Rhodobacter sphaeroides supplementation improves defense ability of Chinese mitten crab Eriocheir sinensis against Shewanella putrefaciens infection via intestinal flora and metabolism regulation.

Shewanella putrefaciens is a vital bacterial pathogen implicated in serious diseases in Chinese mitten crab Eriocheir sinensis. Yet the use of probiotics to improve the defense ability of E. sinensis against S. putrefaciens infection remains poorly understood. In the present study, the protective effect of dietary R. sphaeroides against S. putrefaciens infection in E. sinensis was evaluated through antioxidant capability, immune response, and survival under bacterial challenge assays, and its protective mechanism was further explored using a combination of intestinal flora and metabolome assays. Our results indicated that dietary R. sphaeroides could significantly improve immunity and antioxidant ability of Chinese mitten crabs, thereby strengthening their disease resistance with the relative percentage survival of 81.09% against S. putrefaciens. In addition, dietary R. sphaeroides could significantly alter the intestinal microbial composition and intestinal metabolism of crabs, causing not only the reduction of potential threatening pathogen load but also the increase of differential metabolites in tryptophan metabolism, pyrimidine metabolism, and glycerophospholipid metabolism. Furthermore, the regulation of differential metabolites such as N-Acetylserotonin positively correlated with beneficial Rhodobacter could be a potential protection strategy for Shewanella infection. To the best of our knowledge, this is the first study to illustrate the protective effect and mechanism of R. sphaeroides supplementation to protect E. sinensis against S. putrefaciens infection.

Shewanella putrefaciens as an emerging pathogen of hepatopancreas necrosis disease in Chinese mitten crab Eriocheir sinensis.

Shewanella putrefaciens has been recognized as an emerging important pathogen in aquaculture. However, scarce information is available on the characterization and microbial control of S. putrefaciens as a causal agent of hepatopancreas necrosis disease in Chinese mitten crab Eriocheir sinensis. In this study, a multi-resistant S. putrefaciens isolate (DZ-A) was identified as a causal pathogen of hepatopancreas necrosis disease in Chinese mitten crabs. It showed a lethal dose (LD50) of 2.20 × 105 CFU ml-1 in Chinese mitten crabs, and multiple resistance to aminoglycoside, chloramphenicol, macrolide, penicillin, peptide, and tetracycline antimicrobials. In addition, Bdellovibrio powder exhibited a significant antibacterial effect against the pathogenic S. putrefaciens, and conferred significant protection to challenged Chinese mitten crabs with relative percentage survivals of 80.00% to 93.33% via significant improvement in their immune response and antioxidant capability. The findings of this study provide valuable insights into the phenotypic characterization and biological control of pathogenic S. putrefaciens in Chinese mitten crabs.

Tomato protein Rx4 mediates the hypersensitive response to Xanthomonas euvesicatoria pv. perforans race T3.

Bacterial spot, which is caused by several Xanthomonas species, is an economically important disease in tomato (Solanum lycopersicum). Great efforts have been made for the identification of resistant sources and the genetic analysis of resistance. However, the development of resistant commercial varieties is slow due to the existence of multiple species of the pathogen and a poor understanding of the resistance mechanism in tomato. The current study revealed that the Rx4 gene encodes a nucleotide-binding leucine-rich repeat protein in the wild tomato species Solanum pimpinellifolium and specifically recognizes and confers a hypersensitive response (HR) to Xanthomonas euvesicatoria pv. perforans race T3 expressing the AvrXv3 avirulence protein. Complementation of the Rx4 gene in the susceptible tomato line Ohio 88119 using a transgenic approach resulted in HR, whereas knockout of the gene through CRISPR/Cas9 editing in resistant lines Hawaii 7981 and PI 128216 led to non-HR to race T3. Transcription of Rx4 was not induced by the presence of race T3. Furthermore, the Rx4 protein did not show physical interaction with AvrXv3 but interacted with SGT1-1 and RAR1. Virus-induced gene silencing of SGT1-1 and RAR1 in the resistant line PI128216 suppressed the HR to race T3. Taken together, our study confirms Rx4 is the gene conferring the HR to bacterial spot race T3 and reveals the potential roles of SGT1-1 and RAR1 as signals in the Rx4-mediated HR. This discovery represents a step forward in our understanding of the mechanism of resistance to bacterial spot in tomato and may have important implications for understanding plant-bacterial interactions.

