C-type lectin 4 of Toxocara canis activates NF-ĸB and MAPK pathways by modulating NOD1/2 and RIP2 in murine macrophages in vitro.

Toxocara canis is a parasitic zoonose that is distributed worldwide and is one of the two pathogens causing toxocariasis. After infection, it causes serious public health and safety problems, which pose significant veterinary and medical challenges. To better understand the regulatory effects of T. canis infection on the host immune cells, murine macrophages (RAW264.7) were incubated with recombinant T. canis C-type lectin 4 (rTc-CTL-4) protein in vitro. The quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot were used to analyze the nucleotide-binding oligomerization domain-containing protein 1/2 (NOD1/2), receptor-interacting protein 2 (RIP2), nuclear factor kappa-light-chain enhancer of activated B cells (NF-κB), and mitogen-activated protein kinase (MAPK) on mRNA level and protein expression level in macrophages. Our results indicated that 10 µg/mL rTc-CTL-4 protein could modulate the expression of NOD1, NOD2, and RIP2 at both the transcriptional and translational levels. The protein translation levels of NF-κB, P-p65, p38, and P-p38 in macrophages were also modulated by rTc-CTL-4 protein. Macrophages were co-incubated with rTc-CTL-4 protein after siRNA silencing of NOD1, NOD2, and RIP2. The expression levels of NF-κB, P-p65, p38, and P-p38 were significantly changed compared with the negative control groups (Neg. Ctrl.). Taken together, rTc-CTL-4 protein seemed to act on NOD1/2-RIP2-NF-κB and MAPK signaling pathways in macrophages and might activate MAPK and NF-κB signaling pathways by regulating NOD1, NOD2, and RIP2. The insights from the above studies could contribute to our understanding of immune recognition and regulatory mechanisms of T. canis infection in the host animals.

Molecular characterization of clathrin heavy chain (Chc) in Rhipicephalus haemaphysaloides and its effect on vitellogenin (Vg) expression via the clathrin-mediated endocytic pathway.

Clathrin plays an important role in arthropods, but its function in ticks has not been explored. Here, we describe the molecular characteristics of the clathrin heavy chain of the tick Rhipicephalus haemaphysaloides and its effects on yolk development. The open reading frame of the clathrin heavy chain (Chc) (Rh-Chc) gene consists of 5103 nucleotides encoding 670 amino acids, which is most closely related to that of Ixodes scapularis and relatively close to Homo sapiens and Drosophila melanogaster. Real-time qPCR revealed that Rh-Chc was expressed at all developmental stages and organs. After Rh-Chc is silenced, ticks did not feed and mortality rate was 100%. Moreover, Rh-Chc co-localized with Vitellogenin receptor (VgR) on oocyte membrane. Immunofluorescence showed that the expression of Vitellogenin (Vg) (Rh-Vg) was also closely related to Rh-Chc. Immunofluorescence showed that the expression of Vg was also closely related to Rh-Chc, Rh-Chc silencing slowed the development of oocytes in tick, and culture of ovary in vitro silenced Rh-Chc, the development of oocytes in ticks also slowed down. Overall, the results of this study indicated that Rh-Chc is a vital gene in the tick R. haemaphysaloides that plays an important role in its growth, development, and reproduction.

Tissue expression pattern of ABCG transporter indicates functional roles in reproduction of Toxocara canis.

Toxocara canis is a zoonotic parasite with worldwide distribution. ATP-binding cassette (ABC) transporters are integral membrane proteins which involve in a range of biological processes in various organisms. In present study, the full-length coding sequence of abcg-5 gene of T. canis (Tc-abcg-5) was cloned and characterized. A 633 aa polypeptide containing two conserved Walker A and Walker B motifs was predicted from a continuous 1902 nt open reading frame. Quantitative real-time PCR was employed to determine the transcriptional levels of Tc-abcg-5 gene in adult male and female worms, which indicated high mRNA level of Tc-abcg-5 in the reproductive tract of adult female T. canis. Tc-abcg-5 was expressed to produce rabbit polyclonal antiserum against recombinant TcABCG5. Indirect-fluorescence immunohistochemical assays were carried out to detect the tissue distribution of TcABCG5, which showed predominant distribution of TcABCG5 in the uterus (especially in the germ cells) of adult female T. canis. Tissue transcription and expression pattern of Tc-abcg-5 indicated that Tc-abcg-5 might play essential roles in the reproduction of this parasitic nematode.

