| The protozoa under the genus Cryptosporidium is a zoonotic apicomplexan obligate intracellular parasite. Cryptosporidiosis, the term used to designate infection caused by Cryptosporidium, is considered as one of the most common food and waterborne diseases with worldwide spread, acting as a common cause of diarrhoea in animals and man. In immunocompetent individuals, Cryptosporidium typically induces self-limiting diarrhoea, which may resolve on its own after 2-3d. However, cryptosporidiosis may turn life-threatening and subsequently lead to death in immunocompromised person. The diagnosis for Cryptosporidium infection is usually carried out through examination of stool for the presence of oocysts which measured 4-6 μm with spherical appearance. However, identification of Cryptosporidium species must also be combined with morphological, molecular biology and host specificity data.Feces were collected from 34 Tibetan sheep of Xikang,584 rabbits of Ya’an,656 cattle of Dazhou, Yibin, Qionglai and Hongyuan, and examined for the presence of Cryptosporidium. The results showed that the infection of Cryptosporidium from Tibetan sheep, rabbits and cattle were 2.94%(1/34),3.6%(21/584) and 2.13% (14/656), respectively. The highest incidence was found in< 1 month age group of rabbits and 2-6 age group of cattle.The positive sample of Tibeten sheep was subjected to molecular analysis by nested polymerase chain reaction (nested PCR) of the 18S rRNA, HSP70 and CpA135 genes. The secondary 18S rRNA PCR product was analysed by restriction fragment length polymorphism (RFLP) of using the conventional Ssp I and Vsp I restriction enzymes. Phylogenetic tree was constructed to determine the infected species. The homologies of 99.9% and 98.7% were noticed compared to C. xiaoi (GU553016 and FJ896041) in the 18S rRNA and HSP70 genes, respectively. The maximum similarity between the isolate and C. ubiquitum (HM358023) was 99.3% at the CpA135 locus. Digestion using Ssp I and Vsp I consisted of three bands of 493 bp, 262 bp and 103 bp, and three bands of 508 bp,181 bp and 104 bp, respectively, Phylogenetic analysis indicated that the present isolate shared the same evolution branch in the 18S rRNA and HSP70 loci. These analyses demonstrated that the Tibetan sheep isolate belonged to C. xiaoi.The 21 isolates of Cryptosporidium from rabbits were analysed by nested PCR of the 18S rRNA, HSP70, CpA135, COWP and GP60 genes. Phylogenetic trees were constructed. The results showed that all 21 isolates were C. cuniculus. The homologies of 100% were noticed compared to C. cuniculus (GU097631, GU097646, KC157564 and GU097635) in the 18S rRNA, HSP70, CpA135 and COWP genes, respectively. This C. cuniculus was named VaA31.The 14 isolates of Cryptosporidium from cattle were analysed by nested PCR of the 18S rRNA and phylogenetic tree were constructed. The results showed that the similarity between DZ1, DZ2 and C. ryanae (HQ179574), YB1 and C. andersoni (AB777194), QL1, QL3-QL5, QL8-QL11 and C. andersoni (KF271480) and QL2, QL6, QL7 and C. bovis (JX515546) were 100%. Digestion using Ssp I and Vsp I of DZ1, DZ2, QL2, QL6 and QL7 consisted of three bands of 430bp,270bp, 100bp, and three bands of 620bp,115bp,105bp, respectively. YB1、QL1、QL3-QL5 and QL8-QL11 consisted of two bands of 450bp,400bp and 730bp,115bp, respectively. These analyses demonstrated that DZ1, DZ2 were C. ryanae, YB1, QL1, QL3-QL5, QL8-QL11 were C. andersoni and QL2, QL6, QL7 were C. bovis.The results showed that Tibetan sheep, rabbits and cattle were all infected with Cryptosporidium. This is the first report of C. xiaoi infecting Tibetan sheep; All the 21 isolates of rabbits were C. cuniculus and VaA31 was a new genetic subtypes; 14 isolates of cattle were C. ryanae, C. andersoni and C. bovis. |
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