Rhodobacter azotoformans supplementation improves defense ability of Chinese mitten crab Eriocheir sinensis against citrobacteriosis.

Rhodobacter probiotics are considered as good alternatives to antibiotics for aquaculture. Yet the beneficial effects of Rhodobacter on Chinese mitten crab Eriocheir sinensis are still unclear, and more functions of Rhodobacter supplementation need to be clarified. In this study, a 60-day feeding trial was performed to investigate the protective effects of R. azotoformans against citrobacteriosis in E. sinensis by growth performance, serum immunity, hepatopancreatic antioxidant capability, intestinal flora, and resistance to Citrobacter freundii challenge assays. The results showed that R. azotoformans supplementation significantly and dose-dependently increased weight gain and specific growth rate as well as activities of serum immune and hepatopancreatic antioxidant enzymes, leading to notable improvement in the growth performance, serum immunity and hepatopancreatic antioxidant status of E. sinensis. Besides, R. azotoformans supplementation significantly enhanced intestinal microbial abundance and diversity in E. sinensis, and conferred significant protection of the crabs against C. freundii challenge with seven-day survival rates of 70.0%-100.0%. To the best of our knowledge, this is the first study to reveal the protective effects of R. azotoformans against citrobacteriosis in E. sinensis.

Protective effects of Bacillus licheniformis against Citrobacter freundii infection in Chinese mitten crab Eriocheir sinensis.

Citrobacter freundii is an important bacterial pathogen that causes serious diseases in Chinese mitten crab Eriocheir sinensis. However, scarce information is available on the use of Bacillus licheniformis to protect E. sinensis from C. freundii infection by improving the immunity, antioxidant ability and intestinal flora. In the present study, a 60-day feeding trial was conducted to examine the potential effects of dietary supplementation with antagonistic B. licheniformis on the non-specific immunity, antioxidant capability, intestinal flora and resistance of E. sinensis to C. freundii infection. The results indicated that dietary supplementation of B. licheniformis could boost non-specific immunity and antioxidant capability of E. sinensis in a significant dose-dependent manner respectively by increasing serum lysozyme, superoxide dismutase, alkaline phosphatase, and catalase activities and hepatopancreatic superoxide dismutase, catalase, acid phosphatase activities. In addition, crabs fed diets with B. licheniformis displayed increased composition and diversity of the intestinal flora, and exhibited significant defense against C. freundii infection with the relative percentage survivals ranging from 61.9% to 100.0% for seven days. To our knowledge, this is the first report of antagonistic B. licheniformis as a supplement in Chinese mitten crab feed to effectively resist C. freundii infection by improving the non-specific immunity, antioxidant status and intestinal flora.

Effect of copper sulfate on Bdellovibrio growth and bacteriolytic activity towards gibel carp-pathogenic Aeromonas hydrophila.

The use of bdellovibrios has been regarded as an alternative to control multidrug-resistant pathogens and fish bacteriosis. However, scarce information is available on the potential of bdellovibrios in the presence of copper sulfate, which is an algicide widely used to treat cyanobacterial blooms in aquaculture. In the present study, the effects of copper sulfate at sublethal and lethal levels (0.1 and 1.0 mg·L-1) on Bdellovibrio sp. strain BDF-H16 were evaluated. The growth of Bdellovibrio sp. strain BDF-H16 was significantly promoted by both concentrations of copper sulfate, but less so by the lethal concentration. The bacteriolysis of gibel carp-pathogenic Aeromonas hydrophila by Bdellovibrio sp. strain BDF-H16 was also stimulated by copper sulfate in both solid and liquid media. However, Bdellovibrio sp. strain BDF-H16 with 0.1 mg·L-1 copper sulfate clearly inhibited infection of gibel carps by A. hydrophila better than Bdellovibrio sp. strain BDF-H16 with 1.0 mg·L-1 copper sulfate did.

