[Genetic variation of sequences of partial mitochondrial nicotinamide adenine dinucleotide dehydrogenase subunit 1 (pnad1) and pnad5 genes of Toxascaris leonina isolates from foxes and dogs in Jiuquan City of Gansu Province].

OBJECTIVE: To investigate the genetic variations of Toxascaris leonina isolates from different hosts in Jiuquan City, Gansu Province. METHODS: The mitochondrial sequences of partial mitochondrial nicotinamide adenine dinucleotide dehydrogenase subunit 1 (pnad1) and pnad5 of eleven T. leonina isolates from domestic dogs, foxes and pet dogs in Jiuquan City, Gansu Province, were amplified using PCR, and the amplification product was sequenced. The genetic variations of pnad1 and pnad5 genes in T. leonina isolates were analyzed. RESULTS: The sequences of T. leonina pnad1 and pnad5 genes measured 530 bp and 550 bp in size, respectively. The nucleotide sequence homology was 99.4% to 100.0% for T. leonina pnad1 gene and 99.5% to 99.8% for T. leonina pnad5 gene, and the sequences of T. leonina pnad1 and pnad5 genes shared 99.2% to 99.9% and 99.1% to 99.9% with corresponding sequences of known T. leonina isolates. In addition, there were 19 and 24 polymorphic sites detected in the sequences of T. leonina pnad1 and pnad5 genes, with 10 and 9 haplotypes, haplotype diversity of 0.982 and 0.964 and nucleotide diversity of 0.039 4 and 0.034 2, respectively. Phylogenetic analysis based on pnad1 and pnad5 gene sequences showed that the eleven T. leonina isolates and known T. leonina isolates were clustered into the same branch with a random distribution, which were close to the branch where Toxocara canis was clustered, and far from the branch where other Ascaris species were clustered. CONCLUSIONS: There is a minor genetic variation in pnad1 and pnad5 genes of T. leonina isolates from different hosts in Jiuquan City, Gansu Province, and the pnad1 gene is more suitable as a molecular marker than pnad5 gene for analysis of genetic variations in T. leonina.

Molecular characterization of ascaridoid parasites from captive wild carnivores in China using ribosomal and mitochondrial sequences.

BACKGROUND: Despite the public health importance of toxocariasis/toxascariasis, only a few species of these ascaridoid parasites from wild canine and feline carnivores have been studied at the molecular level so far. Poor understanding of diversity, host distribution and the potential (zoonotic) transmission of the ascaridoid species among wild animals negatively affects their surveillance and control in natural settings. In this study, we updated previous knowledge by profiling the genetic diversity and phylogenetic relationships of ascaridoid species among eleven wild canine and feline animals on the basis of a combined analysis of the ribosomal internal transcribed spacer region (ITS) gene and the partial mitochondrial cytochrome c oxidase subunit 2 (cox2) and NADH dehydrogenase subunit 1 (nad1) genes. RESULTS: In total, three genetically distinct ascaridoid lineages were determined to be present among these wild carnivores sampled, including Toxocara canis in Alopex lagopus and Vulpes vulpes, Toxocara cati in Felis chaus, Prionailurus bengalensis and Catopuma temmincki and Toxascaris leonina in Canis lupus, Panthera tigris altaica, Panthera tigris amoyensis, Panthera tigris tigris, Panthera leo and Lynx lynx. Furthermore, it was evident that T. leonina lineage split into three well-supported subclades depending on their host species, i.e. wild felids, dogs and wolves and foxes, based on integrated genetic and phylogenetic evidence, supporting that a complex of T. leonina other than one species infecting these hosts. CONCLUSIONS: These results provide new molecular insights into classification, phylogenetic relationships and epidemiological importance of ascaridoids from wild canids and felids and also highlight the complex of the taxonomy and genetics of Toxascaris in their wild and domestic carnivorous hosts.

Phylogenetic relationships among Toxocara spp. and Toxascaris sp. from different regions of the world.

