Exploitation of multiple host-derived nutrients by the yellow catfish epidermal environment facilitates Vibrio mimicus to sustain infection potency and susceptibility.

Infection with Vibrio mimicus in the Siluriformes has demonstrated a rapid and high infectivity and mortality rate, distinct from other hosts. Our earlier investigations identified necrosis, an inflammatory storm, and tissue remodeling as crucial pathological responses in yellow catfish (Pelteobagrus fulvidraco) infected with V. mimicus. The objective of this study was to further elucidate the impact linking these pathological responses within the host during V. mimicus infection. Employing metabolomics and transcriptomics, we uncovered infection-induced dense vacuolization of perimysium; Several genes related to nucleosidase and peptidase activities were significantly upregulated in the skin and muscles of infected fish. Concurrently, the translation processes of host cells were impaired. Further investigation revealed that V. mimicus completes its infection process by enhancing its metabolism, including the utilization of oligopeptides and nucleotides. The high susceptibility of yellow catfish to V. mimicus infection was associated with the composition of its body surface, which provided a microenvironment rich in various nucleotides such as dIMP, dAMP, deoxyguanosine, and ADP, in addition to several amino acids and peptides. Some of these metabolites significantly boost V. mimicus growth and motility, thus influencing its biological functions. Furthermore, we uncovered an elevated expression of gangliosides on the surface of yellow catfish, aiding V. mimicus adhesion and increasing its infection risk. Notably, we observed that the skin and muscles of yellow catfish were deficient in over 25 polyunsaturated fatty acids, such as Eicosapentaenoic acid, 12-oxo-ETE, and 13-Oxo-ODE. These substances play a role in anti-inflammatory mechanisms, possibly contributing to the immune dysregulation observed in yellow catfish. In summary, our study reveals a host immune deviation phenomenon that promotes bacterial colonization by increasing nutrient supply. It underscores the crucial factors rendering yellow catfish highly susceptible to V. mimicus, indicating that host nutritional sources not only enable the establishment and maintenance of infection within the host but also aid bacterial survival under immune pressure, ultimately completing its lifecycle.

Corrigendum: Transcriptome reveals the role of the htpG gene in mediating antibiotic resistance through cell envelope modulation in Vibrio mimicus SCCF01.

[This corrects the article DOI: 10.3389/fmicb.2023.1295065.].

The cAMP Receptor Protein (CRP) of Vibrio mimicus Regulates Its Bacterial Growth, Type II Secretion System, Flagellum Formation, Adhesion Genes, and Virulence.

Vibrio mimicus is a serious pathogen in aquatic animals, resulting in significant economic losses. The cAMP receptor protein (CRP) often acts as a central regulator in highly pathogenic pathogens. V. mimicus SCCF01 is a highly pathogenic strain isolated from yellow catfish; the crp gene deletion strain (Δcrp) was constructed by natural transformation to determine whether this deletion affects the virulence phenotypes. Their potential molecular connections were revealed by qRT-PCR analysis. Our results showed that the absence of the crp gene resulted in bacterial and colony morphological changes alongside decreases in bacterial growth, hemolytic activity, biofilm formation, enzymatic activity, motility, and cell adhesion. A cell cytotoxicity assay and animal experiments confirmed that crp contributes to V. mimicus pathogenicity, as the LD50 of the Δcrp strain was 73.1-fold lower compared to the WT strain. Moreover, qRT-PCR analysis revealed the inhibition of type II secretion system genes, flagellum genes, adhesion genes, and metalloproteinase genes in the deletion strain. This resulted in the virulence phenotype differences described above. Together, these data demonstrate that the crp gene plays a core regulatory role in V. mimicus virulence and pathogenicity.

Unraveling extracellular protein signatures to enhance live attenuated vaccine development through type II secretion system disruption in Vibriomimicus.

