Population genetic structure of Neoschongastia gallinarum in South China based on mitochondrial DNA markers.

The genetic diversity and differentiation of four geographic populations of Neoschongastia gallinarum were evaluated using concatenated mitochondrial gene sequences (pCOI, pCOII, and pND5). Based on the results, the N. gallinarum populations had high genetic diversity and strong ecological adaptability. Genetic differentiation among paired populations calculated using concatenated mitochondrial gene sequences revealed that geographic isolation resulted in genetic differentiation among the populations of N. gallinarum, and gene flow between populations associated with human trade activities. Systematic development and molecular variance based on haplotypes revealed that genetic variation existed in different haplotypes; however, no clear rule related to geographic region was found. Further, genetic variation was mainly derived from individuals within the population. A neutral test based on concatenated mitochondrial gene sequences and nucleotide pair differences revealed that N. gallinarum did not experience an obvious population expansion in recent historical periods. Accordingly, the population size was relatively stable.

A PCR assay with high sensitivity and specificity for the detection of swine toxoplasmosis based on the GRA14 gene.

Toxoplasma gondii, an intracellular apicomplexan protozoan parasite, can infect all warm-blooded animals. Infected swine are considered one of the most important sources of T. gondii infection in humans. Rapidly and effectively diagnosing T. gondii infection in swine is essential. PCR-based diagnostic tests have been fully developed, and very sensitive and specific PCR is crucial for the diagnosis of swine toxoplasmosis. In this study, we used the T. gondii dense granule protein 14 (GRA14) gene as a target to design specific primers and established a high-specificity and high-sensitivity PCR detection method for swine toxoplasmosis. Notably, this PCR method could detect T. gondii tachyzoite DNA in the acute infection phase. The GRA14 gene PCR assay detected a minimum of 2.35 tachyzoites of T. gondii and can be used for T. gondii detection in blood, tissue, semen, urine and waste feed specimens. A total of 5462 blood specimens collected from pigs in 5 provinces and autonomous regions in southern China during 2016-2017 were assessed by the newly established GRA14 gene PCR method. The overall T. gondii infection rate was 18.9 % (1033/5462). According to the statistical analysis of different regions in China, the positive rates of swine toxoplasmosis from 2016 to 2017 were highest in the Shaanxi, Fujian and Guangdong areas, at 31.7 % (44/139), 21.9 % (86/391) and 18.8 % (874/4645), respectively. Specimens collected in 2017 had a higher positive rate (19.1 %) than those collected in 2016 (16.1 %). In addition, specimens collected in autumn (39.4 %), spring (22.8 %) and winter (18.2 %) had higher positive rates than those collected in summer (3.8 %). These results indicate that the new PCR method based on the T. gondii GRA14 gene has utility for the diagnosis of swine toxoplasmosis and can facilitate the diagnosis of toxoplasmosis in clinical laboratories.

Characterization of Neoschoengastia gallinarum from subtropical China by rDNA and identification of two genotypes based on mitochondrial cox1.

Genetic variations in the 18S ribosomal DNA (18S), 28S ribosomal DNA (28S), second internal transcribed spacer of ribosomal DNA (ITS2), and mitochondrial cytochrome c oxidase subunit 1 (cox1) of Neoschoengastia gallinarum collected from subtropical China were examined. First, a portion of the 18S (p18S), a portion of the 28S (p28S), and the complete ITS2 were separately amplified from individual mites and sequenced. The lengths of the sequences of p18S, p28S, and ITS2 were found to be 1379 bp, 3465~3468 bp, and 200 bp, respectively. The intraspecific sequence variation was 0~0.1% for p28S and 0~1.6% for ITS2, though no variation was observed for p18S, suggesting conservation of rDNA sequences. Second, a portion of the mitochondrial cox1 gene (pcox1) of N. gallinarum was analyzed. The length of the pcox1 sequence is 460 bp, and two distinct groups were observed in N. gallinarum. All pcox1 sequences in group I were identical, and there was only one nucleotide transition observed in group II; however, 7.0~7.2% variations between the two groups were observed, suggesting that two genotypes of N. gallinarum: genotype I and genotype II. Phylogenetic analyses based on pcox1 sequences indicated that N. gallinarum isolates (genotype I or genotype II) clustered into one branch; according to cox1 sequence analysis of Trombiculidae, Walchia hayashii is the closest species. The present study shows that ITS2 rDNA sequence can act as marker for the identification of N. gallinarum samples. Furthermore, analysis of the mitochondrial pcox1 sequence suggests the existence of two genotypes, which has implications for further studies of the ecology and population genetic structures of N. gallinarum.

