Dynamic pathology and pathogen distribution of the yeast Metschnikowia bicuspidata in the Chinese mitten crab (Eriocheir sinensis).

In this study, the "milky disease" model of Eriocheir sinhensis was constructed via intramuscular injection with the pathogenic yeast Metschnikowia bicuspidata. The dynamic pathological changes of E. sinensis after injection were elucidated with two staining methods (haemotoxylin-eosin and alcian blue periodic acid-Schiff) and fluorescence in situ hybridization technology. Anatomical observation revealed three stages of the "milky disease": no clinical signs (1-4 days after infection), the appearance of signs of disease (5-7 days), and significant liquefaction (10 days). Histological observation also revealed three stages of the disease: yeast diffusion (1-2 days after infection), yeast slow development (3-4 days), and yeast rapid proliferation (5 days). And FISH technique was suitable for the early detection of infection with M. bicuspidata in E. sinensis. We found that M. bicuspidata spread to the whole body of the crab through the haemolymph and developed into fungal septicaemia. These results elucidate the systemic pathological characteristics of "milky disease" in E. sinensis and suggest the pathogenic mechanism of M. bicuspidata.

Antiparasitic efficacy of flavonoids identified from Psoralea corylifolia against Tetrahymena piriformis in guppy (Poecilia reticulate).

Tetrahymena piriformis belongs to the ciliated protists (ciliates), causing severe economic losses in aquaculture. Chemical drugs currently used usually have toxic side effects, and there is no specific drug against Tetrahymena. Therefore, it is an urgent need to identify new antiparasitic lead compounds. In the present study, the in vitro parasiticidal activity of ethyl acetate (EtOAc) extracts and water extracts from 22 selected traditional Chinese medicines (TCMs) were evaluated against T. piriformis. The EtOAc extract of P. corylifolia turned out to be the most active with the minimum parasiticidal concentration of 100 mg/L within 3 h. Thus, it was separated into 12 fractions by the first-dimensional (D1) normal phase liquid chromatography (NPLC), meanwhile combining with in vitro antiparasitic tests for activity tracking. Subsequently, 8 flavonoids were identified in the active fractions by the second-dimensional (D2) reverse phase liquid chromatography (RPLC) tandem high-resolution mass spectrometry. According to the results, 5 flavonoids were selected for in vitro antiparasitic test, of which isobavachalcone showed the minimum parasiticidal concentration of 3.125 mg/L in 2 h. Bathing treatment of infected guppies with isobavachalcone could significantly reduce the burden of T. piriformis, obtaining a 24-h median effective concentration (24-h EC50) value of 1.916 mg/L. And the concentration of isobavachalcone causing guppies to die within 24 h is 39 times than that of 24-h EC50. The results demonstrated that isobavachalcone has the potential to be developed into a novel commercial fish drug against T. piriformis.

The complete mitochondrial genome sequence of the Metschnikowia bicuspidata (Metschnikoff, 1884), an emerging pathogen of 'milky disease' in Chinese mitten crab.

The outbreak of milky disease of Chinese mitten crab caused by M. bicuspidata seriously restricted the development of the crab industry. In this study, the mitochondrial genome sequence of M. bicuspidata was assembled, annotated, and further analyzed. The results indicated that the complete mitochondrial genome of M. bicuspidata was 75,095 bp, which contained two rRNAs, 23 tRNAs, and 13 protein-coding genes. The phylogenetic tree of 13 yeasts based on the complete mitochondrial genome was constructed which showed that M. bicuspidata (accession number OK514652) and M. bicuspidata (accession number MW147605.1) were clustered in a clade. To sum up, our research results would further provide essential data for the systematics and evolution study of M. bicuspidata.

First case of Coxiella burnetii infection in Palaemonetes sinensis cultured in Liaoning, China.

The Palaemonetes sinensis aquaculture industry in Panjin City, Liaoning Province, China, experienced heavy losses in October 2018. Morbidity of cultured shrimp reached 50% and was characterized by cloudiness of muscle and the gradual spread of disease within the population. When the infection was mild, histopathological examinations revealed that the muscle cells contained a considerable number of microorganisms. In extreme cases, the structure of the hepatopancreatic glandular and muscle fiber was obscured or even vanished. Electron microscope observations revealed the presence of granular cytoplasmic inclusions in cells from hepatopancreas and muscle tissues. The 16S rDNA sequence of the intracytoplasmic organism was 94.7% identity to that of Coxiella burnetii. This is the first report of infection by C. burnetii in P. sinensis.

