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SUMMARYThe role of the protozoan parasite Toxoplasma gondii in the pathogenesis of liver disease has recently gained much interest. The aim of this study was to determine the prevalence and risk factors associated with T. gondii infection in patients with liver disease from three cities in Shandong and Henan provinces, China. A case–control study was conducted from December 2014 to November 2015 and included 1142 patients with liver disease and 1142 healthy controls. Serum samples were collected from all individuals and were examined with enzyme-linked immunosorbent assay for the presence of anti-T. gondii IgG and IgM antibodies. Information on the demographics, clinical, and lifestyle characteristics of the participants was collected from the medical records and by the use of a questionnaire. The prevalence of anti-T. gondii IgG was 19·7% in patients with liver disease compared with 12·17% in the controls. Only 13 patients had anti-T. gondii IgM antibodies compared with 12 control individuals (1·14% vs. 1·05%, respectively). The highest seroprevalence was detected in patients with liver cancer (22·13%), followed by hepatitis patients (20·86%), liver cirrhosis patients (20·42%), and steatosis patients (20%). Multivariate logistic regression analysis indicated that consumption of raw meat (odds ratio (OR) = 1·32; 95% confidence interval (CI) 1·01–1·71; P = 0·03) and source of drinking water from wells (OR = 1·56; 95% CI 1·08–2·27; P = 0·01) were independent risk factors for T. gondii infection in liver disease patients. These findings indicate that T. gondii infection is more likely to be present in patients with liver disease. Therefore, efforts should be directed toward health education of populations at high risk of T. gondii infection and measures should be taken to protect vulnerable patients with liver disease.

SUMMARYParrots are one of the most popular pet birds in China, and can harbour Chlamydia which has significance for human and animal health. We investigated, by indirect haemagglutination assay, the seroprevalence of Chlamydia infection in four species of parrots, namely budgerigars (Melopsittacus undulatus), lovebirds (Agapornis sp.), cockatiels (Nymphicus hollandicus) and Alexandrine parakeets (Psittacula eupatria) that were collected from Weifang and Beijing cities, North China and explored the association between potential risk factors and chlamydial seropositivity. We further determined the genotype of Chlamydia in 21 fresh faecal samples based on the ompA sequence by reconstruction of phylogenetic relationships. Of the 311 parrots examined, 35·37% (95% confidence interval 30·06–40·68) were seropositive, and species, gender, age, season and geographical location were identified as risk factors. Two PCR-positive samples represented Chlamydia psittaci genotype A. The occurrence of C. psittaci genotype A in the droppings of two pet parrots in China suggests potential environmental contamination with Chlamydiaceae and may raise a public health concern.

The present study examined sequence variation in three mitochondrial DNA (mtDNA) genes, namely cytochrome c oxidase subunit 3 (cox3) and NADH dehydrogenase subunits 1 and 4 (nad1 and nad4), among Ascaridia galli isolates from different geographical localities in China. A portion of cox3 (pcox3), nad1 (pnad1) and nad4 (pnad4) genes were amplified by polymerase chain reaction (PCR) separately from adult A. galli individuals and the amplicons were subjected to sequencing from both directions. The length of the sequences of pcox3, pnad1 and pnad4 were 408 bp, 471 bp and 333 bp, respectively. The intraspecific sequence variations within A. galli were 0–1.7% for pcox3, 0–2.8% for pnad1 and 0–3.4% for pnad4. The A+T contents of the sequences were 67.16–67.65% (pcox3), 67.09–67.94% (pnad1) and 69.91–71.77% (pnad4). The interspecific sequence differences among members of the Ascaridida were significantly higher, being 13.2–30.9%, 12.8–29.0% and 15.1–34.1% for pcox3, pnad1 and pnad4, respectively. Phylogenetic analyses using combined sequences of pcox3, pnad1 and pnad4, with three different computational algorithms (Bayesian analysis, maximum likelihood and maximum parsimony), all revealed distinct groups with high statistical support. These findings demonstrated the existence of intraspecific variation in mitochondrial DNA (mtDNA) sequences among A. galli isolates from different geographical regions in China, and have implications for studying molecular epidemiology and population genetics of A. galli.

