X. Q. Zhu

Moving towards an integrated approach to molecular detection and identification of Toxoplasma gondii

The development of simple, sensitive and rapid methods for the detection and identification of Toxoplasma gondii is important for the diagnosis and epidemiological studies of the zoonotic disease toxoplasmosis. In the past 2 decades, molecular methods based on a variety of genetic markers have been developed, each with its advantages and limitations. The application of these methods has generated invaluable information to enhance our understanding of the epidemiology, population genetics and phylogeny of T. gondii. However, since most studies focused solely on the detection but not genetic characterization of T. gondii, the information obtained was limited. In this review, we discuss some widely used molecular methods and propose an integrated approach for the detection and identification of T. gondii, in order to generate maximum information for epidemiological, population and phylogenetic studies of this key pathogen.

Sensitive and rapid detection of Schistosoma japonicum DNA by loop-mediated isothermal amplification (LAMP).

In this study, a loop-mediated isothermal amplification (LAMP) assay was established to detect Schistosoma japonicum DNA in faecal and serum samples of rabbits, and serum samples of humans infected with S. japonicum. This LAMP assay was based on the sequence of highly repetitive retrotransposon SjR2, and was able to detect 0.08 fg S. japonicum DNA, which is 10(4) times more sensitive than conventional PCR. The LAMP assay was also highly specific for S. japonicum and able to detect S. japonicum DNA in rabbit sera at 1 week p.i. Following administration of praziquantel, detection of S. japonicum DNA in rabbit sera became negative at 12 weeks post-treatment. These results demonstrated that LAMP was effective for early diagnosis of, and evaluation of therapy effectiveness for, S. japonicum infection. Both PCR and LAMP assays were then used to detect S. japonicum DNA in 30 serum samples from S. japonicum-infected patients and 20 serum samples from healthy persons. The percentage sensitivity of LAMP was 96.7%, whereas that of PCR was only 60%, indicating that LAMP was more sensitive than conventional PCR for clinical diagnosis of schistosomiasis cases in endemic areas. The established LAMP assay should provide a useful and practical tool for the routine diagnosis and therapeutic evaluation of human schistosomiasis.

Genetic variability among Schistosoma japonicum isolates from different endemic regions in China revealed by sequences of three mitochondrial DNA genes

The present study examined sequence variation in three mitochondrial DNA (mtDNA) regions, namely cytochrome c oxidase subunit 3 (cox3), NADH dehydrogenase subunits 4 and 5 (nad4 and nad5), among Schistosoma japonicum isolates from different endemic regions in China, and their phylogenetic relationships were re-constructed. A portion of the cox3 gene (pcox3), a portion of the nad4 and nad5 genes (pnad4 and pnad5) were amplified separately from individual trematodes by polymerase chain reaction (PCR) and the amplicons were subjected to direct sequencing. In the mountainous areas, sequence variations between parasites from Yunnan and those from Sichuan were 0.3% for pcox3, 0.0-0.1% for pnad4, and 0.0-0.2% for pnad5. In the lake/marshland areas, sequence variations between male and female parasites among different geographical locations were 0.0-0.3% for pcox3, 0.0-0.7% for pnad4, and 0.0-1.6% for pnad5. Sequence variations between S. japonicum from mountainous areas and those from lake/marshland areas were 0.0-0.5% for pcox3, 0.0-0.7% for pnad4, and 0.0-1.6% for pnad5. Phylogenetic analyses based on the combined sequences of pcox3, pnad4 and pnad5 revealed that S. japonicum isolates from mountainous areas (Yunnan and Sichuan provinces) clustered together. For isolates from the lake/marshland areas, isolates from Anhui and Jiangsu provinces clustered together and was sister to samples from Jiangxi province, while isolates from Hubei and Zhejiang province clustered together. However, isolates from different geographical locations in Hunan province were in different clades. These findings demonstrated the usefulness and attributes of the three mtDNA sequences for population genetic studies of S. japonicum, and have implications for studying population biology, molecular epidemiology, and genetic structure of S. japonicum, as well as for the effective control of schistosomiasis.

Prevalence of helminthes in adult dogs in Heilongjiang Province, the People's Republic of China.