Type II secretion systems (T2SS) are important molecular machines used by bacteria to transport a wide range of proteins across the outer membrane from the periplasm. Vibrio mimicus is an epidemic pathogen threats to both aquatic animals and human health. Our previous study demonstrates that T2SS deletion reduced virulence by 307.26 times in yellow catfish. However, the specific effects of T2SS-mediated extracellular protein secretion in V. mimicus, including its potential role in exotoxin secretion or other mechanisms, require further investigation. Through proteomics and phenotypic analyses, this study observed that the ΔT2SS strain exhibited significant self-aggregation and dynamic deficiency, with a notable negative correlation with subsequent biofilm formation. The proteomics analysis revealed 239 different abundances of extracellular proteins after T2SS deletion, including 19 proteins with higher abundance and 220 proteins with lower and even absent in the ΔT2SS strain. These extracellular proteins are involved in various pathways, such as metabolism, virulence factors expression, and enzymes. Among them, purine, pyruvate, and pyrimidine metabolism, and the Citrate cycle, were the primary pathways affected by T2SS. Our phenotypic analysis is consistent with these findings, suggesting that the decreased virulence of ΔT2SS strains is due to the effect of T2SS on these proteins, which negatively impacts growth, biofilm formation, auto-aggregation, and motility of V. mimicus. These results provide valuable insights for designing deletion targets for attenuated vaccines development against V. mimicus and expand our understanding of the biological functions of T2SS.

Integrated bioinformatics identifies key mediators in cytokine storm and tissue remodeling during Vibrio mimicus infection in yellow catfish (Pelteobagrus fulvidraco).

Introduction: The pathogenesis of Vibrio mimicus infection in yellow catfish (Pelteobagrus fulvidraco) remains poorly understood, particularly regarding the impact of infection with the pathogen on primary target organs such as the skin and muscle. Methods: In this study, we aim to analyze the pathological intricacies of the skin and muscle of yellow catfish after being infected with V. mimicus using a 1/10 LC50 seven-day post-infection model. Furthermore, we have utilized integrated bioinformatics to comprehensively elucidate the regulatory mechanisms and identify the key regulatory genes implicated in this phenomenon. Results: Our histopathological examination revealed significant pathological changes in the skin and muscle, characterized by necrosis and inflammation. Moreover, tissue remodeling occurred, with perimysium degeneration and lesion invasion into the muscle along the endomysium, accompanied by a transformation of type I collagen into a mixture of type I and type III collagens in the perimysium and muscle bundles. Our eukaryotic transcriptomic and 4D label-free analyses demonstrated a predominantly immune pathway response in both the skin and muscle, with downregulation observed in several cell signaling pathways that focused on focal adhesion-dominated cell signaling pathways. The upregulated genes included interleukins (IL)-1 and -6, chemokines, and matrix metallopeptidases (mmp)-9 and -13, while several genes were significantly downregulated, including col1a and col1a1a. Further analysis revealed that these pathways were differentially regulated, with mmp-9 and mmp-13 acting as the potential core regulators of cytokine and tissue remodeling pathways. Upregulation of NF-κB1 and FOSL-1 induced by IL-17C and Nox 1/2-based NADPH oxidase may have held matrix metallopeptidase and cytokine-related genes. Also, we confirmed these relevant regulatory pathways by qPCR and ELISA in expanded samples. Discussion: Our findings unequivocally illustrate the occurrence of a cytokine storm and tissue remodeling, mediated by interleukins, chemokines, and MMPs, in the surface of yellow catfish infected with V. mimicus. Additionally, we unveil the potential bidirectional regulatory role of MMP-9 and MMP-13. These results provide novel perspectives on the intricate immune response to V. mimicus infection in yellow catfish and highlight potential targets for developing therapies.

Transcriptome reveals the role of the htpG gene in mediating antibiotic resistance through cell envelope modulation in Vibrio mimicus SCCF01.

