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Sequence variability in three mitochondrial DNA (mtDNA) regions, namely portions of cytochrome c oxidase subunit 1 (p cox1), NADH dehydrogenase subunit 1 (p nad1) and NADH dehydrogenase subunit 4 (p nad4), for Toxocara canis. Baylisacaris transfuga. Ascaris suum and Parascaris equorum from Canis lupus. Ursus thibetanus. Sus scrofa and Equus burchelli in China were examined. The lengths of the sequences of p cox1, p nad1 and p nad4 were 711 bp, 648 bp and 666 bp, respectively. No intra-species differences were detected in p cox1 for the four examined ascarid species, in p nad1 for T. canis. A. suum and P. equorum, and in p nad4 for B. transfuga and P. equorum. Sequence differences in p nad4 for six roundworm samples of T. canis and P. equorum were 0-0.1% and 0-0.3%, respectively, and were 0-0.3% in p nad1 for six roundworm samples isolate of B. transfuga. The inter-specific sequence differences among four species were 8.7-12.4% for p cox1, 13.9-17.7% for p nad1, and 14.0-25.7% for p nad4. Phylogenetic analyses suggested that the three mtDNA fragments could be used to identify ascarid species in families Ascaridiae and Toxocaridae. [ABSTRACT FROM AUTHOR]

The present study examined genetic variability among Clonorchis sinensis isolates from four different geographical localities (Guangzhou, Nanning, Jiamusi and Daqing) and host species (cats, dogs, human and rabbits) in Mainland China by sequence analyses of two mitochondrial DNA (mtDNA) genes, namely NADH dehydrogenase subunits 2, 5 ( nad2 and nad5) and ribosomal internal transcribed spacer 1 (ITS1). A portion of the ITS1, nad2 (p nad2) and nad5 (p nad5) was amplified by polymerase chain reaction separately from adult C. sinensis individuals and the amplicons were subjected to sequencing from both directions. The length of the sequences of ITS1, p nad2 and p nad5 was 643, 666 and 771 bp, respectively. The intraspecific sequence variations within C. sinensis were 0-1.7% for ITS1, 0-1.4% for p nad2 and 0-0.9% for p nad5. The interspecific sequence variations within other zoonotic trematodes, which were published previously, were 4.5-84.9% for ITS1, 21.9-43.6% for p nad2 and 19.2-48.9% for p nad5. The A+T contents of the sequences were 45.26-45.88% (ITS1), 62.91-63.51% (p nad2) and 58.24-58.63% (p nad5). Phylogenetic analyses using ribosomal and mitochondrial sequence data set, with three different computational algorithms (Bayesian inference, maximum parsimony and maximum likelihood), all revealed distinct groups with high statistical support. These findings demonstrated the existence of low-level intraspecific variations in ribosomal DNA (rDNA) and mtDNA sequences among C. sinensis isolates from four different regions and hosts in China and elucidated that mtDNA sequences and rDNA sequences provided reliable genetic markers for phylogenetic studies of zoonotic trematodes. [ABSTRACT FROM AUTHOR]

The article presents a study which characterizes the genomic DNA of Cryptosporidium from the feces of preweaned calves in Jilin and Daqing China through 18S rRNA gene nested PCR-RFLP assay.

The complete mitochondrial genome of Fimbriaria fasciolaris is reported for the first time in this study. Its entire mitogenome is 13,543 bp in length, contained 12 protein-coding genes, 2 ribosomal RNA genes, 22 transfer RNA genes, and 2 non-coding regions. The arrangement of the twelve PCGs was identical to that of most cestode mtDNA sequence. Among all the cestodes mitochondrial genomes consulted from GenBank database, there existed a tiny difference in the order of trnY, trnL1, trnL2, and trnS2. The phylogenetic analysis by Bayesian inference method shows that F. fasciolaris of this study was confirmed to be a member of family Hymenolepididae. [ABSTRACT FROM AUTHOR]

The complete mitochondrial genome of Haemaphysalis concinna is reported for the first time in this study. Its entire mitogenome is 14,675 bp in length, contained 13 protein-coding genes, two ribosomal RNA genes, 22 transfer RNA genes, and two non-coding regions. Among the 13 protein-coding genes, apart from the nad1, nad4, nad4L, and nad5 gene encoded on the L-strand, the remaining protein-coding genes were encoded on the H-strand. The phylogenetic analysis by Bayesian inference method shows that Amblyomma sphenodonti and H. parva formed one clade, while H. concinna and other species of genus Haemaphysalis formed the other clade, indicating that H. concinna belong to the genus Haemaphysalis. [ABSTRACT FROM AUTHOR]

