Enhanced effectiveness in preventing Nocardia seriolae infection utilizing heterologous prime-boost approach in orange-spotted grouper Epinephelus coioides.

Persistent nocardiosis has prompted exploration of the effectiveness of heterologous approaches to prevent severe infections. We have previously reported the efficacy of a nucleic acid vaccine in protecting groupers from highly virulent Nocardia seriolae infections. Ongoing research has involved the supplementation of recombinant cholesterol oxidase (rCho) proteins through immunization with a DNA vaccine to enhance the protective capacity of orange-spotted groupers. Recombinant rCho protein exhibited a maturity and biological structure comparable to that expressed in N. seriolae, as confirmed by Western blot immunodetection assays. The immune responses observed in vaccinated groupers were significantly higher than those observed in single-type homologous vaccinations, DNA or recombinant proteins alone (pcD:Cho and rCho/rCho), especially cell-mediated immune and mucosal immune responses. Moreover, the reduction in N. seriolae occurrence in internal organs, such as the head, kidney, and spleen, was consistent with the vaccine's efficacy, which increased from approximately 71.4 % to an undetermined higher percentage through heterologous vaccination strategies of 85.7 %. This study underscores the potential of Cho as a novel vaccine candidate and a heterologous approach for combating chronic infections such as nocardiosis.

An Integrated in silico and in vivo study of nucleic acid vaccine against Nocardia seriolae infection in orange-spotted grouper Epinephelus coioides.

Nocardiosis in aquatic animals caused by Nocardia seriolae is a frequently occurring serious infection that has recently spread to many countries. In this study, DNA vaccines containing potential bacterial antigens predicted using the reverse vaccinology approach were developed and evaluated in orange-spotted groupers. In silico analysis indicated that proteins including cholesterol oxidase, ld-transpeptidase, and glycosyl hydroxylase have high immunogenicity and are potential vaccine candidates. In vitro assays revealed the mature and biological configurations of these proteins. Importantly, when compared to a control PBS injection, N. seriolae DNA-based vaccines showed significantly higher expression of IL1ß, IL17, and IFNγ at 1 or 2 days, in line with higher serum antibody production and expression of other cellular immune-related genes, such as MHCI, CD4, and CD8, at 7 days post-immunization. Remarkably, enhanced immune responses and strong protective efficacy against a highly virulent strain of N. seriolae were recorded in DNA vaccine-cholesterol oxidase (pcD::Cho) injected fish, with a relative survival rate of 73.3%. Our results demonstrate that the reverse vaccinology approach is a valid strategy for screening vaccine candidates and pcD::Cho is a promising candidate that can boost both innate and adaptive immune responses and confer considerable protection against N. seriolae infection.

Differential Expression Genes of the Head Kidney and Spleen in Streptococcus iniae-Infected East Asian Fourfinger Threadfin Fish (Eleutheronema tetradactylum).

Streptococcus iniae is a Gram-positive bacterium and is considered a harmful aquaculture pathogen worldwide. In this study, S. iniae strains were isolated from East Asian fourfinger threadfin fish (Eleutheronema tetradactylum) reared on a farm in Taiwan. A transcriptome analysis of the head kidney and spleen was performed in the fourfinger threadfin fish 1 day after infection using the Illumina HiSeq™ 4000 platform for RNA-seq to demonstrate the host immune mechanism against S. iniae. A total of 7333 genes based on the KEGG database were obtained after the de novo assembly of transcripts and functional annotations. Differentially expressed genes (DEGs) (2-fold difference) were calculated by comparing the S. iniae infection and phosphate-buffered saline control group gene expression levels in each tissue sample. We identified 1584 and 1981 differentially expressed genes in the head kidney and spleen, respectively. Based on Venn diagrams, 769 DEGs were commonly identified in both the head kidney and spleen, and 815 and 1212 DEGs were specific to the head kidney and spleen, respectively. The head-kidney-specific DEGs were enriched in ribosome biogenesis. The spleen-specific and common DEGs were found to be significantly enriched in immune-related pathways such as phagosome, Th1, and Th2 cell differentiation; complement and coagulation cascades; hematopoietic cell lineage; antigen processing and presentation; and cytokine-cytokine receptor interactions, based on the KEGG database. These pathways contribute to immune responses against S. iniae infection. Inflammatory cytokines (IL-1ß, IL-6, IL-11, IL-12, IL-35, and TNF) and chemokines (CXCL8 and CXCL13) were upregulated in the head kidney and spleen. Neutrophil-related genes, including phagosomes, were upregulated post-infection in the spleen. Our results could offer a strategy for the treatment and prevention of S. iniae infection in fourfinger threadfin fish.