Changes of cecal microflora in chickens following Eimeria tenella challenge and regulating effect of coated sodium butyrate.

Eimeria tenella, one of the most important parasitic protozoa in the genus Eimeria, is responsible for chicken caecal coccidiosis resulting in huge economic losses to poultry industry. The present study investigated the changes in caecal microflora of E. tenella-infected chickens and the regulating effect of coated sodium butyrate, a potential alternative to antibiotics. Using high-throughput sequencing of 16S rRNA V3-V4 region of bacteria we found significant changes in caecal microflora of E. tenella-infected chickens indicated by an increase of Firmicutes (mainly Ruminococcaceae, Lachnospiraceae and vadin BB60) and Proteobacteria (mainly Enterobacteriaceae) and a decrease of Bacteroidetes (predominantly Bacteroidaceae). Inclusion of coated sodium butyrate in the diet of chickens per se had no significant effect on caecal microflora of normal healthy chickens but significantly prevented the increase in Firmicute abundance and decrease of Bacteroidetes abundance in E. tenella-infected birds. No significant changes to caecal microflora were observed at the phylum level between control and E. tenella-infected birds given coated sodium butyrate. In conclusion, our results show that coated sodium butyrate can balance the disorders of cecal microflora caused by E. tenella; thus, it can be a useful supplement for the control of avian coccidiosis.

Tissue distribution and functional analysis of vitellogenin-6 of Toxocara canis.

Toxocara canis is an common intestinal nematode of canids and the principal causative agent of human toxocariasis. Vitellogenin (Vg), a source of amino acids and lipids in the eggs, are considered to play an important role in embryo development of a wide range of organisms. In the present study, the transcriptional levels of Tc-vit-6 gene in male and female adult T. canis were determined by quantitative real-time PCR, which indicated high transcription of Tc-vit-6 in the intestine, reproductive tract and body wall of male and female adult T. canis. The fragment of Tc-vit-6 encoding a vWD domain, was cloned and expressed to produce a rabbit anti-TcvWD polyclonal antibody. Tissue distribution of TcVg6 was detected by immunohistochemical assays, which showed predominant distribution of TcVg6 in the tissues of intestine, as well as reproductive tract (including some of the germ cells) and musculature of male and female adult worms. Collectively, these results indicated multiple biological roles of TcVg6 apart from that in the reproduction of T. canis.

Characterization and differential expression analysis of Toxocara canis aquaporin-1 gene.

Toxocara canis is an intestinal nematode of canids with a worldwide distribution, causing an important but neglected parasitic zoonosis in humans. Aquaporins (AQP) are a family of water channel proteins, which function as membrane channels to regulate water homeostasis. In this study, the coding sequence of aquaporin-1 gene of T. canis (Tc-aqp-1) was cloned and characterized. The obtained Tc-aqp-1 coding sequence was 933 bp in length, which predicted to encode 311 amino acids. Two conserved asparagine-proline-alanine (NPA) motifs were identified in the multiple sequence alignments. Phylogenetic analysis revealed the closest relationship between T. canis and Opisthorchis viverrini based on aquaporin-1 amino acid sequence. A structure was predicted with ligand binding sites predicted at H93, N95, N226, L94, I79, and I210 and with active sites predicted at I256 and G207. Gene Ontology (GO) annotations predicted its cellular component term of integral component of plasma membrane (GO: 0005887), molecular function term of channel activity (GO: 0015250), and biological process term of water transport (GO: 0006833). Tissue expression analysis revealed that the Tc-aqp-1 was highly expressed in the intestine of adult male. The findings of the present study provide the basis for further functional studies of T. canis aquaporin-1.