Toxocara and Toxascaris are parasitic nematodes that infect canids and felids although species of the genus Toxocara also infect humans. This work aimed to establish the phylogenetic and phylogeographic relationship between specimens of T. canis, T. cati, T. malaysiensis and Toxascaris leonina and to evaluate the degree of host specificity. In total, 437 samples (adults and pools of eggs) were collected from canids and felids from eight countries. Parasites were identified by morphology, PCR linked Restriction Fragment Length Polymorphism (PCR-RFLP) and partial sequencing of the mitochondrial gene cox1. Phylogenetic trees were constructed and genetic distance among isolates was estimated. Based on the molecular characterization all worms were identified in agreement with their respective hosts with the exception of three samples; two from cats and one from dogs identified as T. canis and T. cati, respectively. There was no clear geographical clustering of the samples despite this study including parasites from three continents. This is the first study, to our knowledge, to use molecular methods to identify T. canis in cats and T. cati in dogs with host specificity being the most common finding. Our developed PCR-RFLP method was found to be a facile and reliable method for identifying Toxocara species.

Ancient parasitic DNA reveals Toxascaris leonina presence in Final Pleistocene of South America.

Parasitological analysis of coprolites has allowed exploring ecological relationships in ancient times. Ancient DNA analysis contributes to the identification of coprolites and their parasites. Pleistocene mammalian carnivore coprolites were recovered from paleontological and archaeological site Peñas de las Trampas 1.1 in the southern Puna of Argentina. With the aim of exploring ancient ecological relationships, parasitological analysis was performed to one of them, dated to 16 573-17 002 calibrated years BP, with 95.4% probability. Parasite eggs attributed to Toxascaris sp. by morphological characters were isolated. DNA of coprolite and eggs was extracted to molecular identification. Ancient mitochondrial DNA analysis confirmed the zoological origin of the coprolite as Puma concolor and that of parasite eggs as Toxascaris leonina. This is the oldest molecular parasite record worldwide, and it supports the presence of this parasite since the Pleistocene in America. These findings have implications for the biogeographic history of parasites and for the natural history of the region.

Comparative analysis of mitochondrial DNA datasets indicates that Toxascaris leonina represents a species complex.

BACKGROUND: Toxascaris leonina is one of the most common intestinal parasites of canids and felids. In this study, we characterised the entire mitochondrial (mt) genome sequence of T. leonina from the cheetah and compared it with that of T. leonina from the dog. RESULTS: The entire mt genome sequence of T. leonina from the cheetah is 14,685 bp in size, which is 375 bp longer than that from the dog, and it is 408 bp longer than that from the South China tiger. The overall nucleotide sequence (except for the non-coding region) identity was 92.8% between the two mt genomes of T. leonina from the cheetah and the dog. For the 12 protein-coding genes, sequence difference between T. leonina from the cheetah and the dog was 5.0-9.7% at the nucleotide level and 1.0-7.2% at the amino acid level. Moreover, comparison of mt cox1 sequences among T. leonina isolates (n = 23) from different hosts revealed substantial nucleotide differences (10.6%). Phylogenetic analysis showed the separation of T. leonina from canid and felid hosts into three distinct clades. CONCLUSIONS: Taken together, these mtDNA datasets indicate that T. leonina from canid and felid hosts represents a species complex. Our results have implications for further studies of the molecular epidemiology, systematics and population genetics of this nematode.

Molecular data reveal cryptic speciation and host specificity in Toxascaris leonina (Nematoda: Ascarididae).

Toxascaris leonina (Ascarididae) is a cosmopolitan and polyxenical parasite whose host are canids and felids. To date, molecular phylogenetic studies included toxascarid representatives collected only from dogs or felids, therefore the intra-species differences between T. leonina collected from different host species has not been noticed. In this paper, we test the hypothesis of cryptic speciation in the T. leonina complex based on extended sequence data (ITS1, nad1, cox1) and individuals collected from dogs, felids and foxes. Phylogenetic analysis clustered T. leonina representatives into three well-supported clades depending on their host species, i.e. dogs and wolves, wild felids and foxes. Both genetic distances and the barcoding-gap analysis strongly support the species status of populations inhabiting different hosts. The results suggest additional genetic separation in felids. However, to determine the actual size of the Toxascaris complex, it would be necessary to analyse individuals collected from other canid and felid Toxascaris leonina host species.

Structural Basis for Carbohydrate Recognition and Anti-inflammatory Modulation by Gastrointestinal Nematode Parasite Toxascaris leonina Galectin.