PCR Identification and Phylogenetic Analysis of Trichomonas gallinae from Domestic Pigeons in Guangzhou, China.

Avian trichomoniasis caused by Trichomonas gallinae is a serious protozoan disease worldwide. The domestic pigeon (Columba livia domestica) is the main host for T. gallinae and plays an important role in the spread of the disease. Based on the internal transcribed spacers of nuclear ribosomal DNA of this parasite, a pair of primers (TgF2/TgR2) was designed and used to develop a PCR assay for the diagnosis of T. gallinae infection in domestic pigeons. This approach allowed the identification of T. gallinae, and no amplicons were produced when using DNA from other common avian pathogens. The minimum amount of DNA detectable by the specific PCR assay developed in this study was 15 pg. Clinical samples from Guangzhou, China, were examined using this PCR assay and a standard microscopy method, and their molecular characteristics were determined by phylogenetic analysis. All of the T. gallinae-positive samples detected by microscopic examination were also detected as positive by the PCR assay. Most of the samples identified as negative by microscopic examination were detected as T. gallinae positive by the PCR assay and were confirmed by sequencing. The positive samples of T. gallinae collected from Guangzhou, China, were identified as T. gallinae genotype B by sequencing and phylogenetic analyses, providing relevant data for studying the ecology and population genetic structures of trichomonads and for the prevention and control of the diseases they cause.

Evaluation of the protective effect of pVAX-EtMIC3-recombined plasmid against E. tenella in chicken.

Coccidiosis caused by protozoan parasites of the genus Eimeria has a severe economic impact on commercial production worldwide. Micronemes of Eimeria play important roles in invading intestinal cell processes. In this study, the DNA vaccine expressing Eimeria tenella microneme protein 3 (EtMIC3) was constructed to evaluate its immune protective effect against E. tenella infection in chickens. The results demonstrated that chickens immunized with pVAX-EtMIC3 produced strong immune responses in the body, as shown by significant lymphocyte proliferation, cytokine production, and antibody responses. The average body weight gains of chickens in all the vaccinated groups were higher than those of non-vaccinated and challenged groups. In general, oocyst shedding was reduced, and bloody feces and gut lesion scores decreased. In addition, the survival rate of the immunized chickens increased compared to that of the unvaccinated and challenged control chickens. In summary, this study indicated that pVAX-EtMIC3 could induce protective immune effects against coccidiosis and that EtMIC3 is a potential vaccine candidate against coccidiosis.

Observation of the excretion pattern of a precocious line of Eimeria necatrix and the strengthening of immune homogeneity.

The excretion frequencies of cecal and intestinal droppings of Chinese Lingnan yellow chickens were observed for 10 consecutive days. The chickens were then orally inoculated with a precocious line of Eimeria necatrix, and the oocysts present in the cecal and intestinal droppings were separately collected and monitored using the McMaster method. The results showed that the excretion frequency of cecal droppings was significantly lower than that of intestinal droppings, and the oocysts of E. necatrix were distributed primarily in the cecal droppings. This distribution affects the homogeneity of the second and third generation of oocysts ingested by the chickens and therefore affects the immune effect observed during E. necatrix immunization. To artificially strengthen the immunologic homogeneity against E. necatrix, a method of artificially strengthening the second immunization was applied, and the immune effect was evaluated based on oocyst excretion, body weight gain, fecal scores, intestinal lesion scores and survival percentages. The results showed that no significant intestinal damage was caused by immunization reactions in the chickens. In addition, the number of excreted oocysts in the immunized chicken groups could be significantly increased, and the immunologic homogeneity of the immunized chickens could be improved by artificially strengthening the second immunization, which could in turn improve the immune protective effect.

