Ming-Wei Li

The complete mitochondrial genomes for three Toxocara species of human and animal health significance

BackgroundStudying mitochondrial (mt) genomics has important implications for various fundamental areas, including mt biochemistry, physiology and molecular biology. In addition, mt genome sequences have provided useful markers for investigating population genetic structures, systematics and phylogenetics of organisms. Toxocara canis, Toxocara cati and Toxocara malaysiensis cause significant health problems in animals and humans. Although they are of importance in human and animal health, no information on the mt genomes for any of Toxocara species is available.ResultsThe sizes of the entire mt genome are 14,322 bp for T. canis, 14029 bp for T. cati and 14266 bp for T. malaysiensis, respectively. These circular genomes are amongst the largest reported to date for all secernentean nematodes. Their relatively large sizes relate mainly to an increased length in the AT-rich region. The mt genomes of the three Toxocara species all encode 12 proteins, two ribosomal RNAs and 22 transfer RNA genes, but lack the ATP synthetase subunit 8 gene, which is consistent with all other species of Nematode studied to date, with the exception of Trichinella spiralis. All genes are transcribed in the same direction and have a nucleotide composition high in A and T, but low in G and C. The contents of A+T of the complete genomes are 68.57% for T. canis, 69.95% for T. cati and 68.86% for T. malaysiensis, among which the A+T for T. canis is the lowest among all nematodes studied to date. The AT bias had a significant effect on both the codon usage pattern and amino acid composition of proteins. The mt genome structures for three Toxocara species, including genes and non-coding regions, are in the same order as for Ascaris suum and Anisakis simplex, but differ from Ancylostoma duodenale, Necator americanus and Caenorhabditis elegans only in the location of the AT-rich region, whereas there are substantial differences when compared with Onchocerca volvulus,Dirofiliria immitis and Strongyloides stercoralis. Phylogenetic analyses based on concatenated amino acid sequences of 12 protein-coding genes revealed that the newly described species T. malaysiensis was more closely related to T. cati than to T. canis, consistent with results of a previous study using sequences of nuclear internal transcribed spacers as genetic markers.ConclusionThe present study determined the complete mt genome sequences for three roundworms of human and animal health significance, which provides mtDNA evidence for the validity of T. malaysiensis and also provides a foundation for studying the systematics, population genetics and ecology of these and other nematodes of socio-economic importance.

Clear genetic distinctiveness between human- and pig-derived Trichuris based on analyses of mitochondrial datasets.

The whipworm, Trichuris trichiura, causes trichuriasis in ∼600 million people worldwide, mainly in developing countries. Whipworms also infect other animal hosts, including pigs (T. suis), dogs (T. vulpis) and non-human primates, and cause disease in these hosts, which is similar to trichuriasis of humans. Although Trichuris species are considered to be host specific, there has been considerable controversy, over the years, as to whether T. trichiura and T. suis are the same or distinct species. Here, we characterised the entire mitochondrial genomes of human-derived Trichuris and pig-derived Trichuris, compared them and then tested the hypothesis that the parasites from these two host species are genetically distinct in a phylogenetic analysis of the sequence data. Taken together, the findings support the proposal that T. trichiura and T. suis are separate species, consistent with previous data for nuclear ribosomal DNA. Using molecular analytical tools, employing genetic markers defined herein, future work should conduct large-scale studies to establish whether T. trichiura is found in pigs and T. suis in humans in endemic regions.

Sparganosis in mainland China

Sparganosis is an infection of humans and animals caused by the plerocercoid larvae (spargana) of various diphyllobothroid tapeworms belonging to the genus Spirometra. Sparganosis has been reported sporadically around the world, and a higher prevalence of the disease occurs in several Asian countries, including South Korea, Japan, Thailand, and China. To date, a total of more than 1000 cases of human sparganosis have been reported in 25 provinces in mainland China. Sparganosis is emerging in mainland China because of food consumption habits and the unusual practice of treating wounds or other lesions with poultices of frog or snake flesh. This article reviews the current status of sparganosis in mainland China. Increased public awareness about the risks associated with eating raw food and strengthened food safety measures are needed.

