Molecular Identification Of Giardia Intestinal In Dairy Calves And Analysis Of The Genetic Diversity Of Assemblage E

Giardia intestinalis（G.intestinalis）is an important zoonotic parasite that can infect many mammals,including humans.Dairy cattle are an important economic animal.G.intestinalis infections can cause diarrhea and dysplasia in cattle,resulting in economic losses,and pose a potential threat to human health.The evolutionary potential of parasites depends on the distribution,flow,and degree of genetic diversity,which can be better understood by molecular methods.At present,although there have been many literatures about the infection of G.intestinalis in dairy calves at home and abroad,there is no relevant research in Sichuan area in China,and there is also a lack of research on population genetic information.This study investigated the infection of G.intestinalis of dairy calves in intensive feeding farms and free-ranging farms in Sichuan Province.Based on the multilocus genotyping method（MLG）,the molecular identification of G.intestinalis of dairy calves was carried out.Combined with the existing data in China,the population genetic diversity analysis of G.intestinalis assemblage E from dairy cattle was studied and provided theoretical basis for the prevention and control of giardiasis in dairy cattle.This experiment was conducted from June 2016 to November 2018,centering on Chengdu,Sichuan Province,radiating 13 surrounding areas（including 7 intensive feeding farms and 6 free-ranging farms）,and 584 fresh faeces（278 from pre-weaned and 306 from post-weaned）were collected from dairy calves（age<12 months）.After DNA extraction,β-giardin（bg）gene,glutamate dehydrogenase（gdh）gene,and triose phosphate isomerase（tpi）,for detection of Giardia spp.,were amplified by nested PCR,then the amplified products were sequenced and phylogenetic analysis was carried out.The results showed that the total infection rate of G.intestine was 26%（152/584）,and the infection rate of pre-weaned dairy calves was 9.4%（26/278）,which was significantly lower than that of41.2%（126/306）in post-weaned calves（P<0.01,X²=76.623,df=1）.The infection rates of pre-weaned dairy calves in intensive feeding farms and free-ranging farms were 10.3%（20/195）and 7.2%（6/83）respectively.The infection rates of post-weaned dairy calves in intensive feeding farms and free-ranging farms were 45.3%（63/139）and 37.8%（63/167）respectively.There was no significant difference in the infection rate between different breeding patterns in pre-weaned（p=0.428,X²=0.629,df=1）and post-weaned dairy calves（P=0.179,X²=1.809,df=1）.Giardia intestinal positive samples were mainly detected as livestock-specific assemblage E（92.8%,141/152）,accompanied with zoonotic assemblage A（subtype AI;2%,3/152）,assemblage A/E mixed infection（5.2%,8/152）.Assemblage A and mixed infection were detected only in post-weaned calves（8.7%,11/126）.Based on assemblage,15,14,18 subtypes were detected at the three gene loci of bg,tpi and gdh,seven new genotypes（E14-E20）were found at bg locus,three new genotypes（E23-E25）at tpi locus,and seven new genotypes（E19-E25）at gdh locus,showed a high diversity.Multi-locus sequence typing（MLG）showed that 118 samples were successfully amplified at three loci,forming 60 MLG subtypes,including 52 MLG E subtypes,1 MLG A subtype,and 7 MLG A/E mixed subtypes.In order to understand the genetic diversity of G.intestinal assemblage E from dairy cattle in China,110 MLG E identified from dairy calves in Sichuan and 541 MLG E sequences of G.intestines reported in other regions of China（Jiangsu,Xinjiang,Henan,Gansu,Ningxia,Shaanxi,Guangdong and Shanghai,a total of 8 regions）were genetically analyzed.The results showed that 220 haplotypes were detected in the G.intestinal assemblage E population of dairy cattle in China,which showed high haplotype diversity（Hd=0.97225）and low nucleotide diversity（π=0.00259）feature.The overall neutral test values（Tajima’s D and Fu’s Fs）in nine regions of China were all significantly negative,and the mismatch distribution showed a unimodal curve,indicating that the G.intestinal assemblage E population in dairy cattle in China has experienced population expansion.The genetic differentiation index(F_ST)and gene flow（Nm）results showed a low degree of genetic differentiation of the G.intestinal assemblage E in dairy cattle in China,with a frequent genetic communication.Combined with the analysis of molecular variance（AMOVA）,genetic variation within the population（81.7%）was higher than the genetic variation among populations（18.3%）,indicating a low degree of genetic differentiation between populations.In the results of haplotype network and phylogenetic tree,there is no specific genetic differentiation structure between different groups due to different geographical regions,and no geographical isolation phenomenon was observed.In summary,this study showed that there existed G.intestinalis infection in dairy calves in surveyed area of Sichuan province.The infection rate of pre-weaned calves was significantly lower than that of post-weaned calves.There was no significant difference in infection rates between different breeding patterns.Livestock-specific assemblage E,zoonotic assemblage A and A/E mixed infection were detected.There was a high gene flow among G.intestinalis assemblage E populations in dairy cattle in China.The genetic differentiation of the entire population was not obvious,and no obvious geographical isolation was observed.