Metschnikowia bicuspidata isolated from milky diseased Eriocheir sinensis: Phenotypic and genetic characterization, antifungal susceptibility and challenge models.

A severe disease occurred in farmed Eriocheir sinensis characterized by milky liquid accumulation in the pectoral cavity, in the province of Liaoning, China, during October 2018-April 2019. Diseased crabs moved sluggishly, exhibited appetite loss and readily lost legs. Under the microscopic analysis, it was observed that the milky liquid contained a large number of yeastlike microorganisms (0.8-1.2 µm × 1.5-1.9 µm), which were also present in the muscle, hepatopancreas and gills. A dominant strain was isolated from the milky liquid and other tissues of diseased crabs. It grew on nutrient agar and formed 1- to 3-mm white opaque colonies, each with a protuberance in the centre. Besides, the results of TEM and SEM also demonstrate a typical multilateral budding model of the yeast clearly. We identified the strain, which we named 2EJM001, as Metschnikowia bicuspidata based on 18S rDNA, ITS and 26S rDNA sequence analyses and on its morphological, physiological and biochemical characteristics. Phylogenetic analysis revealed that 26S rDNA of 2EJM001 was clustered with M. bicuspidata (LNES0119) as reported by Bao et al. In addition, unlike Bao et al., two challenge experiments (injection and immersion) were used in this study. The results of challenge experiments show that 2EJM001 was pathogenic to E. sinensis and caused signs similar to those found in the naturally infected crabs. At the same time, the minimum inhibitory concentrations (MIC80 and MIC90 ) were determined. This study further confirms that M. bicuspidata 2EJM001 was the pathogen responsible for 'milky disease' in E. sinensis from Liaoning Province.

First case of non-O1/O139 Vibrio cholerae infection in the pink-tailed chalceus Chalceus macrolepidotus in China.

A pathogen was isolated from diseased pink-tailed chalceus Chalceus macrolepidotus during a high-mortality outbreak in a freshwater culture farm in Liaoyang, China. The diseased fish were characterized by disoriented behaviors, exophthalmos, and redness and swelling of the top of the head. A Gram-negative, pure strain of bacteria (CM0428) was isolated from the brain, kidney, and liver. The isolate was identified as Vibrio cholerae based on ompW gene amplification and 16S rRNA and gyrB gene sequence analysis. Serogroup testing indicated that CM0428 was a non-O1/O139 strain of V. cholerae. The challenge test showed that CM0428 exhibited strong virulence to pink-tailed chalceus, and hlyA and toxR virulence-related genes were detected. The isolate was sensitive to multi-class antibiotics, but resistant to tetracycline. Histological examination revealed that V. cholerae CM0428 infection caused multiple organ and tissue lesions, and typical pathological features were cell degeneration and necrosis.

RNA-Sequencing Analysis of the Spleen and Gill of Takifugu rubripes in Response to Vibrio harveyi Infection.

Takifugu rubripes is commonly subjected to the disease-causing bacterium, Vibrio harveyi. However, the mechanism involved in the immune response of T. rubripes to V. harveyi infection is unclear. We conducted a transcriptomic analysis of the spleen and gill from T. rubripes infected with V. harveyi. We obtained 60,981,357 and 60,760,550 clean reads from the control and infected spleens, and 57,407,586 and 57,536,651 clean reads from the control and infected gills, respectively. We also identified 1,560 and 1,213 differentially expressed genes in the spleen and gill, respectively. Gene ontology analysis revealed that the most enriched biological process in both the spleen and gill was "immune response". The most enriched Kyoto Encyclopedia of Genes and Genomes immune response-related pathways were the NOD-like receptor signaling pathway in the spleen and cytokine-cytokine receptor interaction in the gill. We found 10 candidate immune-related genes in the spleen and gill. These putative immune pathways and candidate genes will provide insight into the immune response mechanisms of T. rubripes against V. harveyi.