In the present study, the second nuclear internal transcribed spacer (ITS-2) rDNA of Schistosoma japonicum isolates in mainland China was amplified, sequenced, and assessed for inferring the intra- and inter-species phylogenetic relationships of trematodes in the order Strigeata. The fragment containing ITS-2 rDNA was obtained from 24 S. japonicum isolates from eight epidemic provinces in mainland China. The length polymorphisms were observed among these ITS-2 rDNA sequences, ranging from 343 to 346 bp, and the intra- and inter-population variations in ITS-2 sequence were 0.0–2.1 % among S. japonicum isolates in China. Phylogenetic analyses using the maximum parsimony and maximum likelihood methods revealed that the ITS-2 rDNA sequence is not a suitable marker for studying inter- and intra-population variation in S. japonicum. However, phylogenetic analysis of trematodes in the order Strigeata indicated that the ITS-2 rDNA sequence provides an effective molecular marker for studying inter-species phylogenetic relationships among trematodes in this order.

Enzootic pneumonia caused by Mycoplasma hyopneumoniae is a severe disease of pigs, causing significant economic losses to the pig industry worldwide, including the tropical and subtropical regions. In order to obtain the baseline prevalence of M. hyopneumoniae in pigs from intensive farms in southern China, double-sandwich enzyme-linked immunosorbent assay (ELISA) was used to detect M. hyoneumoniae antibodies in 460 pig serum samples collected from 12 administrative cities in China's southern Guangdong province. According to the proportions of the infected animals, among the 12 intensive farms, only two of them showed no infection of M. hyoneumoniae and the seroprevalence ranged from 0% to 90%, with an averaged prevalence of 45.7%. The highest prevalence was found in breeding boars (68.8%), followed by sows (54.5%). These data showed that the infection of pigs with M. hyopneumoniae is severe, and boars might be more important carriers and transfers of M. hyoneumoniae than sows. Integrated strategies and measures should be taken to control the infection of pigs with M. hyopneumoniae in southern China.

The prevalence of spargana infection in frogs (Rana nigromaculata) was investigated in China's central Hunan Province, from March 2007 to October 2009. 59 of 292 (20.2%) wild-caught frogs were found to be infected with plerocercoids (spargana) of the genus Spirometra. Spargana were recovered from the skeletal muscle of the hind limb. The infection rate ranged from 4.5% to 27.4%, and the infection intensity was 1-15 spargana per frog. To identify the species identity of the collected spargana, a portion of the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene was amplified, sequenced, and analyzed. Sequence variations for cox1 among all the examined spargana were 0.0-3.1%, with 14 variable sites being identified in sequences obtained (3.1%, 14/446), representing 6 different cox1 sequences. Phylogenetic analysis showed that all the spargana isolates in Hunan province represented Spirometra erinaceieuropaei. This is the first report of S. erinaceieuropaei infection in frogs in Hunan province, China.

The present study studied the genetic variation among Schistosoma japonicum isolates from different endemic regions in mainland China and examined the phylogenetic relationships of zoonotic trematodes using the combined mitochondrial 16S and 12S ribosomal DNA sequences. The fragments of 16S and 12S rDNA were amplified from 22 S. japonicum isolates, and sequenced, and the relevant sequences of other nine trematode species belonging to six genera in four families were downloaded from GenBank, and their phylogenetic relationships were re-constructed by unweighted pair-group method with arithmetic averages analyses using the combined 16S and 12S rDNA sequences, with Trichinella spiralis as outgroup. The results showed that the partial sequences of mitochondrial 16S and 12S rDNA of S. japonicum were 757 and 797 bp, respectively, and they were quite conserved among the S. japonicum isolates. Phylogenetic analysis revealed that the combined 16S and 12S rDNA sequences were not able to distinguish S. japonicum isolates in mountainous areas from those in lake/marshland areas in mainland China. However, the combined sequences could distinguish different species of zoonotic trematodes. Therefore, the combined mitochondrial 16S and 12S rDNA sequences provide an effective molecular marker for the inter-species phylogenetic analysis and differential identification of zoonotic trematodes.

