Monoclonal antibodies against extra small virus show that it co-localizes with Macrobrachium rosenbergii nodavirus

Piscirickettsiosis or salmonid rickettsial septicaemia (SRS) caused by Piscirickettsia salmonis constitutes one of the main problems in farmed salmonid and marine fishes. Since the first reports of the disease, it has been successfully isolated and maintained in eukaryotic cell--culture systems, but these systems are time-consuming, the media are costly, and eliminating heavily contaminated host cell debris is difficult. In this report, we describe a marine-based broth supplemented with L-cysteine, named AUSTRAL-SRS broth, that facilitates superior growth of P. salmonis strains. Strains reached an optical density of approximately 1.8 when absorbance was measured at 600 nm after 6 d incubation at 18°C. Several passages (n = 6) did not alter the culture kinetics. We report for the first time the purification of DNA, lipopolysaccharide (LPS) and whole membrane protein obtained from P. salmonis grown in this liquid medium, and thus provide a suitable platform to simplify the preparation of P. salmonis cells for genetic and serological studies. Moreover, the results of the cytopathic effect test showed that P. salmonis grown in AUSTRAL-SRS broth maintained their virulence properties, inducing apoptosis after 3 d. This makes the medium a good candidate for the successful growth of P. salmonis and an excellent basis for the development of low cost vaccines.     

Liver fluke-induced hepatic oxysterols stimulate DNA damage and apoptosis in cultured human cholangiocytes

Oxysterols are cholesterol oxidation products that are generated by enzymatic reactions through cytochrome P450 family enzymes or by non-enzymatic reactions involving reactive oxygen and nitrogen species. Oxysterols have been identified in bile in the setting of chronic inflammation, suggesting that biliary epithelial cells are chronically exposed to these compounds in certain clinical settings. We hypothesized that biliary oxysterols resulting from liver fluke infection participate in cholangiocarcinogenesis. Using gas chromatography/mass spectrometry, we identified oxysterols in livers from hamsters infected with Opisthorchis viverrini that develop cholangiocarcinoma. Five oxysterols were found: 7-keto-cholesta-3,5-diene (7KD), 3-keto-cholest-4-ene (3K4), 3-keto-cholest-7-ene (3K7), 3-keto-cholesta-4,6-diene (3KD), and cholestan-3β,5α,6β-triol (Triol). Triol and 3K4 were found at significantly higher levels in the livers of hamsters with O. viverrini-induced cholangiocarcinoma. We therefore investigated the effects of Triol and 3K4 on induction of cholangiocarcinogenesis using an in vitro human cholangiocyte culture model. Triol- and 3K4-treated cells underwent apoptosis. Western blot analysis showed significantly increased levels of Bax and decreased levels of Bcl-2 in these cells. Increased cytochrome c release from mitochondria was found following treatment with Triol and 3K4. Triol and 3K4 also induced formation of the DNA adducts 1,N(6)-etheno-2'-deoxyadenosine, 3,N(4)-etheno-2'-deoxycytidine and 8-oxo-7,8-dihydro-2'-deoxyguanosine in cholangiocytes. The data suggest that Triol and 3K4 cause DNA damage via oxidative stress. Chronic liver fluke infection increases production of the oxysterols Triol and 3K4 in the setting of chronic inflammation in the biliary system. These oxysterols induce apoptosis and DNA damage in cholangiocytes. Insufficient and impaired DNA repair of such mutated cells may enhance clonal expansion and further drive the change in cellular phenotype from normal to malignant.     

Feeding habits of two sympatric loliginid squids,Uroteuthis (Photololigo) chinensis (Gray, 1849) and Uroteuthis (Photololigo) duvaucelii (d’Orbigny, 1835), in the lower part of the South China Sea

The feeding habits of two sympatric squid species, Uroteuthis (Photololigo) chinensis and Uroteuthis (Photololigo) duvaucelii from the southwestern Gulf of Thailand were studied. They fed on low numbers of food types (AF) and had a low diet breadth; 1.18 and 0.01 for U. (P.) chinensis and 1.49 and 0.05 for U. (P.) duvaucelii, respectively. Three major prey types (fishes, crustaceans and molluscs) were always detected and cannibalism was observed. Fish was the greatest contributor to the diet of both species, contributing 89.5% for U. (P.) chinensis and 69.9% for U. (P.) duvaucelii. Fish size significantly affected fullness index (FL) and AF for U. (P.) chinensis (P?<?0.001) and U. (P.) duvaucelii (P?<?0.001). Depth affected the FL of U. (P.) chinensis (P?<?0.001) but not of U. (P.) duvaucelii (P?>?0.05). Maturity stages of both male and female U. (P.) chinensis influenced FL (male: P?<?0.001; female: P?<?0.05) and AF (male: P?<?0.05; female: P?<?0.01). The FL of squid from cast nets was higher than those from trawls. The multivariate results showed dietary grouping between size classes of both species.     

