Detection of Mycobacterium avium subsp. paratuberculosis in bovine milk from the state of Pernambuco, Brazil

Abstract The aim of this study was to detect the IS900 region of Mycobacterium avium subsp. paratuberculosis (MAP) in bovine milk samples using real-time polymerase chain reaction (qPCR) and conventional PCR, and to study the agreement between these tests. A total of 121 bovine milk samples were collected from herds considered positive for MAP, from the State of Pernambuco, Brazil. MAP DNA was detected in 20 samples (16.5%) using conventional PCR and in 34 samples (28.1%) using qPCR. MAP DNA was detected in all of the 6 animal farms studied. Moderate agreement was found between qPCR and conventional PCR results, where the sensitivity and specificity of conventional PCR in relation to qPCR were 50% and 96.6%, respectively. Thus, the IS900 region of MAP was found in bovine milk samples from the State of Pernambuco. To the best of our knowledge, this is the first report of MAP DNA found in bovine milk in Northeast Brazil. We also demonstrated the qPCR technique is more sensitive than conventional PCR with respect to detection of MAP in milk samples.

Mycobacterium paratuberculosis detection in cow's milk in Argentina by immunomagnetic separation-PCR

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.

Molecular typing of Argentinian Mycobacterium avium subsp. paratuberculosis isolates by multiple-locus variable number-tandem repeat analysis

Multiple-locus variable number-tandem repeat analysis (MLVA) of Mycobacterium avium subspecies paratuberculosis (MAP) isolates may contribute to the knowledge of strain diversity in Argentina. Although the diversity of MAP has been previously investigated in Argentina using IS900-RFLP, a small number of isolates were employed, and a low discriminative power was reached. The aim of the present study was to test the genetic diversity among MAP isolates using an MLVA approach based on 8 repetitive loci. We studied 97 isolates from cattle, goat and sheep and could describe 7 different patterns: INMV1, INMV2, INMV11, INMV13, INMV16, INMV33 and one incomplete pattern. INMV1 and INMV2 were the most frequent patterns, grouping 76.3% of the isolates. We were also able to demonstrate the coexistence of genotypes in herds and co-infection at the organism level. This study shows that all the patterns described are common to those described in Europe, suggesting an epidemiological link between the continents.

Evaluation of ‘cattle’ and ‘Indian Bison’ type antigens of Mycobacterium avium subspecies paratuberculosis for diagnosis of Bovine Johne’s Disease using ‘indigenous ELISA’ and AGPT .

Two antigens (‘cattle’ type and ‘Indian Bison’ type) of Mycobacterium avium subspecies paratuberculosis were evaluated for diagnosis of Johne’s disease (JD) in a gaushala (cattle herd). Of the 160 cows of Sahiwal and Hariana breeds screened, 81 (50.6%) tested positive in ELISA and 66 (41.8%) in AGPT test. Using the two antigens, 33.5% tested positive in both the tests while 41.1% tested negative. Exclusively, only 8.2% tested positive in ELISA while 17.1% tested positive in AGPT. Two antigens together detected 58.9% prevalence of MAP in the gaushala. Individually, indigenous ELISA using antigen from native source of MAP proved superior to AGPT in the diagnosis of JD in cows.

First identification of Mycobacterium avium paratuberculosis sheep strain in Argentina

We here identified for the first time the presence of Mycobacterium avium paratuberculosis (MAP) sheep (S) strain in Argentina. IS900 polymerase chain reaction (PCR) was positive. The S strain was compared with MAP cattle (C) strains by using IS1311 PCR-restriction endonuclease analysis (PCR-REA), multiplex PCR and restriction fragment length polymorphism (RFLP) analysis.

Obtención y evaluación de un derivado proteico purificado de una cepa argentina de Mycobacterium avium subsp. paratuberculosis / Production and evaluation of a purified protein derivative from an Argentine strain of Mycobacterium avium subsp. paratuberculosis (MAP)