Delineating the key virulence factors and intraspecies divergence of Vibrio harveyi via whole-genome sequencing.

Vibrio harveyi is one of the major pathogens in aquaculture. To identify the key virulence factors affecting pathogenesis of V. harveyi towards fish, we conducted a field investigation for three representative fish farms infected with V. harveyi. Multilocus sequence typing (MLST) and whole-genome sequencing were conducted to delineate the phylogenetic relationship and genetic divergence of V. harveyi. A total of 25 V. harveyi strains were isolated from the diseased fish and groundwater and were subtyped into 12 sequence types by MLST. Five virulence genes, mshB, pilA, hutR, ureB, and ureG, were variably presented in the sequenced strains. The virulence gene profiles strongly correlated with the distinct pathogenicity of V. harveyi strains, with a strain harboring all five genes exhibiting the highest virulence towards fish. Phenotype assay confirmed that reduced virulence correlated with decreased motility and biofilm formation ability. Additionally, three types of type VI secretion system, namely T6SS1, T6SS2, and T6SS3, were identified in V. harveyi strains, which can be classified into six, four, and 12 subtypes, respectively. In conclusion, the results indicated that the virulence level of V. harveyi is mainly determined by the above virulence genes, which may play vital roles in environmental adaptation for V. harveyi.

A novel research on isolation and characterization of Photobacterium damselae subsp. damselae from Pacific white shrimp, Penaeus vannamei, displaying black gill disease cultured in China.

In June 2019, massive mortalities of cultured Penaeus vannamei occurred in a local farm in Hainan Province, China. The diseased shrimp displayed evident black gills. Three bacterial strains 20190611001, 20190611007 and 20190611022 were isolated from hepatopancreas and gills of the diseased shrimp and identified as Photobacterium damselae subsp. damselae based on the sequence analysis of 16S rRNA and toxR genes. These three isolates showed haemolytic activities. Of them, strain 20190611022 isolated from hepatopancreas was selected and processed for pathogenic analysis. The calculated median lethal dose (LD50 ) was 9.75 ± 4.29 × 105 CFU/g (body weight) by challenging P. vannameivia reverse gavage. The diseased shrimp displayed enlarged hepatopancreatic tubules and sloughing of epithelial cells in tubular lumens. The strain 20190611022 was also characterized by the testing of API 20NE systems and antibiotic susceptibility. The results of disc diffusion test showed that strain 20190611022 was sensitive to chloramphenicol, compound sulfamethoxazole, cefoperazone, ceftriaxone, ceftazidime and cefuroxime. To our knowledge, this is the first report of isolation and characterization of Photobacterium damselae subsp. damselae from natural diseased P. vannamei. Our findings can serve as a basis for further studies of its pathogenicity and provide technological support for disease controlling in shrimp aquaculture.

Long-distance transmission of pathogenic Vibrio species by migratory waterbirds: a potential threat to the public health.

A potential mechanism for the global distribution of waterborne pathogens is through carriage by the migratory waterbirds. However, this mode of transmission has yet been confirmed epidemiologically. Here, we conducted whole genome sequencing of Vibrio spp. collected from waterbirds, sediments, and mollusks in the estuary of the Liaohe River in China to investigate this transmission mode. We found that a V. parahaemolyticus strain isolated from a waterbird was clonally related to the other V. parahaemolyticus strains obtained from the sediments and mollusks, and three V. mimicus strains isolated from bird feces were genomically related to those found in the mollusks and upstream groundwater, suggesting that the bird-carried Vibrio strains were acquired through the direct predation of the local mollusks. Surprisingly, two bird-carried V. parahaemolyticus strains belonging to the same clone were identified in Panjin and Shanghai, which are over 1,150 km apart, and another two were found at two locations 50 km apart, further supporting that waterbirds are capable of carrying and disseminating these pathogens over long distances. Our results provide the first evidence of direct transmission from mollusks to waterbirds and confirm that waterbirds act as disseminating vehicles of waterborne pathogens. Effective surveillance of migratory waterbirds along their routes will be valuable for predicting future epidemics of infectious diseases.

A Human Intestinal Infection Caused by a Novel Non-O1/O139 Vibrio cholerae Genotype and Its Dissemination Along the River.

