Identification of Resistant Gene Against <I>Escherichia coli <?I> Causing Diarrhea in Pre-weaned Pigs Using DDRT-PCR Technique

Abstract

Diarrhea due to Escherichia coli (E. coli) infection is an important problem in pig production especially in pre-weaned period. It is responsible for considerable high economic losses. The E. coli susceptible or resistant pigs were presented or absent specific receptor on brush border of small intestine and associated with adhesion or non adhesion of bacteria to the receptors, respectively. In this study,  brush border adhesion test was used to classify susceptible and resistant phenotype of piglets.   Differential display reverse transcription polymerase chain reaction (DDRT-PCR) was performed with 12 combination primers (dT₁₂VG anchored and aribitrary primers) to identify differentially expressed genes in brush border between two phenotypic groups. The results showed that adhesion values of the susceptible and resistant piglet group were 32% and 7%, respectively. The results of DDRT–PCR analysis showed a total of 512 cDNA fragments and had band size from 150 to 570 bp. Average cDNA fragment were 42.67 cDNA bands per a primer combination. Twelve specific bands revealed differential expression between two piglet groups. Out of these, 7 bands were only expressed in susceptible phenotype where as 5 bands were only expressed in the resistant piglet group. Five cDNA fragments were successfully cloned and sequenced. Three clones (ZP18B3, ZP24B2 and ZP18B1) showed homology with Dipeptidyl-peptidase I precursor (DPPI),      S-Adenosylhomocysteine hydrolase (AHCY) and liver porcine EST (AK232914), respectively. Two clones (ZP01B1 and ZP17B1) did not show any homology and were identified as novel gene. These ESTs could be used as candidate genes for further investigating of the resistant and susceptible to diarrhea in pre-weaned pigs.