ISMap02 element targeted nested polymerase chain in the detection of Mycobacterium avium subsp. paratuberculosis in fecal samples of cattle and buffaloes
Background and Aim: Johne’s disease is chronic granulomatous enteritis which affects ruminants. There are many diagnostic approaches for the detection of Mycobacterium avium subsp. paratuberculosis (MAP) of which molecular detection methods using various elements are less time consuming and more accurate. The present study was conducted using ISMap02 element for nested polymerase chain reaction (nPCR) based detection of MAP in fecal samples. The aim was to test the sensitivity and specificity of the ISMap02 element and also to use this element for the detection of MAP in fecal samples of cattle and buffaloes. Materials and Methods: A total of 211 fecal samples of cattle and buffaloes from different herds around Ludhiana aged between 2 and 13 years were collected, and DNA extraction was done from these samples. The nPCR was carried out for the detection of MAP in fecal samples. Results: The ISMap02 element was specific for the detection of MAP only and showed a sensitivity of detection of 7.6 fg/µL of the standard genomic DNA. Among the 211 fecal samples of cattle and buffaloes tested for the ISMap02 element, 18 samples (8.5%) were positive for MAP. Conclusion: The ISMap02 element is specific and sensitive for the detection of MAP in various samples, and when used in nPCR format, it can increase the sensitivity of detection.
Comparative evaluation of PCR assays for the robust molecular detection of Mycobacterium avium subsp. paratuberculosis.
Mycobacterium avium subsp. paratuberculosis (MAP) can cause a very serious, often-fatal disease, namely paratuberculosis, in several animal species, especially ruminants. Recently, it has also been implicated in the pathogenesis of Infectious Bowel Disease of man. The aim of this study was to develop a molecular method for the routine detection and identification of MAP, from tissue samples of animal origin. The proposed assay would have to combine optimum performance and cost, with high reproducibility. To this goal, three laboratories in Greece and the Czech Republic undertook different parts of a study that involved evaluation of DNA extraction procedures, and PCR assays, for MAP detection. For DNA extraction we used one in-house, and one commercial method, and for the PCR we assessed a number of different assays, starting with the evaluation of primer specificity with an extended GenBank database search. Based on these results, we chose to assess a one-tube nested, 2 two-tube nested, and a single PCR assay, targeted to different genomic regions of the IS900 element. These four methods were applied on positive and negative control samples, consisted of pure bacterial cultures and formalin-fixed paraffin-embedded (FFPE) tissue samples collected from cattle with paratuberculosis and chickens with M. avium subsp. avium infection. Based on the criteria of reliability and cost, the procedure that performed better was the one-tube nested PCR assay combined with the in-house DNA extraction method. The agreement of the results obtained by the three collaborating laboratories indicates the reliability of the proposed assay even under different laboratory conditions.
Effect of Soybean Cyst Nematode (Heterodera glycines) on Yield of Resistant and Susceptible Soybean Cultivars Grown in Ohio.
Soybean (Glycine max) producers in Ohio rarely use soybean cyst nematode (Heterodera glycines, SCN)-resistant cultivars because of concerns over limited yield potential and lack of resistance to Phytophthora sojae. A two-year study was initiated to determine grain yield and nematode population increase on soybean cyst nematode-resistant cultivars in maturity groups II and III in production fields. Sites differed in soil texture, nematode densities, and P. sojae infestation at a number of locations in Ohio. Soil was assayed for nematode densities before planting and at harvest. Yields of resistant cultivars averaged 0% to 18% higher than those of susceptible cultivars in fine-textured soils with average preplant populations ranging from 463 to 14,330 SCN eggs/100 cm(3) soil. In coarse-textured soils, yields of susceptible cultivars were 21% to 56% less than the resistant cultivars with average preplant densities ranging from 1,661 to 15,558 SCN eggs/100 cm(3) soil. The reproductive index ranged from 0.1 to 5.5 for resistant cultivars and 0.4 to 112 for susceptible cultivars. In 1993, yield of P. sojae-susceptible, nematode-resistant 'Asgrow A 3431' was as high as yield of the P. sojae-resistant, nematode-susceptible cultivar 'Resnik' in a Phytophthora-infested field. The nematode-resistant cultivars Madison Experimental 131527 and Asgrow A3431 had higher yields than AgVenture AV1341 and susceptible cultivars Resnik and Kenwood when compared over five nematode-infested sites. Nematode-resistant cultivars were found to be excellent alternatives to currently grown susceptible cultivars for managing SCN where group III cultivars are used. However, better cultivar alternatives may be needed for sites with combined Phytophthora root rot and cyst nematode problems.