HtpG, a bacterial homolog of the eukaryotic 90 kDa heat-shock protein (Hsp90), represents the simplest member of the heat shock protein family. While the significance of Hsp90 in fungal and cancer drug resistance has been confirmed, the role of HtpG in bacterial antibiotic resistance remains largely unexplored. This research aims to investigate the impact of the htpG gene on antibiotic resistance in Vibrio mimicus. Through the creation of htpG gene deletion and complementation strains, we have uncovered the essential role of htpG in regulating the structural integrity of the bacterial cell envelope. Our transcriptomics analysis demonstrates that the deletion of htpG increases the sensitivity of V. mimicus to antimicrobial peptides, primarily due to upregulated lipopolysaccharide synthesis, reduced glycerophospholipid content, and weakened efflux pumps activity. Conversely, reduced sensitivity to ß-lactam antibiotics in the ΔhtpG strain results from decreased peptidoglycan synthesis and dysregulated peptidoglycan recycling and regulation. Further exploration of specific pathway components is essential for a comprehensive understanding of htpG-mediated resistance mechanisms, aiding in the development of antimicrobial agents. To our knowledge, this is the first effort to explore the relationship between htpG and drug resistance in bacteria.

The Antibacterial Activity of Thymol Against Drug-Resistant Streptococcus iniae and Its Protective Effect on Channel Catfish (Ictalurus punctatus).

Streptococcus iniae is a zoonotic pathogen, which seriously threatens aquaculture and human health worldwide. Antibiotics are the preferred way to treat S. iniae infection. However, the unreasonable use of antibiotics leads to the enhancement of bacterial resistance, which is not conducive to the prevention and treatment of this disease. Therefore, it is urgent to find new efficient and environmentally friendly antibacterial agents to replace traditional antibiotics. In this study, the antibacterial activity and potential mechanism of thymol against S. iniae were evaluated by electron microscopy, lactate dehydrogenase, DNA and protein leakage and transcriptomic analysis. Thymol exhibited potent antibacterial activity against S. iniae in vitro, and the MIC and MBC were 128 and 256µg/mL, respectively. SEM and TEM images showed that the cell membrane and cell wall were damaged, and the cells were abnormally enlarged and divided. 2MIC thymol disrupted the integrity of cell walls and membranes, resulting in the release of intracellular macromolecules including nucleotides, proteins and inorganic ions. The results of transcriptomic analysis indicated that thymol interfered with energy metabolism and membrane transport, affected DNA replication, repair and transcription in S. iniae. In vivo studies showed that thymol had a protective effect on experimental S. iniae infection in channel catfish. It could reduce the cumulative mortality of channel catfish and the number of S. iniae colonization in tissues, and increase the activities of non-specific immune enzymes in serum, including catalase, superoxide dismutase, lysozyme and acid phosphatase. Taken together, these findings suggested that thymol may be a candidate plant agent to replace traditional antibiotics for the prevention and treatment of S. iniae infection.

Regional analysis of the characteristics and potential risks of bacterial pathogen resistance under high-pressure antibiotic application.

The study aimed to perform a regional investigation of the antibiotic resistance characteristics (ARCHs) of zoonotic pathogens in environments of high antibiotic pressure to observe the future trend of antibiotic application. In this study, an ARCH analysis of the animal pathogens was conducted in the Sichuan Basin with an area of about 180,000 km2 and an estimated high antibiotic application exceeding 2000 tons. A total of 388 bacterial strains from nine species were isolated during 2013-2021. The results showed a dynamic change in the pathogen resistance in the Sichuan Basin with no apparent temporal trend. Fifty-two of 54 antibiotic resistance phenotypes (ARPs) and 180/218 antibiotic resistance genes (ARGs) were detected in this region. The antibiotic resistance in the classification of ß-lactam, sulfanilamide, and tetracycline had a relatively high detective rate, with 33-58% of ARPs and about 29.7% of ARGs. The isolates from terrestrial animals generally had higher ARPs and ARGs than aquatic animals. Most pathogens carried 5-11 ARPs, and each isolate carried 19.7 ARGs on average. Our result showed that there was a complicated accumulation of ARGs under high antibiotic pressure. Besides, the unique strain in the Sichuan Basin did not show higher resistance rates compared with the World Health Organization data, possibly due to fitness cost. However, the complex ARCH under high pressure still deserves attention to prevent the emergence of super-resistant bacteria.