The non-glycosylated protein of Toxocara canis MUC-1 interacts with proteins of murine macrophages.

Toxocariasis is a neglected parasitic disease caused predominantly by larvae of Toxocara canis. While this zoonotic disease is of major importance in humans and canids, it can also affect a range of other mammalian hosts. It is known that mucins secreted by larvae play key roles in immune recognition and evasion, but very little is understood about the molecular interactions between host cells and T. canis. Here, using an integrative approach (affinity pull-down, mass spectrometry, co-immunoprecipitation and bioinformatics), we identified 219 proteins expressed by a murine macrophage cell line (RAW264.7) that interact with prokaryotically-expressed recombinant protein (rTc-MUC-1) representing the mucin Tc-MUC-1 present in the surface coat of infective larvae of T. canis. Protein-protein interactions between rTc-MUC-1 and an actin binding protein CFL1 as well as the fatty acid binding protein FABP5 of RAW264.7 macrophages were also demonstrated in a human embryonic kidney cell line (HEK 293T). By combing predicted structural information on the protein-protein interaction and functional knowledge of the related protein association networks, we inferred roles for Tc-MUC-1 protein in the regulation of actin cytoskeletal remodelling, and the migration and phagosome formation of macrophage cells. These molecular interactions now require verification in vivo. The experimental approach taken here should be readily applicable to comparative studies of other ascaridoid nematodes (e.g. T. cati, Anisakis simplex, Ascaris suum and Baylisascaris procyonis) whose larvae undergo tissue migration in accidental hosts, including humans.

Construction of a cDNA library from female adult of Toxocara canis, and analysis of EST and immune-related genes expressions.

Toxocara canis is a widespread intestinal nematode parasite of dogs, which can also cause disease in humans. We employed an expressed sequence tag (EST) strategy in order to study gene-expression including development, digestion and reproduction of T. canis. ESTs provided a rapid way to identify genes, particularly in organisms for which we have very little molecular information. In this study, a cDNA library was constructed from a female adult of T. canis and 215 high-quality ESTs from 5'-ends of the cDNA clones representing 79 unigenes were obtained. The titer of the primary cDNA library was 1.83×10(6)pfu/mL with a recombination rate of 99.33%. Most of the sequences ranged from 300 to 900bp with an average length of 656bp. Cluster analysis of these ESTs allowed identification of 79 unique sequences containing 28 contigs and 51 singletons. BLASTX searches revealed that 18 unigenes (22.78% of the total) or 70 ESTs (32.56% of the total) were novel genes that had no significant matches to any protein sequences in the public databases. The rest of the 61 unigenes (77.22% of the total) or 145 ESTs (67.44% of the total) were closely matched to the known genes or sequences deposited in the public databases. These genes were classified into seven groups based on their known or putative biological functions. We also confirmed the gene expression patterns of several immune-related genes using RT-PCR examination. This work will provide a valuable resource for the further investigations in the stage-, sex- and tissue-specific gene transcription or expression.

Activation of Interleukin-1ß Release and Pyroptosis by Transmissible Gastroenteritis Virus Is Dependent on the NOD-Like Receptor Protein 3 Inflammasome in Porcine Intestinal Epithelial Cell Line.

Transmissible gastroenteritis virus (TGEV) is a coronavirus, which causes fatal severe diarrhea and leads to high mortality in newborn piglets. Inflammasomes are hub molecules that induce proinflammatory cytokine production and maturation to initiate innate immune defenses upon cellular infection. To date, the potential role of inflammasome in TGEV infection in porcine intestinal epithelial cells has not been elucidated. The present study aims to investigate the function of the inflammasome in response to TGEV infection in porcine intestinal epithelial cells. Our results revealed that TGEV infection induced the production of pro-interleukin-1ß (pro-IL-1ß) and enhanced its processing and maturation in porcine intestinal epithelial cells through caspase-1 activation. In addition, TGEV infection in porcine intestinal epithelial cells induced pyroptosis, indicated by cell death and the production and cleavage of gasdermin D (GSDMD). Meanwhile, TGEV infection sufficiently activated the expression and assembly of the NOD-like receptor protein 3 (NLRP3) inflammasome in porcine intestinal epithelial cells, and inhibition of NLRP3 blocked TGEV-induced IL-1ß release. We also found that inhibition of NLRP3 enhanced the replication of TGEV without inducing cell death. In conclusion, these data demonstrated that activation of IL-1ß release and pyroptosis is dependent on NLRP3 inflammasome, thus NLRP3 inflammasome may play a central role in the innate immune response to TGEV infection.

