An-Xing Li

Clonorchiasis: a key foodborne zoonosis in China

The oriental liverfluke, Clonorchis sinensis, is of major socioeconomic importance in parts of Asia, including China, Japan, Korea, Taiwan, and Vietnam. The parasite is transmitted via snails to freshwater fish, and then to human beings and other piscivorous mammals, and causes substantial clinical or subclinical disease, known as clonorchiasis. There is considerable evidence for an aetiological relation between clonorchiasis and cholangiocarcinoma in human beings. It is estimated that about 35 million people are infected globally, of whom approximately 15 million are in China. Although very little information from China has been published in the English language, recent analyses of epidemiological data sets suggest that clonorchiasis is having an increased human-health impact due to the greater consumption of raw freshwater fish. To gain an improved insight into clonorchiasis in China, this review provides a background on the parasite and its life cycle, summarises key aspects regarding the pathogenesis, diagnosis, and treatment of clonorchiasis, describes the geographic distribution and prevalence of clonorchiasis, and makes some recommendations for future research and the control of this important disease.

Streptococcus suis: an emerging zoonotic pathogen

Streptococcus suis is a major porcine pathogen worldwide, and can be transmitted to human beings by close contact with sick or carrier pigs. S suis causes meningitis, septicaemia, endocarditis, arthritis, and septic shock in both pigs and human beings, and mortality is high. Human infection with S suis occurs mainly among certain risk groups that have frequent exposure to pigs or pork. Outbreaks of human S suis infection are uncommon, although several outbreaks have occurred in China in recent years. In July, 2005, the largest outbreak of human S suis infection occurred in Sichuan province, China, where 204 people were infected and 38 of them died. There have been 409 cases of human S suis infection worldwide, most of which have occurred in China, Thailand, and the Netherlands, and these infections have led to 73 deaths. This review provides background information on the biology and molecular characteristics of this Gram-positive bacterium, and describes the clinical signs, pathology, epidemiology, diagnosis, and treatment of human infection with S suis.

Immune responses and immune-related gene expression profile in orange-spotted grouper after immunization with Cryptocaryon irritans vaccine

In order to elucidate the immune-protective mechanisms of inactivated Cryptocaryon irritans vaccine, different doses of C. irritans theronts were used to immunize orange-spotted grouper (Epinephelus coioides). We measured serum immobilization titer, blood leukocyte respiratory burst activity, serum alternative complement activity, and serum lysozyme activity weekly. In addition, the expression levels of immune-related genes such as interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), major histocompatibility complexes I and II (MHC I and II), and transforming growth factor-β1 (TGF-β1) were determined in spleen and gills. The results showed that the immobilization titer, respiratory burst activity, and alternative complement activity of immunized fish were significantly increased, and the levels of the last two immune parameters in the high-dose vaccine group were significantly higher than in the low-dose vaccine group. Serum lysozyme activity in the high-dose vaccine group was significantly higher than in the PBS control group. Vaccination also regulated host immune-related gene expression. For example, at 2- and 3- weeks post immunization, IL-1β expression in the high-dose vaccine group spleen was significantly increased. At 4-weeks post immunization, the fish were challenged with a lethal dose of parasite, and the survival rates of high-dose vaccine group, low-dose vaccine group, PBS control group, and adjuvant control group were 80%, 40%, 0%, and 10% respectively. These results demonstrate that inactivated C. irritans vaccination improves specific and nonspecific immune responses in fish, enhancing their anti-parasite ability. These effects are vaccine antigen dose-dependent.