Transcriptome analysis of immune- and iron-related genes after Francisella noatunensis subsp. orientalis infection in Nile tilapia (Oreochromis niloticus).

Francisella noatunensis subsp. orientalis (Fno) is a gram-negative intracellular bacterium identified in many fish species worldwide, including cultured Nile tilapia (Oreochromis niloticus) in Taiwan. To investigate the gene expression responses to Fno infection, we performed transcriptome analysis of the head kidney and spleen in Nile tilapia using RNA-seq. Total RNA was extracted from the head kidney and spleen of infected (Fno-injected) and uninfected (control) tilapia at 1-day and 2-days post-infection, and RNA-seq was performed using the Illumina HiSeq™ 4000 platform. After de novo assembly, a total of 106,534 transcripts were detected. These transcripts were annotated and categorized into a total of 7171 genes based on the KEGG pathway database. Differentially expressed genes (DEGs) were significantly (2-fold difference comparing Fno and PBS groups at each time point) enriched in the immune-related pathways, including the following: complement and coagulation cascades, cytokine-cytokine receptor interaction, hematopoietic cell lineage, lysosome, phagosome. We identified the upregulation of inflammatory cytokine-, apoptosis-, and neutrophil-related genes, and downregulation of complement- and lymphocyte-related genes. Additionally, we found the induction of natural resistance-associated macrophage protein 1 (NRAMP1) and heme responsive gene-1 (HRG1). Anemia of inflammation, caused by intracellular iron storage in spleen after Fno infection, was also observed. This study provides natural disease control strategies against Fno infection in tilapia. It is suggested that intercellular iron storage is a host protection strategy.

ASC-deficiency impairs host defense against Aeromonas hydrophila infection in Japanese medaka, Oryzias latipes.

Apoptosis-associated speck-like protein containing a caspase-recruitment domain (ASC) is a component of inflammasome, which plays crucial roles in the inflammatory response. In mammals, ASC regulates caspase-1 activation, thereby inducing pyroptosis and producing activated inflammatory cytokines. In addition, ASC also interacts with receptor-interacting protein kinase 2 (RIPK2) and induces nuclear factor-κB (NF-κB) activation. However, the role of ASC remains poorly understood in fish. In this study, we focused on elucidating the role of ASC in fish that were infected with Aeromonas hydrophila using Japanese medaka (Oryzias latipes) as fish model, and ASC-knockout (KO) medaka was established using CRISPR-Cas9 system. ASC-KO and wild type (WT) medakas were infected with A. hydrophila, and mortality was observed. ASC-KO medaka demonstrated higher mortality than WT. Moreover, the expression of immune-related genes in the kidney and intestine of the ASC-KO and WT medakas challenged with A. hydrophila were analyzed. Following A. hydrophila infection, the kidney of ASC-KO medaka exhibited significantly lower expression of NF-κB regulated genes (e.g., IL-1ß, IL-6, IL-8 and TNF-α) and RIPK2 gene than in WT kidney. Moreover, to investigate the immune response against A. hydrophila via ASC in the medaka, bacterial burden, superoxide anion production, and lactate dehydrogenase release in the kidney cells of ASC-KO medaka were measured. After infection, these responses in ASC-KO medaka were significantly decreased compared to those in WT. These results suggest that the medaka ASC plays a critical role against A. hydrophila infection by inducing inflammatory responses and cell death for bacterial clearance.

Interleukin-17A/F1 from Japanese pufferfish (Takifugu rubripes) stimulates the immune response in head kidney and intestinal cells.