The complete mitochondrial genome of Pseudoterranova azarasi and comparative analysis with other anisakid nematodes.

Anisakiasis/anisakidosis caused by anisakid nematodes is an emerging infectious disease that can cause a wide range of clinical syndromes and are difficult to diagnose and treat in humans. In spite of their significance as pathogens, the systematics, genetics, epidemiology and biology of these parasites remain poorly understood. In the present study, we sequenced the complete mitochondrial (mt) genome of Pseudoterranova azarasi, which is one of the most important zoonotic anisakid parasites. The circular mt genome is 13,954 bp in size and encodes of 36 genes, including 12 protein-coding, 2 ribosomal RNA and 22 transfer RNA genes. The mt gene order of P. azarasi is the same as those of Ascaris spp. (Ascarididae), Toxocara spp. (Toxocaridae) and Anisakis simplex (Anisakidae), but distinct from those of Ascaridia spp. (Ascaridiidae) and Cucullanus robustus (Cucullanidae). Phylogenetic analyses based on concatenated amino acid sequences of 12 protein-coding genes by Bayesian inference (BI) showed that Pseudoterranova were more closely related to Anisakis than they were to Contracaecum with strong a posterior probability support. This mt genome provides a novel genetic markers for exploring cryptic/sibling species and host affiliations, and should have implications for the diagnosis, prevention and control of anisakidosis in humans.

Protective efficacy of recombinant canine adenovirus type-2 expressing TgROP18 (CAV-2-ROP18) against acute and chronic Toxoplasma gondii infection in mice.

BACKGROUND: The use of recombinant viral vectors expressing T. gondii antigens is a safe and efficient approach to induce immune responses against the parasite, as well as a valuable tool for vaccine development. We have previously prolonged the survival time of mice challenged with the RH strain of T. gondii by immunizing the mice with a eukaryotic vector expressing the protein ROP18 of T. gondii. We are now looking for ways to improve this vaccination strategy and enhance protection. METHODS: In this study, we constructed and characterized a novel recombinant canine adenovirus type 2 expressing ROP18 (CAV-2-ROP18) of T. gondii by cytopathic effect (CPE) and indirect immunofluorescence assay (IFA) following transfection into MDCK cells. Intramuscular immunization of Kunming mice with CAV-2-ROP18 was carried out to evaluate humoral and cellular immune responses. RESULTS: The vaccination of experimental mice with CAV-2-ROP18 elicited antibody production against ROP18, including high levels of a mixed IgG1/IgG2a and significant production of IFN-γ or IL-2, and displayed a significant bias towards a helper T cell type 1 (Th1) profile. Furthermore, the presence of T. gondii-specific IFN-γ-production and TNF-α-production T cells was elicited in both CD4+ and CD8+ T cell compartments. Significantly higher survival rates (40%) occurred in the experimental group, and a reduction in brain cyst burden was detected in vaccinated mice. CONCLUSION: These results demonstrate the potential use of a CAV vector harboring the ROP18 gene in the development of a vaccine against acute and chronic toxoplasmosis.

Biological characteristics of Chinese precocious strain of eimeria acervulina and its immune efficacy against different field strains.