The prevalence of helminthes in adult dogs was investigated in Heilongjiang Province, the People’s Republic of China, between 1996 and 2004. A total of 178 adult farm dogs from representative geographical locations in Heilongjiang Province were killed and examined for the presence of helminthes using a helminthological approach. The worms were examined, counted, and identified to species according to existing keys and descriptions. A total of 17 species of helminthes were found to infect dogs, and they represented two phyla, three orders, 13 families, and 15 genera. All dogs were infected by more than one helminth species. Clonorchis sinensis (26.4%), Paragonimus westermani (7.9%), and Metagonimus yokogawai (6.2%) were the most common trematode species; Mesocestoides lineatus (20.2%), Taenia hydatigena (19.7%), and Dipylidium caninum (14.6%) were the most common cestodes species; and Ancylostoma caninum (66.3%), Toxocara canis (36.5%), and Trichinellanativa (21.9%) were the most common nematode species. The results of the present investigation provide relevant “base-line” data for assessing the effectiveness of future control strategies against helminth infection in dogs in Heilongjiang Province, China.

Identification of anisakid nematodes with zoonotic potential from Europe and China by single-strand conformation polymorphism analysis of nuclear ribosomal DNA

Using genetic markers defined previously in the second internal transcribed spacer (ITS-2) of nuclear ribosomal DNA (rDNA), isotopic, and non-isotopic polymerase-chain-reaction-coupled single-strand conformation polymorphism (SSCP) were utilized to identify each of three anisakid species [Anisakis simplex (s.l.), Contracaecumosculatum (s.l.), and Hysterothylacium aduncum] from different host species and geographical locations in Poland and Sweden. While subtle microheterogeneity was observed within each of Anisakis simplex (s.l.) and H. aduncum, distinct SSCP profiles were displayed for each of the three species, allowing identification and differentiation of the three taxa. Subsequent sequencing of the ITS-1 and ITS-2 rDNA revealed that A. simplex (s.l.) represented Anisakis simplex s.s. and Contracaecum osculatum (s.l.) represented C. osculatum C. Application of the non-isotopic SSCP assay of ITS-2 to larval anisakid samples from different hosts and geographical locations in China revealed three distinct SSCP profiles, one of which was consistent with that of A. simplex (s.l.), and the other two had different SSCP profiles from that of C. osculatum C and H. aduncum. Sequencing of the ITS-1 and ITS-2 rDNA for representative Chinese anisakid samples examined revealed three anisakid species in China, i.e., Anisakis typica, Anisakis pegreffii, and Hysterothylacium sp. These molecular tools will be useful for identification and investigation of the ecology of anisakid nematodes in China and elsewhere.

Skin-scale genetic structure of Sarcoptes scabiei populations from individual hosts: Empirical evidence from Iberian ibex-derived mites

The objective of the present study was to examine the extent of genetic diversity among Sarcoptes scabiei individuals belonging to different skin subunits of the body from individual mangy hosts. Ten microsatellite primers were applied on 44 individual S. scabiei mites from three mangy Iberian ibexes from Sierra Nevada Mountain in Spain. Dendrograms of the mites from the individual Iberian ibexes, showing the proportion of shared alleles between pairs of individual mites representing three skin subpopulations (head, back, and abdomen subunits), allowed the clustering of some mite samples up to their skin subunits. This genetic diversity of S. scabiei at skin-scale did not have the same pattern in all considered hosts: for the first Iberian ibex (Cp1), only mites from the head subunit were grouped together; in the second individual (Cp2), the clustering was detected only for mites from the abdomen subunit; and for the third one (Cp3), only mites from the back subunit were clustered together. Our results suggest that the local colonization dynamics of S. scabiei would have influenced the nonrandom distribution of this ectoparasite, after a single infestation. Another presumable explanation to this skin-scale genetic structure could be the repeated infestations. To our knowledge, this is the first documentation of genetic structuring among S. scabiei at individual host skin-scale. Further studies are warranted to highlight determining factors of such trend, but the pattern underlined in the present study should be taken into account in diagnosis and monitoring protocols for studying the population genetic structure and life cycle of this neglected but important ectoparasite.