The objective of the present investigation was to estimate the seroprevalence T. gondii infection in breeding sows in Western Fujian Province, the People's Republic of China. Sera collected from breeding sows during 2006-2007 from 6 different regions in Western Fujian Province were assayed for antibodies to T. gondii by an indirect hemagglutination antibody (IHA) test. Antibodies to T. gondii were detected in 87 (14.38%) of 605 breeding sows. Differences in seroprevalence were observed between sampling regions, ranging from 10.14% to 37.50%. The present investigation demonstrated that the prevalence of T. gondii infection in breeding sows in Fujian Province was high. Integrated control strategies and measures should be implemented to prevent and control T. gondii infection in breeding sows, which in turn will have significant implications for the control of human infection with T. gondii in this province.

In the present study, polymerase chain reaction (PCR) assay was used to detect haemoplasmas (haemotropic bacteria) in 40 clinically healthy pet dogs in Foshan city, Guangdong Province, China, and one dog was found positive. Comparison of its 16S ribosomal DNA (rDNA) sequence with relevant sequences showed that the isolated haemoplasma had greater sequence identity to feline species "Candidatus Mycoplasma haemominutum" (99%) than to "Candidatus Mycoplasma haematoparvum" (95%). This result, for the first time, indicates the presence of the feline "Candidatus Mycoplasma haemominutum" in Chinese dogs and it represents the first survey of its kind in China by using PCR assay. The results indicated that dog may represent one of the hosts for the feline "Candidatus Mycoplasma haemominutum".

The prevalence of helminths in the Asian water buffaloes (Bubalus bubalis) was investigated in Hunan Province, People's Republic of China between April 2005 and October 2007. A total of 359 adult buffaloes slaughtered at local abattoirs in 12 representative geographical locations in Hunan Province were examined for the presence of helminths. The worms were examined, counted and identified to species according to existing keys and descriptions. A total of 13 helminth species were found representing one phyla, two classes, eight families and nine genera. All buffaloes were infected by more than one helminth species. 61.8% of the examined buffaloes were infected with Haemonchus contortus, 44.7% with Fasciola hepatica, 24.9% with Fasciola hepatica, 23.5% with Homalogaster paloniae and 23.2% with Setaria labiatopapillosa, whereas the infection of adult buffaloes with cestodes was not detected in the present investigation. The results of the present investigation indicated that the prevalence of nematodes and trematodes in buffaloes is quite severe, some of which pose significant zoonotic public health problems (eg., schistosomiasis). It is imperative that integrated strategies and measures be taken to control helminth infections in buffaloes in Hunan Province and elsewhere.

Based on the sequences of the internal transcribed spacers (ITS-1 and ITS-2) of nuclear ribosomal deoxyribonucleic acid (DNA) of the porcine nodule worms Oesophagostomum dentatum and O. quadrispinulatum, a pair of specific primers (OdspF/OdspR2) for O. dentatum and a pair of specific primers (OqspF/OqspR) for O. quadrispinulatum were designed and used to develop a multiplex polymerase chain reaction (PCR) assay for the identification and differentiation of the two porcine nodule worms. This approach allowed the specific identification and differentiation of O. dentatum and O. quadrispinulatum, with no amplicons being amplified from heterogeneous DNA samples, and sequencing confirmed the identity of the sequences amplified. The minimum amount of DNA detectable using this multiplex PCR assay was 0.1 ng for both O. dentatum and O. quadrispinulatum. The identity of 53 porcine nodule worms collected from pigs from different geographical localities in mainland China was ascertained as O. dentatum or O. quadrispinulatum, respectively, by this multiplex PCR method. This multiplex PCR assay is useful for the simultaneous identification of eggs of O. dentatum and O. quadrispinulatum and should provide a useful tool for the diagnosis and molecular epidemiological investigation of Oesophagostomum spp. infection in pigs.

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.), Contracaecum osculatum (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.