In this study, the biologic characteristics of one experimental precocious strain of Eimeria acervulina and seven field isolates from different geographic locations in China were compared, and the immune efficacy of two precocious strains against coccidiosis in chickens was assessed to explore their potential use as coccidiosis vaccines. All the different strains were purified by single oocyst separation and their monospecificity was confirmed using E acervulina-specific PCR assays. The average sizes of E. acervulina oocysts were 18.28-20.19 X 14.09-14.79 microm and the shape indexes were from 1.28 to 1.40. The prepatent periods ranged from 93 to 115 hr, except for the Heyuan precocious strain (HYP; 75 hr). Chickens infected with Huadu field strain (GHD) produced the highest oocyst output whereas HYP induced the lowest level. When inoculated with 50,000 sporulated oocysts or more, the average weight gains of infected chickens were reduced, with apparent clinical symptoms. To assess the immunogenicity of precocious strains HYP and Baoding (BDP), birds were orally immunized and challenged with seven different field strains of E. acervulina. Body weight gain, fecal oocyst output, and gut lesion scores were compared to evaluate their vaccine potential. The results showed that the average body weight gains of chickens in all the vaccinated and challenged groups were higher than those of nonvaccinated and challenged groups. In general, oocyst shedding was reduced 34.39%-95.31% and gut lesion scores decreased 31.03%-86.21% compared with unvaccinated and challenged control chickens. In summary, this study indicated that the precocious strains of E. acervulina could induce a protective immune effect with various responses against coccidiosis caused by different field strains.

Characterization of the intergenic spacer rDNAs of two pig nodule worms, Oesophagostomum dentatum and O. quadrispinulatum.

The characteristics of the intergenic spacer rDNAs (IGS rDNAs) of Oesophagostomum dentatum and O. quadrispinulatum isolated from pigs in different geographical locations in Mainland China were determined, and the phylogenetic relationships of the two species were reconstructed using the IGS rDNA sequences. The organization of the IGS rDNA sequences was similar to their organization in other eukaryotes. The 28S-18S IGS rDNA sequences of both O. dentatum and O. quadrispinulatum were found to have variable lengths, that is, 759-762 bp and 937-1128 bp, respectively. All of the sequences contained direct repeats and inverted repeats. The length polymorphisms were related to the different numbers and organization of repetitive elements. Different types and numbers of repeats were found between the two pig nodule species, and two IGS structures were found within O. quadrispinulatum. Phylogenetic analysis showed that all O. dentatum isolates were clustered into one clade, but O. quadrispinulatum isolates from different origins were grouped into two distinct clusters. These results suggested independent species and the existence of genotypes or subspecies within pig nodule worms. Different types and numbers of repeats and IGS rDNA structures could serve as potential markers for differentiating these two species of pig nodule worms.

Prevalence of Sarcoptes scabiei infection in pet dogs in southern China.

Little is known about the prevalence of Sarcoptes scabiei infection in pet dogs in China. In the present study, the prevalence of S. scabiei infection in pet dogs in Guangzhou, southern China, was investigated between January and December, 2009. A total of 3,977 pet dogs admitted to animal hospitals were examined for the presence of S. scabiei using a parasitological approach. The average prevalence of S. scabiei infection in pet dogs is 1.18% (95% confidence interval (CI): 0.85-1.52%). The prevalence of S. scabiei was higher in winter (1.42%; 95% CI: 0.29-2.55%), summer (1.39%; 95% CI: 0.83-1.96%), and autumn (1.1%; 95% CI: 0.53-1.68%) than in spring (0.63%; 95% CI: 0.02-1.25%). Furthermore, the prevalence of S. scabiei was the highest in Pekingese (21.88%; 95% CI: 7.55-36.2%), followed by Papillon (5.26%; 95% CI: 0-11.06%) and Bichon Frise (3.19%; 95% CI: 0-6.75%). The results of the present investigation indicate that S. scabiei infection is prevalent in pet dogs in Guangzhou, China, which provides relevant "baseline" data for conducting control strategies and measures against scabies in this region and elsewhere in China. To our knowledge, this is the first comprehensive report of S. scabiei prevalence in pet dogs in China.