The occurrence of Toxocara malaysiensis in cats in China, confirmed by sequence-based analyses of ribosomal DNA

Non-isotopic polymerase chain reaction (PCR)-based single-strand conformation polymorphism and sequence analyses of the second internal transcribed spacer (ITS-2) of nuclear ribosomal DNA (rDNA) were utilized to genetically characterise ascaridoids from dogs and cats from China by comparison with those from other countries. The study showed that Toxocara canis, Toxocara cati, and Toxascaris leonina from China were genetically the same as those from other geographical origins. Specimens from cats from Guangzhou, China, which were morphologically consistent with Toxocara malaysiensis, were the same genetically as those from Malaysia, with the exception of a polymorphism in the ITS-2 but no unequivocal sequence difference. This is the first report of T. malaysiensis in cats outside of Malaysia (from where it was originally described), supporting the proposal that this species has a broader geographical distribution. The molecular approach employed provides a powerful tool for elucidating the biology, epidemiology, and zoonotic significance of T. malaysiensis.

Sequence variability in two mitochondrial DNA regions and internal transcribed spacer among three cestodes infecting animals and humans from China.

Sequence variability in two mitochondrial DNA (mtDNA) regions, namely cytochrome c oxidase subunit 1 (cox1) and NADH dehydrogenase subunit 4 (nad4), and internal transcribed spacer (ITS) of rDNA among and within three cestodes, Spirometra erinaceieuropaei, Taenia multiceps and Taenia hydatigena, from different geographical origins in China was examined. A portion of the cox1 (pcox1), nad4 genes (pnad4) and the ITS (ITS1+5.8S rDNA+ITS2) were amplified separately from individual cestodes by polymerase chain reaction (PCR). Representative amplicons were subjected to sequencing in order to estimate sequence variability. While the intra-specific sequence variations within each of the tapeworm species were 0-0.7% for pcox1, 0-1.7% for pnad4 and 0.1-3.6% for ITS, the inter-specific sequence differences were significantly higher, being 12.1-17.6%, 18.7-26.2% and 31-75.5% for pcox1, pnad4 and ITS, respectively. Phylogenetic analyses based on the pcox1 sequence data revealed that T. multiceps and T. hydatigena were more closely related to the other members of the Taenia genus, and S. erinaceieuropaei was more closely related to the other members of the Spirometra genus. These findings demonstrated clearly the usefulness of mtDNA and rDNA sequences for population genetic studies of these cestodes of socio-economic importance.

Advances in molecular identification, taxonomy, genetic variation and diagnosis of Toxocara spp.

The genus Toxocara contains parasitic nematodes of human and animal health significance, such as Toxocara canis, Toxocara cati and Toxocara vitulorum. T. canis and T. cati are among the most prevalent parasites of dogs and cats with a worldwide distribution. Human infection with T. canis and T. cati, which can cause a number of clinical manifestations such as visceral larva migrans (VLMs), ocular larva migrans (OLMs), eosinophilic meningoencephalitis (EME), covert toxocariasis (CT) and neurotoxocariasis, is considered the most prevalent neglected helminthiasis in industrialized countries. The accurate identification Toxocara spp. and their unequivocal differentiation from each other and from other ascaridoid nematodes causing VLMs and OLMs has important implications for studying their taxonomy, epidemiology, population genetics, diagnosis and control. Due to the limitations of traditional (morphological) approaches for identification and diagnosis of Toxocara spp., PCR-based techniques utilizing a range of genetic markers in the nuclear and mitochondrial genomes have been developed as useful alternative approaches because of their high sensitivity, specificity, rapidity and utility. In this article, we summarize the current state of knowledge and advances in molecular identification, taxonomy, genetic variation and diagnosis of Toxocara spp. with prospects for further studies.

Human sparganosis, a neglected food borne zoonosis

Human sparganosis is a food borne zoonosis caused by the plerocercoid larvae (spargana) of various diphyllobothroid tapeworms of the genus Spirometra. Human infections are acquired by ingesting the raw or undercooked meat of snakes or frogs, drinking untreated water, or using raw flesh in traditional poultices. More than 1600 cases of sparganosis have been documented worldwide, mostly in east and southeast Asia. Sporadic cases have been reported in South America, Europe, and Africa, and several cases have been described in travellers returning from endemic regions. Epidemiological data suggest that the increased effect of sparganosis on human health is because of greater consumption of raw meat of freshwater frogs and snakes. This Review provides information about the Spirometra parasites and their lifecycles, summarises clinical features, diagnosis, and treatment of human sparganosis, and describes geographical distribution and infection characteristics of Spirometra parasites in host animals.

