The Exploration Of Culture Method And Establishment Of Examination Method For Lawsonia Intracellularis In Pigs

Porcine proliferative enteritis is one kind of infectious intestinal disease primary infect ileum,caused growth arrest and feed conversion decline which is caused by lawsonia intracellularis. LI was gram-negative, curved, comma-shaped or S-sharp bacteria, lenth about1.25-1.75pm, width of0.25-0.43pm. The isolated strains is very limited in the world because of its special culture condition which was parasitic on intracellular strictly. Lawsonia intracellularis was a worldwide epidemic, consided to be an important enteric pathogen, which can cause significant economic losses. However, due to the mixed infection with other pathogens, and we often overlooked the loss. Until now, only a little research related to its clinical infection situations in our country, it is necessary to carry out further research. The detailed experimentations were described as follow.1The establishment of nest PCR assay and preliminary investigation of infection situation for lawsonia intracellularisIn order to understand the epidemic dynamics of lawsonia intracellularis, since October2010to Apirl2012, we collect porcine ileum, stool and rectal swab amount to541from the Shanghai Animal Disease Prevention and Control Center clinic, slaughterhouses and scale farms in Shanghai, Zhejiang, Henan. We detected all the samples by nest PCR method. Statistical analysis showed that the bacteria was an important pathogen cause diarrhes because the significant differences between no diarrhea symptoms and diarrhea herds, which positive rates of LI were18.6%and29.0%.The positive rates among different ages showed the significant differences which2-5weeks,6-9weeks,10-13weeks,14-17weeks, above18weeks and sows were14.3%、16.9%、26.6%、39.3%、38.8%and39.3%of LI respectively.It may be concerned with the ditective of maternal antibody. Also existed in different parts of herds, we should pay more attention to LI and establish the general control measures to reduce the losses.2Development and application of a TaqMan real time PCR assay for the detect of Lawsonia intracellularisA real time PCR assay was developed to detect and quantify LI in this study, the assay utilized a pair of specific primers and a TaqMan probe which was based on the16SrRNA sequence of LI. The method showed high specificity, sensitivity and good reproducibility. The correlation coefficient of the standard curve was0.999094in the assay; the detection limit of the method was1.2copies of initial templates. The established assay successfully applied for the detection of173clinically positive samples, and the results were basic agreement with nest PCR. In comparison, although the detection sensitivity of both methods are equivalent, but the real time PCR assay take about1h, the nest PCR may take5-6h. So, it is easily and quickly in the detection of large number samples.3The establishment and detection of culture method for Lawsonia intracellularisAccording to the culture method of related foreign literature, the positive strain of LI infected and proliferative on the IEC-18cells successfully. Real time PCR assay was used to detect the bacterium DNA content in different raised time, the result demonstrated that the right culture time is5-6d due to the DNA content is the highest in this time. Using Immunofluorescence and Immunohistochemistry to detect the infection of LI respectively, both of the two methods confirmed the presence of bacterial existence. Meantime, we dealed with the positive samples of LI and infect IEC-18cells. Although not separated LI successfully, we accumulated some work experience and lay the foundation for the further study.