Genetic evidence for the existence of sibling species within Contracaecum rudolphii (Hartwich, 1964) and the validity of Contracaecum septentrionale (Kreis, 1955) (Nematoda: Anisakidae)

Specimens of Contracaecum rudolphii sensu lato (s.l.) (Nematoda: Anisakidae) from Phalacrocorax carbo sinensis from northeastern and central Italy were characterised genetically and compared with those from Phalacrocorax aristotelis from Galician coasts, Spain (identified as C. rudolphii A by multilocus enzyme electrophoresis) and with specimens of C. septentrionale from Alca torda from the Galician coasts, Spain. The first (ITS-1) and second (ITS-2) internal transcribed spacers (ITS) of ribosomal DNA (rDNA) were amplified by polymerase chain reaction (PCR) from individual nematodes and the amplicons subjected to single-strand conformation polymorphism (SSCP) analysis and/or sequencing. For each ITS region, C. septentrionale specimens were distinct from those of C. rudolphii (s.l.) and C. rudolphii A based on SSCP profiles and ITS sequences. Some specimens of C. rudolphii (s.l.) had the same SSCP profiles and ITS sequences as C. rudolphii A, whereas the others had distinct SSCP profiles and ITS sequences and were suggested to represent C. rudolphii B based on host and geographical origins and genetic similarity to C. rudolphii A. While no length or nucleotide variation in the ITS-1 and ITS-2 sequences was detected within each taxon, nucleotide differences of 1.8–5.5% (ITS-1) and 5.1–12.2% (ITS-2) were detected among them. The results support the hypothesis that C. rudolphii represents a complex of at least two sibling species and provide support for the validity of C. septentrionale as a separate species. The definition of genetic markers in the ITS rDNA provides opportunities for investigating the life cycles, transmission patterns and ecology of the anisakid nematodes studied herein.

Expression profiles of toll-like receptors in channel catfish (Ictalurus punctatus) after infection with Ichthyophthirius multifiliis

Toll-like receptors (TLRs) play a crucial role in the innate immune system, but to date the roles of fish TLRs in response to parasitic infection are still poorly understood. In the present study, we used channel catfish (Ictalurus punctatus) and the ciliate parasite Ichthyophthirius multifiliis as a model to investigate whether and which fish TLRs play important roles in the immune response against parasitic pathogens by detecting the expression profiles of a complete set of TLRs in catfish at different time points after infection with I. multifiliis. The expression profiles of TLR1 and TLR2 were similar, and both were significantly up-regulated in the skin and head kidney at most time points after infection. Furthermore, the expression of TLR2 was also up-regulated in the gill and spleen. TLR9 was induced in the skin and gill, whereas TLR21 was induced in the head kidney and spleen after infection. For TLR19, significant up-regulation was observed in the skin and gill, but significant down-regulation was detected in the head kidney and spleen. In contrast to TLR19, TLR25 was significantly up-regulated in the head kidney and spleen at some time points. No significant changes were observed for the rest of the TLRs at most time points. The results indicated that some TLRs may play essential roles in catfish defense against I. multifiliis infection.

The radiation of Haliotrema (Monogenea: Dactylogyridae: Ancyrocephalinae): molecular evidence and explanation inferred from LSU rDNA sequences

The D1-D2 domains of LSU rDNA were used to reconstruct the phylogenetic relationships within the Ancyrocephalinae (Monogenea: Dactylogyridae) utilizing maximum-parsimony (MP), maximum-likelihood (ML), minimum evolution (ME) and neighbour-joining (NJ) methods. A total of 32 monogenean taxa were examined in the present study, including 9 Haliotrema species and 13 other species representing the Ancyrocephalinae, 4 Thaparocleidus species representing the Ancylodiscoididae, and 6 species representing the Diplectanidae which were used as multiple outgroups. All 4 analyses (i.e. MP, ML, ME and NJ) inferred the same interrelationship pattern: (Diplectanidae, (Ancylodiscoididae, Dactylogyridae)) with high bootstrap support. However, 9 Haliotrema species were dispersed to form 4 clades together with species from other genera, indicating the apparent non-monophyly of Haliotrema. Three major groups were defined based on reconstructed phylogenetic trees to explain the radiation of Haliotrema species. The morphology of the reproductive organ, particularly the male copulatory organ (MCO), was discussed to further understand the formation of each group. (1) Results of the present study indicated an intimate relationship among Metahaliotrema (2 species), Protogyrodactylus (4 species) and Haliotrema (2 of 9 species), and notably, all these species share vagina-absence. (2) Based on the present molecular analyses and the morphological characters of the MCO, we propose to transfer H. spirotubiforum and the undetermined Haliotrema sp. ZHDDb to Euryhaliotrema as new combinations. (3) We propose to erect a new genus to accommodate the Haliotrema species with horn-like shaped MCO. Taxonomic implications of the present molecular phylogenetic analyses are discussed. A wider range of taxa and more DNA markers displaying various evolutionary rates should be used to estimate phylogenetic relationships among species within the Ancyrocephalinae and Ancylodiscoididae in further studies.