Autophagy induced by largemouth bass virus inhibits virus replication and apoptosis in epithelioma papulosum cyprini cells.

Autophagy and apoptosis play important roles in the occurrence and development of diseases. Largemouth bass virus (LMBV) is a primary agent that causes infectious skin ulcerative syndrome in largemouth bass and threatens the aquaculture of the species. We investigated the relationship between LMBV and autophagy, as well as the effect of autophagy on apoptosis induced by LMBV. Results showed that LMBV could induce autophagy in epithelioma papulosum cyprinid (EPC) cells. There was also an increase in LC3-II protein and decrease in p62 protein, along with autophagosome-like membranous vesicles and punctate autophagosomes fluorescent spots being observed in EPC cells. Enhancing autophagy inhibited the replication of LMBV and apoptosis in EPC cells while inhibiting autophagy produced the opposite effect. These results offer new insights into the pathogenesis of LMBV and anti-LMBV strategies.

The potential risk of antibiotic resistance of Streptococcus iniae in sturgeon cultivation in Sichuan, China.

Sichuan, located in the upper reaches of the Yangtze River, is the gathering place of many rivers and plays an important role in sturgeon aquaculture and wild sturgeon protection in China, where it suffered the severe influence of Streptococcus iniae infection in sturgeon. However, the annual thousands of tons of antibiotic usage in Sichuan may accumulate in water and cause obstacles to the prevention of S. iniae infection. In contrast, the regional antibiotic resistance characteristics have been rarely unknown. Seventeen S. iniae strains were collected from the major sturgeon culture areas in Sichuan, and the genotyping and the distribution of antibiotic resistance profiles (ARPs) and genes (ARGs) of S. iniae were established in this study. The results showed that the isolates could be divided into four subtypes by pulsed-field gel electrophoresis analysis. Besides, most isolates showed multiple resistance to the antibiotic such as amikacin, neomycin, enrofloxacin, lincomycin, and sulfamethoxazole. Also, sturgeon-derived S. iniae has a relatively low similarity with other fish-derived S. iniae in the world but high similarity with three animal-derived pathogens from Sichuan in previous studies. Moreover, a total of 37 ARGs were detected positively based on 95 ARGs detection, in which aac(6')-Ib(aka aacA4)-01, aac(6')-Ib(aka aacA4)-02, aadA1, floR, blaTEM, sulA/folP-03, and tetA-02 were most prevalent. Our study indicated that the ARGs of sturgeon-derived S. iniae were significantly enhanced compared with the ATCC29178 strains and have a risk of accessing more ARGs from other bacteria in water in Sichuan. This study claimed that sturgeon has a potential risk in the prevention and control of Streptococcosis in Sichuan, the upper reaches of Yangtze River, based on the antibiotic resistance analysis of S. iniae, and it may also increase the risk of highly resistant S. iniae transmission into the middle and lower reaches.

Nocardia seriolae: a serious threat to the largemouth bass Micropterus salmoides industry in Southwest China.