Characterization of the complete mitochondrial genome of Spirocerca lupi: sequence, gene organization and phylogenetic implications

BackgroundSpirocerca lupi is a life-threating parasitic nematode of dogs that has a cosmopolitan distribution but is most prevalent in tropical and subtropical countries. Despite its veterinary importance in canids, the epidemiology, molecular ecology and population genetics of this parasite still remain unexplored.MethodsThe complete mitochondrial (mt) genome of S. lupi was amplified in four overlapping long fragments using primers designed based on partial cox 1, rrn S, cox 2 and nad 2 sequences. Phylogenetic re-construction of 13 spirurid species (including S. lupi) was carried out using Bayesian inference (BI) based on concatenated amino acid sequence datasets.ResultsThe complete mt genome sequence of S. lupi is 13,780 bp in length, including 12 protein-coding genes, 22 transfer RNA genes and two ribosomal RNA genes, but lacks the atp 8 gene. The gene arrangement is identical to that of Thelazia callipaeda (Thelaziidae) and Setaria digitata (Onchocercidae), but distinct from that of Dracunculus medinensis (Dracunculidae) and Heliconema longissimum (Physalopteridae). All genes are transcribed in the same direction and have a nucleotide composition high in A and T. The content of A + T is 73.73% for S. lupi, in accordance with mt genomes of other spirurid nematodes sequenced to date. Phylogenetic analyses using concatenated amino acid sequences of the 12 protein-coding genes by BI showed that the S. lupi (Thelaziidae) is closely related to the families Setariidae and Onchocercidae.ConclusionsThe present study determined the complete mt genome sequence of S. lupi. These new mt genome dataset should provide novel mtDNA markers for studying the molecular epidemiology and population genetics of this parasite, and should have implications for the molecular diagnosis, prevention and control of spirocercosis in dogs and other canids.

Oesophagostomum dentatum and Oesophagostomum quadrispinulatum: Characterization of the complete mitochondrial genome sequences of the two pig nodule worms

In the present study, the complete mitochondrial DNA (mtDNA) sequences of the pig nodule worm Oesophagostomum quadrispinulatum were determined for the first time, and the mt genome of Oesophagostomum dentatum from China was also sequenced for comparative analysis of their gene contents and genome organizations. The mtDNA sequences of O. dentatum China isolate and O. quadrispinulatum were 13,752 and 13,681 bp in size, respectively. Each of the two mt genomes comprises 36 genes, including 12 protein-coding genes, two ribosomal RNA and 22 transfer RNA genes, but lacks the ATP synthetase subunit 8 gene. All genes are transcribed in the same direction and have a nucleotide composition high in A and T. The contents of A+T are 75.79% and 77.52% for the mt genomes of O. dentatum and O. quadrispinulatum, respectively. Phylogenetic analyses using concatenated amino acid sequences of the 12 protein-coding genes, with three different computational algorithms (maximum likelihood, maximum parsimony and Bayesian inference), all revealed that O. dentatum and O. quadrispinulatum represent distinct but closely-related species. These data provide novel and useful markers for studying the systematics, population genetics and molecular diagnosis of the two pig nodule worms.

Sequence variability in three mitochondrial genes between the two pig nodule worms Oesophagostomum dentatum and O. quadrispinulatum

In this study, sequence variation in three mitochondrial DNA regions, namely cytochrome c oxidase subunit (cox1) and NADH dehydrogenase subunits 1 and 4 (nad1 and nad4), between Oesophagostomum dentatum and O. quadrispinulatum isolated from pigs in different geographical origins in Mainland China was examined, and their phylogenetic relationships were reconstructed. A partial of the cox1 (pcox1), nad1, and nad4 genes (pnad1 and pnad4) were amplified separately from individual nodule worms by PCR and were subjected to direct sequencing in order to define sequence variations. While the intraspecific sequence variations within each of the two species were 0.3–5.2% for pcox1, 0–4.9% for pnad1, and 0–7.1% for pnad4, the interspecific sequence differences were significantly higher, being 10.7–13.4% for pcox1, 11–14.6% for pnad1, and 14.9–18% for pnad4, respectively. There were a number of nucleotide positions in the pcox1, pnad1, and pnad4 sequences with no apparent intraspecific variation but distinct interspecific differences among those samples of Oesophagostomum spp. examined, which may be used as genetic makers for the identification and differentiation of the Oesophagostomum spp. Phylogenetic analyses using three inference methods, namely Bayesian inference, maximum likelihood, and maximum parsimony based on the combined sequences of pcox1, pnad1, and pnad4 revealed that the O. dentatum and O. quadrispinulatum form monophyletic groups, respectively. These findings demonstrated clearly the usefulness of the three mitochondrial sequences for studying systematics, population genetic structures, and the molecular ecology of Oesophagostomum spp.