| Hookworm is a globally distributed zoonotic parasite,mainly parasitic in the digestive tracts of mammals such as dogs,cats and humans.It causes some mild and local symptoms such as skin inflammation,eye infections and enteritis,but also may cause grave symptoms like hemorrhagic anemia,even lead to the death of the host.In this study,the hookworm from stray dogs and cats in Guangdong were identified by established a specific PCR method based on the ITS sequence of ribosomal DNA.The epidemiological investigation of stray dogs and cats in Guangdong province was conducted by this method to elucidate the status of hookworm infections and the dominant species in the area.At the same time,several SNPs from the most conserved ITS1 sequences of hookworms were selected to establish a Tm-shift detection method for dog-derived and cat-derived hookworms,which provides a new technique for clinical detection of hookworms in dogs and cats.Finally,homology and phylogenetic analysis of cox1 gene of three species of hookworm isolates in Guangdong were conducted to provide basic data for the risk assessment of zoonotic hookworms from dogs and cats in China.Firstly,a total of 702 canine faecal samples from eight regions?Guangzhou,Foshan,Zhongshan,Shenzhen,Huizhou,Shaoguan,Shantou and Chaozhou?of Guangdong Province,and a total of 308 feline faecal samples from five regions?Guangzhou,Foshan,Zhongshan,Shaoguan,and Shantou?of Guangdong Province were examined by the flotation technique with saturated zinc sulfate.The results showed that the total infective rate of canine hookworm was 20.23%?142/702?.The total infective rate of feline hookworm was 15.26%?47/308?.The infection rates of hookworms in the eight regions were significantly different,with the highest in Shaoguan,reaching 34.38%,and the lowest in Huizhou,only 6.67%.The infection rates of feline hookworms in the five regions were not significantly different,ranging from 10.94%to 19.64%.By comparison,young dogs??1 year?are more susceptible to hookworm infection than adult dogs?>1 year?,and dogs raised outdoors are more susceptible to hookworm than indoor dogs.Secondly,using the ITS sequence of the hookworm from dogs and cats as genetic marker,specific PCR detection method was established by designing specific primers for three hookworms Ancylostoma ceylanicum,A.caninum and A.tubaeforme,and their specificity and sensitivity experiments were performed.The results showed that the method can specifically amplify the ITS sequences of the three hookworms in dogs and cats,and their amplified fragments were 268 bp,427 bp and 170 bp,respectively.There was no cross-reaction with genomic DNA from Toxocara canis,Toxocara cati,Isospora and Giardia lamblia.The minimum detectable DNA concentration was 11.26×10-6 ng/?L?A.ceylanicum?,10.27×10-6ng/?L?A.caninum?,and 8.30×10-6 ng/?L?A.tubaeforme?,respectively.The results indicate that the PCR method is specific and sensitive,and can effectively compensate for the deficiency of microscopic examination in accurate identification of hookworm species.Thirdly,three SNP locis?ITS71,ITS197,and ITS296?from highly conserved ITS1sequences of A.ceylanicum and A.caninum and two SNP locis?ITS101,and ITS296?from hookworm A.ceylanicum and A.tubaeforme were selected to design specific primers,where GC-rich tails of unequal length attached to their 5'-end.Then a Tm-shift method for detection of dogs and cats hookworm was developed according to different peaks of melting curve in different Tm value.The detection effect of the Tm-shift method was assessed through the stability,sensitivity,accuracy test,and clinical detection.The results showed that these three sets of primers could distinguish accurately between dog-derived A.ceylanicum and A.caninum,and these two sets of primers could distinguish accurately between cat-derived A.ceylanicum and A.tubaeforme,each SNP loci corresponded to a standard curve with two peaks.The coefficient of variation in their Tm values of all the SNP loci were between0.07%0.18%.Dilution of the standard plasmid to 10-7 concentration still could identify the two hookworm species.The Tm-shift results of twenty DNA samples from hookworms were consistent with their known species.It is concluded that the Tm-shift method is efficient,sensitive and accurate,which can provide a new molecular detecting technology for the clinical detection and epidemiological investigation of the dog-and cat-derived hookworms.Using the universal primers JB3 and JB4.5 to amplify 12 hookworm isolates?dog-derived A.ceylanicum and A.caninum,cat-derived A.ceylanicum and A.tubaeforme?by PCR method.And the sequences were analyzed by MegAlign and Mega 5.1 softwares for homology and phylogenetic relationship.Results showed that the similarity of cox1 between the same species of hookworm was between 96.6%and 99.8%,while the similarity between different species was generally lower than 94%.Phylogenetic tree showed that three isolates from cat-derived A.ceylanicum and human-derived A.ceylanicum were clustered into a small group,indicating that there was a certain risk of zoonotic infection.Moreover,the A.ceylanicum from different regions and hosts had their own branches,showing the existence of inter-species diversity in the world. |
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