Nocardia seriolae is the causative agent of nocardiosis in both marine and freshwater fish. Here, we report on multiple outbreaks of nocardiosis associated with elevated mortality (23-35%) in farmed largemouth bass in Sichuan, China, from 2017 to 2018. A total of 9 strains isolated from diseased largemouth bass were identified as N. seriolae by phenotypic characterization, 16S rRNA and hsp65 gene sequence analysis. The clinical signs of infected largemouth bass included hemorrhage, skin ulcers and prominent tubercles varying in size in the gill, liver, spleen and kidney. Experimental infection indicated that these isolates were the pathogens responsible for the mortalities. In vitro antibacterial activities of 12 antibiotics against N. seriolae isolates were determined as minimum inhibitory concentrations. Histopathological observation of diseased fish infected with N. seriolae showed necrotizing granulomatous hepatitis, nephritis, splenitis, epithelial hypertrophy and hyperplasia with degenerative changes of the epithelium in the gill. Large quantities of bacterial aggregates were found in the necrotic area of the granuloma by Lillie-Twort Gram stain and immunocytochemistry. Our findings indicated that N. seriolae is a serious threat to the largemouth bass Micropterus salmoides industry in Southwest China.

Molecular and Functional Characterization of Inhibitor of Apoptosis Proteins (IAP, BIRP) in Echinococcus granulosus.

The larval stage of Echinococcus granulosus sensu lato, resulting in cystic echinococcosis, a parasitic zoonosis, causes huge economic losses to the livestock industry and poses a threat to public health. Inhibitor of apoptosis proteins (IAPs) is a class of endogenous anti-apoptotic family, which plays a significant functional role in the regulation of organism's development. Herein, to explore potential functions of IAPs in E. granulosus, two members of IAPs from E. granulosus (Eg-IAP and Eg-BIRP) were cloned, expressed, and molecularly characterized. Eg-IAP and Eg-BIRP encoded putative 331 and 168 residue proteins, respectively. Bioinformatic analysis showed that both proteins contained a type II BIR domain-the essential functional domain of IAPs. Fluorescence immunohistochemistry revealed that both proteins were ubiquitously localized in all life-cycle stages of E. granulosus. Our fluorescent quantitative PCR (RT-qPCR) results revealed relatively higher transcription levels of two Eg-IAPs in protoscoleces (PSCs) compared to the 18-day strobilated worms. We further used different concentrations of LCL161, a Smac-mimetic pan-IAPs inhibitor, to induce the apoptosis in PSCs in vitro, and revealed that the survival rate of PSCs and transcription levels of both genes were negatively correlated with the concentration of LCL161. While the results of light microscopy, transmission electron microscopy (TEM), and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay also showed a higher apoptotic rate in PSCs with the increasing concentrations of LCL161. Taken together, our findings provide the reasonable evidence that both Eg-IAP and Eg-BIRP have potential implication in critical anti-apoptotic roles during the development of E. granulosus.

Characterization and genomic analysis of a ranavirus associated with cultured black-spotted pond frogs (Rana nigromaculata) tadpoles mortalities in China.

Ranaviruses are globally emerging pathogens causing mass mortality in both farmed and wild populations. In this study, we confirmed that a novel ranavirus was related to mass die-offs of black-spotted pond frogs (Rana nigromaculata) in Sichuan Province, China. Histological examination revealed marked degeneration or necrosis in parenchymatous organs, and intracytoplasmic inclusions can be observed in the liver and intestine cells. The virus was isolated from diseased tadpoles (tentatively named the virus as RNRV), and the ranavirus-like particles could be observed in infected EPC cells by the electron microscope. The tadpoles challenged with the isolated virus displayed similar clinical symptoms and pathogenicity as those naturally infected. Physicochemical characteristics showed that it was sensitive to chloroform treatment, trypsin treatment, heating treatment, acidity and alkalinity. Biological characteristics showed that RNRV can induce cytopathic effect (CPE) in various fish cell lines and the optimum growth temperature is 25~28°C. In addition, the complete genome sequence of RNRV was determined (GenBank accession number MG791866) and analysed. The results showed that the genome of RNRV consists of 104,286 bp containing 55.2% GC content and 104 predicted ORFs were identified in the genome. Phylogenetic analysis of the complete genome revealed that RNRV should belong to the FV3-like group in genus Ranavirus, and is more closely related to RGV and STIV. These studies confirmed that the RNRV was the causative agent of this natural epizootic event and genome analysis indicated that it belongs to the FV3-like group. In addition, viral physicochemical and biological characteristics will provide a scientific basis for prevention and control.