Cats are important in the epidemiology of Toxoplasma gondii infection because they are the only hosts that can excrete the environmentally resistant oocysts. In the present study, prevalence of T. gondii was determined in serum, feces, and tissues of 34 cats from People's Republic of China. Antibodies to T. gondii were assayed by the modified agglutination test and found in 27 of 34 (79.4%) cats with titers of 1:40 in one, 1:80 in one, 1:160 in three, 1:320 in three, 1:640 in eight, and 1:1280 or higher in 11 cats. T. gondii oocysts were not found in feces of any cat as ascertained by bioassay in mice. Tissues (brain, heart, and tongue) of 27 seropositive cats were pooled and bioassayed in mice (8 cats) or cats (19 cats). T. gondii was isolated from tissues of 17 of 27 seropositive cats. Genotyping of these 17 T. gondii isolates using polymorphisms at 10 nuclear markers including SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1 and a new SAG2, and an apicoplast marker Apico revealed two genotypes. This is the first report of genetic typing of T. gondii isolates from cats from China.

The present study examined sequence variability in four mitochondrial genes, namely cytochrome c oxidase subunit (cox1), cytochrome b (cytb) and NADH dehydrogenase subunits 1 and 5 (nad1 and nad5), among Bunostomum trigonocephalum isolates from four different geographic regions in China. Ten B. trigonocephalum samples were collected from each of the four provinces (Heilongjiang, Jilin, Shaanxi and Yunnan), China. A part of the cox1 (pcox1), cytb (pcytb), nad1 and nad5 genes (pnad1 and pnad5) were amplified separately from individual hookworms by polymerase chain reaction (PCR) and were subjected to direct sequencing in order to define sequence variations and their phylogenetic relationships. The intra-specific sequence variations within B. trigonocephalum were 0–1.9% for pcox1, 0–2.0% for pcytb, 0–1.6% for pnad1 and 0–1.7% for pnad5. The A+T contents of the sequences were 69.6–70.4% (pcox1), 71.9–72.7 (pcytb), 70.4–71.1% (pnad1) and 72.0–72.6% (pnad5). However, the inter-specific sequence differences among members of the family Ancylostomatidae were significantly higher, being 12.1–14.2% for pcox1, 13.7–16.0 for cytb, 17.6–19.4 for nad1 and 16.0–21.6 for nad5. Phylogenetic analyses based on the combined partial sequences of cox1, cytb, nad1 and nad5 using three inference methods, namely Bayesian inference (Bayes), maximum likelihood (ML) and maximum parsimony (MP), revealed that all the B. trigonocephalum samples form monophyletic groups, but samples from the same geographical origin did not always cluster together, suggesting that there was no obvious geographical distinction within B. trigonocephalum based on sequences of the four mtDNA genes. These results demonstrated the existence of low-level intra-specific variation in mitochondrial DNA (mtDNA) sequences among B. trigonocephalum isolates from different geographic regions.

The present study examined sequence variation in four mitochondrial (mt) genes, namely cytochrome c oxidase subunits 1 (cox1) and 2 (cox2), and NADH dehydrogenase subunits 1 and 2 (nad1 and nad2) among Clonorchis sinensis isolates from different endemic regions in China, and their phylogenetic relationships with other zoonotic trematodes were reconstructed. A portion of the cox1 and cox2 genes (pcox1 and pcox2), and nad1 and nad2 genes (pnad1 and pnad2) were amplified separately from individual liver flukes by polymerase chain reaction (PCR) and the amplicons were subjected to sequencing from both directions. The intra-specific sequence variations within C. sinensis were 0–1.6% for pcox1, 0–1.4% for pcox2, 0–0.9% for pnad1 and 0–1.0% for pnad2. Phylogenetic analyses based on the combined sequences of pcox1, pcox2, pnad1 and pnad2 revealed that all the C. sinensis isolates grouped together and were closely related to Opisthorchis felineus. These findings revealed the existence of intra-specific variation in mitochondrial DNA (mtDNA) sequences among C. sinensis isolates from different geographic regions, and demonstrated that mtDNA sequences provide reliable genetic markers for phylogenetic studies of zoonotic trematodes.