The complete mitochondrial genome ofDrepanidotaenia lanceolatawas sequenced. The complete mtDNA sequence is 13 573 bp long and contains 12 protein-coding genes (cox1–3,nad1–6,nad4L,atp6, andcytb), two ribosomal RNA (rRNA) genes, 22 transfer RNA (tRNA) genes, and 2 non-coding regions (NCR). Phylogenetic analysis using three methods (Bayesian inference, maximum parsimony, and maximum likelihood) demonstrates with high statistical support thatD. lanceolatais closely related to the genusHymenolepis. This study reports the first complete mt genome sequence of a representative species of the genusDrepanidotaeniaand should provide novel genetic data for further studies of the taxonomy, systematics, and population genetics of these and other related cestodes of socio-economic significance. [ABSTRACT FROM PUBLISHER]

In the present study, we determined the complete mitochondrial DNA (mtDNA) sequence ofEimeria magnafrom rabbits for the first time, and compared its gene contents and genome organizations with that of sevenEimeriaspp. from domestic chickens. The size of the complete mt genome sequence ofE. magnais 6249 bp, which consists of 3 protein-coding genes (cytb,cox1 andcox3), 12 gene fragments for the large subunit (LSU) rRNA, and 7 gene fragments for the small subunit (SSU) rRNA, without transfer RNA genes, in accordance with that ofEimeriaspp. from chickens. The putative direction of translation for three genes (cytb,cox1 andcox3) was the same as those ofEimeriaspecies from domestic chickens. The content of A + T is 65.16% forE. magnamt genome (29.73% A, 35.43% T, 17.09 G and 17.75% C). TheE. magnamt genome sequence provides novel mtDNA markers for studying the molecular epidemiology and population genetics ofEimeriaspp. and has implications for the molecular diagnosis and control of rabbit coccidiosis. [ABSTRACT FROM PUBLISHER]

Sequences of the complete nuclear ribosomal DNA (rDNA) gene from five individual Paramphistomum cervi were determined for the first time. The five complete rDNA sequences, which included the 18S rDNA, the internal transcribed spacer 1 (ITS1), the 5.8S rDNA, the internal transcribed spacer 2 (ITS2), the 28S rDNA, and the intergenic spacer (IGS) regions, had a length range of 8,493-10,221 bp. The lengths of the investigated 18S, ITS1, 5.8S, ITS2, and 28S rDNA sequences, which were 1,994 bp, 1,293 bp, 157 bp, 286 bp, and 4,186 bp, respectively, did not vary. However, the IGS rDNA sequences had a length range of 577-2,305 bp. The 5.8S and ITS-2 rDNA sequences had 100% identity among the five investigated samples, while the identities among the IGS had a range of 53.7-99.8%. A comparative analysis revealed that different types and numbers of repeats were found within each ITS1 and IGS region, which may be related to the length polymorphism of IGS. The phylogenetic position of P. cervi in Paramphistomatidae was analyzed based on the 18S rDNA sequences. These results will aid in studying the intra- and interspecific variation of the Paramphistomatidae and the systematics and phylogenetics of Digenea. [ABSTRACT FROM AUTHOR]