Sequence variation in four mitochondrial genes among sibling species within Contracaecum rudolphii sensu lato.

The present study investigated sequence variability in four mitochondrial DNA (mtDNA) regions, namely cytochrome c oxidase subunit (cox1), NADH dehydrogenase subunits 1 and 4 (nad1 and nad4), and small subunit of rRNA (rrnS), among Contracaecum rudolphii A, C. rudolphii B, C. rudolphii C and Contracaecum septentrionale from different hosts and geographical origins in China, Italy, Spain and the USA. Regions in the cox1, nad1, nad4 and rrnS genes (designated pcox1, pnad1, pnad4 and prrnS, respectively) were amplified separately from individual nematodes by PCR, sequenced and compared to estimate sequence variability. While sequence variation within each of the Contracaecum species was 0-2.6% for pcox1, 0.3-2.5% for pnad1, 0-1.9% for pnad4 and 0-2.9% for prrnS, differences between species was significantly higher, being 3.3-12%, 9.8-15.2%, 9.6-18.3% and 3.5-11.12% for these regions, respectively. Phylogenetic analyses of pcox1, pnad1, pnad4 and prrnS sequence data using maximum likelihood (ML), maximum parsimony (MP) and neighbour joining (NJ) showed that the specimens of each Contracaecum species clustered together. These results provide additional genetic evidence for the existence of sibling species within C. rudolphii sensu lato.

Identification and characterization of new major sperm protein genes from Oesophagostomum dentatum and Oesophagostomum quadrispinulatum from pigs in China.

The present study identified and characterized new major sperm protein (MSP) genes from the two nodule worms Oesophagostomum dentatum and Oesophagostomum quadrispinulatum collected from pigs in China. Total genomic DNA was extracted individually from 10 male nematode samples representing O. dentatum, and 4 male nematode samples representing O. quadrispinulatum. A pair of primers (OMSP1F/MSP1R) was designed based on the MSP gene sequences of Ascaris suum and O. dentatum available in GenBank, and used to amplify the MSP genes from the two porcine nodule worms. The PCR products were purified and subsequently cloned into pGEM-T Easy vector. Recombinants were identified by PCR and sequenced. Sequence analysis revealed that there were two different types of MSP sequences in O. dentatum and O. quadrispinulatum, one contained intron, and the other did not. The lengths of the MSP sequences containing introns were 433 bp or 439 bp in O. dentatum, and 436 bp, 439 bp or 446 bp in O. quadrispinulatum, containing 1 or 2 introns. Five and three new members of the MSP multigene family were identified in O. dentatum and O. quadrispinulatum in this study, respectively. The MSP sequences without introns were 381 bp in length, and can be deduced into 126 amino acids. The sequences of MSP genes containing introns seem to be more conserved than those without introns. The identities of deduced amino acid sequences of the MSP genes containing introns were 96.0-100% within and between the two nodule worms, and were 81.1-93.7% compared with other published MSP sequences of the representative nematodes. The present study identified new MSP genes with introns from O. dentatum and O. quadrispinulatum for the first time. The identification and characterization of newly described MSP genes from O. dentatum and O. quadrispinulatum have implications for further studies of molecular biology and reproduction control of Oesophagostomum spp.

First report of Toxoplasma gondii seroprevalence in peafowls in Yunnan Province, Southwestern China.

