Sequence Analysis Of Ribosomal ITS And Mitochondrial Cox1 Of Toxocara Cati And The Establishment Of The Lamp Assay For T.cati

The genus Toxocara mainly parasitize canines and felines.The most common species are Toxocara canis and Toxocara cati.T.cati is among the most prevalent parasite of cats with a worldwide distribution,and it is also considered the most prevalent neglected helminthiasis.Although the larvae are unable to develop into mature adult worms in humans,but they may migrate to a range of tissues,causing damage to whatever tissue they happen to enter,resulting in a number of clinical manifestations such as visceral larva migrans?VLMs?,ocular larva migrans?O LMs?,eosinophilic meningoencephalitis?EME?,covert toxocariasis and neurotoxocariasis.Therefore,it is important to conduct accurate and effective identification of species as well as the prevention and control of human infection with T.cati.The parasites collected from cat intestines in different areas of Guangdong Province were firstly identified by morphological observation,and then PCR was used for further identification.The results showed that there were 142 cat intestine s detected,102 cats were infected with intestinal parasites,and the infection rate was 71.8%.According to morphological and molecular methods,six species of parasites,i.e.,Spirometra mansoni,Dipylidium caninum,Taenia taeniaformis,Clonorchis sinensis,T.cati,and Ancylostoma tubaeforme were identified.The infection rates were 30.3%,21.1%,19.7%,3.5%,21.1%,and 4.9%,respectively,and the highest infection rate was cat cestodes,followed by T.cati,C.sinensis,and A.tubaeforme.Most of these parasites were widespread zoonotic parasites,and human infection was a great security risk.Genomic DNA was then extracted from 26 individual T.cati.The mitochondrial pcox1 and ribosomal internal transcribed spacers?ITS?were amplified individually and subjected to agarose gel electrophoresis.First,the complete ITS?ITS1+5.8S rDNA+ITS2?was amplified separately from individual worms with PCR,and amplicons were then subjected to sequencing from both directions.The results were submitted to GenBank and given accession numbers.The sequences of ITS-1 and ITS-2 were 462 bp and 335 bp in length,respectively.The A+T contents of the sequences were 48.1%⁴8.9%?ITS-1?and51.6%⁵3.2%?ITS-2?,respectively,which was consistent with those of previous studies.The intra-specific sequence variations within T.cati were 0%².4%for ITS-1 and 0%².7%for ITS-2.However,compared with T.canis,the inter-specific sequence differences were significantly higher,i.e.,10.7%¹5.9%and 11.3%¹7.9%for ITS-1 and ITS-2,respectively,which was consistent with those of previous studies.The pcox1 was amplified separately from individual worms with PCR,and amplicons were then subjected to sequencing from both directions,submitted to GenBank and were given accession numbers.The sequence of pcox1 was 308 bp in length.The A+T contents of the sequences were 62.5%⁶5.8%,respectively,which was consistent with those of previous studies.The intra-specific sequence variations within T.cati were 0%³.6%for pcox1.However,compared with T.canis,the inter-specific sequence differences were significantly higher,bening 8.6%¹3.2%for pcox1,which was consistent with those of previous studies.In the present study,based on the pcox1 sequences,the phylogenetic relationships among the four Toxocara species were also reconstructed.These results indicated that all the isolates in Guangdong province?the same origin?were in genus Toxocara,which confirmed that these parasites represent T.cati.From the phylogenetic tree,T.catiwere sister to the T.canis,T.malaysiensis and T.vitulorum.In the third part of the study,a species-specific loop-mediated isothermal amplification?LAMP?assay for the rapid detection and discrimination of T.cati was developed.Based on the pcox1 and ITS sequences of T.cati,10 sets of primers were designed and the specific and sensitive one named T-5 was screened out.Temperature optimization,reaction time optimization,specificity and sensitivity tests were carried out.The LAMP assay was inexpensive,easy to perform and provided a rapid reaction,such that the amplification could be obtained in approximately one hour?65 min?under isothermal conditions at 63°C.The optimal assay conditions with no cross-reaction with other closely related worms?Toxascaris leonine,Spirometra mansoni,Dipylidium caninum,Taenia taeniaformis,Clonorchis sinensis,and Ancylostoma tubaeforme?were established.The minimum detectable DNA concentration was 10^-5 ng/?L.The results indicated that the LAMP assay was approximately 10³ times more sensitive than the conventional specific PCR assays.The LAMP method was tested using 82 stray cat feces from Guangdong Province.There were 13 stray cat feces that had T.cati eggs detected via microscope observation,and there were 19 stray cats feces that were positive detected by the established LAMP assay.The positive rate was 23%.These findings indicate that this T.cati species-specific LAMP assay may have potential clinical application for detection.In the present study,genetic variation in the mt pcox1 gene and the r DNA ITS region among T.cati in Guangdong Province was assessed.We also developed a species-specific loop-mediated isothermal amplification?LAMP?assay for the rapid detection and discrimination of T.cati.These results not only provided novel sequences information of rDNAand mt DNA for the study of nematodes but also provided important implications for the molecular taxonomy and population genetic struc tures of nematodes,and themolecularepidemiologicalstudyof T.cati.