Non-O1/O139 Vibrio cholerae is increasingly reported in the clinical settings. However, intestinal infections via the consumption of non-O1/O139 V. cholerae-carrying seafood are rarely documented in China. In this study, we reported a case of mild watery diarrhea in a young male, caused by non-O1/O139 V. cholerae in the downstream of Liaohe River. Epidemiological investigation showed that this intestinal infection potentially associated with the raw consumption of mollusc. Prior to this finding, we conducted a 6-month pathogen surveillance of three locations along the Liaohe River and identified three environmental non-O1/O139 V. cholerae strains. To confirm the epidemiological links between clinical and environmental strains, high-resolution genomic typing was employed and revealed that V. cholerae isolated from human stool sample was genomically related to the one found in local mollusc and shared a common ancestor with other environmental strains obtained in the upstream sites of the Liaohe River. This fact suggests that the river is a natural reservoir for non-O1/O139 V. cholerae which poses a potential threat to the public health. In summary, our results deepened the insights on the transmission of non-pandemic V. cholerae strains and underscored the significance of genomic surveillance for drinking water along the river sites.

Delineating the origins of the multidrug-resistant pathogens in ornamental fish farms by multilocus sequence typing and identification of a novel multidrug-resistant plasmid.

To evaluate the overuse of antibiotics and to identify the origin of pathogens in the ornamental fish industry, we conducted a field investigation of three representative fish farms in Liaoning province, China. Drug-resistant pathogens in the fishponds and groundwater were isolated and subtyped by multilocus sequence typing (MLST). In total, 33 pathogenic strains, including Aeromonas veronii and five other pathogens, were isolated from diseased fish and from groundwater. MLST revealed that A. veronii obtained from diseased fish in three fish farms can be subtyped into four sequence types, which were also identified in the corresponding groundwater. All of the isolates obtained from diseased fish showed resistance to at least four antibiotics. Notably, Citrobacter freundii JY-17 exhibited resistance to the majority of the antibiotics and was a carrier of a megaplasmid with 15 drug resistance genes. PCR assays targeting ß-lactam, kanamycin, macrolide, phenicol, sulfonamide, and trimethoprim resistance genes in the pathogens from the diseased fish and groundwater were also conducted. The results revealed strong correlations between antibiotic treatment and increased antimicrobial resistance in fish pathogens. The results suggested that groundwater is the origin of the pathogens in ornamental fish. Antibiotic treatment of ornamental fish promoted the emergence of resistant pathogens.

Mitochondrial genome sequencing of the monogenean Heterobothrium okamotoi isolated from the tiger puffer Takifugu rubripes in North China.

In this study, monogenean Heterobothium okamotoi was isolated and identified from the gill of diseased Tiger puffer (T. rubripes) at an industrial farm in Liaoning, North China (121.3459 E, 38.9861 N). With the completion of H. okamotoi mitochondrial genome sequencing, the full-length mitochondrial genome of H. okamotoi was assembled and analyzed. All results indicate that the complete mitochondrial genome of H. okamotoi was 14,643 bp. There were 2 rRNAs, 20 tRNAs, and 12 protein-coding genes (PCGs) all located at the heavy (H) strand. Besides, the phylogenetic tree of 19 monogeneans was constructed. The results showed that H. okamotoi and Pseudochauhanea macrorchis were clustered in a clade. To sum up, our research results would further provide essential data for systematics and evolution study of H. okamotoi.

Localization and characterization of hematopoietic tissues in adult sea cucumber, Apostichopus japonicus.

