Epidemiology Investigation On Intestinal Parasites And Phylogenetic Relationship Of Cryptosporidium Isolates Derived From Dogs

In the present study, the epidemiology investigation on intestinal parasites from dogs were conducted in Henan province, then the Cryptosporidium isolates obtained were identified from the following aspects, i.e., the morphology of oocysts, cross-transmission study, PCR amplification of 18S rRNA, actin and 70kDa heat shock protein (HSP70) genes and bioinformation analyses using phylogenetic softwares, moreover, the biology character studies of parasitic site, parasite characterization and ultrastruction were also involved.A total of 309 dogs with differnet age and different species, in dog breeding farms, the pet market in the urban area of Zhengzhou, the experimental animal house of Henan Agricultural University, pet hospitals and the rural areas of Zhengzhou, were surveyed for the prevalence of intestinal parasites using the Sheather's sugar flotation technique. As a result, 6 species parasites were found, namely, 2 species of protozoon (Coccidian, Cryptosporidium), 3 species of nematode (Toxocara canis, Strongyloides stercoralis, Trichuris vulpis) and 1 specie of Cestode (unnamed). In present investigation, 96 fecal samples were positive and therefore the average infection rate was 31.07%(96/309). Thereinto, the infectioin rate of Isospora sp. was 28.16%(87/309), Cryptosporidium sp. was 2.59% (8/309), Toxocara canis was 11.33%(35/305), Trichuris vulpis was 1.94%(6/309), Strongyloides stercoralis was 4.21%(13/309), and Cestode was 0.65%(2/309). The results showed that more than 66 % parasite species found were zoonotic species. Isospora sp. and T. canis were commonly found in lower than 6-month-old dogs.105 fecal samples of dog from partial area in Zhengzhou were examined by Sheather's sugar flotation technique. 33 samples were positive and the general infection rate was 31.4%(33/105). The positive dogs detected were all less than 6-month-old. Two species were discovered, namely, Isospora ohioenais and Isospora burrowsi. The average infection intensity (OPG) was 1.28×10~3. At the same time, SMZ-SD-TMP was used to treat coccidiosis and shown a better effect. 309 fecal samples, collected from dog farms, the pet market in the urban area of Zhengzhou, the experimental animal house of Henan Agricultural University, pet hospitals and the rural areas of Zhengzhou, were examined using the Sheather's sugar flotation technique and the modified acid-fast stain technique to study the prevalence of Cryptosporidium in dogs. The results showed that the average infection rate was 2.59% (8/309). Thereinto, The infection rate of dog farms, the suburban areas, the experimental animal house were 0.56%(1/179), 10.53%(2/19), 16.67%(5/30), respectively. No positive case was found in the pet market and pet hospitals. The mean size of oocysts was 4.81μm(4.210μm～5.263μm)×4.39μm(4.210μm～5.263μm), and the ratio of length to width was 1.09.The results of cross-transmission showed that C.canis can infect immunosuppressed dogs, however, can not infect 35-day-old SCID mice and immunocompetent dogs. The latent period of Cryptosporidium infection in dog was 3～5 days, two peak periods were found, the first peak was at DPI 10 days and lasts 2 days, and DPI 17 was the second peak. The duration of oocyst excretion was 22 days.By the routine histotomy and the H.E staining technique and the scanning electron microscope technique, Cryptosporidium was only found in the small intestine of dogs, including duodenum and jejunum. There was a large amount of Cryptosporidium that mainly located in the middle section and the last section of the duodenum, and in the front section and the middle section of the jejunum. There was little Cryptosporidium in the front section of the duodenum, and no Cryptosporidium was found in the other parts.The PCR methods were developmented according to the sequences of 18S rRNA, actin and 70-kDa heat shock protein (HSP70) genes published previously. The partial sequence of 18S rRNA, HSP70 and actin genes were amplified and sequenced. Sequence data were analyzed with BLAST online. Then, sequences alignment, phylogenetic relationships and homological analysis were performed using Clustal X 1.81, Phylip3.64 and DNAstar 4.0 respectively. Based on the phylogenetic analyses of the 18S rRNA, HSP70 and actin genes, the Cryptosporidium isolates from Henan were identified as Cryptosporidium canis.Cryptosporidium canis is one of the zoonotic Cryptosporidium species. It is a potential threat to humans'public health. To study Cryptosporidium canis is of important public health significance. No report on cryptosporidiosis in dogs in China, therefore, the present study provides an important data for further studying the biological characteristics, molecular epidemiology and the evolution relationship of different isolates, etc. in the future.