Expression and serodiagnostic potential of antigen B and thioredoxin peroxidase from Taenia multiceps.

Coenurosis is a serious parasitic disease of herbivorous animals caused by the metacestode of Taenia multiceps (Coenurus cerebralis). Accordingly, a significant amount of research is currently dedicated to the development of appropriate antigens for use in rapid and accurate coenurosis diagnosis kits. In the present study, antigen B (AgB) and thioredoxin peroxidase (TPx) from T. multiceps were cloned and expressed using a prokaryotic system, molecular characterization of Tm-AgB was determined by bioinformatical analyses. The serological diagnostic potentials of rTm-AgB and rTm-TPx were evaluated by indirect ELISA and compared with those of previously reported rTm-AnxB2, rTm-HSP70, and rTm-GST. The results showed that Tm-AgB is a specific lipoprotein of cestodes with good thermal stability. The ELISA assay showed that rTm-AgB exhibited a sensitivity of 95.8% and a specificity of 87.5%, indicating its strong potential for serological diagnosis of T. multiceps.

Multiplex genome editing by natural transformation in Vibrio mimicus with potential application in attenuated vaccine development.

Vibrio mimicus (V. mimicus) is a significant pathogen in freshwater catfish, though knowledge of virulence determinants and effective vaccine is lacking. Multiplex genome editing by natural transformation (MuGENT) is an easy knockout method, which has successfully used in various bacteria except for V. mimicus. Here, we found V. mimicus strain SCCF01 can uptake exogenous DNA and insert it into genome by natural transformation assay. Subsequently, we exploited this property to make five mutants (△Hem, △TS1, △TS2, △TS1△TS2, and △II), and removed the antibiotic resistance marker by Flp-recombination. Finally, all of the mutants were identified by PCR and RT-PCR. The results showed that combination of natural transformation and FLP-recombination can be applied successfully to generate targeted gene disruptions without the antibiotic resistance marker in V. mimicus. In addition, the five mutants showed mutant could be inherited after several subcultures and a 668-fold decrease in the virulence to yellow catfish (Pelteobagrus fulvidraco). This study provides a convenient method for the genetic manipulation of V. mimicus. It will facilitate the identification and characterization of V. mimicus virulence factors and eventually contribute to a better understanding of V. mimicus pathogenicity and development of attenuated vaccine.

Cloning, expression, characterization, and immunological properties of citrate synthase from Echinococcus granulosus.

The larval stages of the tapeworm Echinococcus granulosus (Cestoda: Taeniidae) are the causative agent of cystic echinococcosis, one of the most important parasitic zoonoses worldwide. E. granulosus has a complete pathway for the tricarboxylic acid cycle (TCA), in which citrate synthase (CS) is the key enzyme. Here, we cloned and expressed CS from E. granulosus (Eg-CS) and report its molecular characterization. The localization of this protein during different developmental stages and mRNA expression patterns during H2O2 treatment were determined. We found that Eg-CS is a highly conserved protein, consisting of 466 amino acids. In western blotting assays, recombinant Eg-CS (rEg-CS) reacted with E. granulosus-positive sheep sera and anti-rEg-CS rabbit sera, indicating that Eg-CS has good antigenicity and immunoreactivity. Localization studies, performed using immunohistochemistry, showed that Eg-CS is ubiquitously expressed in the larva, germinal layer, and adult worm sections of E. granulosus. Eg-CS mRNA expression levels increased following H2O2 exposure. In conclusion, citrate synthase might be involved in the metabolic process in E. granulosus. An assessment of the serodiagnostic potential of rEg-CS based on indirect ELISA showed that, although sensitivity (93.55%) and specificity (80.49%) are high, cross-reactivity with other parasites precludes its use as a diagnostic antigen.