Characterization of a new Lactobacillus salivarius strain engineered to express IBV multi-epitope antigens by chromosomal integration.

To obtain adhesive and safe lactic acid bacteria (LAB) strains for expressing heterologous antigens, we screened LAB inhabitants in intestine of Tibetan chickens by analyzing their adhesion and safety properties and the selected LAB was engineered to express heterologous antigen (UTEpi C-A) based on chromosomal integration strategy. We demonstrated that a new Lactobacillu salivarius TCMM17 strain is strongly adhesive to chicken intestinal epithelial cells, contains no endogenous plasmids, is susceptible to tested antimicrobials, and shows no toxicities. In order to examine the potential of TCMM17 strain as heterogenous antigen delivering vehicle, we introduced a UTEpi C-A expression cassette in its chromosome by constructing a non-replicative plasmid (pORI280-UUTEpi C-AD). The recombinant TCMM17 strain (∆TCMM17) stably was found to keep the gene cassette through 50 generations, and successfully displayed EpiC encoded by the cassette on its surface. This work provides a universal platform for development of novel oral vaccines and expression of further antigens of avian pathogens.

Development of a multi-epitope antigen of S protein-based ELISA for antibodies detection against infectious bronchitis virus.

An indirect enzyme-linked immunosorbent assay (ELISA) method based on a novel multi-epitope antigen of S protein (SE) was developed for antibodies detection against infectious bronchitis virus (IBV). The multi-epitope antigen SE protein was designed by arranging three S gene fragments (166-247 aa, S1 gene; 501-515 aa, S1 gene; 8-30 aa, S2 gene) in tandem. It was identified to be approximately 32 kDa as a His-tagged fusion protein and can bind IBV positive serum by western blot analysis. The conditions of the SE-ELISA method were optimized. The optimal concentration of the coating antigen SE was 3.689 µg/mL and the dilution of the primary antibodies was identified as 1:1000 using a checkerboard titration. The cut-off OD450 value was established at 0.332. The relative sensitivity and specificity between the SE-ELISA and IDEXX ELISA kit were 92.38 and 89.83%, respectively, with an accuracy of 91.46%. This assay is sensitive and specific for detection of antibodies against IBV.

Development of an ELISA based on a multi-fragment antigen of infectious bronchitis virus for antibodies detection.

OBJECTIVES: To develop a cost-effective ELISA for detection of antibodies against infectious bronchitis virus (IBV) by using a multi-fragment protein as coating antigen. RESULTS: A multi-fragment antigen, termed BE, which was composed of eight antigenic fragments selected from the three major proteins (S, M, and N) of IBV, was expressed in Escherichia coli. The entire protein had a molecular weight of 61.5 kDa. In addition to it, a smaller truncated protein was also produced; both could react with IBV-positive serum. Next, an indirect ELISA (BE-ELISA) was developed. Coefficients of variation of this assay were lower than 10 %, and no cross-reactivity between the coated antigen BE and antiserum against newcastle disease virus, avian influenza virus, or infectious bursal disease virus was observed. The performance of BE-ELISA was evaluated, and showed 95.4 % coincidence ratio with the whole virus based-ELISA (IDEXX). CONCLUSIONS: The multi-fragment antigen (BE) may represent a promising alternative to the whole virus without safety problems, and this newly established ELISA provides an effective method for anti-IBV antibody detection.

Vibrio cholerae pathogen from the freshwater-cultured whiteleg shrimp Penaeus vannamei and control with Bdellovibrio bacteriovorus.