Trichinella spiralis is an important zoonotic nematode causing trichinellosis which is associated with human diseases such as malaise, anorexia, nausea, vomiting, abdominal pain, fever, diarrhea, and constipation. microRNAs (miRNAs) are endogenous small non-coding RNAs that play important roles in the regulation of gene expression. The objective of the present study was to examine the miRNA expression profile of the larvae of T. spiralis by Solexa deep sequencing combined with stem-loop real-time polymerase chain reaction (PCR) analysis. T. spiralis larvae were collected from the skeletal muscle of naturally infected pigs in Henan province, China, by artificial digestion using pepsin. The specific identity of the T. spiralis larvae was confirmed by PCR amplification and subsequent sequence analysis of the internal transcribed spacer of ribosomal DNA. A total of 17,851,693 reads with 2,773,254 unique reads were obtained. Eleven conserved miRNAs from 115 unique xsmall RNAs (sRNAs) and 12 conserved miRNAs from 130 unique sRNAs were found by BLAST analysis against the known miRNAs of Caenorhabditis elegans ( ftp://ftp.ncbi.nih.gov/genomes/Caenorhabditis_elegans ) and Brugia malayi dataset ( http://www.ncbi.nlm.nih.gov/genomeprj?Db=genomeprjcmd=ShowDetailViewTermToSearch=9549 ) in miRBase, respectively. One novel miRNA with 12 precursors were identified and certified using the reference genome of B. malayi, while no novel miRNA was found when using the reference genome of C. elegans. Nucleotide bias analysis showed that the uracil was the prominent nucleotide, particularly at the 1st, 6th, 18th, and 23th positions, which were almost at the beginning, middle, and the end of the conserved miRNAs. The identification and characterization of T. spiralis miRNAs provides a new resource to study regulation of genes and their networks in T. spiralis.

The accurate characterization of Schistosoma japonicum has important implications for analyzing genetic variation and would provide basic data for disease control. Previous studies using proteins, coding sequences, and especially antigen-coding genes showed lower genetic variation among S. japonicum isolates from mainland China. Therefore, the present study focused on variations in intron sequences of housekeeping and antigen-coding genes, which may be more informative for genetic analysis. We compared sequence variation between introns of two housekeeping genes and two antigen-coding genes. All 4 genes were polymorphic among all the S. japonicum isolates in mainland China, with 103, 158, 47, and 19 polymorphic (segregating) sites per kilobase in intron sequences of Actin, FBPA, 22.6kDa antigen and GST-26, respectively. Introns of housekeeping genes were slightly more polymorphic than coding and non-coding regions of antigen-coding genes examined in the present study within or among lake/marshland and mountainous types. Phylogenetic analysis based on sequences of single gene or combined sequences of multiple genes showed no specific clustering comprising parasites from single geographical or endemic regions. These results demonstrated that introns of housekeeping and antigen-coding genes were polymorphic, but the intron sequences examined in the present study were not suitable markers for examining genetic relationship among different isolates from endemic regions in mainland China.

Little information on epidemiology of Toxoplasma gondii infection in pigeons was available in People's Republic of China. In the present study, sera from 275 pigeons raised in different commercial flocks in Guangdong Province of southern China were evaluated using modified agglutination test (MAT). Specific antibodies were found in sera of 8.7% of 275 pigeons (MAT titer ≥ 1:5), and the seropositivity of eight herds we surveyed varied ranging from 0 to 18.2%. The results demonstrated the circulation of T. gondii in the examined pigeon farms, which poses potential risk for human infection with T. gondii. To our knowledge, this is the first seroprevalence survey of pigeons infected by T. gondii in People's Republic of China.

The prevalence of coccidial infection in sheep and goats was examined in Heilongjiang Province, northeastern China between January 2007 and June 2009. A total of 508 faecal samples (309 from sheep and 199 from goats) were collected from eight sheep and goat rearing administrative regions. Oocysts in faecal samples were detected using a flotation technique, sporulated and identified to species based on morphological features of the sporulated oocysts. The overall prevalence of coccidial infection was 90.9% (462/508), with prevalence of 92.9% (287/309) for sheep and 87.9% (175/199) for goats. 13 species of Eimeria were identified, namely, E. bakuensis, E. faurei, E. parva, E. ahsata, E. crandallis, E. granulosa, E. intricata, E. pallida, E. christenseni, E. caprina, E. alijevi, E. apsheronica and E. arloingi. In sheep, the most common Eimeria species were E. ahsata (67.2%), E. parva (59.9%) and E. bakuensis (44.3%). In goats, the more prevalent species were E. christenseni (78.3%), E. alijevi (73.7%), E. caprina (62.3%) and E. arloingi (44.6%). Concurrent infection with two to eight species was common. However, no clinical symptoms were observed in the examined sheep and goats. The results of the present investigation have implications for the control of coccidial infections in sheep and goats in northeastern China.

