Characterization of chicken IFI35 and its antiviral activity against Newcastle disease virus.

Interferon-induced protein-35 kDa (IFI35) was an antiviral protein induced by interferon (IFN)-γ, which plays an important role in the IFN-mediated antiviral signaling pathway. Here, we cloned and identified IFI35 in the chicken for the first time. Chicken IFI35 (chIFI35) contains an open reading frame (ORF) of 1,152 bp encoding a protein of 384 amino acids containing two conserved Nmi/IFI35 domain (NID) motifs. Tissue distribution analysis of chIFI35 in healthy and Newcastle disease (ND) virus-infected chickens indicated a positive correlation between chIFI35 mRNA transcription and ND viral loads in various tissues. The role of chIFI35 in regulation NDV replication were further assessed by up- or down-regulated chIFI35 expression in DF-1 cells transfected with plasmid harboring chIFI35, pCMV-3HA-chIFI35 or shRNA targeting chIFI35, pshRNA-chIFI35 plasmids. NDV replications in DF-1 cells were significantly reduced or slightly increased by over- or under-expression of the chIFI35 protein, respectively, indicating the role of chIFI35 in anti-NDV infection. Moreover, chIFI35 also involved in regulation of viral gene transcription and IFNs expression. The collected data were meaningful for research of chicken antiviral immunity and shed light on the pleiotropic antiviral effect of chIFI35 during NDV infection.

Common microRNA⁻mRNA Interactions in Different Newcastle Disease Virus-Infected Chicken Embryonic Visceral Tissues.

To investigate the roles and explore the altered expression of microRNAs (miRNAs) and mRNAs in chicken embryos in response to Newcastle disease virus (NDV) infection, deep sequencing was performed. Then, a conjoint analysis of small RNA-seq and mRNA-seq was performed to screen interactional miRNA⁻mRNA pairs during NDV infection. In total, 15 and 17 up- and downregulated miRNAs were identified that potentially targeted 4279 and 6080 mRNAs in NDV-infected chicken embryonic tissues, respectively; in addition, 595 upregulated and 480 downregulated mRNAs were identified. The conjoint analysis of the obtained data identified 1069 miRNA⁻mRNA pairs. Among these pairs, 130 pairs were related to immune or inflammatory responses. The relationship between gga-miR-203a and its target transglutaminase 2 (TGM2) was confirmed using a dual-luciferase reporter system and a real time quantitative polymerase chain reaction (RT-qPCR) assay. Overall, the discovery of miRNAs, mRNAs, and their potential pairing relationships, which may be involved in the regulation of NDV infection, will facilitate our understanding of the complex regulatory relationship between the host and the virus.

Pseudanoplocephala crawfordi is a member of genus Hymenolepis based on phylogenetic analysis using ribosomal and mitochondrial DNA sequences.

Pseudanoplocephala crawfordi is one of the important zoonotic cestodes causing economic significance and public health concern. In the present study, the phylogenetic position of P. crawfordi isolated from pigs was re-inferred using molecular markers of internal transcribed spacer ribosomal DNA (ITS rDNA) and partial NADH dehydrogenase subunit 1 (pnad1) mitochondrial DNA. The lengths of ITS1, ITS2 rDNA and pnad1 were 757 bp, 628 bp and 458 bp, respectively. Sequence differences in the ITS1, ITS2 rDNA and pnad1 between P. crawfordi and Hymenolepis species were smaller than that between cestodes within genus Hymenolepis. Phylogenetic analyses based on three gene fragments showed that P. crawfordi was grouped into cluster of Hymenolepis species. These results suggested that P. crawfordi would be one member of genus Hymenolepis but not in a new genus Pseudanoplocephala.

The complete mitochondrial genome of the gullet worm Gongylonema pulchrum: gene content, arrangement, composition and phylogenetic implications.

BACKGROUND: Gongylonema pulchrum (Nematoda: Gongylonematidae), a thread-like spirurid gullet worm, infects a range of mammalian definitive hosts, including cattle, pigs, equines, goats, primates and humans, and can cause gongylonemiasis. METHODS: In the present study, the complete mitochondrial (mt) genome of G. pulchrum was obtained using Long-range PCR and subsequent primer walking. The phylogenetic position of G. pulchrum within the Spiruromorpha was established using Bayesian analyses of the protein-coding genes at the amino acid level. RESULTS: The length of this AT-rich (75.94%) mt genome is 13,798 bp. It contains 12 protein-coding genes, two ribosomal RNA genes, 22 transfer RNA genes and one non-coding region. The gene arrangement is the same as those of Thelazia callipaeda (Thelaziidae) and Setaria digitata (Onchocercidae), but distinct from that of Heliconema longissimum (Physalopteridae). Phylogenetic analyses, based on the concatenated amino acid sequence data for all 12 protein-coding genes using Bayesian inference (BI) method, showed that G. pulchrum (Gongylonematidae) was more closely related to Spirocerca lupi (Spiruroidea) than other members of the infraorder Spiruromorpha. CONCLUSIONS: The present study represents the first mt genome sequence for the family Gongylonematidae, which provides the opportunity to develop novel genetic markers for studies of epidemiology, population genetics and systematics of this nematode of human and animal health significance.