Sea cucumber Apostichopus japonicus rely on the efficient innate immune mechanisms against invaders, in which the consumption and regeneration of coelomocytes take place at the same time. In the present study, histological features of putative hematopoietic tissues (HPTs) including the rete mirabile, the respiratory tree, the polian vesicle and the coelomic epithelium were characterized. The distribution of transcription factor GATA1 in coelomocytes and putative HPTs was examined by immunohistochemistry. In addition, cell proliferation using EdU labeling and coelomocyte distribution in different tissues using monoclonal antibody labeling were analyzed to further confirm the HPTs. The results showed that two homologs of GATA1 were detected with molecular weight of 43 and 90 kDa in coelomocytes, rete mirabile, respiratory tree and polian vesicle, whereas no signals were detected in the coelomic epithelium. A few cells were detected to be EdU-positive for coelomocytes, which accounted for approximately 9.5%. In the rete mirabile and the respiratory tree, the EdU signals were strong in cells of the tube wall. In the polian vesicle, numerous EdU-positive cells were detected in the cyst wall. In the coelomic epithelium, little EdU signaling was detected. Immunohistochemistry analysis by mAb 3F6 against A. japonicus coelomocytes showed that positive signals were observed in the tube wall of the rete mirabile, respiratory tree, cyst wall of the polian vesicle and in the coelomocyte antrum of coelomic epithelium. These results suggest that the rete mirabile, respiratory tree and polian vesicle are the HPTs of A. japonicus.

Regeneration of coelomocytes after evisceration in the sea cucumber, Apostichopus japonicus.

Sea cucumber, Apostichopus japonicus, is one of the most important holothurian species cultured in China. Severe evisceration induced by various natural and artificial factors commonly occurs during transport and culture of A. japonicus. Evisceration causes higher mortality and lower yield. Along with the visceral regeneration process, sea cucumbers also regenerate coelomocytes in order to recover immune function. In this study, evisceration of A. japonicus was induced by intracoelomic injection of 0.35 M KCl. Regeneration of coelomocytes was investigated by time course cell counting as well as detection of DNA replication by the EdU labeling technique. Coelomic fluid volume was restored to the pre-evisceration level within 2 h after evisceration. Total coelomocyte count (TCC) reached a peak at 6 h post-evisceration, followed decreased and then increased with a slight fluctuation, restored to the pre-evisceration level at 35 d post-evisceration. The change in different subtypes of coelomocytes was consistent with that of total coelomocytes. However, there were some variations in the regeneration of coelomocyte subtypes. At the end of the study, only the counts of amoebocytes and morula cells recovered to the pre-evisceration level. DNA replication assay showed EdU-positive cells accounted for 9.5% before evisceration and 4.7% at 6 h post-evisceration. However, the percentage of EdU-positive cells significantly increased, reaching 18.6% at 3 d after evisceration, then declined. Therefore, we analyzed the observed increase in coelomocytes at 6 h post-evisceration, which may be due to coelomocyte migration from the water-vascular system into the coelom rather than de novo cell proliferation.

Development of a Colloidal Gold-Based Immunochromatographic Assay for the Rapid Detection of Edwardsiella Ictaluri.

Edwardsiella Ictaluri is known as the etiological agent of enteric septicaemia of channel catfish, causing heavy economic losses in the aquaculture industry. In this study, a colloidal gold-based immunochromatography assay (GICA) was developed for rapid detection of E. ictaluri. Briefly, monoclonal antibody (MAbs) and polyclonal antibody (PAbs) against E. ictaluri were prepared. Sensitivity of MAbs and PAbs to E. ictaluri was analyzed by Dot ELISA. Mouse MAb5D11 against E. ictaluri was conjugated with the 20 nm colloidal gold particles as the detector. Rabbit PAbs of E. ictaluri and goat anti-mouse IgG antibody was sprayed on nitrocellulose membranes as test line (T) and control line (C) respectively. The minimum detectable amount of this method to E. ictaluri was 5 × 106 CFU/mL. Cross reactions wouldn't occur when detecting E. tarda, Aeromonas hydrophila, V. parahaemolyticus and other several common standard strains. The result could be got in only 5 to 10 minutes. It didn't need professional technologies and testing experience. So this assay was very suitable for basic departments of aquatic product companies.

Mitochondrial genome sequencing and analysis of scuticociliates (Uronema marinum) isolated from Takifugu rubripes.

Scuticociliates are dangerous parasitic pathogens for in worldwide mariculture. Scuticociliates cause high mortality to marine fish. After an outbreak of scuticociliatosis in Takifugu rubripes in Liaoning Province, northern China, Uronema marinum, a scuticociliate, was identified. In this study, using Illumina MiSeq next-generation sequencing, the mitochondrial genome of U. marinum was assembled and analysed phylogenetically using mitochondrial genomes of other scuticociliates. The complete U. marinum mitochondrial genome was 39,845 bp; it contained two rRNAs, six tRNAs, and 39 protein-coding genes (PCGs). From the 39 PCGs, 15 PCGs were located on the heavy strand, and 24 PCGs on the light strand of U. marinum mitogenome. The phylogenetic tree showed that there were two main clades, Oligohymenophorea and Spirotrichea. Nine ciliate species were clustered together within Oligohymenophorea; Uronema marinum was a separate cluster sharing a relatively close ancestry with Hymenostomatida. The results of this study will help advance the systematics, and studies of evolution and molecular epidemiology of scuticociliates.