Aquaporin 1 is located on the intestinal basolateral membrane in Toxocara canis and might play a role in drug uptake.

BACKGROUND: Aquaporins (AQPs) are a family of integral membrane channel proteins that facilitate the transport of water and other small solutes across cell membranes. AQPs appear to play crucial roles in parasite survival and represent possible drug targets for novel intervention strategy. In this work, we investigated the tissue distribution and biological roles of an aquaporin TcAQP1 in the neglected parasitic nematode Toxocara canis. METHODS: Recombinant C-terminal hydrophilic domain of AQP1 of T. canis (rTcAQP1c) and polyclonal antibody against rTcAQP1c were produced to analyse the tissue expression of native TcAQP1 in adult (female and male) worms using an immunohistochemical approach. RNA interference (RNAi), quantitative real-time PCR (qRT-PCR) and nematocidal assays were performed to investigate the functional roles of TcAQP1 in the adult stage of T. canis. RESULTS: Immunofluorescence analysis showed that TcAQP1 was localised predominantly in the epithelial linings of the reproductive tract and basolateral membrane of the intestine in the adult stage (female and male) of T. canis, indicating important roles in reproduction, nutrient absorption and/or osmoregulation. Treatment with silencing RNA for 24 h resulted in a significant reduction of Tc-aqp-1 mRNA level in adult T. canis, though no phenotypical change was observed. The efficient gene knockdown compromised the nematocidal activity of albendazole in vitro, suggesting the role of TcAQP1 in drug uptake. CONCLUSIONS: The findings of this study provide important information about tissue expression and functional roles of TcAQP1 protein in adult T. canis. Understanding the biological functions of this protein in other developmental stages of T. canis and related parasitic nematodes would contribute to the discovery of novel diagnostic or anthelmintic targets.

Molecular and serological prevalence of Toxoplasma gondii and Anaplasma spp. infection in goats from Chongqing Municipality, China.

Toxoplasmosis and anaplasmosis are severe zoonotic diseases, the former caused by Toxoplasma gondii and the latter by Anaplasma spp. In the present study, 332 goat blood samples were randomly collected from Chongqing Municipality, China to screen for T. gondii and Anaplasma spp. We used a polymerase chain reaction (PCR) to detect DNA, and enzyme-linked immunosorbent assay (ELISA) to test for T. gondii antibodies. The prevalence of T. gondii and Anaplasma spp. was 38% and 35% respectively by PCR, and 42% for T. gondii antibodies by ELISA. The co-infection rate by T. gondii and Anaplasma was 13%, where the two predominant pathogens co-infecting were Anaplasma phagocytophilum + A. bovis (10%), followed by T. gondii + A. phagocytophilum (9.64%). While co-infection by three pathogens varied ranging from 1.81% to 5.72%, less than 1% of goats were found to be positive for four pathogens. This is the first investigation of T. gondii and Anaplasma spp. infection in goats from Chongqing.

Comparative transcriptomic analyses of male and female adult Toxocara canis.