Culicidae, the mosquito family, includes more than 3600 species subdivided into the subfamilies Anophelinae and Culicinae. One-third of mosquitoes belong to the Aedini tribe, which is subordinate to the subfamily Culicinae, which comprises common vectors of viral zoonoses. The tribe of Aedini is extremely diverse in morphology and geographical distribution and has high ecological and medical significance. However, knowledge about the systematics of the Aedini tribe is still limited owing to its large population and the similar morphological characteristics of its species. This study provides the first description of the complete mitochondrial (mt) genome sequence of Aedes vexans and Ochlerotatus caspius belonging to the Aedini tribe. The mt genomes of A. vexans and O. caspius are circular molecules that are 15,861 bp and 15,954 bp in size, with AT contents of 78.54% and 79.36%, respectively. Both the circular mt genomes comprise 37 functional subunits, including 13 protein-coding genes (PCGs), two ribosomal RNA genes, 22 transfer RNA genes (tRNAs), and a control region (also known as the AT-rich region). The most common start codons are ATT/ATG, apart from cox 1 (TCG) and nad 5 (GTG), while TAA is the termination codon for all PCGs. All tRNAs have a typical clover leaf structure, except tRNA Ser1. Phylogenetic analysis of the concatenated, aligned amino acid sequences of the 13 PCGs showed that A. vexans gathered with Aedes sp. in a sister taxon, and O. caspius gathered with Ochlerotatus sp. in a sister taxon. The findings from the present study support the concept of monophyly of all groups, ratify the current taxonomic classification, and provide vital molecular marker resources for further studies of the taxonomy, population genetics, and systematics of the Aedini tribe. [Display omitted] • The mitochondrial genomes of Aedes vexans and Ochlerotatus caspius was performed for the first time. • Evolutionary analysis showed that the obtained species belong to Aedes and Ochlerotatus genus, respectively. • The present study provides vital molecular marker resources for the taxonomy and systematics of the Aedini tribe. [ABSTRACT FROM AUTHOR]

Crytosporidium spp., Giardia duodenalis , and Enterocytozoon bieneusi are important diarrheal pathogens with a global distribution that threatens the health of humans and animals. Despite cattle being potential transmission hosts of these protozoans, the associated risks to public health have been neglected. In the present study, a total of 1155 cattle fecal samples were collected from 13 administrative regions of Heilongjiang Province. The prevalence of Cryptosporidium spp., G. duodenalis , and E. bieneusi were 5.5% (64/1155; 95% CI: 4.2-6.9), 3.8% (44/1155; 95% CI: 2.7-4.9), and 6.5% (75/1155; 95% CI: 5.1-7.9), respectively. Among these positive fecal samples, five Cryptosporidium species ( C. andersoni , C. bovis , C. ryanae , C. parvum , and C. occultus ), two G. duodenalis assemblages (E and A), and eight E. bieneusi genotypes (BEB4, BEB6, BEB8, J, I, CHS7, CHS8, and COS-I) were identified. Phylogenetic analysis showed that all eight genotypes of E. bieneusi identified in the present study belonged to group 2. It is worth noting that some species/genotypes of these intestinal protozoans are zoonotic, suggesting a risk of zoonotic disease transmission in endemic areas. The findings expanded our understanding of the genetic composition and zoonotic potential of Cryptosporidium spp., G. duodenalis , and E. bieneusi in cattle in Heilongjiang Province.

Tetrameres grusi is a significant parasitic nematode of cranes that is classified into suborder Spirurina. However, for more than a century, this classification has been controversial. Mitochondrial genomes are valuable resources for parasite taxonomy, population genetics and systematics studies. Here, the mitochondrial genome of T. grusi was determined and subsequently compared with those from Spirurina species using concatenated datasets of amino acid sequences predicted from mitochondrial protein-coding genes. The complete mitochondrial genome of T. grusi is circular with 13,709 bp, and it contains 12 protein-coding genes, 22 transfer RNA genes, two ribosomal RNA genes and one non-coding region. All of the protein-coding genes are transcribed in the same direction. There were 18 intergenic spacers of 1-44 bp, and six locations with gene overlaps, ranging from 1 bp to 28 bp, in the mitochondrial genome of T. grusi . The AT content of this mitochondrial genome was 71.56%. This was similar to mitochondrial genomes of other Spirurina species, which also exhibited strong AT content bias, not only in the nucleotide composition but also in codon usage. The sequenced mitogenomes of the 25 Spirurina nematodes showed three classes of gene arrangements based on the 12 protein-coding genes, and the gene arrangement of the T. grusi mitochondrial genome belonged to the Class I. Phylogenetic analyses using mitochondrial genomes of 25 Spirurina nematodes revealed that T. grusi (Habronematoidea) was closer to Gongylonema pulchrum (Spiruroidea) than Spirocerca lupi (Thelazioidea). The availability of the complete mitochondrial genome sequence of T. grusi provides new and useful genetic markers for further studies on Spirurina nematodes., Competing Interests: The authors report no conflicts of interests., (© 2021 The Authors.)