Mitochondrial genome sequencing of notorious scuticociliates (Pseudocohnilembus persalinus) isolated from Turbot (Scophthalmus maximus L.).

Previously, a pathogenic ciliate was isolated from the surface ulcer of a diseased Turbot (Scophthalmus maximus L.) at an aquaculture farm in North China. After morphological and molecular biological identification based on 18rRNA, the ciliated was identified as the notorious scuticociliates (Pseudocohnilembus persalinus). In this study, the whole sequence of the mitochondrial genomic gene of P. persalinus was carried out. The sequencing results showed that the complete sequence of P. persalinus mitogenome was 38,375 bp. There were 2 rRNAs, 4 tRNAs, and 34 protein-coding genes (PCGs), respectively, located on both the heavy strand and the light stand. 15 PCGs were on the heavy strand, and 19 PCGs on the light strand. Besides, phylogenetic trees among 11 ciliates were constructed based on the sequences of 17 PCGs located in mitogenome using BI methods. The results of clustering showed that P. persalinus and Uronema marinum was the first cluster belonging to the order Scuticociliatida. Our research results will further provide primary data for evolution and classified study of scuticociliates.

First characterization of three cyclophilin family proteins in the oyster, Crassostrea ariakensis Gould.

Cyclophilins (CyPs) are a family of proteins that bind the immunosuppressive agent cyclosporin A (CsA) with high-affinity and belong to one of the three superfamilies of peptidyl-prolyl cis-trans isomerases (PPIase). In this report, three cyclophilin genes (Ca-CyPs), including Ca-CyPA, Ca-CyPB and Ca-PPIL3, were identified from oyster, Crassostrea ariakensis Gould in which Ca-CyPA encodes a protein with 165 amino acid sequences, Ca-CyPB encodes a protein with 217 amino acid sequences and Ca-PPIL3 encodes a protein with 162 amino acid sequences. All of the three Ca-CyPs genes contain a typical CyP-PPIase domain with its signature sequences and Ca-CyPB contains an N-signal peptide sequences. Tissue distribution study revealed that Ca-CyPs were ubiquitously expressed in all examined tissues and the highest levels were observed in hemocytes. RLO incubation upregulated the mRNA expression levels of Ca-CyPs, indicating that three Ca-CyPs might be involved in oyster immune response against RLO infection.

Characterization and expression analysis of a caspase-2 in an invertebrate echinoderm sea cumber Apostichopus japonicus.

Caspase-2 is the most evolutionarily conserved member of the caspase family which mediates the programmed cell death and plays crucial roles in key cellular processes. In this study, a caspase-2 homolog was identified and functionally characterized in sea cucumber Apostichopus japonicus, which we named AjCASP. The full-length cDNA consists of 2100 bp with an ORF encoding a protein of 378 amino acids. The deduced amino acid sequence shows that AjCASP consists of a conserved CARD-CASP2 domain and a CASs domain containing two active residues, two proteolytic cleavage residues, a substrate pocket and a dimer interface as well. In addition, a p20 large subunit with a characteristic five-peptide motif (QACRG) and a p10 small subunit in C-terminal were identified in CASs domain. Above data demonstrated that AjCASP is similar to CED-3 (the caspase-2 homolog of nematode Caenorhabditis elegans), which is further confirmed by phylogenetic tree analysis. AjCASP was ubiquitously expressed in sea cucumber and the obviously higher expression level was observed in coelomocyte, respiratory tree and intestine. Real-time PCR analyses further demonstrated that AjCASP was significantly induced by LPS. Taken together, these results strongly suggest that AjCASP is a caspase-2 homolog and it may be involved in invertebrate immune response, especially in eliminating and degrading invading pathogens.