Molecular identification of Pomacea canaliculata and P. insularum from rice paddy in different origins in China using mitochondrial adenosine triphosphate subunit 6 gene.

To identify Pomacea canaliculata and P. insularum using a molecular approach, the partial sequences of mitochondrial (mt) adenosine triphosphate subunit 6 (patp6) genes of two apple snails species from eight provinces of China were obtained. The intra-specific variations in mt patp6 within P. canaliculata were 0-4.5%, and no sequence difference in this region was observed within P. insularum. However, high inter-specific variations between P. canaliculata and P. insularum were detected, with sequence differences of 8.9-10.1%. Phylogenetic analyses based on sequences of mt patp6 revealed that P. canaliculata and P. insularum were grouped in different clades, but the genetic trees could not reveal geographically genetic relationships of P. canaliculata isolates from different origins. These findings will provide basic information for further study of molecular epidemiology and control of Pomacea invasion in China as well as in the world.

Characterization of the complete mitochondrial genomes of Nematodirus oiratianus and Nematodirus spathiger of small ruminants.

BACKGROUND: Nematodirus spp. are among the most common nematodes of ruminants worldwide. N. oiratianus and N. spathiger are distributed worldwide as highly prevalent gastrointestinal nematodes, which cause emerging health problems and economic losses. Accurate identification of Nematodirus species is essential to develop effective control strategies for Nematodirus infection in ruminants. Mitochondrial DNA (mtDNA) could provide powerful genetic markers for identifying these closely related species and resolving phylogenetic relationships at different taxonomic levels. METHODS: In the present study, the complete mitochondrial (mt) genomes of N. oiratianus and N. spathiger from small ruminants in China were obtained using Long-range PCR and sequencing. RESULTS: The complete mt genomes of N. oiratianus and N. spathiger were 13,765 bp and 13,519 bp in length, respectively. Both mt genomes were circular and consisted of 36 genes, including 12 genes encoding proteins, 2 genes encoding rRNA, and 22 genes encoding tRNA. Phylogenetic analyses based on the concatenated amino acid sequence data of all 12 protein-coding genes by Bayesian inference (BI), Maximum likelihood (ML) and Maximum parsimony (MP) showed that the two Nematodirus species (Molineidae) were closely related to Dictyocaulidae. CONCLUSIONS: The availability of the complete mtDNA sequences of N. oiratianus and N. spathiger not only provides new mtDNA sources for a better understanding of nematode mt genomics and phylogeny, but also provides novel and useful genetic markers for studying diagnosis, population genetics and molecular epidemiology of Nematodirus spp. in small ruminants.

The expression dynamics of IL-17 and Th17 response relative cytokines in the trachea and spleen of chickens after infection with Cryptosporidium baileyi.

BACKGROUND: Cryptosporidium baileyi is the dominant Cryptosporidium species in birds causing emerging health problems in the poultry industry, and is also a model to study the biology of Cryptosporidium spp.. IL-17 (also called IL-17A) is a hallmark pro-inflammatory cytokine of Th17 cells that plays an important role in several human autoimmune diseases and microbial infection disease of many animals, and it may play a role in Cryptosporidium infection. METHODS: The present study examined the mRNA level of IL-17 and Th17 response relative cytokines in the trachea and spleen of C. baileyi-infected chickens at different time points using real-time quantitative PCR (qPCR). RESULTS: All examined cytokines in the trachea were up-regulated in the infected chickens compared with the uninfected control during C. baileyi infection. Significant increased IL-17 mRNA level in the trachea was observed as early as 12 h post infection (pi), peaking at 24 h pi and 10 d pi, and declining thereafter. The transcription levels of IL-17 and Th17 response relative cytokines in spleen were also significantly increased at different time points during the infection. CONCLUSIONS: IL-17 was indicated to participate in the induction of inflammation during infection of some intracellular protozoan parasites. The results in the present study suggest that IL-17 may play a role in immunity against Cryptosporidium infection, and provide basic information for determining the role of Th17 cell in Cryptosporidium infection.

Genetic variability in the three mitochondrial genes among Oesophagostomum asperum isolates from different regions in Shaanxi and Hunan Provinces, China.

The present study examined sequence variations in three mitochondrial DNA (mtDNA) regions, namely, NADH dehydrogenase subunit 5 (nad5), adenosine triphosphate subunit 6 (atp6) and cytochrome c oxidase subunit 3 (cox3), among Oesophagostomum asperum isolates from different regions in Shaanxi and Hunan provinces, China. The lengths for partial sequences of nad5 (pnad5), atp6 (patp6) and cox3 (pcox3) were 427 bp, 381 bp and 337 bp, respectively. The intra-specific sequence variations among all O. asperum samples were 0-2.11%, 0-1.84% and 0-1.48% for pnad5, patp6 and pcox3, respectively, while the inter-specific sequence differences among Oesophagostomum species in pig and small ruminants were 18-21.3% for pnad5, 18.3-24.5% for patp6 and 10.6-13.7% for pcox3. A phylogenetic analysis based on combined sequences of three mtDNA fragments indicated that all O. asperum isolates were grouped in one solid clade, and the Oesophagostomum spp. from pig were located in another clade. However, these mtDNA fragments could not reveal genetic relationships of geographical isolates of O. asperum in China. These results provided valuable information for studying population genetics of Oesophagostomum spp., and for controlling Oesophagostomum infection in animals as well as humans.