Toxascaris leonina galectin (Tl-gal) is a galectin-9 homologue protein isolated from an adult worm of the canine gastrointestinal nematode parasite, and Tl-gal-vaccinated challenge can inhibit inflammation in inflammatory bowel disease-induced mice. We determined the first X-ray structures of full-length Tl-gal complexes with carbohydrates (lactose, N-acetyllactosamine, lacto-N-tetraose, sialyllactose, and glucose). Bonds were formed on concave surfaces of both carbohydrate recognition domains (CRDs) in Tl-gal. All binding sites were found in the HXXXR and WGXEER motifs. Charged Arg61/Arg196 and Glu80/Glu215 on the conserved motif of Tl-gal N-terminal CRD and C-terminal CRD are critical amino acids for recognizing carbohydrate binding, and the residues can affect protein folding and structure. The polar amino acids His, Asn, and Trp are also important residues for the interaction with carbohydrates through hydrogen bonding. Hemagglutination activities of Tl-gal were inhibited by interactions with carbohydrates and mutations. We found that the mutation of Tl-gal (E80A/E215A) at the carbohydrate binding region induced protein aggregation and could be caused in many diseases. The short linker region between the N-terminal and C-terminal CRDs of Tl-gal was very stable against proteolysis and maintained its biological activity. This structural information is expected to elucidate the carbohydrate recognition mechanism of Tl-gal and improve our understanding of anti-inflammatory mediators and modulators of immune response.

Ascarid infestation in captive Siberian tigers in China.

The Siberian tiger is endangered and is listed by the International Union for the Conservation of Nature; the captive environment is utilized to maintain Siberian tiger numbers. Little information regarding the prevalence of parasites in Siberian tigers is available. A total of 277 fecal samples of Siberian tigers were analyzed in this study. The microscopic analysis indicated the presence of ascarid eggs of Toxascaris leonina and Toxocara cati. The ascarid infection rate was 67.5% in Siberian tigers. The internal transcribed spacer-1 (ITS-1) phylogenetic analysis indicated that T. leonina belonged to Toxascaris and that Toxo. cati belonged to Toxocara. The infestation rate and intensity of T. leonina were higher than those of Toxo. cati. One-way analysis of variance showed that the presence of T. leonina was significantly associated with age (P<0.05). Temperature changes also influenced T. leonina and Toxo. cati infestation, and a rise in temperature caused an increase in the number of T. leonina and Toxo. cati eggs. This study provides a better understanding of ascarid infestation among the captive Siberian tigers and is helpful for the prevention of the spread of infectious parasitic diseases among other tigers in the zoo.

Sequence Analysis of Mitochondrial Genome of Toxascaris leonina from a South China Tiger.

Toxascaris leonina is a common parasitic nematode of wild mammals and has significant impacts on the protection of rare wild animals. To analyze population genetic characteristics of T. leonina from South China tiger, its mitochondrial (mt) genome was sequenced. Its complete circular mt genome was 14,277 bp in length, including 12 protein-coding genes, 22 tRNA genes, 2 rRNA genes, and 2 non-coding regions. The nucleotide composition was biased toward A and T. The most common start codon and stop codon were TTG and TAG, and 4 genes ended with an incomplete stop codon. There were 13 intergenic regions ranging 1 to 10 bp in size. Phylogenetically, T. leonina from a South China tiger was close to canine T. leonina. This study reports for the first time a complete mt genome sequence of T. leonina from the South China tiger, and provides a scientific basis for studying the genetic diversity of nematodes between different hosts.

Sequence variation in mitochondrial cox1 and nad1 genes of ascaridoid nematodes in cats and dogs from Iran.

The study was conducted to determine the sequence variation in two mitochondrial genes, namely cytochrome c oxidase 1 (pcox1) and NADH dehydrogenase 1 (pnad1) within and among isolates of Toxocara cati, Toxocara canis and Toxascaris leonina. Genomic DNA was extracted from 32 isolates of T. cati, 9 isolates of T. canis and 19 isolates of T. leonina collected from cats and dogs in different geographical areas of Iran. Mitochondrial genes were amplified by polymerase chain reaction (PCR) and sequenced. Sequence data were aligned using the BioEdit software and compared with published sequences in GenBank. Phylogenetic analysis was performed using Bayesian inference and maximum likelihood methods. Based on pairwise comparison, intra-species genetic diversity within Iranian isolates of T. cati, T. canis and T. leonina amounted to 0-2.3%, 0-1.3% and 0-1.0% for pcox1 and 0-2.0%, 0-1.7% and 0-2.6% for pnad1, respectively. Inter-species sequence variation among the three ascaridoid nematodes was significantly higher, being 9.5-16.6% for pcox1 and 11.9-26.7% for pnad1. Sequence and phylogenetic analysis of the pcox1 and pnad1 genes indicated that there is significant genetic diversity within and among isolates of T. cati, T. canis and T. leonina from different areas of Iran, and these genes can be used for studying genetic variation of ascaridoid nematodes.