Toxoplasma gondii is widely distributed in humans and other animals including domestic poultry throughout the world, but little is known of the prevalence of T. gondii in chickens and ducks in People's Republic of China. In the present study, antibodies to T. gondii were investigated in 349 domestic ducks (Anas spp.), 361 free-range, and 244 caged chickens (Gallus domesticus) raised in commercial flocks in Southern China's Guangdong Province using the modified agglutination test (MAT). Antibodies to T. gondii (MAT titer of 1:5 or higher) were found in 56 (16%) of 349 ducks, 41 (11.4%) of 361 free-range, and 10 (4.1%) of 244 caged chickens. The results indicate soil contamination due to T. gondii oocysts because free-range chickens feed from the ground, and suggest that the meat from the domestic poultry may be an important source for human infection by T. gondii in People's Republic of China.

Summary The prevalence of anti-Toxoplasma gondii specific IgG in stray and household cats in Guangzhou, China was determined by ELISA on serum samples from 206 cats (117 strays and 89 households) and the overall infection rate was 25.24%. The infection rate in stray cats (30.77%) was significantly higher (P

The present study examined sequence variation in three mitochondrial DNA (mtDNA) regions, namely cytochrome c oxidase subunit 3 ( cox 3), NADH dehydrogenase subunits 4 and 5 ( nad 4 and nad 5), among Schistosoma japonicum isolates from different endemic regions in China, and their phylogenetic relationships were re-constructed. A portion of the cox 3 gene (p cox 3), a portion of the nad 4 and nad 5 genes (p nad 4 and p nad 5) 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 p cox 3, 0.0–0.1% for p nad 4, and 0.0–0.2% for p nad 5. In the lake/marshland areas, sequence variations between male and female parasites among different geographical locations were 0.0–0.3% for p cox 3, 0.0–0.7% for p nad 4, and 0.0–1.6% for p nad 5. Sequence variations between S. japonicum from mountainous areas and those from lake/marshland areas were 0.0–0.5% for p cox 3, 0.0–0.7% for p nad 4, and 0.0–1.6% for p nad 5. Phylogenetic analyses based on the combined sequences of p cox 3, p nad 4 and p nad 5 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.

The prevalence of helminths in adult dogs was investigated in Hunan Province, the People's 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.

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 Trichinella nativa (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.

Seven isolates of Cryptocaryon irritans from different host species and geographical locations in Mainland China were characterized by the first (ITS-1) and second (ITS-2) internal transcribed spacers (ITS) of nuclear ribosomal DNA (rDNA) using two isolates of Ichthyophthirius multifiliis for comparative purposes. The rDNA region including the ITS-1, 5.8S, ITS-2, and flanking 18S and 28S sequences were amplified by polymerase chain reaction and the amplicons were sequenced directly. The ITS-1, 5.8S, and ITS-2 sequences were 129, 160, and 190 bp in length, respectively, for all seven C. irritans isolates, whereas the corresponding sequences for the two I. multifiliis isolates were 142, 153, and 194 bp, respectively. While sequence variation among the seven C. irritans isolates ranged from 0 to 1.6% in both the ITS-1 and ITS-2, and the two I. multifiliis isolates differed by 1.4% in the ITS-1 and 1.0% in the ITS-2; C. irritans differed from I. multifiliis by 57.1-60.9% in the ITS-1 and 79.4-83.0% in the ITS-2, indicating that ITS sequences provide reliable genetic markers for the identification and differentiation of the two species. Phylogenetic analysis using the sequence pairwise-distance data using the neighbor-joining method inferred that the seven C. irritans isolates from Mainland China and two other isolates (T.A and Aus.C) from other countries clustered together to show monophyly, which could be readily distinguished from the other monophyletic group all from other regions. Therefore, ITS sequence data and phylogenetic analysis provided strong support that C. irritans isolates from Mainland China represent a single species. The definition of genetic markers in the ITS rDNA provide opportunities for studying the ecology and population genetic structures of the C. irritans from Mainland China and elsewhere and is also relevant to the diagnosis and control of fish diseases they cause.

