Application of different methods for the diagnosis of experimental paratuberculosis in goats.

The diagnosis of subclinical paratuberculosis is still considered a major problem worldwide. As part of investigating diagnostic strategies for the paratuberculosis infection, sequential results of various diagnostic methods in a progressive experimental infection in goats were evaluated. Twenty-three goat kids were divided into three groups: the infected, contact and control, comprising 10, five and eight goats respectively. Animals of the infected group were orally inoculated on seven occasions with 5 ml of inoculum containing 2 x 10(9)Mycobacterium avium ssp. paratuberculosis per ml. Lymphoycte proliferation test using johnin PPD detected paratuberculosis infection from 60 days post-infection (DPI) onwards. The johnin PPD was found to be a better antigen for the proliferative assays as compared with the sonicated antigen. The faecal smear examination with acid-fast staining detected more goats as positive than bacterial culture and polymerase chain reaction (PCR). Lipoarabinomannan enzyme-linked immunosorbent assay (ELISA) started detecting infected goats from 150 DPI onwards followed by indirect ELISA and agar gel immunodiffusion from 180 DPI onwards. Histological examination was confirmatory and detected five infected goats as positive.

Comparison of IS900 tissue PCR, bacterial culture, johnin and serological tests for diagnosis of naturally occurring paratuberculosis in goats.

Comparative efficacy of an IS900 tissue PCR, bacterial culture, johnin, agar-gel immunodiffusion (AGID) and absorbed-ELISA tests was investigated in 43 goats naturally infected with paratuberculosis. On histological examination, tissue sections from all animals showed typical granulomatous inflammatory changes. The lesions were classified as multibacillary (MB) (n=30), which had diffuse granulomatous lesions with abundant acid-fast bacilli (AFB), and paucibacillary (PB) (n=13), which had focal or multifocal granulomatous lesions with few AFB. The sensitivities of johnin test, tissue culture, faecal culture, tissue PCR, AGID and ELISA were 68% (17/25), 100% (30/30), 84.6% (22/26), 100% (30/30), 96.2% (25/26) and 100% (26/26) in MB goats, and 88.8 (8/9), 46.1% (8/13), 40% (4/10), 61.5% (8/13), 50% (5/10), and 70% (7/10) in PB goats, respectively. Except for the johnin test, which showed higher sensitivity in PB goats, all other tests displayed significantly higher sensitivities in MB goats. The results indicate the usefulness of tissue PCR, culture and serological tests in the diagnosis of clinically affected paratuberculous goats, especially with multibacillary pathology.

Progressive immunopathological changes during early stages of experimental infection of goats with Mycobacterium avium subspecies paratuberculosis.

A dose of 10(10) Mycobacterium avium subspecies paratuberculosis was administered orally on seven occasions to produce experimental paratuberculosis infection in 10 5-8-week-old goat kids. Bacteriological, immunological, and histopathological changes, their relationships, and the efficacy of the commonly used diagnostic methods were studied during the progressive disease up to 270 days postinfection (DPI). Significant lymphocyte proliferative responses in the peripheral blood of five goats were detected as early as 60 DPI. A lymphoproliferative test was also performed on lymphocytes purified from different compartments of the guts of five infected and five control goats. Significant proliferative responses were observed in lymphocytes of jejunal compartments of all five goats, of which four had also significant lymphocyte proliferation in the blood. The ileal lymphocytes from two goats, one each at 120 and 270 DPI, had significant proliferation. The histological lesions were mainly observed in the gut-associated lymphoid tissues of the ileocecal valve, the ileum, and the terminal jejunum. Acid-fast bacilli were demonstrated in the lesions of two goats at 60 and 210 DPI. Bacterial culture showed poor sensitivity, detecting positive results for only one goat in the fecal and tissue samples at 210 DPI, whereas polymerase chain reaction (PCR) detected one goat in fecal sample at 210 DPI and two goats in tissue samples at 60 and 210 DPIs, respectively. Enzyme-linked immunosorbent assay and agar gel immunodiffusion test were found to be 100% sensitive from 180 and 210 DPI onwards, respectively.

Detection of classical swine fever virus in archival formalin-fixed tissues by reverse transcription-polymerase chain reaction.

Classical swine fever (CSF) is an economically important Office International des Epizooties list A disease of swine. Inadequate diagnostic infrastructure in developing countries may sometimes make the conduit of viable samples to diagnostic laboratories difficult and at times formalin-fixed tissues may be the only morbid materials available which are not amenable to most laboratory tests. The potential diagnostic abilities in such situations would be enhanced if a highly sensitive and specific method as reverse transcription-polymerase chain reaction (RT-PCR) could be applied to such samples. In the present study, archival formalin-fixed CSF samples were subjected to successful RT-PCR amplification of a 172-bp fragment from E2/NS2 region of CSFV genome. The fidelity of amplification was established by a dot-blot hybridization assay employing a homologous biotinylated cDNA probe. The optimized procedure may be resorted to as a diagnostic tool if viable samples are unavailable and may have the potential to facilitate retrospective studies on archival samples.