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.

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.

Toxocarosis of the organ of sight - the complex pathological and diagnostic problem.

Selected parasitological and epidemiological aspects of Toxocara spp. invasion were discussed. Pathomechanism of lesions and involvement of the organ of sight in toxocarosis were presented. It was stressed that pathological lesions may involve various structures of the eyeball. Moreover, the pattern of lesions may vary depending on the early or late stage of the invasion. Diagnostic techniques were presented, indispensable in confirmation of toxocarosis and establishing its duration. In diagnosis of the ocular form of toxocarosis the significance of differential diagnostic analysis was stressed, particularly essential in oligosymptomatic cases and upon coexistence of other diseases progressing with involvement of the organ of sight. This is important for further specialised management and in selection of an appropriate therapy.

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.

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.

Detection and molecular characterization of ascarid nematode infection (Toxascaris leonina and Toxocara cati) in captive Asiatic lions (Panthera leo persica).

The objective of this study was to investigate the ascarid infection in Asiatic lions using scat samples, based on microscopic analysis, PCR amplification of the ITS-2 region of ribosomal DNA and sequence analysis of the amplicons. Microscopic analysis indicated the presence of eggs of Toxascaris leonina in eleven of the sixteen scat samples analysed and in one of these eleven scats eggs of Toxocara cati were also detected. In five of the scats eggs were not detectable. The presence of T. leonina in all the infected samples was also confirmed by PCR amplification of the ITS-2 of ribosomal RNA gene and five of these also showed amplicons corresponding to T. cati, respectively. Toxocara canis infection was not observed in any of the scat samples. Nucleotide sequence analysis of the ITS-2 region indicated 97% to 99% similarity with T. leonina and T. cati, respectively. To our knowledge, this is the first molecular characterization of ascarid infection in captive Asiatic lions from a zoological garden of India. This study also indicates that Asiatic lions are more prone to infection either with T. leonina or T. cati and the parasite is not host specific.

Biochemical and immunopathological changes in experimental neurotoxocariasis.

Toxocariasis is a widespread soil-transmitted parasitic disease. Toxocara canis larvae migrate through the tissues with a special predilection for the central nervous system. Recently, neurotoxocariasis is being diagnosed in humans with increasing frequency due to improved diagnostic tools. The present study aimed at exploring the biochemical and immunopathological alterations in the brain in experimental T. canis infection. For this purpose, 75 Toxocara-infected mice were sacrificed at 2, 5, and 16 weeks post-infection. The brains were removed and assayed for total larval count, pro-inflammatory cytokines (TNF-alpha, IL-6), and central neurotransmitters (gamma-aminobutyric acid, glutamate, dopamine, norepinephrine, and serotonin). Brain sections were also stained for histopathological study, and for assessment of the expression of inducible nitric oxide synthase (iNOS), and glial fibrillary acidic protein (GFAP) by immunohistochemical methods. We found that larval recovery showed progressive increase over the course of infection. Furthermore, the infected mice displayed increased expression of pro-inflammatory cytokines and iNOS, as well as significant disturbances in neurotransmitter profile. Astrocytic activation, evidenced by enhanced expression of GFAP, was also manifest in infected animals. These changes were maximal in the chronic stage of infection or intensified over time. In conclusion, experimental neurotoxocariasis is associated with significant biochemical, immunological, and pathological changes.

Characterization of antigenic property of Toxocara canis and Toxascaris leonina adults and larvae through immunodiagnostic electrophoresis (SDS-PAGE) and western blot technique.

Differential molecular studies were performed by sodium dodecyle sulphate polyacrylamide gel electrophoresis--SDS-PAGE--between somatic antigen of Toxocara canis and Toxascaris leonina, adults and larvae, recovered from dogs. SDS-PAGE of both adults somatic antigen showed two closely similar bands (90.00, 91.95 KDa and 69.25-70.56 KDa). Each parasite had characteristic bands clustered in different molecular weights. While for their larval antigen, T. canis showed a very different antigenic profile when analysed in comparison to T. leonina antigen except at one band (66.85-66.89 KDa). The Western blot analysis showed four prominent bands represented immunoreaction between the separated somatic antigen of T. canis adults and experimentally immunized rabbit with the corresponding parasite (125.37, 117.73, 90.00 and 69.25 KDa). While separated antigen of T. leonina adults immune reacted with the corresponding hyperimmune rabbit sera at 119.04, 91.95 and 70.56 KDa. The Western blot showed cross reactive immune bands between T. canis and T. leonina adults somatic antigen at two bands (90.00, 91.95 KDa and 69.25-70.56 KDa). The polypeptide bands reacted at 125.37 KDa and 117.73 KDa can be used as specific finger print for T. canis adults while that at 119.04 KDa was specific for T. leonina adult worm.

Efficacy of fenbendazole and piperazine against developing stages of toxocara and toxascaris in dogs.

In a series of controlled trials involving 59 naturally infected greyhounds, fenbendazole at a dose rate of 50 mg/kg/day for three consecutive days reduced the overall numbers of third and fourth stage Toxocara canis by 94.0 per cent and third stage, fourth stage and immature adult stages of Toxascaris leonina by 92.4 per cent. In contrast, piperazine at 100 mg/kg had little or no useful effect against the larval stages of T canis and T leonina and variable efficacy against immature adult T leonina. Fenbendazole was also 100 per cent effective against immature Trichuris vulpis. In a separate controlled experiment, puppies in three litters exposed to reinfection with T canis were treated with fenbendazole at two weeks old and again only after their mean faecal egg counts exceeded 200 epg. Between one and three doses were required to suppress the output of eggs during the puppies' first 12 weeks of life.