In mammals, interleukin (IL)-17A and IL-17F, mainly produced by Th17 cells, are hallmark inflammatory cytokines that play important roles in the intestinal mucosal immune response. In contrast, three mammalian IL-17A and IL-17F counterparts (IL-17A/F1-3) have been identified in teleosts, and most of their functions have been described in the lymphoid organs. However, their function in the intestinal mucosal immune response is poorly understood. In this study, a recombinant (r) tiger puffer fish fugu (Takifugu rubripes) IL-17A/F1 was produced and purified using a mammalian expression system, and was used to stimulate cells isolated from fugu head kidney and intestines. The gene expression levels of TNF-α, IL-1ß, IL-6, and ß-defensin-like protein-1 (BD-1) genes were evaluated at 0, 3, 6 and 12 h post-stimulation (hps). Phagocytic activity and superoxide anion production were evaluated at the same time points using an NBT assay. The rIL-17A/F1 protein was shown to induce the expression of pro-inflammatory cytokines and antimicrobial peptides in both head kidney and intestinal cells. Expression levels for IL-1ß, TNF-α, and IL-6 were all up-regulated between 3 and 12 hps. In addition, stimulation with rIL-17A/F1 enhanced phagocytic activity at 24 hps. Superoxide anion production was increased at 48 hps in the head kidney cells and moderately increased at 48 hps in intestinal cells. This study suggests that fugu IL-17A/F1 plays an important role in promoting the innate immune response and may act as a bridge between innate and adaptive immunity in the head kidney and intestine.

Group C Streptococcus dysgalactiae infection in fish.

Streptococcus dysgalactiae subsp. dysgalactiae (GCSD) is a Gram-positive, facultative anaerobic bacterium and mostly non-ß-haemolytic with Lancefield group C antigen. GCSD infection has been identified in various vertebrates. From 2002 to the present, GCSD infection of fish has been reported to cause severe economic losses in aquaculture farms around the world. Moreover, GCSD isolates from teleosts have been identified in patients with ascending upper limb cellulitis. Therefore, the economic and clinical significance of GCSD has increased in aquaculture, livestock and human health. Many studies have been presented, from the first report of isolated GCSD in fish, to the pathogenesis, characterization, immune responses and vaccine development. In this review, we present the current knowledge of GCSD in teleosts.

Differential expression of immune-related genes in head kidney and spleen of cobia (Rachycentron canadum) having Streptococcus dysgalactiae infection.

Streptococcus dysgalactiae is a gram-positive bacterium and a harmful aquaculture pathogen. To investigate the immune response against S. dysgalactiae, we performed transcriptome analysis of the head kidney and spleen of cobia (Rachycentron canadum) using RNA-seq. Total RNA was extracted from the head kidney and spleen of cobia, 1 and 2 days after treatment with S. dysgalactiae or control PBS. After RNA purification and cDNA library generation, sequencing was performed using the Illumina HiSeq™ 4000 platform. The filtering and de novo assembling transcripts were annotated using several databases. To identify differentially expressed genes (DEGs) between the S. dysgalactiae and PBS groups, the mapped values of fragments per kilobase of transcripts per million fragments were calculated. After de novo assembly, a total of 106,984 transcripts were detected, with an N50 of 3020 bp. These transcripts were annotated and categorised into a total of 7608 genes based on the KEGG pathway database. DEGs (2-fold difference) were calculated by comparing the S. dysgalactiae and PBS control group gene expression levels at each time point. The DEGs were mainly annotated into signal transduction and immune system categories, based on the KEGG database. The DEGs were significantly enriched in the immune-related pathways - "cytokine-cytokine receptor interaction", "complement and coagulation cascades", and "hematopoietic cell linage". In this study, immune-related genes responding to S. dysgalactiae were detected, and several immune system pathways were categorized. We identified the IL17C-related pathway for inducing the expression of pro-inflammatory cytokine genes (IL-1ß, IL-6, and IFNγ). Additionally, neutrophil-related genes (CSF3, CD121, and CD114) were induced in the spleen after S. dysgalactiae infection. It was suggested that these pathways contribute to immune responses against S. dysgalactiae infection. The data revealed in this study may offer improved strategies against S. dysgalactiae infection in cobia.