Toxoplasma gondii , an intracellular zoonotic parasite, can infect humans and various animals worldwide. Wild rodents plan an important role as intermediate hosts of T. gondii . Some studies on T. gondii from wild rodents have been published, but the investigation data of T. gondii in wild rodents in China are limited. Therefore, brain tissue samples from 382 wild rodents in four provinces and one autonomous region of China were screened by PCR amplification of T. gondii B1 gene. Furthermore, the wild rodents were identified as five species based on their morphological characteristics, including Citellus dauricus ( n  = 35 from Heilongjiang), Lasiopodomys brandti ( n  = 81 from Inner Mongolia), Apodemus agrarius ( n  = 12 from Heilongjiang), Rattus norvegicus ( n  = 2 from Heilongjiang; n  = 99 from Zhejiang; n  = 54 from Shanxi), and Mus musculus ( n  = 99 from Guangxi). The overall prevalence of T. gondii in these wild rodents was 5.24% (20/382) in this study. At different regions, the highest prevalence of T. gondii was in Guangxi (12.12%) compared with other regions (0% in Heilongjiang; 2.47% in Inner Mongolia; 2.02% in Zhejiang; 7.41% in Shanxi). At different climates, the higher prevalence was found in temperate/mesothermal climates (7.07%) compared with continental/microthermal climates (3.26%). Also, the results showed that M. musculus had the highest prevalence of T. gondii infection (12.12%) among the rodent species sampled. Moreover, sampling year was significantly related to the prevalence of T. gondii in investigated wild rodents ( p  = 0.0117). This is the first report of T. gondii in wild rodents from Zhejiang, Guangxi, Shanxi, Heilongjiang provinces and Inner Mongolia autonomous region in China, providing the fundamental information for further prevention and control of toxoplasmosis in China.

Toxoplasma gondii is an important protozoan parasite that can infect a range of animals. Cats are the most important definitive hosts for T. gondii . Therefore, an assessment of the level of T. gondii infection in cats is of public health importance. As limited information about seroprevalence of T. gondii infection in cats in northeastern China was available, 854 serum samples, including 235 cats in Jilin province, 267 cats in Liaoning province, and 352 cats in Heilongjiang province, were examined by an indirect hemagglutination assay (IHA) test, between September 2013 and July 2019. The overall seroprevalence of T. gondii was 19.09% (163/854) at the cutoff 1:64, with 19.60% (69/352), 18.29% (43/235), and 19.10% (51/267) in Heilongjiang, Jilin and Liaoning, respectively. Logistic regression analysis showed that mode of life was highly related to T. gondii infection in cats at the investigation areas. These results could provide foundation data for prevention and control of T. gondii prevalence in these regions. Moreover, the results also suggested that the effective control strategies should continue to be performed.

Introduction: Toxoplasma gondii is the pathogen of toxoplasmosis, which affects a wide range of animal species and humans., Materials and Methods: In the present study, a serological investigation was undertaken to determine the seroprevalence of T. gondii infection in free-ranging pigs in northeastern China. A total of 806 serum samples were collected from free-ranging pigs in 6 cities in Liaoning, Jilin, and Heilongjiang Provinces., Results: Among them, 163 serum samples (20.22%) were detected T. gondii positive by indirect hemagglutination test (IHA). In each administrative district, the prevalence ranges from 12.99 to 22.22%. It was found that sample collection year and season were associated with T. gondii infection. Samples collected in 2015 and 2016 had had higher positive rate compared to those collected in 2017. Samples collected in summer and autumn also have higher positive rate than those in winter., Conclusion: This is the first report on T. gondii seroprevalence in free-ranging pigs in northeastern China, which adds the epidemiological data of T. gondii infection in free-ranging pigs in China. These results indicated that free-ranging pigs in Liaoning, Jilin, and Heilongjiang Provinces are exposed to T. gondii, which is a significant threat to local pig industry as well as public health. It is necessary to take relevant comprehensive measures to prevent and control T. gondii infection in free-ranging pigs in these regions.

