Correlation between Herrold Egg Yolk Medium Culture and Real-Time Quantitative Polymerase Chain Reaction Results for Mycobacterium Avium Subspecies Paratuberculosis in Pooled Fecal and Environmental Samples

Abstract

Abstract. Real-time quantitative polymerase chain reaction (qPCR) testing for Mycobacterium avium subspecies paratuberculosis (MAP) in fecal samples is a rapid alternative to culture on Herrold egg yolk medium (HEYM), the traditional antemortem reference test for MAP. Although the sensitivity and specificity of these 2 tests have been estimated based on dichotomized test results, the correlation between real-time qPCR threshold cycle (Ct) values and colony-forming units (CFU) on HEYM for fresh and thawed samples has not been evaluated. The objectives of the present study were to estimate the correlation and association between Ct and CFU in fresh and thawed pooled fecal and environmental samples. Results of HEYM culture of 1,997 pooled fecal samples from cows in 14 herds, and 802 environmental samples from 109 dairies nationwide were negatively (inversely) correlated with their respective real-time qPCR results. The Spearman's rank correlation between Ct and CFU was good (20.66) in fresh and thawed pooled fecal samples, and excellent (20.76) and good (20.61) in fresh and thawed environmental samples, respectively. The correlation varied from good (20.53) to excellent (20.90) depending on the number of samples in a fecal pool. Truncated regression models indicated a significant negative association between Ct and CFU in fecal pools and environmental samples. The use of real-time qPCR instead of HEYM can yield rapid, quantitative estimates of MAP load and allow for incorporation of real-time qPCR results of pooled and environmental samples in testing strategies to identify dairy cow groups with the highest MAP shedding.

Paratuberculose em ruminantes no Brasil

“…Por exemplo, Douarre et al (2010) compararam a metodologia convencional de cultivo em HEYM e nested-PCR para dois alvos moleculares diferentes, IS900 e ISMAP02, e constataram que a PCR pode produzir resultados rápidos e confiáveis, inclusive, para análise de "pools" de amostras para estimar o "status" da infecção em um rebanho. Igualmente, Aly et al (2010) utilizaram "pool" de amostras fecais para identificar grupos de vacas que estariam eliminando grande quantidade de Map no ambiente e constataram a eficiência do real-time PCR em comparação ao cultivo bacteriano em HEYM. Desta forma, a rapidez, sensibilidade e especificidade dos testes baseados na PCR torna esta metodologia confiável para a confirmação do diagnóstico rotineiro da paratuberculose.…”

Cost-effectiveness of diagnostic strategies to identify Mycobacterium avium subspecies paratuberculosis super-shedder cows in a large dairy herd using antibody enzyme-linked immunosorbent assays, quantitative real-time polymerase chain reaction, and bacterial culture

“…Quantitative real-time polymerase chain reaction (qPCR) testing has been shown to yield results that are highly correlated with culture results on HEYM. 1,2 Hence, qPCR may be preferable for identification of super-shedders because it yields rapid quantitative results (threshold cycle values [Ct]) at similar cost to HEYM culture 5 without the need for serial dilution of fecal samples.…”

Detection of Mycobacterium avium subspecies paratuberculosis excretion in bovine feces, using quantitative real time polymerase chain reaction (Q-PCR)

“…Nevertheless, mistakes in DNA extraction or sample contamination could also explain the results and it cannot be excluded (Khol et al, 2010). Correlation between bacteriological culture and Q-PCR depends on the stage of the infection present in the individual at the time of sample collection which has an effect on the amount of bacilli shed (Kralik et al, 2011;Aly et al, 2010;Pinedo et al, 2008). The ELISA used in the study detected less number of animals as positive, this is because, in early infection stages the predominant immune response is cell type, and the elimination of bacillus in feces begins in an intermediate way.…”

“…Feces from all animals were cultured in duplicate following the technique described by Aly et al (2010). All samples were decontaminated using 0.5% Zephiran and sowns in Herrold's egg yolk solid media with and without 2 mg/L mycobactin J (Allied Monitor INC) and incubated at 37°C for 6 months.…”