Application of different methods for the diagnosis of Mycobacterium avium subsp paratuberculosis in a dairy cattle herd in Argentine

Universidad Nacional de Mar del Plata, Mar de Plata, Buenos Aires, Argentina
Journal of Veterinary Medicine Series B (Impact Factor: 1.57). 03/2003; 50(1):20-6. DOI: 10.1046/j.1439-0450.2003.00606.x
Source: PubMed

ABSTRACT Paratuberculosis (Ptbc) has a high prevalence in Argentina, that affects dairy and beef cattle. The culture is the gold standard to the diagnosis of the disease. Mycobacterium avium ssp. paratuberculosis (M. paratuberculosis), the aetiological agent, is difficult to isolate and grow in culture. In this study, 24 randomly selected cows of the Fresian breed from a dairy herd with a history of Ptbc were used to evaluate the performance of different diagnostic techniques. These animals did not show clinical signs of the disease. However, another animal from this herd presented evidence of clinical disease at the moment of the present study. This animal was necropsied and one strain of M. paratuberculosis was isolated from faeces, lymph nodes and intestine. Serum for indirect absorbed enzyme-linked immunosorbent assay (ELISA) and agar gel immunodiffusion (AGID) tests and whole blood samples to perform gamma interferon (gammaIFN) release assays were obtained from each animal. Faeces and milk samples to carry out bacteriological cultures, PCR identification of M. paratuberculosis, and direct examinations of smears with Ziehl-Neelsen's (ZN) stain were also collected. Tuberculin test with bovine purified protein derivative (PPD) in the caudal fold was performed. The results showed that 10 out of 24 animals (41.6%) were positive to ELISA. Eight strains of M. paratuberculosis were isolated, six from faeces, two from milk. Five of the animals that excreted the bacteria through faeces were ELISA-positive, whereas the excreters through milk were negative to ELISA. No positive samples by AGID were obtained in clinical asymptomatic animals. Seven samples gave positive gammaIFN results with avian PPD, but only two of these animals were confirmed with culture. Direct PCR, to detect IS900 (M. paratuberculosis) in faeces and milk samples, was negative, but PCR using material taken from faecal and milk cultures gave positive results before visualizing the colonies. No sample was positive by PCR directed to IS6110 (M. tuberculosis complex). There was not always agreement between isolations and ZN in the studied samples. In conclusion, the absorbed ELISA was useful to detect positive animals and excreters through faeces but not through milk. PCR applied to cultures with incipient development before the visualization of colonies was effective to specifically determine the presence of M. paratuberculosis. The gammaIFN test was not able to detect the most positive animals confirmed by culture. The importance of using ELISA and cultures is emphasized by this study but it is necessary to continue with the gammaIFN test development for early detection of the disease.

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Available from: Angel Cataldi, Jul 05, 2014
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    • "Recent advances in diagnostics, prophylaxis/vaccination alternative therapeutic modalities (Paolicchi et al., 2003; Tripathi et al., 2006; Kumar et al., 2007, 2014; Munjal et al., 2007; Deb et al., 2011; 2013; Dhama Singh et al (2014) "
    Advances in Animal and Veterinary Sciences 01/2014; 1(1S):1-11.
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    • "Samples from the ileum or the ileocaecal lymph node, of six cows were used. Four corresponded to adult animals with clinical signs of PTB, and Map isolate from fecal samples (Paolicchi et al., 2003), while the other two were from young animals belonging to a free herd with no clinical signs of disease which were used as negative controls (Table 1). Tissues were fixed in 10% formalin solution and embedded in paraffin following the standard histological technique. "
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    ABSTRACT: Paratuberculosis or Johne's disease is a chronic infectious disorder caused by Mycobacterium avium subsp. paratuberculosis (Map). The disease produces diarrhea and weight loss in cattle and other animal species, and it is characterized by granulomatous enteritis and lymphadenitis. Histopathology and in situ techniques can be used as a diagnostic test, but the performance of these methods was not previously compared. The aim of this paper was to evaluate the ability of immunohistochemistry and in situ hybridization to detect Map in formalin-fixed tissue samples from infected cattle. Samples (ileum or ileocecal lymph node) from four animals that had positive Map culturing, lesions and detectable acid fast bacilli, as well as from two control animals, were tested by immunohistochemistry and in situ hybridization. Immunostaining and positive hybridization were observed in areas with lesions from infected animal samples, inside the cytoplasm of macrophages, epithelioid and giant cells. Immunostaining was intense in three samples and weak in one, while hybridization was weak in all cases. In situ hybridization was positive in negative areas of tissues analyzed by immunohistochemistry, which could be related to spheroplast detection as it was previously described for this method. Control samples resulted negative by these two methods. Both techniques were able to detect Map in formalin fixed and paraffin embedded tissues, however immunohistochemistry produced higher intensity staining and was easier to perform. Therefore, we believe that immunohistochemistry and in situ hybridization to be useful for the post-mortem diagnosis and research of Paratuberculosis.
    Veterinary Microbiology 09/2008; 134(3-4):383-7. DOI:10.1016/j.vetmic.2008.08.023 · 2.73 Impact Factor
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    • "Several studies with MAPTB antigens have been performed to set up diagnostic tests of paratuberculosis in livestock. Although several antigenic proteins have been described in the pathogen (De Kesel et al., 1992; Gilot et al., 1993; Cameron et al., 1994; White et al., 1994; el-Zaatari et al., 1994, 1995; Mutharia et al., 1997; Coetsier et al., 1998; Silbaq et al., 1998; Cobb and Frothingham, 1999; Liu et al., 2001; Olsen et al., 2001; Mullerad et al., 2003; Paolicchi et al., 2003), identification and cataloguing of antigens of MAPTB is well behind that of M. bovis, the agent of bovine tuberculosis. The complete genomic sequencing of both, MAPTB and M. avium subsp. "
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    ABSTRACT: A Mycobacterium avium subsp. paratuberculosis expression library in lambda ZAP was screened with immunized mice sera. One clone was selected, sequenced and further characterized. The sequence analysis of the hypothetical open-reading frame (ORF) predicts a protein of 20.8 kDa with a probable signal sequence compatible with Cys-acylation at Cys24, characteristic of lipoproteins. In consequence, the protein was termed Lpp34. Recombinant expression of Lpp34 was achieved by cloning the lpp34 gene into the histidine-tag expression vector pRSET-A. Western blot analysis showed a protein band with a molecular weight of 34 kDa. The native protein was localized in the membrane fraction of M. avium subsp. paratuberculosis and extracted in the detergent phase of Triton X-114. Southern blot and polymerase chain reaction showed that the gene is absent from all the non-M. avium complex mycobacterial genomes tested. Humoral reactivity using bovine sera demonstrated that this protein is widely recognized by both the infected and non-infected animals. This could partly be due to the conserved sequence in close-related environmental bacteria such as M. avium subsp. avium and to the presence of a conserved epitope in other bacteria such as Escherichia coli. In conclusion, these findings show that Lpp34 is a membrane protein and a putative lipoprotein present in M. avium complex mycobacteria and absent in the M. tuberculosis complex.
    Journal of Veterinary Medicine Series B 03/2006; 53(1):34-41. DOI:10.1111/j.1439-0450.2006.00905.x · 1.57 Impact Factor
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