BACKGROUND: Toxoplasma gondii is an intracellular protozoan parasite infecting almost all warm-blooded animals, including birds, with a worldwide distribution. Surveys of T. gondii infection in wild birds have been reported extensively in the world, but little is known of T. gondii infection in peafowls worldwide. This study was performed to determine the seroprevalence of T. gondii infection in peafowls in Yunnan Province, southwestern China. METHODS: Sera from 277 peafowls, including 272 blue peafowls (Pavo cristatus) and 5 green peafowls (Pavo muticus) originated from two geographic areas in Yunnan Province were assayed for T. gondii antibodies using the modified agglutination test (MAT). RESULTS: Specific T. gondii antibodies were detected in 35 of 277 (12.64%) peafowls (MAT titer ≥ 1:5). Seropositive birds were found in both species, 33 in 272 blue peafowls and 2 in 5 green peafowls. There was no significant difference in T. gondii seroprevalence between the adolescent birds (6.74%) and the adult birds (6.67%) (P > 0.05). The geographical origins of peafowls was found to be highly associated with T. gondii infection in the present study, a statistically significant difference in T. gondii seropositivity was observed between peafowls from Kunming (31.08%) and those from Xishuangbanna Dai Autonomous Prefecture (5.91%) (OR = 10.956, 95% CI = 1.632-73.545, P = 0.014). Statistical analyses showed that there were no significant interactions between ages and geographical origins of peafowls (P > 0.05). CONCLUSIONS: The results of the present survey indicated that infection of peafowls with T. gondii is widespread in Yunnan Province, which has significant public health concerns and implications for prevention and control of toxoplamosis in this province. To our knowledge, this is the first seroprevalence report of T. gondii infection in China's southwestern Yunnan Province.

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.

The complete mitochondrial genome sequence of Eimeria mitis (Apicomplexa: Coccidia).

In this study the complete mitochondrial DNA (mtDNA) sequence of Eimeria mitis was sequenced, and its gene contents and genome organizations were compared with that of other Eimeria spp. The complete mt genome sequence of E. mitis is 6407 bp in size. It consists of 3 protein-coding genes (cox1, cox3, and cytb), 12 gene fragments for the large subunit (LSU) rRNA, and 7 gene fragments for the small subunit (SSU) rRNA, but no transfer RNA genes, similar to that of Eimeria spp. The putative direction of translation for three genes (cox1, cox3, and cytb) was the same as those of six other Eimeria spp. The A+T content of the E. mitis mt genome was 67.30%. The E. mitis mt genome sequence provides novel mtDNA marker for studying the molecular epidemiology and population genetics of E. mitis and has implications for the molecular diagnosis of chicken coccidiosis caused by E. mitis.

A specific PCR assay for the identification and differentiation of Schistosoma japonicum geographical isolates in mainland China based on analysis of mitochondrial genome sequences.

In the present study, near-complete mt genome sequences for eight representative Schistosoma japonicum samples from seven endemic provinces in mainland China were analyzed. Sequence differences among the eight mt genomes of S. japonicum samples were 0.20-2.51%. Variation in protein-coding genes was greater than that in rRNA genes. The mt DNA sequences of S. japonicum samples from south-western (SW) China were 2 bp [position 11727-11728 within tRNA-Cys, microsatellite (AG) indel] longer than those of the parasites from the lower Yangtze/Zhejiang areas. Representative DNA sequencing confirmed that such (AG) indel could be exploited for identification and differentiation of S. japonicum populations in SW China's Yunnan and Sichuan province which have two (AG) repeats from those in all remaining endemic provinces along the Yangtze River below the Three Gorges regions or close to the east coast of China (e.g., Zhejiang) which have only one (AG) repeat. Phylogenetic analyses based on the concatenated amino acids of 12 protein-coding genes also showed that samples from SW China (Sichuan and Yunnan provinces), above the Three Gorges Dam, formed a distinct cluster. Based on this indel polymorphism, a pair of specific primers was designed and used to develop a specific-PCR polyacrylamide gel detection assay. There was an obvious length difference in the amplified PCR products between S. japonicum samples from the two endemic types. The specific-PCR assay allowed the specific identification of S. japonicum, with no amplicons being amplified from other closely related trematodes, and the minimum amount of DNA detectable was 0.05 ng. This approach is inexpensive, easy to perform and the whole detection process can be completed within 4h. Examination of 81 S. japonicum samples from SW China's Yunnan and Sichuan provinces, and 264 samples from the lower Yangtze provinces (Hubei, Jiangsu, Jiangxi, Anhui and Hunan) and from Zhejiang validated the value of the specific PCR assay and proved its reliability. These findings indicate that the specific PCR assay would provide a useful tool for the epidemiological surveillance and for tracing the source of S. japonicum infection in humans and animals in China.