Molecular characterization and functional analysis of TRAF6 in orange-spotted grouper (Epinephelus coioides)

Tumor necrosis factor receptor (TNFR)-associated factor 6 (TRAF6) is a crucial signal transducer in both the TNFR superfamily and Toll-like receptor/interleukin 1R family. Although significant progress has been made in clarifying the role of TRAF6 in mammals, the function of TRAF6 in fish is still poorly understood. In this study, we cloned the orange-spotted grouper (Epinephelus coioides) TRAF6 (EcTRAF6) cDNA, with an open reading frame of 1713bp encoding 570 amino acids. Sequence analysis indicated that EcTRAF6 contains the four characteristic domains conserved in the TRAF family, including an N-terminal RING finger, two zinc fingers, a coiled-coil domain, and a C-terminal MATH domain. Homology alignment and phylogenetic analysis demonstrated that EcTRAF6 shares high sequence identity with TRAF6 of other fish species. The EcTRAF6 gene contains seven exons and six introns, which is similar to the organization in ayu, but not in the common carp, human, or mouse (six exons and five introns). EcTRAF6 transcripts were broadly expressed in all tissues tested, and increased after infection with Cryptocaryon irritans. Intracellular localization showed EcTRAF6 was distributed mainly in the cytoplasm. Over-expression of wild type (WT) EcTRAF6, truncated forms of EcTRAF6, including ΔZinc finger 2 and ΔMATH, and a mutant of C78A activated NF-κB strongly in HEK293T cells; whereas truncations, including ΔRING, ΔZinc finger 1 and Δcoiled-coiled, and a mutant of K132R induced the activity of NF-κB slightly compared to WT EcTRAF6, implying the latter has a more crucial role in downstream signal transduction. Together, these results suggested EcTRAF6 functions like that of mammals to activate NF-κB, and it might have an important role in host defense against parasitic infections.

Molecular and morphological evidence indicates that Pseudorhabdosynochus lantauensis (Monogenea: Diplectanidae) represents two species

Sequences of the first internal transcribed spacer (ITS-1) and the D1-D3 domains of the large subunit (LSU) of the ribosomal DNA (rDNA) were determined for multiple specimens of 4 operational taxonomic units (OTUs) of the monogenean, Pseudorhabdosynochus lantauensis. OTUs were defined based on their collecting localities, host and/or morphological characteristics. All P. lantauensis specimens of one group (OTUs 1 and 3) differed in their sequences of the ITS-1 and partial LSU rDNA when compared with specimens of a second group (OTUs 2 and 4) by 12% and 2%, respectively. Results of the phylogenetic analyses of the LSU rDNA sequence data showed total (100%) bootstrap support for the separation of P. lantauensis into 2 distinct clades. At least 11 of the 18 nucleotide differences in the LSU sequence between the two P. lantauensis clades were derived (i.e. autapomorphic) characters when the morphologically distinct species, P. epinepheli and P. coioidesis, were used as outgroups. Furthermore, there were several autapomorphic character states for each P. lantauensis clade. This provides sufficient evidence to reject the null hypothesis that P. lantauensis represents a single species. Morphological and morphometric differences between these two clades provided additional strong support for the separation of P. lantauensis into two species. These two parasite species were found to co-exist on one of the two species of serranid fish (i.e. Epinephelus coioides) examined in the South China Sea (Guangdong Province, China).

