Detection And Typing Of Enterocytozoon Bieneusi And Rotavirus In Pre-weaned Dairy Calves In Shanghai

Enterocytozoon bieneusi and rotavirus are important pathogens causing diarrhea in dairy calves,mainly infect epithelial cells of small intestinal causing persistent diarrhea and acute gastroenteritis.Severe infection can cause failure and dehydration,which are the main causes of host death.Among them,bovine specific group 2 of E.bieneusi and zoonotic group A(RVA)of rotavirus are the most commonly detected groups in dairy calves.To further understand the association between E.bieneusi or rotavirus with occurrence of diarrhea,as well as to trace the origin of these two pathogens,the present study collected specimens of dairy calves from five farms in Shanghai,China.A total of 809 fecal specimens of pre-weaned dairy calves were collected on five farms in Shanghai.Based on the internal transcribed spacer(ITS)in small subunit ribosomes(SSU rRNA)gene and multilocus sequence typing,E.bieneusi genotypes and subtypes were identified by using nested polymerase chain reaction(nested PCR)and DNA sequencing techniques.Also,multilocus genotyping(MLG)technique was used to analyze the subtype distribution and the intragenotypic diversity within dominant genotypes.The results showed that the overall infection rate of E.bieneusi was 26.5%(214/809)in Shanghai,and the occurence on farms varied from 12.6%(Farm 5)to 38.5%(Farm 4).Infection rates increased with age during early life,with the peak infection rate(43.0%or 43/100)occurring at six weeks.Four E.bieneusi genotypes were detected in the present study,including J,BEB4,CHN4 and CHN15.In addition,differences were present in the distribution of the dominant genotypes J and BEB4 among five study farms.Altogether,10 multilocus genotypes(MLGs)were identified in the two dominant ITS genotypes,including MLG-J1 to MLG-J8 of genotype J and MLG-B1 to MLG-B2 of genotype BEB4.MLG-B1 and MLG-B2 were seen on Farms 1,2 and 5,whereas MLG-J1 to MLG-J5 and MLG-J6 to MLG-J8 were seen on Farms 3 and 4,respectively.There is extensive genetic heterogeneity within E.bieneusi in dairy calves in Shanghai,China,and MLST should be used in molecular epidemiological studies of E.bieneusi in cattle.Also,There was no relationship between E.bieneusi infection and diahhrea.To further clarify the association between rotavirus infection and diarrhea in calves,this study was going to do RVA detection and typing.At present,rotavirus can be divided into different P/G types based on its highly variable structural proteins VP4 and VP7 which can generate different P/G-specific antibodies independently.However,current methods cannot successfully amplify the experimental specimens.Therefore,the present study aimed to establish and optimize a set of RVA detection and typing methods.88 fecal specimens were selected from pre-weaned dairy calves specimens in Shanghai and Jiangsu for RVA detection using ELISA,VP6 RT-PCR and qRT-PCR.VP6 is a major structural protein of rotavirus,and can be used for group discrimination because of its different nucleotide sequences in different groups.By comparing the detection results of these three methods and optimizing the amplification primers and reaction condition,a set of RVA detection and typing methods was successfully established.The specific methods are as follows:using ELISA as a preliminary screening tool for RVA detection,using VP6 RT-PCR for verification,using one step RT-PCR to amplify the full length of rotavirus VP4/VP7 genes with specific primers,using semi-nested PCR for typing amplification with different P/G specific typing primers,and then the specific P/G types were identified by agarose gel electrophoresis.Optimized Bovine-UK VP7 primers were used for VP7 full-length amplification.And the optimal annealing temperature for P/G typing were 45℃-55℃ and 52℃-48℃,respectively.The set of method can reduce RNA extraction workload and contamination,take less time,be easier to operate,and suitable for strains with large differences in sequence.The results showed that RVA infection rate was 33.0%(29/88)in pre-weaned dairy calves in Shanghai and Jiangsu.RVA mainly infected calves less than 5 weeks of age.Only one P type and one G type was identified(P[11]and G6).Also,RVA infection was associated with watery diarrhea.This study established a RVA detection and typing methods,and further studies are still needed to detect RVA infection and its correlation with diarrhea on the remaining specimens of pre-weaned dairy calves in Shanghai.