Toxocariasis is an important, neglected zoonosis caused mainly by Toxocara canis. Although our knowledge of helminth molecular biology is improving through completed draft genome projects, there is limited detailed information on the molecular biology of Toxocara species. Here, transcriptomic sequencing of male and female adult T. canis and comparative analyses were conducted. For each sex, two-thirds (66-67%) of quality-filtered reads mapped to the gene set of T. canis, and at least five reads mapped to each of 16,196 (87.1%) of all 18,596 genes, and 321 genes were specifically transcribed in female and 1467 in male T. canis. Genes differentially transcribed between the two sexes were identified, enriched biological processes and pathways linked to these genes established, and molecules associated with reproduction and development predicted. In addition, small RNA pathways involved in reproduction were characterized, but there was no evidence for piwi RNA pathways in adult T. canis. The results of this transcriptomic study should provide a useful basis to support investigations of the reproductive biology of T. canis and related nematodes.2.

Genome Sequence of Corynebacterium pseudotuberculosis Strain XH02 Isolated from a Boer Goat in Xuanhan, China.

We report here the genome sequence of Corynebacterium pseudotuberculosis strain XH02, isolated from a Boer goat in China. The genome consists of 2,357,671 bp, with a 52.18% G+C content, 2,263 coding sequences, 21 rRNAs, 49 tRNAs, and 44 predicted pseudogenes.

MicroRNAs of Toxocara canis and their predicted functional roles.

BACKGROUND: Toxocara canis is the causative agent of toxocariasis of humans and other animals. This parasitic nematode (roundworm) has a complex life cycle, in which substantial developmental changes and switches occur. As small non-coding RNAs (sRNAs) are key regulators of gene expression in a wide range of organisms, we explored these RNAs in T. canis to provide a basis for future studies of its developmental biology as well as host interactions and disease at the molecular level. METHODS: We conducted high-throughput RNA sequencing and bioinformatic analyses to define sRNAs in individual male and female adults of T. canis. RESULTS: Apart from snRNA and snoRNA, 560 and 619 microRNAs (miRNAs), including 5 and 2 novel miRNAs, were identified in male and female worms, respectively, without piRNAs being detected in either sex. An analysis of transcriptional profiles showed that, of 564 miRNAs predicted as being differentially transcribed between male and female individuals of T. canis, 218 miRNAs were transcribed exclusively in male and 277 in female worms. Functional enrichment analysis predicted that both male and female miRNAs were mainly involved in regulating embryonic morphogenesis, hemidesmosome assembly and genetic information processing. The miRNAs differentially transcribed between the sexes were predicted to be associated with sex determination, embryonic morphogenesis and nematode larval development. The roles of miRNAs were predicted based on gene ontology (GO) and KEGG pathway annotations. The miRNAs Tc-miR-2305 and Tc-miR-6090 are proposed to have roles in reproduction, embryo development and larval development, and Tc-let-7-5p, Tc-miR-34 and Tc-miR-100 appear to be involved in host-parasite interactions. Together with published information from previous studies, some miRNAs (such as Tc-miR-2861, Tc-miR-2881 and Tc-miR-5126) are predicted to represent drug targets and/or associated with drug resistance. CONCLUSIONS: This is the first exploration of miRNAs in T. canis, which could provide a basis for fundamental investigations of the developmental biology of the parasite, parasite-host interactions and toxocariasis as well as applied areas, such as the diagnosis of infection/disease, drug target discovery and drug resistance detection.

Short communication: Experimental toxocarosis in Chinese Kun Ming mice: Dose-dependent larval distribution and modulation of immune responses.

Toxocarosis is an important parasitic zoonosis which is mainly caused by the infective larvae of Toxocara canis. To identify whether there are correlations among the infectious dose, the larval migrans and immune modulation in inbred Chinese Kun Ming (KM) mice, experimental infections were carried out with a range of dosages of 100, 500, 1000, 2000, and 3000 embryonated eggs (EE). Pathogenic reactions were observed in terms of physical and central nervous symptoms. Distributions of T. canis larvae in liver, lung, kidney, heart and brain organs were respectively detected by scanning tissue sections. Moreover, quantitative real-time PCR was employed to identify the variations of Th2 immune response. The results showed that high inocula resulted in advanced larval emergences and arrested migrations in liver, lung, kidney and brain. However, no larvae were found in any of the histological sections of heart tissues. Higher levels of interleukin (IL)-4, IL-5, and IL-10 were detected along with the increasing inoculation doses, but the heaviest inoculum (3000 EE in this study) resulted in the sharp reduction of these ILs. Although no neurological symptoms or mortalities were noticed, these results indicated dose-dependent distribution patterns and immune regulations of T. canis larvae infection in KM mice.