The prevalence of helminths in adult sheep was investigated in Heilongjiang Province, People's Republic of China between January 1999 and September 2003. A total of 326 adult sheep representing local breeds (Xingjiang Fine Wool Sheep, Dongbei Fine Wool Sheep) as well as introduced breeds (Merino and Charollais) from representative geographical locations in Heilongjiang Province were slaughtered and examined for the presence of helminths. The worms were examined, counted and identified to species according to existing keys and descriptions. A total of 26 helminth species were found representing 2 phyla, 3 classes, 13 families and 20 genera. All sheep were infected by more than one helminth species. Oesophagostomum columbianum, Haemonchus contortus and Trichostrongylus colubriformis were the most common nematode species, and Paramphistomum cervi, Orientobilharzia turkestanica and Fasciola hepatica were the most common trematode species, whereas the infection of adult sheep with cestodes was uncommon. The results of the present investigation provide relevant "base-line" data for Heilongjiang Province, China, for assessing the effectiveness of future control strategies against helminth infections in sheep.

The efficacy and economic benefits of Supercox, a live anticoccidial vaccine were examined and compared with an anticoccidial drug in a trial in broiler chickens under modern commercial conditions in China. In total, 40,660 chickens were used in the present study, half of which were vaccinated with the Supercox vaccine comprising a precocious line of Eimeria tenella and non-attenuated lines of Eimeria maxima and Eimeria acervulina, and the other half were medicated with Diclazuril delivered as feed additive at the dosage of 1mg/kg of feed. The vaccine was administered orally to 7-day-old chickens. No clinical diseases were diagnosed in any of the vaccinated birds. However, clinical coccidiosis occurred in a large proportion of medicated control birds and these chickens had to be treated with anticoccidial drugs (Diclazuril and Toltrazuril). Comparison of production performance between vaccinated birds and medicated control birds revealed that the vaccine Supercox performed better than anticoccidial drugs in terms of mortalities, costs and overall economic benefits (profits). These findings demonstrated that the use of the Supercox vaccine could control clinical coccidiosis in broilers and achieve production performance superior to that using anticoccidial drugs, particularly where drug resistance might result in failure to control clinical diseases.

Isolates of Fasciola (Platyhelminthes: Trematoda: Digenea) from different host species and geographical locations in Mainland China were characterised genetically. The second internal transcribed spacer (ITS-2) of nuclear ribosomal DNA (rDNA) was amplified from individual trematodes by polymerase chain reaction (PCR), and the representative amplicons were cloned and sequenced. The length of the ITS-2 sequences was 361-362bp for all Chinese Fasciola specimens sequenced. While there was no variation in length or composition of the ITS-2 sequences among multiple specimens from France, Sichuan and Guangxi, sequence difference of 1.7% (6/362) was detected between specimens from France and Sichuan, and those from Guangxi. Based on ITS-2 sequence data, it was concluded that the Fasciola from Sichuan represented Fasciola hepatica, the one from Guangxi represented Fasciola gigantica and the one from sheep from Heilongjiang may represent an "intermediate genotype", as its ITS-2 sequences were unique in that two different ITS-2 sequences exist in the rDNA array within a single Fasciola worm. One of the sequences is identical to that of F. hepatica, and the other is almost identical to that of F. gigantica in that nucleotides at five of the six polymorphic positions represent F. gigantica. This microheterogeneity is possibly due to sequence polymorphism among copies of the ITS-2 array within the same worm. Based on the sequence differences, a PCR-linked restriction fragment length polymorphism (PCR-RFLP) assay was established for the unequivocal delineation of the Fasciola spp. from Mainland China using restriction endonuclease Hsp92II or RcaI. This assay should provide a valuable tool for the molecular identification and for studying the ecology and population genetic structures of Fasciola spp. from Mainland China and elsewhere.