Fasciolosis, caused by Fasciola hepatica and Fasciola gigantica, is an important zoonotic disease in the world. It affects livestock, especially for sheep and cattle, causing major economic loss due to morbidity and mortality. Although the excretory and secretory products (ESPs) of F. hepatica have been relatively well studied, little is known about the interaction between the ESP and host, and the mechanism of the key proteins involved in interaction. In this study, buffaloes were infected by Fasciola gigantica, and infection serum was collected at three different periods (42dpi, 70dpi, and 98dpi). The interaction proteins were pulled down with three different period serum by Co-IP assay, respectively, and then identified by LC-MS/MS analysis. A number of proteins were identified; some of them related to the biological function of the parasite, while most of them the functions were unknown. For the annotated proteins, 13, 5, and 7 proteins were pulled down by the infected serum in 42dpi, 70dpi, and 98dpi, respectively, and 18 proteins could be detected in all three periods. Among them, 13 belong to the cathepsin family, 4 proteins related to glutathione S-transferase, and 3 proteins are calcium-binding protein; other proteins related to catalytic activity and cellular process. This study could provide new insights into the central role played by ESPs in the protection of F. gigantica from the host immune response. At the same time, our research provided material for further studies about the interaction between F. gigantica and host.

Gasterophilus spp. (Diptera: Gasterophilidae) has a worldwide distribution; however, no complete mitochondrial (mt) genome data is available for Diptera which has greatly impeded population genetics, phylogenetics, and systematics studies in Gasterophilidae. Mt genome is known to provide genetic markers for investigations in these areas, but complete mt genomic datasets have been lacking for many Gasterophilidae species. Herein, we present the complete mt genome of the third-stage larvae (L3) of Gasterophilus intestinalis from the stomach wall of naturally infected horses in Heilongjiang province (HLJ) and Xinjiang Uygur Autonomous Region (XJ), China. The complete mt genome of G. intestinalis was 15,687 bp (HLJ) and 15,660 bp (XJ) in length and consists of 37 genes, including 13 genes for proteins, 22 genes for tRNA, and 2 genes for rRNA. The gene arrangement is the same as those of Oestroidae species. Phylogenetic analyses using concatenated amino acid sequences of 12 protein-coding genes by Bayesian inference (BI) and maximum likelihood (ML), suggested that the families Gasterophilidae and Oestroidae were more closely related than to Tachinidae. The mt genome of G. intestinalis represents the first mt genome of any member of the family Gasterophilidae. These data provide novel mtDNA markers for studying the molecular epidemiology and population genetics of the G. intestinalis and its congeners.

Passalurus ambiguus (Nematda: Oxyuridae) is a common pinworm which parasitizes in the caecum and colon of rabbits. Despite its significance as a pathogen, the epidemiology, genetics, systematics, and biology of this pinworm remain poorly understood. In the present study, we sequenced the complete mitochondrial (mt) genome of P. ambiguus. The circular mt genome is 14,023 bp in size and encodes of 36 genes, including 12 protein-coding, two ribosomal RNA, and 22 transfer RNA genes. The mt gene order of P. ambiguus is the same as that of Wellcomia siamensis, but distinct from that of Enterobius vermicularis. Phylogenetic analyses based on concatenated amino acid sequences of 12 protein-coding genes by Bayesian inference (BI) showed that P. ambiguus was more closely related to W. siamensis than to E. vermicularis. This mt genome provides novel genetic markers for studying the molecular epidemiology, population genetics, systematics of pinworm of animals and humans, and should have implications for the diagnosis, prevention, and control of passaluriasis in rabbits and other animals.

The complete mitochondrial (mt) genome sequence of Triodontophorus brevicauda, an intestinal equine nematode parasite was determined for the first time. The circular T. brevicauda mt genome is 14,305 bp in length and contains 36 genes, of which 12 code for protein, 22 for transfer RNA, and two for ribosomal RNA, and lacks atp8 mtDNA gene. Phylogenetic analysis based on the concatenated amino acid sequence of the 12 protein-coding genes was performed using three different tree-building methods. The Strongyloidea cluster divides into two large branches, and each nematode family included in our study forms an independent clade, though paraphyly confounds the issue at some nodes. T. brevicauda clusters together with Cylicocyclus insignis with high statistical support. The mtDNA data in this study not only provide a new mtDNA resource for phylogeny, but also become a novel and useful genetic marker for further studies on the identification, population genetics, and molecular epidemiology of the genus Triodontophorus in equine., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Objective: To analyze the immunoreactivity of recombinant cathepsin L-like proteases (CatL) protein of Fasciola hepatica and its immunogenicity in SD rats., Methods: The E. coli BL21(DE3) cells harbouring recombinant plasmid pET30a-FhCatL were inoculated in LB medium, and the protein expression was induced with IPTG. The recombinant protein FhCatL was analyzed by SDS-PAGE and the immunoreactivity was identified by Western blotting with sera from Fasciola hepatica-infected goat as the primary antibody. Twenty SD rats were randomly divided into immunized group and adjuvant control group. SD rats in immunized group were injected subcutaneously with 200 microg of purified FhCatL protein. All the rats received three immunizations at 3-week intervals. The adjuvant control group with 10 SD rats received only adjuvants emulsified with the same amount of PBS. Serum samples were collected at the day before the second and final immunization, 3, 6, and 9 weeks after the final immunization. The IgG antibody of rats' sera was examined by indirect ELISA and spleen lymphocyte proliferation (SLP) was tested by methyl thiazolyl tetrazolium (MTT)., Results: The molecular weight of purified FhCatL was about Mr 42,000. The recombinant FhCatL was recognized by pool sera of goats naturally infected with F. hepatica. The titer of specific antibody IgG in SD rats induced by the recombinant protein against CatL protein was significantly higher than that of the control, and the antibody titer reached the peak at three weeks after the final immunization (1 : 102,400). The stimulation index of splenocytes in immunized group was 2.176 +/- 0.047, which was significantly higher than that of the control (1.171 +/- 0.032) (P<0.05)., Conclusion: The recombinant FhCatL protein bears stronger immnoreactivity and immunogenicity.

