Determination of Cryptosporidium Parvum Subtypes Detected in Calf and Cow

Abstract

Cryptosporidium spp., an intracellular extracytoplasmic localized protozoan, is one of the leading infectious agents in the etiology of neonatal diarrheal syndrome of ruminants. Although cryptosporidiosis is a common disease seen all over the world. However molecular epidemiologic studies are limited on distribution of Cryptosporidium parvum subtypes in the world. To date, nearly 20 subtypes of C. parvum have been described. Gene sequence analysis of the glycoprotein 60 structural protein (GP60) is used to identify subtypes. It is reported that the GP60 gene sequence shows a high degree of genetic polymorphism among the strains. In this study, it was aimed to determine the subtypes of C. parvum parasite in cow and calf stool samples by using GP60 gene sequence analysis. In our study, 125 samples evaluated as Cryptosporidium spp. positive by Kinyoun Acid Fast staining. A total of 16 samples of cows (> 22 months) and 109 calves (< 30 days) from farms in 2 regions of Türkiye (Burdur/Yesilova and Aydin provinces) were used. DNA isolation from feces was done with the QIAamp Stool Mini Kit. In order to identify Cryptosporidium species, PCR based on SSU 18S rRNA gene amplification was performed. The 18S gene sequences were compared to genebank to identify the specie of the infectious agent. Nested PCR was performed to amplify GP60 gene and sequences of amplicons were used to identify subtypes. The results of C. parvum subtyping study showed that IIaA13G2R1 subtype was found to be common. IIaA13G2R1 was detected in 100% of 29 stool samples with C. parvum in Burdur. The diversity was higher among Aydin samples (n=96), 67 (70%) were IIaA13G2R1, 15 (15,5%) IIaA14G1R1, 12(12,5%) IIaA2R1 and one sample (1%) was IIaA16R1 and one sample (1%) IIaA5G1R1. In subtype studies based on the GP60 sequence, IIaA15G2R1 was found to be the most common in the world. In our study, IIaA13G2R1 subtype detected in cattle in Türkiye was found to be common. Our study is the first Cryptosporidium parvum subtyping study in Aydın province. In our study, IIaA2R1 and IIaA5G1R1 subtypes were defined for the first time in the world, IIaA16R1 subtype in Türkiye. Molecular epidemiology studies are important for better understanding the dissemination of subtypes and IIaA13G2R1 is the most common subtype in Turkey and the IIaA15G2R1 the most common subtype in the world were absent among our samples.