Efficacy of recombinant protein vaccines for protection against Nocardia seriolae infection in the largemouth bass Micropterus salmoides.

A reverse vaccinology-based survey of potent antigens associated with fish nocardiosis was conducted using the largemouth bass, Micropterus salmoides, with an aim to develop subunit vaccines. The antigens selected from the virulent strain Nocardia seriolae 961113 include the gene products of NGL2579 (GAPDH), NGL5701 (MMP), NGL4377 (OCTase), NGL4486 (ABC transporter), NGL3372 (LLE), NGL3388 (GHf10), NGL6627 (Antigen-85), NGL6696 (Esterase), and NGL6936 (CBP). These antigens were heterologously expressed in E. coli BL21 (DE3) for recombinant protein production. Then fish were vaccinated was these antigens, boosted at 2 weeks, and challenged with N. seriolae at 6 weeks after vaccination. The relative protection survival assay revealed high and significant protection efficacies of 94.45, 50.00, and 44.45 in fish that received the NGL3388 (GHf10), NGL6936 (CBP), and NGL3372 (LLE) vaccines, respectively. There were no apparent relationships or differences in tissue lesions among the administered vaccines. The serum titers against the bacterial preparations were higher for all vaccinated groups than for the control group at 4 weeks after immunization. However, no significant difference in serum titer was found at 6 weeks after immunization. The results of this study demonstrate that subunit vaccines against fish nocardiosis have differential effects, but are highly promising for nocardial prophylaxis.

Immune-Related Functional Differential Gene Expression in Koi Carp (Cyprinus carpio) after Challenge with Aeromonas sobria.

In order to understand the molecular basis underlying the host immune response of koi carp (Cyprinus carpio), Illumina HiSeqTM 2000 is used to analyze the muscle and spleen transcriptome of koi carp infected with Aeromonas sobria (A. sobria). De novo assembly of paired-end reads yielded 69,480 unigenes, of which the total length, average length, N50, and GC content are 70,120,028 bp, 1037 bp, 1793 bp, and 45.77%, respectively. Annotation is performed by comparison against various databases, yielding 42,229 (non-redundant protein sequence (NR): 60.78%), 59,255 (non-redundant nucleotide (NT): 85.28%), 35,900 (Swiss-Prot: 51.67%), 11,772 (clusters of orthologous groups (COG): 16.94%), 33,057 (Kyoto Encyclopedia of Genes and Genomes (KEGG): 47.58%), 18,764 (Gene Ontology (GO): 27.01%), and 32,085 (Interpro: 46.18%) unigenes. Comparative analysis of the expression profiles between bacterial challenge fish and control fish identifies 7749 differentially expressed genes (DEGs) from the muscle and 7846 DEGs from the spleen. These DEGs are further categorized with KEGG. Enrichment analysis of the DEGs and unigenes reveals major immune-related functions, including up-regulation of genes related with Toll-like receptor signaling, complement and coagulation cascades, and antigen processing and presentation. The results from RNA-Seq data are also validated and confirmed the consistency of the expression levels of seven immune-related genes after 24 h post infection with qPCR. Microsatellites (11,534), including di-to hexa nucleotide repeat motifs, are also identified. Altogether, this work provides valuable insights into the underlying immune mechanisms elicited during bacterial infection in koi carp that may aid in the future development of disease control measures in protection against A. sobria.

Transcriptome analysis of immune response against Vibrio harveyi infection in orange-spotted grouper (Epinephelus coioides).