Sequences of the complete nuclear ribosomal DNA (rDNA) gene from five individual Paramphistomum cervi were determined for the first time. The five complete rDNA sequences, which included the 18S rDNA, the internal transcribed spacer 1 (ITS1), the 5.8S rDNA, the internal transcribed spacer 2 (ITS2), the 28S rDNA, and the intergenic spacer (IGS) regions, had a length range of 8,493-10,221 bp. The lengths of the investigated 18S, ITS1, 5.8S, ITS2, and 28S rDNA sequences, which were 1,994 bp, 1,293 bp, 157 bp, 286 bp, and 4,186 bp, respectively, did not vary. However, the IGS rDNA sequences had a length range of 577-2,305 bp. The 5.8S and ITS-2 rDNA sequences had 100% identity among the five investigated samples, while the identities among the IGS had a range of 53.7-99.8%. A comparative analysis revealed that different types and numbers of repeats were found within each ITS1 and IGS region, which may be related to the length polymorphism of IGS. The phylogenetic position of P. cervi in Paramphistomatidae was analyzed based on the 18S rDNA sequences. These results will aid in studying the intra- and interspecific variation of the Paramphistomatidae and the systematics and phylogenetics of Digenea.

Objective: To analyze the immunogenicity of recombinant glutathione S-transferase protein of Fasciola hepatica (FhGST) in SD rats., Methods: The recombinant expression plasmid pET30a-FhGST was transformed into E. coli BL21 (DE3) cells and induced with IPTG for protein expression. The recombinant protein FhGST was analyzed by SDS-PAGE and identified by Western blotting. Twenty SD rats were randomly divided into two groups: immunized group and adjuvant control group. SD rats in immunized group were injected subcutaneously with 200 microg of purified FhGST protein. The adjuvant control group with 10 SD rats received only adjuvants emulsified with PBS. All the rats received three immunizations at 3-week intervals. Serum samples were collected at pre-immunization, the day after each immunization, 3 weeks and 6 weeks after the final immunization. The IgG antibody of rats' sera was examined by indirect ELISA and spleen lymphocyte proliferation (SLP) was tested by MTT., Results: The molecular weight of purified FhGST was about M(r) 31 300. The recombinant FhGST was recognized by pool sera of goats naturally infected with F. hepatica. The recombinant protein induced specific antibody IgG against GST protein in SD rats significantly higher than that of the control, and the antibody titer reached the peak at 9 weeks after the first immunization (GMT 1:89 144). FhGST protein significantly enhanced the growth and proliferation of rat splenocytes., Conclusion: The recombinant FhGST protein induces specific immune response in SD rats.

