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.

[Observations on the control and eradication of paratuberculosis in dairy herds]. / Uberlegungen zur Kontrolle und Bekämpfung der Paratuberkulose in Milchviehbeständen.

Johne's disease is widely seen in dairy herds in Germany. Estimates based primarily on epidemiological surveys in neighbouring states assume that 5 to 15 % of German herds are infected. In the past three years several authors have reported that the causative agent of Johne's disease, Mycobacterium avium subspecies paratuberculosis (MAP), is found ubiquitously in the environment and can be isolated from a number of different animals, including non ruminants. These results imply that MAP should be considered an environmental pathogen. Based on this assumption a concept for control and eradication of Johne's disease is presented aiming at minimizing the future spread of disease and reducing environmental contamination with the pathogen at low costs. The concept includes the classification of herds based on an bulk milk ELISA followed by a robot-compatible bulk milk PCR in ELISA-positive herds only. Due to the comparatively low costs combined with the high specificity of the approach a detection of heavily infected herds ("tip of the iceberg") all over the country would be possible; based on the eradication of strong shedders in these herds the input of MAP into the environment would be reduced considerably.

Evaluation of a LAM ELISA for diagnosis of paratuberculosis in sheep and goats.

A milk and serum ELISA containing lipoarabinomanan (LAM) antigen was evaluated in sheep and goats versus agar gel immunodiffusion (AGID) test and polymerase chain reaction (PCR) using milk and lymph nodes. Milk and serum samples were obtained from six, two, and four flocks with unknown, negative and positive status of infection, respectively. By comparison of serum ELISA activity and PCR results, the positive and negative predictive values were calculated. Receiver operation characteristic (ROC) analysis was used for calculating the specificity and sensitivity at different cut-offs.

[Considerations concerning diagnostic certainties and cut-off values for a bulk milk ELISA for Mycobacterium avium ssp. paratuberculosis]. / Uberlegungen zu diagnostischen Sicherheiten und Cut-Off-Werten für einen Sammelmilch-ELISA auf Paratuberkulose.

Serological tests for the examination of individual samples from single animals are evaluated based on their ability to detect true positives above a defined threshold value. If results are obtained not from an individual but from a bulk sample this concept usually is adopted such that the threshold is set to allow the detection of a single positive sample within the pool. In conjunction with the development of a diagnostic paratuberculosis ELISA for the examination of bulk milk samples it is discussed which interpolations of this concept are justified when defining the true status of a herd based on the test parameters and the seroprevalence within the herd. Here, bulk milk from up to 50 animals each and the corresponding individual samples of 4241 dairy cows from 28 herds in the state of Brandenburg are investigated, and results are subjected to different evaluation approaches. Based on epidemiological considerations and test parameters a "critical prevalence" is defined which then serves as basis for the deduction of a cut-off value to be used for bulk milk samples. Finally, the practical relevance of this approach is demonstrated by suggesting an initial scheme for paratuberculosis classification of dairy herds with respect to possible control measures.

[Establishment and evaluation of an ELISA for the detection of antibodies in milk against Mycobacterium avium subspecies paratuberculosis]. / Etablierung und Evaluation eines ELISA zum Nachweis von Antikörpern gegen Mycobacterium avium subspecies paratuberculosis in Milch.

Mycobacterium avium ssp. paratuberculosis (M. paratuberculosis) is the etiological agent of paratuberculosis (Johne's Disease), a chronic granulomatous enteritis of ruminants occurring worldwide with increasing frequency and leading to growing economic losses. Continuous surveillance of dairy farms would be advisable, particularly with respect to the increasing economic importance of paratuberculosis and the high tenacity of the pathogen, which can persist in the environment for many months. So far, such measures have not been taken as the cost-intensive collection of serum samples would have been required. Based on these considerations, it was the aim of this study to evaluate an economically viable diagnostic method for antibody detection using milk samples. This objective was reached by establishing a milk-ELISA. A commercially available test (Svanovir-ELISA by Svanova, Sweden) was chosen, because this ELISA has an excellent specificity with respect to cultural examination of the ileocaecal lymph node ("Gold-Standard"). The Svanovir-ELISA could be successfully adapted for testing milk for antibodies against M. paratuberculosis. The milk is skimmed by centrifugation and is diluted 1:10 for testing. The inter-assay-variation was 17%. A comparative antibody analysis done in parallel with milk and serum samples from 601 dairy cows using the Svanovir-ELISA showed a significant correlation between the results obtained with both methods. The optimal "cut-off" for the milk-ELISA of 46 EUMS (> 46 EUMS = positive) resulting in a specificity of 94.6% and a sensitivity of 60.9% was confirmed by receiver-operator characteristics (ROC) analysis. In the meantime the Svanovir-ELISA has been licensed for use with milk samples in Germany.

[Diagnosis of paratuberculosis]. / Diagnostik der Paratuberkulose.

Diagnostics of paratuberculosis infection is a difficult and complex field, that causes confusion, lack of understanding and frustration in practitioners, veterinary officers and last but not least farmers. In this review the various diagnostic approaches with their potentials, advantages and disadvantages and their limits are discussed.

Identification and characterization of a novel extracellular ferric reductase from Mycobacterium paratuberculosis.

A novel extracellular mycobacterial enzyme was identified in the ruminant pathogen Mycobacterium paratuberculosis. The enzyme was capable of mobilizing iron from different sources such as ferric ammonium citrate, ferritin, and transferrin by reduction of the metal. The purified reductase had a calculated Mr of 17,000, was sensitive to proteinase K treatment, and had an isoelectric point of pH 9. Analysis of the amino acid composition revealed glycine, serine, asparagine (or aspartic acid), and glutamine (or glutamic acid) as the most frequently occurring residues. Enzymatic activity was highest at 37 degrees C and between pH 5 and 10. The calculated Km and Vmax for ferric ammonium citrate were 0.213 mM and 0.345 mM min(-1) mg(-1), respectively. Using a specific antireductase antibody in immunoelectron microscopy, we were able to detect the enzyme associated with intracellular mycobacteria in naturally M. paratuberculosis-infected bovine tissue. We prepose that the reductase of M. paratuberculosis represents an alternative strategy of mycobacteria to mobilize ferric iron and discuss its potential role in bacterial evasion of intracellular defense mechanisms.