The radiation of Haliotrema (Monogenea: Dactylogyridae: Ancyrocephalinae): molecular evidence and explanation inferred from LSU rDNA sequences

The D1-D2 domains of LSU rDNA were used to reconstruct the phylogenetic relationships within the Ancyrocephalinae (Monogenea: Dactylogyridae) utilizing maximum-parsimony (MP), maximum-likelihood (ML), minimum evolution (ME) and neighbour-joining (NJ) methods. A total of 32 monogenean taxa were examined in the present study, including 9 Haliotrema species and 13 other species representing the Ancyrocephalinae, 4 Thaparocleidus species representing the Ancylodiscoididae, and 6 species representing the Diplectanidae which were used as multiple outgroups. All 4 analyses (i.e. MP, ML, ME and NJ) inferred the same interrelationship pattern: (Diplectanidae, (Ancylodiscoididae, Dactylogyridae)) with high bootstrap support. However, 9 Haliotrema species were dispersed to form 4 clades together with species from other genera, indicating the apparent non-monophyly of Haliotrema. Three major groups were defined based on reconstructed phylogenetic trees to explain the radiation of Haliotrema species. The morphology of the reproductive organ, particularly the male copulatory organ (MCO), was discussed to further understand the formation of each group. (1) Results of the present study indicated an intimate relationship among Metahaliotrema (2 species), Protogyrodactylus (4 species) and Haliotrema (2 of 9 species), and notably, all these species share vagina-absence. (2) Based on the present molecular analyses and the morphological characters of the MCO, we propose to transfer H. spirotubiforum and the undetermined Haliotrema sp. ZHDDb to Euryhaliotrema as new combinations. (3) We propose to erect a new genus to accommodate the Haliotrema species with horn-like shaped MCO. Taxonomic implications of the present molecular phylogenetic analyses are discussed. A wider range of taxa and more DNA markers displaying various evolutionary rates should be used to estimate phylogenetic relationships among species within the Ancyrocephalinae and Ancylodiscoididae in further studies.

Severe infection of adult dogs with helminths in Hunan Province, China poses significant public health concerns.

The prevalence of helminths in adult dogs was investigated in Hunan Province, the Peoples Republic of China between June 2006 and December 2007. A total of 438 adult farm dogs slaughtered in local abattoirs from 9 representative administrative regions in Hunan Province were examined for the presence of helminths using a helminthological approach. All collected worms were counted and identified to species according to existing keys and descriptions. A total of 11 helminth species were found in the dogs, and they represented 2 phyla, 3 classes, 6 families and 8 genera. All dogs were infected by more than one helminth species. Clonorchis sinensis (29.4%) was the only trematode species found, Dipylidium caninum (42.3%) was the most common cestode species, and Toxocara canis (45.2%) the most common nematode species. 6 of the 11 dog helminths are also transmissible to humans (i.e., zoonotic), and can cause severe clinical human diseases, posing significant public health threats. The results of the present investigation have implications for the ongoing control of helminth infections in dogs and humans in Hunan Province, China.

Genetic variability among Fasciola hepatica samples from different host species and geographical localities in Spain revealed by the novel SRAP marker

A collection of 483 samples representing Fasciola from six naturally infected host species and 16 localities in Spain, previously identified morphologically and genetically as Fasciola hepatica, was characterized by a novel genetic marker, namely sequence-related amplified polymorphism (SRAP), aiming to reveal genetic variability within F. hepatica in Spain. Visualization of amplification fragments was carried out on 6% denaturing polyacrylamide gels, followed by staining with 0.1% AgNO3 solution. Ten SRAP primer combinations were tested—six of them turned out to be polymorphic. Thirty-four representative F. hepatica samples from six host species and 16 geographical localities showed polymorphic banding patterns using SRAP primer combinations and were grouped into four major clusters using the unweighted pair-group method with arithmetic averages, indicating the existence of genetic variability within the examined F. hepatica samples. These four clusters were not related to particular host species and/or geographical origins of the samples. The results of the present study revealed that SRAP markers were useful in revealing sufficient polymorphism in F. hepatica samples from Spain and had implications for studying the population genetic structure of the Spanish F. hepatica. To our knowledge, this is the first application of SRAP marker to study genetic variation in parasites of human and animal health significance.

Some characteristics of host–parasite relationship for Cryptocaryon irritans isolated from South China

A survey on the host range for the parasitic ciliate Cryptocaryon irritans was carried out among the major maricultured fish species in the Huizhou region of Guangdong Province in South China, and some characteristics of its host–parasite relationship were described. The survey showed that all ten investigated species of fish (representing six different families) were infected with C. irritans with similar susceptibility. In chemoattraction assays, sera and mucus collected from investigated fish strongly attracts C. irritans theronts. Sera collected from infected orange-spotted groupers and yellow spotted grunts (Plectorhynchus cinctus) could immobilize C. irritans theronts, and their immobilization titers were 1:40 and 1:6.7, respectively. The surface antigens of C. irritans were demonstrated by indirect immunofluorescence and immunostaining assays using immune orange-spotted grouper serum and a monoclonal antibody against grouper IgM.