Grass carp (Ctenopharyngodon idella) TRAF6 and TAK1: molecular cloning and expression analysis after Ichthyophthirius multifiliis infection.

Ichthyophthirius multifiliis, a pathogenic ciliate parasite, infects almost all freshwater fish species and causes significant economic losses. Tumor necrosis factor receptor-associated factor 6 (TRAF6) and transforming growth factor-β-activated kinase 1 (TAK1) are two important signaling molecules involved in toll-like receptor (TLR) signal transduction. To date, the roles of TRAF6 and TAK1 in host defense against fish parasites are still poorly understood. In the present study, TRAF6 (CiTRAF6) and TAK1 (CiTAK1) were identified from grass carp (Ctenopharyngodon idella). The full-length cDNA sequence of CiTRAF6 (2250 bp) includes an open reading frame (ORF) of 1629 bp, which shows a high similarity to that of Cyprinus carpio TRAF6 and encodes a putative protein of 542 amino acids containing one RING domain, two zinc fingers, one coiled-coil region, and one MATH domain. The full-length CiTAK1 cDNA sequence is 2768 bp and includes an ORF of 1626 bp that encodes a putative protein of 541 amino acids containing a conserved serine/threonine protein kinase catalytic domain and a coiled-coil region. Phylogenetic analysis showed that CiTRAF6 and CiTAK1 were clustered with TRAF6 and TAK1 of other teleosts, respectively. CiTRAF6 and CiTAK1 were both constitutively expressed in all examined tissues but with varied expression levels. The highest expressions of CiTRAF6 and CiTAK1 were in the head kidney and spleen, respectively. The expression profiles of CiTRAF6 and CiTAK1 were detected in grass carp after I. multifiliis infection. Expressions of both genes were significantly up-regulated in the skin, gill, head kidney, and spleen at most time points after infection, indicating that CiTRAF6 and CiTAK1 may play essential roles in grass carp defense against I. multifiliis.

Identification of IRAK-4 in grouper (Epinephelus coioides) that impairs MyD88-dependent NF-κB activation

Interleukin 1 (IL-1) receptor-associated kinase (IRAK) family members are crucial signal transducer in the Toll-like receptor/IL-1R signal pathway, which mediates downstream signal cascades involved in the innate and adaptive immune responses. In this study, we identified an IRAK-4 protein (EcIRAK-4) in the orange-spotted grouper (Epinephelus coioides), with an N-terminal death domain, a proST domain, and a central kinase domain, similar to that of other fishes and mammals. A sequence alignment and phylogenic analysis demonstrated that full-length EcIRAK-4 shares a high degree of sequence identity with those of other fishes, especially the roughskin sculpin, and their death domains and kinase domains share greater identity than their proST domains. A conservation analysis indicated that most of the functional sites in mammalian IRAK-4 are conserved in IRAK-4 of the grouper and other fishes, with the exception of the sites of interaction with IRAK-2 and one autophosphorylation site within the activation loop. EcIRAK-4 is broadly expressed in all the tissues examined, with highest expression in the head kidney and liver. After infection with Cryptocaryon irritans, EcIRAK-4 expression was significantly upregulated, especially in the skin, which suggests that this molecule is involved in the hosts defense against parasitic infection. Surprisingly, after cotransfection with grouper MyD88, EcIRAK-4 significantly impaired the NF-κB activity induced by MyD88. EcIRAK-4 was uniformly distributed throughout the cytoplasm in HeLa cells. These findings suggest that although IRAK-4 is evolutionarily conserved between fish and mammals, its signal transduction function is markedly different.