Los derivados proteicos purificados (PPD) son mezclas antigénicas no definidas obtenidas de distintas micobacterias. Los PPD bovino (PPDb) y PPD aviar (PPDa) son los antígenos que se emplean para evaluar la respuesta inmunitaria celular en infecciones como tuberculosis y paratuberculosis en el bovino. El PPDa comercial se produce a partir de Mycobacterium avium subsp. avium, y no a partir de la subespecie paratuberculosis. En este trabajo se seleccionó una cepa local de Mycobacterium avium subsp. paratuberculosis cuyo patrón molecular por RFLP es el más frecuente entre los aislamientos de nuestro país que han sido estudiados, y a partir de esta, se obtuvo un derivado proteico purificado: PPDj-IB. Se emplearon tanto el PPDa comercial como el PPDj-IB como antígenos en la prueba de liberación de gamma-interferón en animales de un tambo con paratuberculosis y en animales control. Aun cuando ambos PPD fueron capaces de estimular diferencialmente la liberación de la citoquina en el tambo infectado (respecto de los tambos control), no hubo diferencias significativas en los niveles de estimulación producidos y solo dos animales fueron positivos mediante el empleo de PPDj-IB. A partir del análisis por Western blot se demostró que el contenido de lipoarabinomano y del antígeno Apa/ModD era distinto en los PDD evaluados. Estas diferencias podrían explicar, en parte, las diferencias en los niveles de estimulación en términos individuales. Si bien el empleo de PPDj-IB no mejoró significativamente los resultados de la prueba de liberación de ?IFN, es importante destacar que se logró producir en el laboratorio un PPD apto para su empleo en ensayos in vitro.

Purified Protein Derivatives (PPDs) are non-defined antigens prepared from mycobacteria cultures. They are usually employed to evaluate the specific cellular immune response both in animals and humans. Bovine and avian PPDs are usually employed as antigens in mycobacterial infections such as tuberculosis and paratuberculosis. Nevertheless, PPD from Mycobacterium avium subsp. paratuberculosis, (PPDj) is neither commonly used nor frequently available. However, PPD from Mycobacterium avium subsp. avium is in fact used. We aimed to obtain and evaluate the performance of a PPDj from a local isolate of MAP using the ãInterferon-release assay. The stimulation of ãInterferon-release was significantly different between infected and control cattle when this antigen, named PPDj-IB, was used. Stimulation in the infected animals was similar with both antigens (PPDa and PPDj-IB). However, some animals were positively stimulated with PPDj-IB and not with PPDa. We demonstrated by Western blot that two antigenic molecules, lipoarabinoman and APA/ModD antigen were differentially represented in both PPDs. This could explain the difference in stimulation induction of ?IFN observed at individual level. Although PPDj-IB could not improve PPDa performance, we could easily produce an effective purified protein derivative for in vitro assays.

Aspectos clínico-patológicos e controle da paratuberculose em rebanho bovino leiteiro / Clinic-pathological aspects and control of paratuberculosis in a dairy cattle herd

A paratuberculose ou doença de Johne é uma enterite granulomatosa causada por Mycobacterium avium subsp. paratuberculosis. Descrevem-se os aspectos epidemiológicos, clínico-patológicos e laboratoriais da paratuberculose em rebanho bovino leiteiro no município de Rio Claro, região Sul do Estado do Rio de Janeiro. No período de 2006 a 2009, oito vacas adultas da raça Girolanda apresentaram diarreia crônico-intermitente e perda progressiva de peso. À necropsia, observaram-se linfonodos mesentéricos aumentados de volume e úmidos ao corte, vasos linfáticos subserosos das alças intestinais proeminentes, serosa do intestino com aspecto anelado e cerebroide e a mucosa espessada, pregueada e com aspecto microgranular. À microscopia havia, desde o duodeno até o reto, inflamação granulomatosa difusa, marcada dilatação dos vasos linfáticos no ápice das vilosidades, linfangiectasia e linfangite granulomatosa na submucosa, muscular e serosa. A inflamação granulomatosa também foi vista nos linfonodos mesentéricos. A coloração de Ziehl-Neelsen revelou variável quantidade de bacilos álcool-ácido resistentes no interior de macrófagos, de células gigantes de Langhans e livres na mucosa e submucosa dos intestinos delgado e grosso e em linfonodos mesentéricos. Em alguns animais, a lâmina própria da mucosa, principalmente do jejuno e íleo exibia acentuada hipertrofia. Mycobacterium avium subsp. paratuberculosis foi isolado em cultivo bacteriano de Herrold com micobactina, a partir de amostras de fezes, de raspado de mucosa intestinal e de leite e identificado pela técnica de PCR IS900. Através da avaliação sorológica semestral, foram analisadas 298 vacas do mesmo rebanho a partir de três anos de idade, observou-se cerca de 40 por cento de animais reagentes ao teste ELISA indireto no período estudado. O diagnóstico da paratuberculose foi baseado nos dados clínico-patológicos, na sorologia, no isolamento e identificação do agente através de cultivo bacteriano e PCR IS900. Após implementação de medidas de controle, tais como eliminação de animais doentes, abate seletivo dos animais soropositivos, separação dos bezerros ao nascer e utilização de banco de colostro, observou-se, nos três anos de estudo, diminuição da ocorrência de casos clínicos no rebanho, de seis casos por ano para cerca de um caso por ano.