The complete mitochondrial genome of Toxascaris leonina: Comparison with other closely related species and phylogenetic implications.

Adults of Toxascaris leonina (Nematoda: Ascarididae) live in the gastrointestinal tract of both dogs and cats, and cause significant economic losses and potential public health problem worldwide. Although many studies have given insights into this significant pathogen, to date, the complete mitochondrial (mt) genome sequence is still not available for T. leonina. Here, we sequenced the complete mt genome of T. leonina. This AT-rich (71.53%) mt genome (14,310bp) is circular and consists of 36 genes, including 12 genes for proteins, 2 genes for rRNA and 22 genes for tRNA. All mt genes of T. leonina are transcribed in the same direction. The gene order is the same as those of Ascaris spp. (Ascarididae), Toxocara spp. (Toxocaridae), Anisakis simplex and Contracaecum rudolphii B (Anisakidae), but distinct from that of Ascaridia spp. (Ascaridiidae). Phylogenetic analyses using concatenated amino acid sequences of 12 protein-coding genes by Bayesian inference (BI) showed distinct groups with high statistical support, and our data confirm that T. leonina is a member of the Ascarididae, and that this family is more closely related to the Toxocaridae rather than the Anisakidae within the Ascaridoidea. The determination of mt genome sequences of T. leonina provides novel genetic markers for studies into the systematics, population genetics and epidemiology of this parasite.

Comparison of functional gene annotation of Toxascaris leonina and Toxocara canis using CLC genomics workbench.

The ascarids, Toxocara canis and Toxascaris leonina, are probably the most common gastrointestinal helminths encountered in dogs. In order to understand biological differences of 2 ascarids, we analyzed gene expression profiles of female adults of T. canis and T. leonina using CLC Genomics Workbench, and the results were compared with those of free-living nematode Caenorhabditis elegans. A total of 2,880 and 7,949 ESTs were collected from T. leonina and T. canis, respectively. The length of ESTs ranged from 106 to 4,637 bp with an average insert size of 820 bp. Overall, our results showed that most functional gene annotations of 2 ascarids were quite similar to each other in 3 major categories, i.e., cellular component, biological process, and molecular function. Although some different transcript expression categories were found, the distance was short and it was not enough to explain their different lifestyles. However, we found distinguished transcript differences between ascarid parasites and free-living nematodes. Understanding evolutionary genetic changes might be helpful for studies of the lifestyle and evolution of parasites.

Comparison of six simple methods for extracting ribosomal and mitochondrial DNA from Toxocara and Toxascaris nematodes.

Six simple methods for extraction of ribosomal and mitochondrial DNA from Toxocara canis, Toxocara cati and Toxascaris leonina were compared by evaluating the presence, appearance and intensity of PCR products visualized on agarose gels and amplified from DNA extracted by each of the methods. For each species, two isolates were obtained from the intestines of their respective hosts: T. canis and T. leonina from dogs, and T. cati from cats. For all isolates, total DNA was extracted using six different methods, including grinding, boiling, crushing, beating, freeze-thawing and the use of a commercial kit. To evaluate the efficacy of each method, the internal transcribed spacer (ITS) region and the cytochrome c oxidase subunit 1 (cox1) gene were chosen as representative markers for ribosomal and mitochondrial DNA, respectively. Among the six DNA extraction methods, the beating method was the most cost effective for all three species, followed by the commercial kit. Both methods produced high intensity bands on agarose gels and were characterized by no or minimal smear formation, depending on gene target; however, beating was less expensive. We therefore recommend the beating method for studies where costs need to be kept at low levels.

Intraspecific variation between the ITS sequences of Toxocara canis, Toxocara cati and Toxascaris leonina from different host species in south-western Poland.

Some parasitic nematodes can inhabit different definitive hosts, which raises the question of the intraspecific variability of the nematode genotype affecting their preferences to choose particular species as hosts. Additionally, the issue of a possible intraspecific DNA microheterogeneity in specimens from different parts of the world seems to be interesting, especially from the evolutionary point of view. The problem was analysed in three related species - Toxocara canis, Toxocara cati and Toxascaris leonina - specimens originating from Central Europe (Poland). Using specific primers for species identification, internal transcribed spacer (ITS)-1 and ITS-2 regions were amplified and then sequenced. The sequences obtained were compared with sequences previously described for specimens originating from other geographical locations. No differences in nucleotide sequences were established in T. canis isolated from two different hosts (dogs and foxes). A comparison of ITS sequences of T. canis from Poland with sequences deposited in GenBank showed that the scope of intraspecific variability of the species did not exceed 0.4%, while in T. cati the differences did not exceed 2%. Significant differences were found in T. leonina, where ITS-1 differed by 3% and ITS-2 by as much as 7.4% in specimens collected from foxes in Poland and dogs in Australia. Such scope of differences in the nucleotide sequence seems to exceed the intraspecific variation of the species.