Molecular Characterization of a Dirofilaria immitis Cysteine Protease Inhibitor (Cystatin) and Its Possible Role in Filarial Immune Evasion.

Infection with canine heartworm (Dirofilaria immitis), spread via mosquito vectors, causes coughing, asthma, pneumonia, and bronchitis in humans and other animals. The disease is especially severe and often fatal in dogs and represents a serious threat to public health worldwide. Cysteine protease inhibitors (CPIs), also known as cystatins, are major immunomodulators of the host immune response during nematode infections. Herein, we cloned and expressed the cystatin Di-CPI from D. immitis. Sequence analysis revealed two specific cystatin-like domains, a Q-x-V-x-G motif, and a SND motif. Phylogenetic analysis indicates that Di-CPI is a member of the second subgroup of nematode type II cystatins. Probing of D. immitis total proteins with anti-rDi-CPI polyclonal antibody revealed a weak signal, and immunofluorescence-based histochemical analysis showed that native Di-CPI is mainly localized in the cuticle of male and female worms and the gut of male worms. Treatment of canine peripheral blood mononuclear cells (PMBCs) with recombinant Di-CPI induced a Th2-type immune response characterized by high expression of the anti-inflammatory factor interleukin-10. Proliferation assays showed that Di-CPI inhibits the proliferation of canine PMBCs by 15%. Together, the results indicate that Di-CPI might be related to cellular hyporesponsiveness in dirofilariasis and may help D. immitis to evade the host immune system.

Molecular characterisation and expression analysis of two heat-shock proteins in Taenia multiceps.

BACKGROUND: Taenia multiceps is a harmful tapeworm and its larval form (coenurus cerebralis) is the causative agent of coenurosis, a disease affecting the health of herbivores, resulting in great economic loss to animal husbandry. Heat-shock proteins (HSPs), expressed in all prokaryotes and eukaryotes, act as molecular chaperones and can affect pathogenicity. METHODS: Herein, cDNAs of T. multiceps genes Tm-HSP60 and Tm-p36 were cloned and molecularly characterised by bioinformatics analyses. The immunogenicity and immunoreactivity of recombinant rTm-HSP60 and rTm-p36 proteins were investigated by immunoblotting and indirect ELISA was established to evaluate their serodiagnostic potential. Tissue localisation and transcriptional level at different life stages of T. multiceps were determined by immunohistochemical and quantitative real-time PCR analyses. RESULT: The 533 residue rTm-HSP60 and the 314 residue rTm-p36 proteins share typical highly conserved features of HSPs. Tm-p36 shares structural characteristics with metazoan small HSPs, with two N-terminal α-crystallin domains. Compared with Tm-p36, Tm-HSP60 displayed stronger immunogenicity, and the indirect ELISA based on rTm-HSP60 exhibited a sensitivity of 83.3% and a specificity of 87.5%, while rTm-p36 was not suitable to develop indirect ELISA. Tm-HSP60 was widely distributed in all stages of T. multiceps, albeit at relatively low levels, while Tm-p36 was specifically distributed in the protoscolex and oncosphere. CONCLUSIONS: The sequence, structural and functional analyses of these two HSPs indicates that they may play important roles in the life-cycle of T. multiceps as molecular chaperones. Tm-HSP60 displayed stronger immunogenicity compare to Tm-p36, and has the potential for antibody detection. Tm-p36 was strongly associated with the activation of oncospheres and has potential interest for vaccination.

Adjuvant effects of interleukin-2 co-expression with VP60 in an oral vaccine delivered by attenuated Salmonella typhimurium against rabbit hemorrhagic disease.

