Realtime PCR quantification of gastrointestinal microbes in broiler challenge models

Abstract

In this study, we aimed to assess the role of 'Eimeria' infection and fishmeal feeding in the UNE NE challenge model and the capability of real-time PCR quantification of bacterial 16S rDNA as a viable replacement for traditional bacterial culture techniques. The experiments were designed to test the following hypotheses: 1. 'Eimeria' infection and fishmeal feeding are necessary for full NE challenge experiment; and 2. Quantitative real time PCR can improve the accuracy and efficiency of enumeration of bacteria in the intestinal tract of broiler chicken compared to the conventional in vitro culture. The results showed that the combination of 'Eimeria' administration and fishmeal feeding had a significant effect on induction of NE disease and mortality of the birds subjected to Cp challenge. High fishmeal feeding alone did not lead to significant mortality of Cp challenged birds, but showed a significantly higher Cp counts than the control based on the real-time PCR assay. 'Eimeria' administration had a significant effect on bird mortality caused by NE but did not show an impact on the Cp count compared to the corresponding Cp challenged groups with various levels of fishmeal feeding. In accordance with the time course of bird mortality, it can be determined that at least the third Cp inoculation of the 3 successive oral gavage processes did not contribute to the death of the birds, and therefore can be omitted in the challenge procedure. It can be recommended that 25-50% fishmeal feeding, 'Eimeria' ('E. acervulina', 'E. maxima' and 'E. tenella') inoculation, two oral Cp inoculations and appropriate ambient temperatures and diets (stated in methodology) are suitable for an NE challenge experiment as a model. For Cp enumeration, quantitative real-time PCR proved to be compatible to conventional in vitro culture method with higher efficiency and accuracy. The correlation analysis of the means of the treated groups showed that these two methods are highly correlated (R2=0.845). However, the real-time PCR method produced higher accuracy than the culture method as demonstrated by comparison of the relative standard deviations (RSD). The RSD of culture method were as high as 10.8 times of the real-time PCR method.