Comparison of Functional Gene Annotation of Toxascaris leonina and Toxocara canis using CLC Genomics Workbench

The ascarids, Toxocara canis and Toxascaris leonina, are probably the most common gastrointestinal helminths encountered in dogs. In order to understand biological differences of 2 ascarids, we analyzed gene expression profiles of female adults of T. canis and T. leonina using CLC Genomics Workbench, and the results were compared with those of free-living nematode Caenorhabditis elegans. A total of 2,880 and 7,949 ESTs were collected from T. leonina and T. canis, respectively. The length of ESTs ranged from 106 to 4,637 bp with an average insert size of 820 bp. Overall, our results showed that most functional gene annotations of 2 ascarids were quite similar to each other in 3 major categories, i.e., cellular component, biological process, and molecular function. Although some different transcript expression categories were found, the distance was short and it was not enough to explain their different lifestyles. However, we found distinguished transcript differences between ascarid parasites and free-living nematodes. Understanding evolutionary genetic changes might be helpful for studies of the lifestyle and evolution of parasites.

Identification of host immune regulation candidate genes of Toxascaris leonina by expression sequenced tags (ESTs) analysis.

Toxascaris leonina adult worms live in the gastrointestinal tract of dog, cat, and fox, releasing eggs which enter the environment by the fecal route. Previously, we reported that T. leonina adult worm derived protein was able to inhibit OVA-specific Th2 responses, and in particular, immunization with parasite proteins exerts a more profound protective effect than allergen treatment. In order to gain greater insight into the relevant immune evasion mechanisms as well as basic scientific information, we have generated ESTs of T. leonina adult female worm and investigated their functions using euKaryotic Orthologous Groups (KOG) database analysis. From the randomly selected plasmids containing DNA inserts, a total of 487 reads were collected from the T. leonina adult worm cDNA library. The annotated ESTs were classified into 25 KOG categories; the most of ESTs (7.90%) were annotated with energy production and conversion, and the second highly annotated category is translation, ribosomal structure and biogenesis related ESTs (7.69%). We also identified many host-parasite immune related genes including C-type lectin, galectin, SXP, and cathepsin L-like cysteine protease coding genes. It is necessary to get more information regarding these genes for understanding about the mechanisms of immune evasion of Toxascaris.

Electrophoretic analysis of sequence variability in three mitochondrial DNA regions for ascaridoid parasites of human and animal health significance.

Sequence variability in three mitochondrial DNA (mtDNA) regions, namely cytochrome c oxidase subunit 1 (cox1), NADH dehydrogenase subunits 1 and 4 (nad1 and nad4), among and within Toxocara canis, T. cati, T. malaysiensis, T. vitulorum and Toxascaris leonina from different geographical origins was examined by a mutation-scanning approach. A portion of the cox1 gene (pcox1), a portion of the nad1 and nad4 genes (pnad1 and pnad4) were amplified separately from individual ascaridoid nematodes by polymerase chain reaction and the amplicons analyzed by single-strand conformation polymorphism (SSCP). Representative samples displaying sequence variation in SSCP profiles were subjected to sequencing in order to define genetic markers for their specific identification and differentiation. While the intra-specific sequence variations within each of the five ascaridoid species were 0.2-3.7% for pcox1, 0-2.8% for pnad1 and 0-2.3% for pnad4, the inter-specific sequence differences were significantly higher, being 7.9-12.9% for pcox1, 10.7-21.1% for pnad1 and 12.9-21.7% for pnad4, respectively. Phylogenetic analyses based on the combined sequences of pcox1, pnad1 and pnad4 revealed that the recently described species T. malaysiensis was more closely related to T. cati than to T. canis. These findings provided mtDNA evidence for the validity of T. malaysiensis and also demonstrated clearly the usefulness and attributes of the mutation-scanning sequencing approach for studying the population genetic structures of these and other nematodes of socio-economic importance.