Paratuberculosis (Johne's disease) is a granulomatous enteritis of ruminants caused by Mycobacterium avium subsp. paratuberculosis. Epidemiology, clinic-pathological and laboratorial aspects of paratuberculosis in a dairy cattle herd are described. The disease was diagnosed from 2006 to 2009 in eight cows that presented chronic-intermittent diarrhea and chronic weight loss, in the Rio Claro municipality, Rio de Janeiro. At necropsy, the subserosal lymphatic vessels were proeminent and dilated, mesenteric nodes were enlarged and intestinal mucosa was corrugated, thickened and of microgranular aspect. From duodenum to the rectum, histopathology revealed severe and diffuse granulomatous inflammation of the lamina propria and submucosa, broadened and distorted villi, dilatation of the lymphatic vessels in their apex, lymphangioectasia and granulomatous lymphangitis in the submucosa. Ziehl-Neelsen stain showed variable amounts of acid-fast bacilli in macrophages, in Langhans giant cells and freely in the mucosa and submucosa of the small intestine, colon and lymphnodes. In some cows, the lamina propria presented severe hypertrophy, mainly in the jejunum and ileum. Mycobacterium avium subsp. paratuberculosis was isolated through bacterial cultivation of samples taken from feces, intestinal mucosa and milk, and identified through IS900 PCR. From 298 cows older than three years, the percentage of reactive animals was 40 percent by indirect ELISA test. The diagnosis of paratuberculosis was based on clinic-epidemiological data, serology, bacterial isolation in Herrold egg yolk medium with micobactin and on IS900 PCR. After the adoption of control measures, as slaughter of cows with clinical signs, selective slaughter of seropositive cows, removal of the calf from the dam at birth, and use of the colostrum bank, we observed a reduction from six clinical cases to only one case per year, in the last three years of the study.

Strain diversity within Mycobacterium avium subspecies paratuberculosis — A review.

Mycobacterium avium subspecies paratuberculosis (MAP), is the etiological agent of Johne’s disease (or paratuberculosis) in animals and has also been linked with Crohn’s disease of human beings. Extreme fastidious nature of the organism (MAP) has hampered studies on diversity within the organism. Studies based on phenotypic properties like growth rate, pigmentation, lipid profile etc., are unable to provide complete information on diversity of MAP organism in nature. However, with the advent of molecular assays (IS900 RFLP, PFGE, IS1311 PCR-REA, SSR typing, VNTR typing etc.) in last 2 decades, progress has been made to differentiate MAP strains. MAP isolates have been classified into various types and subtypes using these molecular tools. Optimization of these typing assays has led to generation of new information about MAP strains, subtypes, their comparative genomics, relative evolution, comparative virulence etc. Knowledge of strain diversity is important for better understanding of molecular and sero-epidemiology, infection and patho-biology, vaccine development and planning control strategies. The present review provides available information on MAP strains, host adaptations, their virulence, comparative genomics, relative genetic evolution and differentiation.

[Identification of mycobacterium avium subsp paratuberculosis in fecal samples of holstein-friesian cattle using molecular and cultivation methods]

Mycohacterium avium subspecies of Paratuberculosis is the cause of paratuberculosis or Johne's disease. Paratuherculosis is a chronic and progressive infectious enteric disease that affects domestic and wild animals, mostly ruminants. This study was carried out for detection of subclinical forms of Johne's disease using direct PCR and cultivation methods. A total 119 fecal samples were collected Holstein-Friesian cattle herds in Razavi khorasan province of Iran. Among these, 16 samples were taken from cows with advanced clinical signs of Johne's disease. 101 were taken from cows without clinical signs. The fecal samples were tested for presence of Mycobacterium paratuberculosis by directed Nested-PCR and culture on Herrold s media with and without Mycobactin J. The samples of cows with clinical signs, 14[87.5%] and 13[81.3%] were positive by PCR and cultivation assays, respectively. These numberes for the 101 cows without clonical signs of Johne's disease were 10[9.7%] and 12[11.7%]. In conclusion, according to the current report of bacterial detection from different places and the economic importance of John's disease, it is logical and essential that the prevalence rate of the disease in dairy cattle is initially determined by at least two different types of samples and tests and afterwards the control programs are adopted