Genetic variability of Baylisascaris schroederi from the Qinling subspecies of the giant panda in China revealed by sequences of three mitochondrial genes.

The present study examined the variations in three mitochondrial (mt) DNA sequences, namely cytochrome b (cytb), cytochrome c oxidase subunit 3 (cox3) and NADH dehydrogenase subunit 5 (nad5), among Baylisascaris schroederi isolates from the Qinling subspecies of the giant panda in Shaanxi province, northwestern China. No differences in length were detected in the three mt fragments from different isolates. The intra-specific sequence variations within all B. schroederi samples were 0-2.6% for pcytb, 0-1.8% for pcox3 and 0-2.1% for pnad5, while the inter-specific sequence differences among members of the genus Baylisascaris were 8.2-15.2%, 6.2-15.9% and 8.4-16.0% for pcytb, pcox3, pnad5, respectively. A phylogenetic analysis of the combined sequences of pcytb, pcox3 and pnad 5 showed that all B. schroederi samples in the present study were located in two large clusters, with one cluster containing samples from giant pandas in Sichuan province. These findings provide basic information for further study of molecular epidemiology and control of B. schroederi infection in the Qinling subspecies of the giant panda and throughout China.

The complete mitochondrial genomes of Oesophagostomum asperum and Oesophagostomum columbianum in small ruminants.

Oesophagostomum spp., commonly known as 'nodule worms', is one of the important emerging nematode zoonoses worldwide. In the present study, we sequenced the complete mitochondrial (mt) genomes of two small ruminant nodule worms, Oesophagostomum asperum and Oesophagostomum columbianum, and compared them with that of pig nodule worms (Oesophagostomum dentatum and Oesophagostomum quadrispinulatum). The complete mt genomes of O. asperum andO. columbianum were 13,672 and 13,561 bp, respectively. Both mt genomes were circular, and consisted of 36 genes, including 12 genes coding for proteins, 2 genes for rRNA, and 22 genes for tRNA. The gene content and arrangement are identical to that of pig nodule worms. The availability of full mtDNA sequences of O. asperum and O. columbianum provide useful information for studying population genetics of Oesophagostomum spp., molecular epidemiology and control of O. asperum and O. columbianum infection in small ruminants.

Characterization of Haemaphysalis flava (Acari: Ixodidae) from Qingling subspecies of giant panda (Ailuropoda melanoleuca qinlingensis) in Qinling Mountains (Central China) by morphology and molecular markers.

Tick is one of important ectoparasites capable of causing direct damage to their hosts and also acts as vectors of relevant infectious agents. In the present study, the taxa of 10 ticks, collected from Qinling giant pandas (Ailuropoda melanoleuca qinlingensis) in Qinling Mountains of China in April 2010, were determined using morphology and molecular markers (nucleotide ITS2 rDNA and mitochondrial 16S). Microscopic observation demonstrated that the morphological features of these ticks were similar to Haemaphysalis flava. Compared with other Haemaphysalis species, genetic variations between Haemaphysalis collected from A. m. qinlingensis and H. flava were the lowest in ITS2 rDNA and mitochondrial 16S, with sequence differences of 2.06%-2.40% and 1.30%-4.70%, respectively. Phylogenetic relationships showed that all the Haemaphysalis collected from A. m. qinlingensis were grouped with H. flava, further confirmed that the Haemaphysalis sp. is H. flava. This is the first report of ticks in giant panda by combining with morphology and molecular markers. This study also provided evidence that combining morphology and molecular tools provide a valuable and efficient tool for tick identification.

Genetic variability among Dicrocoelium dendriticum isolates from different regions in Shaanxi Province, China revealed by sequences of three mitochondrial genes.

The genetic variations in three mitochondrial DNA (mtDNA) regions, namely portion of cytochrome c oxidase subunit 1 (pcox1), NADH dehydrogenase subunit 1 (pnad1) and cytochrome b (pcytb), were examined for Dicrocoelium dendriticum samples isolated from different origins in Shaanxi Province, northwestern China. The intra-specific sequence differences within D. dendriticum samples were 0-0.52% for pcox1, 0-0.73% for pnad1 and 0-0.58% for pcytb. Phylogenetic analyses based on combined sequences of three mtDNA showed that all D. dendriticum samples were clustered together in same clade of Paragonimus westermani. But the phylogenetic trees could not reveal geographically genetic relationships of D. dendriticum isolates in this province. These findings will provide basic information for further study of molecular epidemiology and control of D. dendriticum infection in this province as well as in China.