The seroprevalence of Toxoplasma gondii in beef cattle and dairy cattle in Heilongjiang Province, northeast China, was surveyed between April 2009 and May 2011. A total of 1803 (693 beef cattle and 1110 dairy cattle) serum samples were collected from 10 administrative regions rearing beef cattle and dairy cattle, and antibodies to T. gondii were examined by indirect hemagglutination (IHA) test. The overall seroprevalence of T. gondii in beef cattle and dairy cattle was 2.6% (46/1803), and the prevalence in beef cattle (3.0%) was slightly higher than that in dairy cattle (2.3%). The prevalence of antibodies in adult animals was higher than that in calves, but the differences among the age groups were not significant (p>0.05). The seroprevalence in female (3.4%) and male (2.5%) beef cattle was not statistically significant (p>0.05). Though the prevalence in intensively reared beef cattle and dairy cattle was lower than that in semi-intensively reared animals, the difference was not statistically significant (p>0.05). The results of this survey indicated the presence of T. gondii infection in beef cattle and dairy cattle in Heilongjiang Province, the coldest province in China, which may cause economic losses to the local livestock industry, and may be a source of T. gondii infection for humans in this region.

Three new naphthoquinones, astropaquinones A-C (1-3), were isolated from cultures of the freshwater fungus Astrosphaeriella papuana YMF 1.01181, together with the known compound, 6-hydroxy-2,4-dimethoxy-7-methylanthraquinone (4). The structures of the compounds were settled mainly by interpretation of their 1D and 2D NMR spectra. Astropaquinone B (2) and C (3) were found to possess a rare pyranonaphthoquinone skeleton containing a lactol ring. Furthermore, compounds 1-4 showed moderate antagonistic activity against nine fungi and four bacterial strains., (Georg Thieme Verlag KG Stuttgart, New York.)

Objective: To investigate the method of constructing small-diameter vascular grafts from xenogenic decellularized arterial matrices and mesenchymal stem cells (MSCs)., Methods: Porcine iliac arteries were decellularized by detergent and trypsin treatment. Histology, mechanical strength and porosity experiments were performed to evaluate the properties of decellularized matrices. MSCs were isolated from bone marrow of dogs and expanded ex vivo. Decellularized matrices were seeded with MSCs and further cultured in a pulsatile bioreactor. Morphological features of the tissue engineered grafts were assayed by HE staining and scanning electron microscopy., Results: After cell extraction, absence of cellular components and preservation of extracellular matrix were verified. Mechanical strength of decellularized matrices was slightly reduced compared with native arteries. Porosity of decellularized matrices was 94.9%. Decellularized matrices were successfully seeded with MSCs, which grew to a near-confluent monolayer under flow conditions and MSCs were highly elongated and oriented to the flow direction., Conclusion: Small-diameter vascular grafts can be constructed by seeding MSCs onto xenogenic decellularized arterial matrices and culturing in a pulsatile bioreactor.

Objective: To clone and express the cathepsin L-like protease gene of Fasciola hepatica (FhCL) and investigate the immunogenicity of the recombinant FhCL protein., Methods: Specific primers were designed according to the reported FhCL gene in GenBank. Using total RNA from adult worms of F. hepatica, FhCL gene was amplified by RT-PCR. The PCR product was cloned into pMD18-T vector and then subcloned into pET30a(+) vector. The recombinant plasmid was transformed into E. coli BL21 (DE3) and followed by expression of the protein induced by IPTG. The expression situation of recombinant FhCL was analyzed by SDS-PAGE. Its immunoresponse to the sera of infected goat and the antisera of SD rats against FhCL was examined by Western blotting analysis., Results: PCR and double enzyme digestion showed that the FhCL gene fragment was about 1,000 bp in length. The constructed recombinant plasmid pET30a (+)-FhCL was identified by sequencing. The recombinant protein (Mr 42,000) was expressed in the form of inclusion body. The protein was recognized respectively by the sera of infected goat and the sera from rat immunized with FhCL., Conclusion: The recombinant plasmid pET30a(+)-FhCL has been constructed, which shows high antigenicity.

Objective: To establish a PCR diagnostic method based on Nc-5 gene of Neospora caninum, for being used to detect Neospora in brain tissues of bovine aborted fetus., Methods: Specific primers were designed and synthesized based on the reported Nc-5 gene of N. caninum (GenBank Accession No. AY459289). Using genomic DNA from N.caninum as templates, Nc-5 gene was amplified by PCR. The PCR product was cloned into pMD18-T vector, transformed into Escherichia coli JM109 and then sequenced. To evaluate the specificity of the PCR, genomic DNA of Theileria annulata, Babesia bovis, Toxoplasma gondii, Leishmania donovani and standard strain of N. caninum were used as a template in the PCR. For determining the detection limit of amplification procedure, PCR was run on a dilution series of genomic DNA from N. caninum (1.562 5-200 ng/ml). Brain tissue samples of 32 aborted fetuses were detected by PCR-based assay, and 23 blood samples from mothers were tested by ELISA., Results: The amplified DNA fragment (350 bp) had a high identity of 98% with the Nc-5 gene sequence of N. caninum (GenBank Accession No. AY459289). The PCR was specific for N. caninum and allowed the detection of 3.125 pg DNA of the parasite, while no amplification occurred with the other four species of protozoa. PCR-based assay and ELISA showed a positive rate of 18.8% (6/32) and 17.4% (4/23) of the samples tested, respectively. Moreover, all the 4 antibody positive samples showed PCR positive. There is no significant difference between the two assays (P > 0.05)., Conclusion: PCR diagnostic method is promising in detecting Neospora infection in brain tissues of aborted bovine.