Vibrio harveyi is a gram-negative bacterium reported as found in many aquaculture species. To increase knowledge of the immune response against V. harveyi, in this study we performed transcriptome analysis of head kidney and spleen in orange-spotted grouper (Epinephelus coioides) at 1 and 2 days post-infection (dpi), using the Illumina sequencing platform. After de novo assembly, a total of 79,128 unigenes was detected with an N50 of 2511 bp. After alignments with sequences recorded in the major databases (NT, NR, Swiss-Prot COG, KEGG, Interpro and GO), based on sequence similarity, 61,208 (77.4%) of the unigene total could be annotated using at least one database. Comparison of gene expression levels between V. harveyi and a control group at each time point revealed differentially expressed genes (DEGs) (P < 0.05): a total of 7918 (5536 upregulated and 2282 downregulated genes) from head kidney at 1 day post infection (dpi), 4260 (1444 upregulated and 2816 downregulated genes) from head kidney at 2 dpi, 7887 (4892 upregulated and 2995 downregulated genes) from spleen at 1 dpi, and 8952 (7388 upregulated and 1564 downregulated genes) from spleen at 2 dpi. The DEGs were mainly annotated into signal transduction and immune system categories, based on the KEGG database. The DEGs were enriched in immune-related pathway functions, NOD-like receptor signaling pathways, Toll-like receptor signaling pathways, NF-κB signaling pathways, and Jak-STAT signaling pathways. Additionally, we selected several DEGs and validated their expression level by RT-qPCR. The data generated in this study may provide a valuable resource for further immune response research and offer improved strategies against V. harveyi infection in teleost fishes.

[A Case of Aortic Cannula Malposition Detected by Prolonged Desaturation of Regional Cerebral Oxygen Index (rcSO2)].

A sixteen-year-old female patient with congenital aortic stenosis underwent Ross procedure. We monitored bilateral regional cerebral saturation of oxygen (rcSO2) on the forehead at the right and left of the midline. After aortic and bicavel cannulation, cardiopulmonary bypass was instituted. On the mor- row of aortic cross clamping, the rcSO2 fell from approximately 55% to below 30%. We searched the cause of this phenomenon, and detected that the tip of aortic cannula was inserted to the left subclavian artery. After repositioning, the bilateral rcSO2 increased to above 65%. We felt keenly that the monitoring of rcSO2 is useful to recognize corrective adjustment of the cannula ori- entation, and the avoidance of cerebral hypoperfusion during the cardiopulmonary bypass period.

Expression and biological activity of two types of interferon genes in medaka (Oryzias latipes).

Type I interferon (IFN) is one of most important cytokines for antiviral responses in fish innate immunity, after the induction pathway following pattern recognition. In this study, 2 types of type I IFN mRNA from a medaka (Japanese rice fish; Oryzias latipes) were identified and classified (phylogenetic analysis) into subgroup-a and -d by (designated olIFNa and olIFNd, respectively). Both olIFNa and olIFNd (encoding 197 and 187 amino acid residues, respectively) contained 2 cysteines. Gene expression pattern of olIFNa, olIFNd and IFN-stimulated genes (ISGs) was assessed (quantitative real-time reverse transcriptase PCR, qRT-PCR) in various organs (i.e., whole kidney, liver and spleen) of medaka stimulated by polyI:C or infected with nervous necrosis virus (NNV). Expression of olIFNa, olIFNd and ISGs, especially the ISG15 gene, were significantly upregulated after NNV-infection. Furthermore, olIFNa, olIFNd and ISGs mRNAs were sufficiently induced in DIT cells (i.e., medaka hepatoma cell line) transfected with polyI:C or infected with NNV. In addition, in vitro biological activities of recombinant olIFNa and olIFNd (rolIFNa and rolIFNd) produced by mammalian cell line HEK293T were also characterized. Expression of GIG1a and ISG15 genes in kidney cells of adult medaka were induced by rolIFNa or rolIFNd. The olIFNs-overexpressing DIT cells had reduced viral titers following NNV infection. Therefore, we inferred that 2 type I IFNs were involved in innate immunity (antiviral response) in medaka fish.

TALENs-mediated gene disruption of myostatin produces a larger phenotype of medaka with an apparently compromised immune system.

