Comparative evaluation of LAMP and Nested-PCR for the diagnosis of bovine paratuberculosis

AbstractIntroductionMycobacterium avium subsp. paratuberculosis causes paratuberculosis (Johne’s disease), a systemic infection and chronic inflammation of the intestine that affects many species, including bovine. Infection is widespread in livestock, and human populations are exposed. A possible association between MAP infection and Crohn’s disease in humans has been also described. Effective control of paratuberculosis has hampered due to lake of rapid and accurate diagnostic test. Range of diagnostic tests is available, but all have inborn limitations. The present study was designed to develop a loop-mediated isothermal amplification (LAMP) assay for the rapid and simple detection of Mycobacterium avium subsp. paratuberculosis (MAP).Materials and methodsSix primers were specially designed for recognizing eight distinct sequence of insertion sequence 900 (IS900). To determine the sensitivity of the LAMP assay, 10-fold serial dilutions were made from 431ng/μl MAP stock solution and compared with Nested-PCR results obtained using similar templates at identical concentrations. Detection limit of the LAMP was defined as the last positive dilution and the reactions were performed four times to examine the reproducibility of the test. The specificity of the assays were evaluated by testing three Gram-positive bacteria including Mycobacterium bovis AN5, Mycobacterium tuberculosis DT and Mycobacterium avium avium.ResultsSensitivity of this assay for detection of DNA of MAP was 4fg/μl and the specificity was 100%. This assay successfully detected MAP not only in the bacterial cultures but also in clinical fecal samples and the specificity of both PCR was 100%. This LAMP method is performed under isothermal conditions and no special apparatus is needed. In addition, its reactivity is directly observed with the naked eye without electrophoresis either as turbidity or in the form of a color change when SYBR Green 1, a fluorescent dsDNA intercalating dye, is employed.ConclusionsThis assay is rapid which requires nearly 1h for detection of MAP, low in cost and simple to perform, sensitive and practical tool for the detection of MAP and will be useful in facilitating the early diagnosis of paratuberculosis (Johne’s disease) caused by the organism

Electrocardiographic changes following experimental hypokalemia in sheep

Hypokalemia in sheep causes cardiac arrhythmia and electrocardiographic changes such as changing the shape of QRS,complex and T wave and the distance between waves. In this study, 20 rams with the age of 12-15 months were divided into two groups. Electrocardiography was used to check the heart rhythm. After recording ECG and sampling, in order to create hypokalemia Isoflupredon acetat at a dose of 4mg was used intramuscularly in oll 10 treatment. All P waves in the control and the experimental group was recorded as positive and their monophasic or diphasic state was also studied. Between days 0 to 4, 30 cases of monophasic and 20 cases of diphasic P wave was recorded is the control group. In the treatment group, these figures were 21 and 29 cases respectively. Different shapes of QRS waves were seen  in both treatment groups .In the control group they were recorded as QRS in 10 cases, qrs in 15 cases and qRs in 25 cases with in the treatment  group it was QRS in 31 cases, Qrs in 1 case, qrs in 4 cases and qRs in 14 cases. By examining the ECG in the control group on days zero to 4th day, sinus arrhythmia and atrioventricular block was observed in 7 and 2 cases respectively and in the treatment group sinus arrhythmia, atrioventricular block, atrial premature contraction and sinus tachycardia was recorded in 14, 12, 3 and 5 cases respectively. The results indicated that various arrhythmias can be observed due to hypokalemia. As physiologic arrhythmias can lead to pathologic arrhythmias, the animals should be treated with anti-arrhythmia drugs