A specific PCR assay for the diagnosis of Clonorchis sinensis infection in humans, cats and fishes.

Clonorchiasis caused by Clonorchis sinensis is a fish-borne parasitic disease which is endemic in a number of countries. Using the sequences of the internal transcribed spacers (ITS-1 and ITS-2) of nuclear ribosomal DNA (rDNA) of C. sinensis as genetic markers, a pair of C. sinensis-specific primers was designed and used to establish a specific PCR assay for the diagnosis of C. sinensis infection in humans, cats and fish. This approach allowed the specific identification of C. sinensis after optimizing amplification conditions, with no amplicons being amplified from related heterogeneous DNA samples, and sequencing of amplicons confirmed the identity of the sequences amplified. The detection limit of this assay was 1.03 pg of adult C. sinensis, 1.1 metacercariae per gram of fish filet, and a single egg in human and cat feces. The PCR assay should provide a useful tool for the diagnosis and molecular epidemiological investigation of clonorchiasis in humans and animals.

Seroprevalence of Toxoplasma gondii infection in household and stray cats in Lanzhou, northwest China.

BACKGROUND: Toxoplasma gondii is an important protozoan parasite infecting humans and almost all warm-blooded animals. As the only definitive host, cats play a crucial role in the transmission of T. gondii infection by shedding parasite oocysts in their feces. However, little information on T. gondii infection in cats was available in Lanzhou, northwest China. This study was performed to determine the seroprevalence of T. gondii infection in household and stray cats in Lanzhou, northwest China. RESULTS: A total of 221 (179 households and 42 strays) blood samples were collected from clinically healthy cats admitted to several pet hospitals located in Lanzhou City, between November 2010 and July 2011 for the serological detection of T. gondii infection. The majority (207) of these cats represented Chinese Lihua cats. 47 of 221 (21.3%) examined cats were seropositive for T. gondii infection using the modified agglutination test (MAT) at the cut-off of 1:25. The seroprevalence in household and stray cats was assessed to be 15.6% and 45.2%, respectively, and the difference was statistically significant (P <0.05). The seroprevalence ranged from 15.1% to 25.8% among different age groups, but the differences were not statistically significant (P >0.05). Studies showed that there was no relationship between seroprevalence and the gender (P >0.05). CONCLUSIONS: The present survey indicated the high seroprevalence of T. gondii in cats in Lanzhou, northwest China, which poses a threat to animal and human health. Therefore, measures should be taken to control and prevent toxoplasmosis of cats in this area.

IRAP: An efficient retrotransposon-based electrophoretic technique for studying genetic variability among geographical isolates of Schistosoma japonicum.

In the present study, a inter-retrotransposon-amplified polymorphism (IRAP) technique, based on retrotransposons, was used to examine genetic variability among Schistosoma japonicum isolates from different provinces in mainland China. Of the 15 primers screened, 5 produced highly reproducible IRAP patterns. Using these primers, 54 discernible DNA fragments were generated with 40 (74.07%) being polymorphic, indicating considerable genetic variation among the examined S. japonicum isolates. The primer LTR-11 was found to be able to differentiate male and female parasites, producing one constant specific band for female S. japonicum isolates. The percentages of polymorphic bands (PPB) among all parasites, among isolates from mountainous provinces and among those from the lake/marshland areas were 74.07, 48.15, and 66.67%, respectively. UPGMA analysis revealed that the IRAP profiles could group S. japonicum isolates in mainland China into two clades (mountainous and lake/marshland types), and samples from the same geographical origins clustered together. These results demonstrated that the IRAP technique is suitable for studying genetic diversity and population structures, and also provides an effective technique for studying sex differentiation of S. japonicum.