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.
Mycobacterium avium subsp. paratuberculosis in cow bulk tank milk in Cyprus detected by culture and quantitative IS900 and F57 real-time PCR.
The possibility that Mycobacterium avium subsp. paratuberculosis (MAP) plays some role in the development of Crohn's disease in humans is attracting attention to milk and milk products originating from infected animals. In this study, we focused on the detection of MAP in 220 bulk tank milk (BTM) samples from all dairy cattle herds in Cyprus. In total, 63 (28.6%) BTM milk samples were found to be positive for MAP using quantitative real-time PCR assays for IS900 and F57. The presence of MAP in BTM was low, and was assessed to be several tens of MAP cells per one ml of BTM. Milk samples examined by cultivation were found to be negative for MAP in all 220 BTM. In two BTM samples cultivation and subsequent sequencing of 16S rRNA revealed two isolates of M. fortuitum.
A comparison of methods to estimate the prevalence of ovine Johne's infection from pooled faecal samples.
OBJECTIVE
To compare estimates of ovine Johne's infection prevalence produced by several alternate methods based on pooled faecal culture (PFC) results with prevalence estimates based on individual faecal culture (IFC).


PROCEDURE
Seven methods for estimating prevalence of infection based on PFC results were incorporated in a computer program, including methods for imperfect test sensitivity and specificity, for variable pool size and a Bayesian method that incorporates prior knowledge about test performance and prevalence. These methods were then used to analyse PFC data at one observation 30 months post-vaccination in a field trial of a killed vaccine for the control of OJD, undertaken on three farms in New South Wales.


RESULTS
Prevalence estimates, for three methods that assume a perfect test, were close to the IFC estimate, whereas for three other methods that assume an imperfect test, the estimated prevalence was generally higher than the IFC estimate. In comparison, the Bayesian approach produced more variable estimates that were substantially higher than the IFC estimate when an inappropriately high prior estimate of prevalence was used.


CONCLUSION
Despite the limitations of each method, two methods provided accurate and reasonable estimates of the prevalence assessed by IFC in all instances, and are appropriate for the analysis of data from this vaccine trial. One of these methods also has the advantage of allowing for variable pool size. However, further research is needed to develop a method that will simultaneously account for variation in pool size and in test sensitivity and specificity.