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.

Ascarid infection in wild Amur tigers (Panthera tigris altaica) in China.

BACKGROUND: Wild Amur tigers are a sparsely populated species, and the conservation of this species is of great concern worldwide, but as an important health risk factor, parasite infection in them is not fully understanding. RESULTS: In this study, sixty-two faecal samples were collected to investigate the frequency and infection intensity of Toxocara cati and Toxascaris leonina in wild Amur tigers. The T. cati and T. leonina eggs were preliminary identified by microscopy, and confirmed by molecular techniques. Infection intensity was determined by the modified McMaster technique. Phylogenetic trees demonstrated that T. cati of wild Amur tiger had a closer relationship with which of other wild felines than that of domestic cats. T. leonina of Amur tiger and other felines clustered into one clade, showing a closer relationship than canines. The average frequency of T. cati was 77.42% (48/62), and the frequency in 2016 (100%) were higher than those in 2013 (P = 0.051, < 0.1; 66.6%) and 2014 (P = 0.079, < 0.1; 72.2%). The infection intensity of T. cati ranged from 316.6 n/g to 1084.1 n/g. For T. leonina, only three samples presented eggs when the saturated sodium chloride floating method was performed, indicating that the frequency is 4.83% (3/62). Unfortunately, the egg number in faecal smears is lower than the detective limitation, so the infection intensity of T. leonina is missed. CONCLUSIONS: This study demonstrated that ascarids are broadly prevalent, and T. cati is a dominant parasite species in the wild Amur tiger population.

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.

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.

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.

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.