Molecular Investigation of Cryptosporidium Spp. in Stray and Pet dog at Chittagong Metropolitan Area

Abstract

Cryptosporidium is a zoonotic pathogen composed of genetically distinct but morphologically identical genotypes. Recent molecular study indicates that dogs may transmit the cattle genotype, which is known to be pathogenic to humans. The aim of the present study was to evaluate the present condition of Cryptosporidium infection in stray and pet dogs at Chittagong metropolitan area in Bangladesh. One hundred and thirty (130) non diarrheic fecal samples obtained from 98 stray dogs and 32 pet dogs were investigated during the period from January 2014 to May 2014. Initial identification of Cryptosporidium spp. was carried out on stool samples by Ziehl-Neelsen (Z-N) acid fast staining method where Cryptosporidium oocysts were revealed as pink to bright red spherical or slightly ovoid bodies. DNA was extracted from Z-N positive fecal samples and Cryptosporidium spp. were confirmed by amplifying 18srRNA gene. The prevalence was determined to be 8.46% in Z-N techniques and 3.85% by nested PCR amplification of 18srRNA gene. Five fecal samples were found positive in PCR where one sample was found positive from pet dog. Four sets of specific oligonucleotide primers were used in this study that amplified products of predicted sizes for each gene in PCR assays [581, 550, 800, 448 bp for 18srRNA(nested), COWP, Gp60(nested) and HSP70 gene respectively] with the use of 1% agarose gel electrophoresis for PCR amplified products. C. parvum was found to be present in one isolates from stray dog which showed successful amplification of a specific DNA fragment at 550 bp with C. parvum outer wall protein (COWP) gene amplicon which may contain cattle genotype of zoonotic significance but it’s unclear up to further sequence study. There was no significant (p>0.05) difference in the prevalence between male and female stray and pet dogs. There was significant (p<0.05) prevalence in young in compared to adult stray dogs. Results indicate that PCR-based diagnostic methods are useful tool for the accurate diagnosis of Cryptosporidium infection in dogs. Significant raise (p<0.05) in lymphocytic and monocyte counts was found in Cryptosporidium infected dogs. This is the first investigation on the presence of C. parvum among stray dogs in Bangladesh based on COWP gene amplification and it has pointed to existence of a genotype that may play an important role as a source of human and farm animal cryptosporidiosis, although gene sequence of the positive isolates is needed for clear identity of the genotypes. The results of this research give an idea that stray dogs may act as reservoirs for zoonotic intestinal protozoan parasites and should be considered important to public health. So that, it is imperative for human to avoid fecal contamination in streets, public gardens and parks. Also stray dog population should be controlled by euthanasia or by performing neutering of male stray dog, specially in urban areas.