Although myostatin, a suppressor of skeletal muscle development and growth, has been well studied in mammals, its function in fish remains unclear. In this study, we used a popular genome editing tool with high efficiency and target specificity (TALENs; transcription activator-like effector nucleases) to mutate the genome sequence of myostatin (MSTN) in medaka (Oryzias latipes). After the TALEN pair targeting OlMyostatin was injected into fertilized medaka eggs, mutant G0 fish carrying different TALENs-induced frameshifts in the OlMSTN coding sequence were mated together in order to transmit the mutant sequences to the F1 generation. Two F1 mutants with frameshifted myostatin alleles were then mated to produce the F2 generation, and these F2 OlMSTN null (MSTN(-/-)) medaka were evaluated for growth performance. The F2 fish showed significantly increased body length and weight compared to the wild type fish at the juvenile and post-juvenile stages. At the post-juvenile stage, the average body weight of the MSTN(-/-) medaka was ∼25% greater than the wild type. However, we also found that when the F3 generation were challenged with red spotted grouper nervous necrosis virus (RGNNV), the expression levels of the interferon-stimulated genes were lower than in the wild type, and the virus copy number was maintained at a high level. We therefore conclude that although the MSTN(-/-) medaka had a larger phenotype, their immune system appeared to be at least partially suppressed or undeveloped.

Development of Erythroid Progenitors under Erythropoietin Stimulation in Xenopus laevis Larval Liver.

Erythroid progenitors that respond to erythropoietin (Epo) are present in the liver of adult Xenopus laevis. However, cells responding to Epo in the larval liver and through the metamorphosis period under hepatic remodeling have not been characterized. In this study, tadpoles were staged using the tables of Nieuwkoop and Faber (NF). Liver cells from pre- (NF56) or post- (NF66) metamorphic stage were cultured in the presence of Epo. ß2-globin mRNA expression peaked at day 7 after the start of culture. Larval ß2-globin was highly expressed in NF56-derived cells, while adult ß2-globinwas detected in those of NF66. In both NF56- and NF66-derived cells, mRNA expression of eporand gata2 peaked at day 5 and days 3-4, respectively. In contrast, gata1 expression peaked at day 6 in NF56 cells and at day 5 in NF66 cells. Half maximal proliferation of erythrocytic blast cells derived from the liver at NF66 was observed at day 3, which was earlier than that of NF56. These results indicate that erythroid progenitors that respond to Xenopus laevis Epo are maintained in pre- and post-metamorphic liver, although the tissue architecture changes dramatically during metamorphosis. Additionally, the globin switching occurred, and/or the erythroid progenitors for larval erythrocytes were replaced by those for adult erythrocytes in the metamorphic liver.

A comparative perspective on erythropoiesis.

The acquisition of fundamental information by the use of recent technologies, including omics-based molecular analyses and total RNA sequencing, has opened the door to further advances in physiological studies on new animal models. Currently, we are endeavoring to develop a comparative hematology protocol in order to build a discovery platform. All vertebrates, with the exception of a few species, have universally peripheral erythrocytes and hemoglobin, suggesting erythropoiesis to be an evolutionary index.

[Anesthetic Management of Single-staged Definitive Repair of Pulmonary Atresia with Ventricular Septum Defect and Major Aorto-pulmonary Collateral Arteries (PA/VSD, MAPCA)in an Adult].

A 25-year-old woman with unrepaired pulmonary atresia, ventricular septal defect and major aorto-pul- monary collateral artery was scheduled for single- staged definitive repair. She was complicated with mod- erate pulmonary hypertension, and had 2 MAPCAs arising from the descending artery. Cardiac catheter- ization demonstrated that right ventricular pressures were approximately equal to the left ventricular pres- sure. Pre-bypass, we maintained her PVR not too high in order to maintain her pulmonary blood flow. Post- bypass, we used hemodynamic support with dopamine, olprinone and nitroglycerin, maintaining her PVR opti- mally low. We inserted a catheter introducer in her jugular vein to deal with massive hemorrhage. After the repair, her right ventricular pressures were 7/10 of systemic pressure, and her postoperative course was uneventful.

Quantification and localization of erythropoietin-receptor-expressing cells in the liver of Xenopus laevis.

