The Distribution Characteristics Of Cryptosporidium Spp.,Giardia Duodenalis,Enterocytozoon Bieneusi And Rotavirus In Dairy Cattles In Guangdong And Jiangsu,China

Cryptosporidium spp.,Giardia duodenalis,Enterocytozoon bieneusi and rotavirus are common zoonotic pathogens,causing diarrhea and other gastrointestinal illness in humans and various animals and are responsible for significant morbidity and mortality.In humans,they are transmitted by direct contact with infected persons(anthroponotic transmission)or animals(zoonotic transmission)or through consumption of contaminated food or water(food-borne or water-borne transmission).Dairy cattles are the most common hosts of zoonotic Cryptosporidium spp.,Giardia duodenalis,Enterocytozoon bieneusi and rotavirus genotype.In case-control studies,the studies of Cryptosporidium spp.,Giardia duodenalis and Enterocytozoon bieneusi in pre-weaned dairy calves are mostly in midwest and northeastern regions and the detection of rotavirus is mostly in humans.To better understand the distribution of pathogens in dairy cattles in Guangdong and Jiangsu in China,the present study collected specimens of dairy cattles and used molecular biology methods.A total of 388 fecal specimens of pre-weaned dairy calves were collected from a farm in Guangdong in China.The prevalence and genotypic identification of Cryptosporidium spp.,Giardia duodenalis and Enterocytozoon bieneusi were identified 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 in Giardia duodenalis.A Chi-square test was used to compare the prevalence of pathogens among sampling dates,age groups,or clinical signs.The results showed that the detection rates of Cryptosporidium spp.,G.duodenalis and E.bieneusi were 24.0%(93/388),74.2%(288/388)and 15.7%(61/388),respectively.Three Cryptosporidium species were detected,including C.bovis(n=73),C.parvum(n=12)and C.ryanae(n=7);one animal had concurrence of C.bovis and C.parvum.Sequence analysis identified the C.parvum as subtype IIdA19G1.The presence of zoonotic C.parvum and the absence of C.andersoni in this study are in stern contrast with observations in the previous study in same area,which indicated that the Cryptosporidium species distribution in pre-weaned dairy calves appears to be different in Guangdong in China.Assemblage E(n=282),assemblage A(n=1),and concurrence of A and E(n=5)were identified among G.duodenalis-positive animals.Altogether,49 assemblage E MLGs were identified,which showed a very high genetic diversity of assemblage E in pre-weaned dairy calves.Sequence analysis identified genotypes J(n=57),D(n=3)and one concurrence of J and D among E.bieneusi-positive animals.C.parvum subtype IIdA19G1,G.duodenalis assemblage E,and E.bieneusi genotype J have been detected in humans,indicating that pre-weaned dairy calves have been the risk of carrying zoonotic pathogens.Also,seasonal differences in detection rates were observed in G.duodenalis and E.bieneusi,but not in Cryptosporidium spp.A higher G.duodenalis detection rate was noted in winte,in contrast,the detection rate of E.bieneusi was higher in spring than in summer and winter.To further identify the distribution of rotavirus in dairy cattles,the present study collected 476 fecal specimens of dairy cattles in Jiangsu in China.Among the 476 specimens,86 had watery diarrhea,149 had diarrhea and 241 had asymptomatic.Based on ELISA technology,the detection rate of rotavirus was 8.6%(41/476)in dairy cattles in Jiangsu.The Chi-square test showed that rotavirus mainly infected calves less than 5 weeks of age.Also,the infection rate of rotavirus in watery diarrhea cattles(17.4%)was higher in diarrhea cattles(6.7%)and asymptomatic cattles(9.1%),which meant that rotavirus infection was mainly associated with watery diarrhea.Based on rotavirus VP4 and VP7 genes,using PCR technology to analyse ELISA-positive specimens,two genotypes were found,including G6P[11](n=25)and P[11](n=10).Genotype G6P[11]was the dominant one,which was different from the results in Gansu and Ningxia in China,indicating the genetic diversity of rotavirus in dairy cattles in China.The genotype G6P[11]has been always detected in humans,showing the zoonotic risk of rotavirus in dairy cattles.The results above help to understand the role of dairy cattles in the transmission of the four human diarrhea pathogens.A large number of zoonotic genotypes were detected in dairy cattles in this study,indicating that dairy cattles have a certain public health significance as an important potential source of infection for human diarrhea pathogens.