Objective: To explore sequence differentiation of ITS and 28S rDNA-LSU of Orientobilharzia turkestanicum from bovine and caprine hosts., Methods: Adult worms of O. turkestanicum from the naturally infected cattle, sheep, cashmere goat and goat were collected and identified morphologically as O. turkestanicum according to existing keys and descriptions. The genomic DNA was extracted from parasites of different hosts. The internal transcribed spacer (ITS, contains ITS-1, 5.8S nuclear ribosomal DNA, ITS-2) and 28S nuclear ribosomal DNA-LSU were amplified by PCR, sequenced and analyzed by Chromans and DNASTAR softwares, and the RNA secondary structure of 28S rDNA-LSU was analyzed by DNAMAN software., Results: ITS-1, 5.8S rDNA, ITS-2 and 28S rDNA-LSU of O. turkestanicum from bovine and caprine hosts were 384, 159, 331 and 1 304 bp, respectively. ITS-1 and 5.8S rDNA of O. turkestanicum from different definitive hosts were identical; ITS-2 of O. turkestanicum from cattle, sheep and cashmere goat were identical, with one nucleotide variation compared with that of goat; 28S rDNA-LSU of O. turkestanicum from sheep and cashmere goat were identical, with two nucleotides variation compared with that of cattle and goat. The RNA secondary structure of 28S rDNA LSU of O. turkestanicum from caprine hosts were identical or similar, but with large variation compared with that of cattle., Conclusion: The rDNA sequence from different definitive hosts shows nucleotide variations to some extent and the RNA secondary structure of 28S rDNA-LSU from caprine hosts shows large variation in comparison to that of bovine.

Five new preussomerin analogues, ymf 1029A (1), B (2), C (3), D (4), and E (5), were isolated from the liquid cultures of an unidentified freshwater fungus YMF 1.01029, along with four known compounds, preussomerin C (6), preussomerin D (7), (4RS)-4,8-dihydroxy-3,4-dihydronaphthalen-1(2H)-one (8), and 4,6,8-trihydroxy-3,4-dihydronaphthalen-1(2H)-one (9). The structures of new metabolites were determined by analysis of NMR and MS data and by analogy with the data for the known bis-spirobisnaphthalene preussomerins. In vitro immersion experiments showed that these metabolites displayed weak nematicidal activity against Bursaphelenchus xylophilus, while compound 7 was the most potent. This is the first report of these compounds, which antagonize the Bursaphelenchus xylophilus nematode.

A living-tissue conduit with strong mechanical properties was used to produce small-diameter vessels. To improve blood compatibility, a shear-resistant confluent monolayer endothelium was formed on the luminal surface of the conduit. Under mechanical stimulation induced by pulsatile flow in a bioreactor, abrupt high-flow shear stress of 15.3 +/- 4.6 dynes/cm2 was applied to endothelial cells (ECs) seeded onto the lumen of a living-tissue conduit after 2 days of static culture. Scanning electron microscopy images revealed that most of the ECs were washed off after 3 days of dynamic culture. When shear stress was increased stepwise from 1.5 +/- 0.8 to 15.3 +/- 4.6 dynes/cm2 and applied to the ECs, scanning electron microscopy images of the luminal surface revealed that the confluent monolayer ECs were highly elongated and oriented to the flow direction, similar to findings in natural arteries in vivo. The results indicated that in vitro flow conditions played a key role in determining the durability of the EC layer. Careful design of the bioreactor and careful selection of the culture conditions will greatly improve the chances of producing a useful anti-thrombogenic surface for tissue-engineered small-diameter vessels.