , Mycobacterium avium subsp. paratuberculosis/genética , Mycobacterium avium subsp. paratuberculosis/química , Leite/química , Fezes/microbiologia
5.
Braz. j. vet. res. anim. sci ; 45(4): 284-288, 2008. tab
Artigo em Inglês | LILACS | ID: lil-489109

RESUMO

Six Doberman Pinscher, between six and eight years of age, were presented to the Veterinary Hospital from Faculty of Veterinary Science of The University of Buenos Aires. Neurological examination revealed tetraparesis with inability to walk, decreased muscle tonus and myotatic reflexes in all dogs. Serum cholesterol levels, creatine kinase and alkaline phosphatase activities were mildly to markedly elevated, and tibial motor nerve conduction velocities were slow in all dogs. Basal measurements of free T4 and TSH were determined by radioimmunoassay. Although fT4 values were within normal range, in all dogs TSH values were elevated. Based on this results, hypothyroidism was diagnosed and a supplementation therapy was established with oral levothyroxine (T4). Two weeks after treatment has been started, all patients had an improvement in clinical signs, and within a month gait became normal, as well as muscular tonus and spinal reflexes.


Seis Dobermans Pinscher, entre seis e oito anos de idade, foram encaminhados ao Hospital Veterinário da Faculdade de Ciências Veterinárias da Universidade de Buenos Aires. O exame neurológico revelou tetraparesia com incapacidade para andar, diminuição do tono muscular e de reflexos miotáticos em todos os cães. Os níveis de colesterol, creatina quinase e fosfatase alcalina encontravam-se de moderada a acentuadamente elevados. As velocidades de condução nervosa do nervo tibial estavam diminuídas em todos os cães. Os níveis séricos basais de T4 livre e TSH foram determinados por radioimunoensaio e, embora os valores de T4 livre estivessem dentro dos limites de normalidade, em todos os cães o TSH estava elevado. Baseado nestes resultados, diagnosticou-se hipotireoidismo e iniciou-se uma suplementação oral com levotiroxina (T4). Duas semanas após o início do tratamento todos os pacientes tiveram melhora clínica e, dentro de um mês a locomoção, o tono muscular e os reflexos medulares tornaram-se normais.


Assuntos
Animais , Cães , Hipotireoidismo/complicações , Polineuropatias/diagnóstico , Tiroxina/análise
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