Erythropoiesis occurs in the African clawed frog, Xenopus laevis and is mediated by erythropoietin (xlEPO), a primary regulator of this process. Previously, we have shown that the xlEPO receptor (xlEPOR), which is expressed by erythroid progenitors that respond to xlEPO, is found predominantly in the liver. The aim of the present study was to determine the dynamics of erythropoiesis in the livers of normal and anemic X. laevis by identifying the number and precise location of mature and immature erythrocytes. We quantified mature and immature erythrocyte numbers by o-dianisidine staining or immunohistochemistry and investigated the dynamics of erythropoiesis in normal, acute hemolytic and blood-loss states by in vivo cell proliferation assays with 5-bromo-2'-deoxyuridine (BrdU). We detected 0.12×10(8) xlEPOR(+) BrdU(+) cells in the liver of the normal X. laevis at 24 h after BrdU injection. Frogs presenting with acute hemolytic anemia and pancytopenia show a 10-fold increase in the number of xlEPOR(+)/BrdU(+) cells (approximately 1.30×10(8) cells) in the liver. The xlEPOR(+) cells are found predominantly on the inner wall of hepatic sinusoids. Hematopoietic progenitors that undergo slow cell cycling were also observed in the hepatic sinusoids. This study clarifies the rate of production of mature and immature erythrocytes per day in the liver of X. laevis and the way that these cell numbers change in response to anemia.

Enhanced erythropoiesis in mice exposed to low environmental temperature.

Hematopoietic responses to environmental factors are not fully characterized. Polycythemia has been reported during exposure to low temperatures in ectothermic animals. The relationship between the causes of polycythemia and erythropoiesis during low temperature exposure is not fully understood. In this study, we exposed C57BL/6 mice to 5°C and monitored the blood cell counts and erythropoiesis. The hematocrit level increased from 45.6 to 52.2% after 14 days. Likewise, the hemoglobin concentration, initially 15.1 g dl(-1), rose to 16.0 g dl(-1). The reticulocyte production index significantly increased from 4 to 8% after 7 days. We examined the anatomy and cell composition of the spleens of the mice. On day 5, the spleens were ∼6 mg g(-1) of body mass, which was twofold greater than the spleens on day 0. Flow cytometry showed fourfold more proerythroblasts on day 5, compared with day 0. Additionally, the number of late-stage mature erythroblasts increased on day 14. Erythropoietin mRNA levels increased in the kidneys, and hypoxia-inducible genes were enhanced in the kidney. Our findings indicated that low ambient temperature is a novel erythropoietic stress, which induces polycythemia by enhanced erythropoiesis.

Recombinant Vibrio harveyi flagellin A protein and partial deletions of middle variable region and D0 domain induce immune related genes in Epinephelus coioides and Cyprinus carpio.

Vibrio harveyi is a Gram-negative bacterium that causes vibriosis in various aquaculture species, including the orange-spotted grouper (Epinephelus coioides). Bacterial flagellin is a potent pathogen-associated molecule that stimulates the innate and adaptive immune systems through toll-like receptor 5 (TLR5) signaling. In this study, we isolated V. harveyi flagellin A (VhFliA) gene from V. harveyi (originated from orange-spotted grouper) and investigated the in vivo activities of recombinant VhFliA protein. Multiple sequence alignment showed that the amino acid sequence of VhFliA has conserved domains of N- and C-terminals (D0 and D1) and a middle variable (MV) region. We produced the VhFliA recombinant protein (wild type (WT)-VhFliA) by Escherichia coli and investigated its in vivo biological activity. Additionally, we prepared the VhFliA recombinant proteins with deletion of domains (ΔMV-VhFliA and ΔD0MV-VhFliA) to identify the domain for biological activity in the orange-spotted grouper. WT and ΔMV-VhFliA induced the expression of inflammatory cytokines (IFNγ, IL-1ß, and IL-8) in groupers. However, ΔD0MV-VhFliA did not induce the expression of inflammatory cytokines. Additionally, to demonstrate the applicability of recombinant VhFliA to teleost species, we performed an in vivo assay of the recombinant proteins in koi carp (Cyprinus carpio). WT-VhFliA stimulates the expression of inflammatory cytokines (IL-1ß, IL-6, and IL-8) in carp. ΔMV-VhFliA did not upregulate IL-1ß and IL-6, whereas ΔD0MV-VhFliA induced expression in carp. These findings showed the potential of VhFliA as an effective immune stimulant adjuvant and comparative studies of flagellin - TLR5 signaling in teleosts.