Phylogenetic analysis of the genus Anaplasma in Southwestern China based on 16S rRNA sequence.

To identify the species within the genus Anaplasma circulating among ruminants in the Southwest of China, we performed the phylogenetic analysis of the 16S rRNA gene of two Anaplasma isolates from cattle and seven from goats. The two sequences obtained from cattle strains belonged to the A. marginale cluster, whereas the other seven sequences from caprine strains formed two Anaplasma spp. clusters, which diverged earlier than the clusters of A. marginale, A. centrale and A. ovis. These results indicate that there are at least two Anaplasma species circulating among ruminants in Southwestern China.

Phylogenetic analysis of Mycoplasma suis isolates based on 16S rRNA gene sequence in China.

To perform phylogenetic analysis of Mycoplasma suis isolates derived from China to define the nature of this pathogen, nearly complete of 16S rRNA genes from Chongqing, Sichuan, Henan and Guangdong isolates were amplified by PCR and sequenced. The four sequences from the blood samples in this study, with other 17 Hemoplasmas sequences and related 3 mycoplasma sequences available in the GenBank, were aligned using Clustal X (version 1.83) sequences alignment program. Maximum parsimony, neighbor-joining and minimum evolution (MEGA 4.0) algorithms were used to create phylogenetic trees. Phylogenetic analysis of these sequences showed that all hemoplasma species were located within a single clade and were most closely related to M. pneumoniae group. The hemoplasma species were further subdivided into two distinct groups, one containing M.wenyonii, M.suis and Candidatus M. haemominutum and the other containing M. haemofelis and M. haemocanis. Within the former clade, four M.suis isolates from Mainland China and other M.suis species formed a monophyletic group in the tree. A tendency of clear geographical grouping of the isolate was evident.

PCR amplification and sequence analyses of ITS-1 rDNA from Cryptosporidium andersoni in dairy cattle.

The internal transcribed spacer 1 (ITS-1) of the ribosomal DNA (rDNA) from GD, HN, and AH strains of Cryptosporidium andersoni was amplified and sequenced to assess whether the ITS-1 rDNA could be used as genetic markers of C. andersoni from different geographic origins in China and to differentiate C. andersoni from other Cryptosporidium species. The result showed that the ITS-1 sequences of GD, HN, and AH strains were basically identical, which were unequivocally different with the sequences of the Cryptosporidium muris and Cryptosporidium parvum registered in the GenBank; however, the ITS-1 sequence of the AH strain differed at three bases compared with that of the other two strains. Our study indicates that the ITS-1 sequences provide useful genetic markers for the identification and differentiation of C. andersoni species and also lay down the foundation for diagnostics of cryptosporidiosis.

Molecular characterization of Cyclospora-like organism from dairy cattle.

Cyclospora cayetanensis was identified as the cause of large outbreaks of diarrhea in many parts of the world, but its host range and reservoirs remains poorly defined. Recently, oocysts resembling the C. cayetanensis were detected in dairy cattle fecal specimens from China. The 18S rDNA from two of these Cyclospora-like oocyst specimens from dairy cattle was amplified and sequenced. Phylogenetic analysis indicated that these cattle-associated Cyclospora-like organisms are nearly identical to each other and belong to the group of primate-derived Cyclospora, which are the closest known relatives of C. cayetanensis; while these cyclosporans constitute a coherent clade within the diverse group of Eimeria species. Moreover, on the basis of our finding that ruminant- and avian-associated Eimeria species are different in MnlI sites, a new PCR-restriction fragment length polymorphism protocol with primers NesCycF and NesCycR was developed to distinguish the Cyclospora species from ruminant-associated Eimeria species.