Rabbit hemorrhagic disease (RHD) is a highly contagious infection that has caused significant damage to the rabbit industry since 1984. Inactivated vaccines, the currently used prevention measures, are effective in controlling RHD. However, these vaccines are derived from the livers of infected rabbits, which constitutes a major concern in terms of animal welfare and safety. Administration of DNA vaccines in collaboration with appropriate adjuvants, in particular, cytokines, to strengthen the immune response presents a novel optimization strategy to generate more efficient vaccines. In this study, the adjuvant effect of interleukin (IL)-2 co-expression with the VP60 gene in a DNA vaccine was evaluated. In total, four groups of 60 RHD virus (RHDV)-free rabbits (30 days old) were orally or subcutaneously administered recombinant SL7207-pVAX1-IL2-VP60, SL7207-pVAX1-VP60, SL7207-pVAX1 bacteria or the commercial inactive vaccine, and the induced immunity evaluated by challenge with the RHDV(Y8504/China) strain on day 56. The Recombinant SL7207-pVAX1-IL2-VP60 induced a higher level of antibodies than the vaccine SL7207-pVAX1-VP60 and inactivated vaccines to a significant extent. The concentrations of interleukin (IL)-4 were markedly higher than those in groups immunized with the naked or inactive vaccine alone. Furthermore, the fusion gene vaccine provided higher protection (93.33%) after virus challenge relative to immunization with the single gene (SL7207-pVAX1-VP60). The collective results indicate that recombinant SL7207-pVAX1-IL-2-VP60 bacteria exert enhanced protective effects against RHDV and therefore present a strong candidate as a potential vaccine. Moreover, IL-2 enhanced both humoral and cellular responses, highlighting the utility of rabbit IL-2 as an effective adjuvant.

Molecular characterization, phylogenetic analysis and adjuvant effect of channel catfish interleukin-1ßs against Streptococcus iniae.

Channel catfish is one of the most extensively cultured species worldwide, which is widely used as a classical model for comparative immunology. Interleukin-1ß (IL1ß) is an immunoregulatory cytokine with the potential to enhance the immune response induced by vaccines in many animals. To characterize the molecular characterization and identify the immunoadjuvant role of channel catfish IL1ß, molecular cloning, phylogenetic analysis, and expression of two IL1ß genes were performed, the bioactivity of their recombinant proteins (rIL1ß1 and rIL1ß2) were detected in vitro and their adjuvant effects on a subunit vaccine encoding C5a peptidase (pSCPI) of Streptococcus iniae were evaluated. The results indicated that two IL1ßs remained highly conserved possessing five conserved motifs compared with other fish IL1ßs, although there were 28 nucleotide differences and 16 amino acid differences between channel catfish IL1ß1 and IL1ß2. Analysis of the ratios of nonsynonymous (dN) and synonymous (dS) substitutions revealed that fish IL1ß genes were subjected to negative/purifying selection with global dN/dS ratios value 0.425. The results of adjuvant effect showed that compared with injection of pSCPI alone, co-injecting pSCPI with both rIL1ß1 and rIL1ß2 significantly enhanced antibody levels, serum bactericidal activity, lysozyme activity, alternative complement hemolytic activity, and the expression of endogenous IL1ß and TNF-α in head kidney and spleen. Although vaccination with rIL1ß1 or rIL1ß2 failed to offer immunoprotection against S. iniae infection, the RPS (relative percent survival) of pSCPI+rIL1ß1 and pSCPI+rIL1ß2 groups were both higher than pSCPI alone (RPS, 50%), with 64.26% and 60.71%, respectively. Moreover, pSCPI+rIL1ß1+rIL1ß2 offered significantly higher (P < 0.05) immunoprotection (RPS, 75%) against S. iniae infection than pSCPI alone. Our present results not only enrich the molecular structure study of fish IL1ßs but also signify that two recombinant channel catfish IL1ßs can be used as potential adjuvants in a subunit vaccine model against bacterial infection, which are of profound importance to prevent and control bacterial disease in channel catfish.