Molecular Identification And Studies Of Mitochondrial Genome Of Toxocara Species

Ascaridoids parasitizing in canine and feline animals are members of the two genera, Toxocara and Toxascaris, Ascarididae, Nematoda. Among them, the most common species are Toxocara canis and Toxocara cati, which have important implications for public health because their larvae are able to invade tissues of non-specific hosts including the human and cause toxocariasis in humans. This thesis includes three parts, which are"Development and applications of species-specific molecular assays for the identification of ascaridoid nematodes from dogs and cats","Studies of DNA polymorphism in three mitochondrial DNA regions for Toxocara and Toxascaris species"and"The complete mitochondrial genome of T. canis: sequence and structure analysis"Part 1: Development and applications of species-specific molecular assays for the identification of ascaridoid nematodes from dogs and catsBased on the published sequences of the internal transcribed spacers (ITS) of ribosomal DNA (rDNA) of Toxocara and Toxascaris species, four forward primers, specific to T. canis, T. cati, T. malaysiensis and Ta. leonina, respectively, were designed in the ITS-1 or ITS-2. With the common conserved reverse primer NC2 and the species-specific forward primers, ITS-1 and/or ITS-2 of rDNA were respectively amplified by PCR from 99 samples representing four ascaridoid species from dogs and cats from China, Australia, Malaysia, England and Holland for the specific identification of these ascaridoid nematodes. The specificity tests indicated that there was no cross-reaction among these ascaridoids, and between them and Toxocara vitulorum, Ascaris suum and Ascaridia galli. The minimum detectable DNA concentrations of the specific assays were 0.14ng/μl for T. canis, 0.54ng/μl for T. cati, 0.38ng/μl for T. malaysiensis and 0.13ng/μl for T. leonina, respectively. The sequencing results of the amplified specific partial ITS1 and/or ITS-2 fragments confirmed the validity of the specific PCR assays. These specific PCR assays could be applied to distinguish these ascaridoid species in tissues, and should provide a complementary tool for the diagnosis and the epidemiologic investigation of toxocariasis in humans.Results of species-specific PCR assays showed that some samples did not represent the species which had been previously identified according to morphological characters. For instance, samples Tmal 41 and Tmal 43 from Malaysia (Tmal represents T. malaysiensis) represented T. cati, instead of T. malaysiensis; Tcat 2-2 from China (Tcat represents T. cati) represented T. canis, but not T. cati; Tcat 1-1 and Tcat 1-2 from China represented T. malaysiensis, not T. cati.Species-specific PCR approaches were applied to identify two individuals of ascaridoid collected from the intestine of a cat from Guangzhou, China, using the ITS of rDNA as genetic markers. With species-specific primer sets, a partial ITS of rDNA was respectively amplified by PCR from the genomic DNA of these two samples and were sequenced. While no DNA fragments were amplified from the two samples using primers specific to T. canis and T. cati, a DNA fragment of expected size was amplified by PCR using primers specific to T. malaysiensis. Sequence similarity in the ITS-2 between the two samples and T. malaysiensis, T. cati and T. canis were 100%, 88.7%~89.0% and 75.8%, respectively. It is concluded that the two ascaridoid samples represent T. malaysiensis, which was found in cat in China the first time.A PCR-based non-isotopic ('cold') single-strand conformation polymorphism (PCR-SSCP) approach was also established for the identification of four ascaridoid nematodes from dogs and cats from different geographical origins. The ITS-2 of rDNA was amplified from individual ascaridoids (n=45) by PCR, and the amplicons were subjected to non-denaturing gel electrophoresis, followed by staining with ethidium bromide. While no heterogeneity in the SSCP banding patterns was observed among individuals of the T. canis, T. cati, and Ta. leonina, and only subtle heterogeneity among individuals of the T. malaysiensis was displayed, the four ascaridoid species displayed distinct interspecific SSCP banding patterns, which allowed their unequivocal identification and differentiation. Compared with the conventional ('hot') SSCP approach using radio-labelled primers, the'cold'SSCP approach has the same advantage of achieving high resolution in the display of sequence variability as'hot'-SSCP, and overcomes the disadvantages of radioactive risk and labour intensity of'hot'-SSCP. The non-isotopic SSCP approach could provide a useful complementary tool to traditional methods for the accurate identification of Toxocara and Toxascaris species from dogs and cats, and for the diagnosis and epidemiological investigation of disease they cause.Part 2: Studies of DNA polymorphism in three mitochondrial DNA regions for Toxocara and Toxascaris speciesIn this part, the objectives were to investigate sequence variation in three regions of mitochondrial DNA (mtDNA) and to examine the genetic relationships among and within Toxocara and Toxascaris species from dogs and cats, and to find better genetic markers for studying systematics and population genetics of these ascaridoids.Total DNA was extracted from ascaridoid nematodes including T. canis, T. cati, T. malaysiensis, T. vitulorum and Ta. leonina from different geographical origins. The partial regions of three mitochondrial