Molecular techniques for the diagnosis of Johne\'s disease based on nucleic acid amplification (regular PCR and/or real-time PCR) have been widely used in animal health diagnostic laboratories. However, the sensitivity of the PCR-based test directly from fecal samples, especially from the low shedders, is still very low. Furthermore, PCR detection in milk and colostrum cannot distinguish between viable and non-viable organisms. Thus, in order to have sufficient material, very long cell culture incubation times are required until a definite answer can be found using PCR. Therefore, we propose to improve the PCR based test by using a novel cell lysis technique, the laser-lysis technology, to improve the yield of DNA extract preparation. The immensely thick lipid layer of MAP is the main obstacle for current lysis methods. Since the laser-lysis technique is based on the local and specific increase of intracellular water molecules, we postulate that it will not be hindered by the presence of the thick lipid layer but will successfully lyse cells without any damage to DNA, RNA and intracellular protein molecules. Our central hypothesis is that the use of the laser lysis technique will increase the template DNA recovery, and therefore, it will increase the sensitivity of PCR test directly from fecal samples, which results in a significantly shorter cell incubation time as required currently. At the same time, biosensor technology will enable us to simplify the detection system and make it inexpensive and even more rapid while keeping its high specificity for the detection of Johne\'s disease.
NON-TECHNICAL SUMMARY: Paratuberculosis (Johne\'s disease) occurs worldwide as a chronic granulomatous enteritis of domestic and wild animals. A sensitive and rapid test is urgently needed. The purpose of this study is: 1) improve the sensitivity of the PCR test directly from fecal samples, milk, and colostrum or liquid media culture system by using a novel laser-induced cell lysis technique to prepare the DNA extracts and (2) develop a rapid, inexpensive, and sensitive biosensor for detection of MAP based on RNA detection. <P>
APPROACH: 1. The use of laser lysis technique will increase the DNA template recovery from fecal sample preparation; thus, it will increase the sensitivity of PCR tests from either fecal sample directly and/or from Trek culture system. If this technique is successful, then, it may be applied to fecal samples directly and/or early detection of Trek liquid culture system. 2. The RNA biosensor based on liposome technology is a rapid, inexpensive and sensitive system to detect the MAP directly from fecal samples and/or Trek culture system. The antibody biosensor is also a rapid, inexpensive and sensitive system to detect the Mycobacterium avium subsp. paratuberculosis (MAP) directly from fecal samples and/or Trek culture system. To accomplish the objective of this study, we will apply two approaches: (1) Increase the PCR sensitivity by the improvement of cell lysis and thus DNA extraction using laser-induced cell lysis. (2) Apply RNA Biosensor for MAP diagnosis.
PROGRESS: 2004/10 TO 2007/09<BR>
OUTPUTS: Mycobacterium avium subsp.paratuberculosis (MAP) is the causative agent of Johne\'s disease (JD), a chronic intestinal granulamatous infection affecting domestic ruminants such as cattle, sheep and goats. Apart from domestic ruminants, wild ruminants like rabbits, deer, bison, and antelopes are also affected by JD. The primary route of infection is through ingestion of contaminated feed and water, colostrum and milk. Although cattle are usually infected early in life, clinical signs do not develop until 2 to 4 years of age which makes early diagnosis of this infection a difficult task. JD is considered as an economically important disease and accounts for an annual loss of $220 million to the US dairy industry due to premature culling and production losses. The need for an effective JD control program has increased as the costs of this infection become more apparent. The proposed, but poorly defined association of MAP with Crohn\'s disease in human beings is also of concern. Effective control of JD is hampered by diagnostic techniques that can not consistently detect infected animals. Currently, JD diagnosis is typically based on either detecting the etiological agent or detecting the host\'s immune responses to the infection. The former method employs culturing of MAP organisms and detecting gene specific sequences from clinical samples. Though, isolation and identification of MAP is the most definitive test for diagnosis, it is time consuming and labor intensive, requiring 8-12 weeks. Contamination is an added problem when MAP is cultured from fecal samples. Although, PCR for IS900 sequences is of diagnostic value, it may lead to false positive amplification due to the presence of environmental bacteria with similar sequences. Novel sequences identified recently in the genome of MAP appear specific and may also be used in nucleic-acid based diagnostic tests. Alternatively, PCR can be coupled with southern blotting to get a definite diagnosis, but again this method too takes more time. Serological tests like agar gel immunodiffusion test, complement fixation test and enzyme linked immunoassay (ELISA) are relatively easy to perform but may lack sensitivity. Serum samples from animals exposed to environmental mycobacteria may also cross react with MAP antigen resulting in false positive reaction. Most diagnostic laboratories continue to use traditional culture methods for identification of MAP, while few laboratories employ molecular methods along with culture methods. Current diagnostic tests have been found to be more useful on a herd basis rather than in testing individual animals. In this scenario, the general feeling is the need for improved, rapid diagnostic techniques for JD. Therefore, we develop a bioanalytical system like biosensors coupled with a reverse transcriptase PCR to achieve low limits of detection for a rapid and accurate detection of MAP. This manuscript has submitted to Journal for publication. We will disseminate our results through publication. <BR> TARGET AUDIENCES: The target audiences are diagnosticians of Johne\'s disease <P>
IMPACT: 2004/10 TO 2007/09<BR>
A simple, membrane-strip-based lateral-flow (LF) biosensor assay and a high-throughput microtiter plate assay have been combined with reverse transcriptase polymerase chain reaction (RT-PCR) for rapid detection of a small number (ten) of viable Mycobacterium avium subsp. paratuberculosis (MAP) cells from fecal samples. The assays are based on the identification of the RNA of the IS900 element of MAP. For the assay, RNA was extracted from fecal samples spiked with known quantity of (101 to 106) MAP cells and amplified using RT-PCR and rapidly identified by the LF biosensor and the microtiter plate assay. While the LF biosensor assay requires only 30 min assay time, the overall process took 10 h for the detection of 10 viable cells. The assays are based on an oligonucleotide sandwich hybridization assay format and use either a membrane flow through system with an immobilized DNA probe that hybridizes with the target sequence or a microtiter plate well. Signal amplification is provided when the target sequence hybridizes to a second DNA probe that has been coupled to liposomes encapsulating the dye sulforhodamine B. The dye in the liposomes provides a signal that can be read visually, quantified with a hand-held reflectometer or with a fluorescence reader. Specificity analysis of the assays revealed no cross reactivity with other mycobacteria such as M.avium complex, M. ulcerans, M.marium, M.kansasii, M.abscessus, M.asiaticum, M.phlei, M.fortutitum, M.scrofalaceum, M.intracellular, M.smegmatis and M.bovis. The overall assay for the detection of MAP in live organisms is less expensive and extremely rapid, especially in comparison to standard MAP detection using culture method requiring 6 - 8 weeks of incubation time, and is significantly less expensive than real-time PCR.