PCR Detection and Phylogenetic Analysis of Cryptosporidium in Dairy Cattle on a Large-scale Introduction Farm in Kashgar Prefecture
[Objective]This study was conducted to characterize the prevalence and species distribution of Cryptosporidium in dairy cattle on a large-scale introduction farm in Kashgar Prefecture,Xinjiang Uygur Autonomous Region.[Method]A total of 190 fecal samples were collected from dairy cattle of different age groups in this farm.Targeting on the small subunit ribosomal RNA gene(SSU rDNA),PCR assay combined with sequencing were used to detect the Cryptosporidium infection.The species of Cryptosporidium were identified through SSU rDNA sequence alignment.Chi-square(x2)test was performed to compare the differences in the Cryptosporidium infection rates among dairy cattle of different age groups.The gp60 genes of C.parvum,C.bovis and C.ryanae were amplified by PCR assay,and the subtypes of the 3 Cryptosporidium species were identified by sequence alignment.In addition,phylogenetic trees were constructed to analyze their genetic evolutionary characteristics.[Result]Thirty-two fecal samples of dairy cattle were positive for Cryptosporidium,with an overall infection rate of 16.84%(32/190).Four Cryptosporidium species,including C.parvum(n=8),C.andersoni(n=10),C.bovis(n=10)and C.ryanae(n=4),were identified.A higher infection rate of Cryptosporidium was observed in the pre-weaned calves under 3 months of age at 28.00%(14/50).There were significant(x2=8.679,P<0.05)differences in the infection rates of Cryptosporidium among dairy cattle of different age groups.Seven gp60 subtype sequences were obtained from the 8 C.parvum positive samples,with 2 subtypes identified asⅡdA15G1(n=3)andⅡdA19G1(n=4).Six gp60 gene subtype sequences were obtained from the 10 C.bovis positive samples,with 3 subtypes identified asⅩⅩⅥb(n=2),ⅩⅩⅥc(n=3)andⅩⅩⅥe(n=1).The 4 C.ryanae positive samples were successfully subtyped with gp60 gene,all of which were identified as subtypeⅩⅪb.The phylogenetic tree analysis showed that the obtainedⅡd subtype sequences of C.parvum in this study were in the same subgroup as the sequences of Ⅱd subtype family of C.parvum derived from different hosts,and were in different subgroups with the sequences ofⅡa subtype family of C.parvum derived from different hosts in New Zealand.Both the obtained subtype sequences of C.bovis and C.ryanae in this study were in the same group as those of cattle derived C.bovis and C.ryanae from different regions of China.[Conclusion]The Cryptosporidium prevalent in this introduction farm was characterized by diverse genetic distribution.The Cryptosporidium infection in dairy cattle occurred after being introduced locally,suggesting that water and forage pollution may be the primary causes of Cryptosporidium infection.
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