Production of monoclonal antibodies specific to Macrobrachium rosenbergii nodavirus using recombinant capsid protein

The gene encoding the capsid protein of Macrobrachium rosenbergii nodavirus (MrNV) was cloned into pGEX-6P-1 expression vector and then transformed into the Escherichia coli strain BL21. After induction, capsid protein-glutathione-S-transferase (GST-MrNV; 64 kDa) was produced. The recombinant protein was separated using SDS-PAGE, excised from the gel, electro-eluted and then used for immunization for monoclonal antibody (MAb) production. Four MAbs specific to the capsid protein were selected and could be used to detect natural MrNV infections in M. rosenbergii by dot blotting, Western blotting and immunohistochemistry without cross-reaction with uninfected shrimp tissues or other common shrimp viruses. The detection sensitivity of the MAbs was 10 fmol μl-1 of the GST-MrNV, as determined using dot blotting. However, the sensitivity of the MAb on dot blotting with homogenate from naturally infected M. rosenbergii was approximately 200-fold lower than that of 1-step RT-PCR. Immunohistochemical analysis using these MAbs with infected shrimp tissues demonstrated staining in the muscles, nerve cord, gill, heart, loose connective tissue and inter-tubular tissue of the hepatopancreas. Although the positive reactions occurred in small focal areas, the immunoreactivity was clearly demonstrated. The MAbs targeted different epitopes of the capsid protein and will be used to develop a simple immunoassay strip test for rapid detection of MrNV.     

The complete mitochondrial DNA sequence of the Mekong giant catfish (Pangasianodon gigas), and the phylogenetic relationships among Siluriformes

The Mekong giant catfish (Pangasianodon gigas) is the largest scale-less freshwater fish of the world, and a critically endangered species. We determined the complete nucleotide sequence (16,533 bp) of the mitochondrial genome of the Mekong giant catfish, and conducted phylogenetic analyses based on mitochondrial protein (the combined amino acid sequences of all 13 mitochondrial protein coding genes) and rRNA (the combined nucleotide sequences of mitochondrial 12S and 16S rRNA genes) data sets in order to further clarify the relative phylogenetic position of P. gigas, and to recover phylogenetic relationships among 15 out of the 33 families of Siluriformes. Phylogenetic analyses (maximum parsimony, minimum evolution, maximum likelihood, Bayesian inference) of the protein data set were congruent with a basal split of the order into Loricarioidei and Siluroidei, and supported a closer relationship of the Mekong giant catfish (family Pangasiidae) to Siluridae than to Bagridae. The rRNA-based Bayesian phylogeny recovered Callichthyidae as the sister group of all other analyzed non-diplomystid catfish families, rendering Loricarioidei paraphyletic. In addition, Loricariidae were recovered as paraphyletic due to the inclusion of Astroblepidae. However, none of the two relationships received bootstrap support in the maximum parsimony, minimum evolution, and maximum likelihood analyses, and should be interpreted with caution. The derived position of Cetopsidae within Siluroidei, the sister group relationship of Pseudopimelodidae and Pimelodidae, and a close relationship of Doradidae and Auchenipteridae to the exclusion of Mochokidae were strongly supported. Pangasiidae was placed as a single lineage without clear affinities.     

Feeding habits and seasonal trophic guilds structuring fish community in the bay mouth region of a tropical estuarine habitat

Understanding trophic relationships of fish in estuarine ecosystem is an important element for sustainable resource management. This study examined the feeding habits of 29 dominant fish species, characterized the trophic guilds, assessed the impact of season and clarified the role of diets in structuring the fish community in the mouth region of Pattani Bay, Thailand. Samples of 5792 fishes collected monthly by gillnets from March 2019 to February 2020 were used for stomach content analyses. It was found that the number of food types and fullness index differed between fish taxa (P < 0.001). Most fishes were specialist feeders feeding on specific food components and were categorized into five trophic guilds: piscivore, shrimp-fish feeder, polychaete feeder, zooplanktivore and planktivore. Six species were piscivorous, considered as apex predators, that fed almost entirely on fishes. High diet overlaps among some species (>0.6) were recorded. Not much variation in seasonal guilds was observed: four guilds in the dry season, three in the moderate rainy season and four in the rainy season. Some species remained in the same guild the whole year round, but some fishes changed seasonally. Two fish communities from different regions of the bay were segregated based on feeding habits. The inner bay community comprised mainly copepod and plankton feeders, but there were more piscivores in the deeper bay mouth area. Results from this study help us to understand the feeding habits and trophic guilds of dominant fish species at the mouth of this tropical estuarine bay.