DNA genes, namely partial cytochrome c oxidase subunit (pcox1), NADH dehydrogenase subunits 1 and 4 (pnad1 and pnad4), were amplified separately from individual nematodes by PCR and analyzed by isotopic single-strand conformation polymorphism (SSCP). Representative samples displaying sequences variation in SSCP were subjected to sequencing in order to find sequence differences.While no heterogeneity among individuals of T. vitulorum and T. leonina and distinct heterogeneity among individuals of T. cati in the SSCP banding patterns were observed, only subtle differences among individuals of T. canis was displayed in the three regions of mitochondrial DNA.Results of selective sequencing supported results of SSCP analyses. While the intraspecific variations in the pcox1 sequence were 2.1%~3.7% for T. canis and 0.2%~0.9% for T. malaysiensis, the interspecific differences were 10.2%~11.3% between T. canis and T. cati, 9.7%~12.2% between T. canis and T. malaysiensis, 9.5%~11.0% between T. canis and T. vitulorum, 10.0%~12.9% between T. canis and T. leonina, 7.9%~8.4% between T. cati and T. malaysiensis, 8.0% between T. cati and T. vitulorum, 8.4%~8.7% between T. cati and T. leonina, 8.5%~8.7% between T. malaysiensis and T. vitulorum, and 10.5%~11.3% between T. malaysiensis and T. leonina, respectively.While the intraspecific variations in the pnad1 sequences were 0.3%~1.9% for T. canis, 2.8% for T. cati, 0.0%~0.3% for T. malaysiensis from different geographical origins, the interspecific differences were 11.0%~14.1%between T. canis and T. cati, 10.7%~12.4% between T. canis and T. malaysiensis, 12.6%~13.7% between T. canis and T. vitulorum, 17.5%~18.2% between T. canis and T. leonina, 12.0%~13.0% between T. cati and T. malaysiensis, 15.1% between T. cati and T. vitulorum, 18.6~21.2% between T. cati and T. leonina, 12.4%~12.8% between T. malaysiensis and T. vitulorum, and 18.6%~19.0% between T. malaysiensis and T. leonine, respectively.While the intraspecific variations in the pnad4 sequences were 0.5%~2.3% for T. canis, 1.0% for T. cati, 0.0%~0.3% for T. malaysiensis from different geographical origins, the interspecific differences were 13.6%~15.2% between T. canis and T. cati, 15.9%~17.6% between T. canis and T. malaysiensis, 14.4%~15.2% between T. canis and T. vitulorum, 20.1%~21.9% between T. canis and T. leonina, 17.9%~18.3% between T. cati and T. malaysiensis, 13.0%~14.3% between T. cati and T. vitulorum, 18.6%between T. cati and T. leonina, 13.5%~13.8% between T. malaysiensis and T. vitulorum, and 20.7%~21.0% between T. malaysiensis and T. leonina.The results of sequencing proved that Tcat1-1, Tcat1-2, MTT1 and Tcat2-2 did not represent T. cati, but T. canis or T. malaysiensis, which was consistent with species-specific PCR assays. In the partial regions of the three mitochondrial DNA genes, the cox1 had lower variability, and the nad4 had higher variability. After pairwise comparisons among sequences representing the five species, 37 nucleotides positions for T. canis, 39 for T. cati, 39 for T. malaysiensis, 33 for T. vitulorum and 68 for Ta. leonina were considered as genetic markers and have diagnostic implications.These findings showed that the average intraspecific variations of the three mitochondrial genes were significantly lower than the interspecific defferences, and should have important implications for studying systematics, population genetic structures and the molecular ecology of Toxocara and Toxascaris species. The results proved also that T. malaysiensis is a valid species. The sequences of three mitochondrial genes could serve as the useful genetic markers for the identification of Toxocara and Toxascaris species.Part 3: The complete mitochondrial genome of T. canis: sequence and structure analysisMitochondrial DNA (mtDNA) is genetic material out of the nucleus of metazoan organisms. Mitochondrial genome sequences provide useful markers for investigating population genetic structures, systematics and phylogenetics because of their maternal inheritance, high evolutionary rates, small size and simple structure. Although Nematoda is the biggest animal group except Insect, mitochondrial genome sequences of less than 10 nematode species have been sequenced. To add more mtDNA data, and to fill gaps of knowledge, mitochondrial genome sequence of T. canis was sequenced. Two fragments, which were approximately 5.5kb and 9.5kb in size, were amplified by PCR using primer sets 39F/42R and 5F/40R. PCR products were sent to Takara Company for sequencing by 3310 sequencing apparatus. The results of sequencing showed that the complete mitochondrial genome of T. canis is 14322 bp in size, comprising 12 protein genes, 2 rRNA genes and 22 tRNA genes. AT rich region is 985 bp in size. No introns were found within genes and there were some spacers among genes (1~11 bases). Gene arrangement of mitochondrial genome in T. canis was same as that of Ascaris suum. The A+T content of mtDNA in T. canis was 68.57%, which is the lowest among the sequenced mitochondrial genomes of other nematodes to date. Two tRNAs, tRNA-Ile and tRNA-Ala, overlaped nad2 and nad5, respectively. Among 12 genes coding for proteins, while TAG, TAA, TA and T used as translation termination codons, TTG, ATT, ATA, GTT and ATG were used as translation initiation codons. The complete mitochondrial genome sequence of T. canis has provided foundation for studying systematics and population genetics of Toxocara species and other nematodes of socio-economic importance.