Vibriosis has become a major global economic problem in freshwater-farmed whiteleg shrimp (Penaeus vannamei). The prevention and control of vibriosis are now priority research topics. In this study, a pathogenic strain (QH) was isolated from vibriosis-infected freshwater-farmed P. vannamei that resulted in leg yellowing and was identified as a Vibrio cholerae isolate through phylogenetic analysis and the API 32GN system. A phylogenetic tree that was constructed using the neighbor-joining method further confirmed the QH isolate as a V. cholerae strain. A virulent outer membrane protein (ompU) gene was found to be present in the QH isolate, which further confirmed its pathogenicity. In addition, Bdellovibrio bacteriovorus conferred significant protection against V. cholerae: B. bacteriovorus exhibited significant bacteriolytic effects on the V. cholerae pathogen, possessed a wide prey range that included Vibrio pathogens, and displayed a positive protective efficacy against experimental V. cholerae infection in P. vannamei. To the best of our knowledge, this is the first report of the control of shrimp pathogen V. cholerae with B. bacteriovorus.

A chimeric transcript containing Psy1 and a potential mRNA is associated with yellow flesh color in tomato accession PI 114490.

Carotenoid content is the primary determinant of fruit color that affects nutritional value and appearance in tomato. Phytoene synthase (PSY) is the key regulatory enzyme in the carotenoid biosynthesis pathway. Absent function of PSY1 in tomato fruit results in yellow flesh phenotype. We, here, report that two different transcripts, a wild-type (Psy1) and a chimeric mRNA (Psy1/Unknown), exist in a yellow-fruited tomato accession PI 114490. Psy1/Unknown is generated by joining exons from two different genes, Psy1 and an unknown gene, transcribed using both complementary DNA strands. The Psy1 shows low expression in the fruit of PI 114490, while the expression of Psy1/Unknown in the fruit of PI 114490 shows the same pattern as Psy1 in red fruit. The PSY1/Unknown has a lower function than PSY1 in a bacterial expression system. Coincidence of one single-nucleotide polymorphism (SNP) in the fourth intron and one simple sequence repeat (SSR) with 19 AT repeats in the downstream sequence of Psy1 gene with Psy1/Unknown in a set of yellow-fruited tomato lines indicates that Psy1/Unknown might be caused by the SNP and/or SSR. One possible explanation of these observations is trans-splicing. Severely reduced Psy1 transcript caused by Psy1/Unknown results in low accumulation of carotenoid and yellow flesh in PI 114490.

Identification of a Proteus penneri isolate as the causal agent of red body disease of the cultured white shrimp Penaeus vannamei and its control with Bdellovibrio bacteriovorus.

Bacteriosis has become a major economic problem in the farming of the Pacific white shrimp Penaeus vannamei. However, no definitive data are available about Proteus penneri infection in cultured P. vannamei and its control. In this study, a virulent strain NC was isolated from diseased P. vannamei suffering from red body disease and identified as a P. penneri isolate through phylogenetic analysis and ATB 32GN system. A phylogenetic constructed tree using the neighbour-joining method identified the NC isolate as a P. penneri strain. In addition, Bdellovibrio bacteriovorus conferred significant protection against P. penneri: it exhibited significant bacteriolytic effects on the pathogenic P. penneri, had a wide prey range towards Proteus pathogens, and displayed a good protective efficacy on experimental P. penneri infection in P. vannamei. To the best of our knowledge, this is the first report of farmed P. vannamei infected with P. penneri and its control with B. bacteriovorus.

Identification of up-regulated proteins potentially involved in the antagonism mechanism of Bacillus amyloliquefaciens G1.

The use of Bacillus probiotics has been demonstrated as a promising method in the biocontrol of bacterial diseases in aquaculture. However, the molecular antibacterial mechanism of Bacillus still remains unclear. In order to explore the antibacterial mechanism of the potential antagonistic Bacillus amyloliquefaciens strain G1, comparative proteomics between B. amyloliquefaciens strain G1 and its non-antagonistic mutant strain was investigated. The 2-dimensional electrophoresis gel maps of their total extracted proteins were described and 42 different proteins were found to be highly expressed in strain G1 in comparison with those in the mutant strain. 35 of these up-regulated proteins were successfully identified using MALDI-TOF-TOF MS and databank analysis, and their biological functions were analyzed through the KEGG database. The increased expression of these proteins suggested that high levels of energy metabolism, biosynthesis and stress resistance could play important roles in strain G1's antagonism. To our knowledge, this is the first report on the proteins involved in the antagonism mechanism of B. amyloliquefaciens using a proteomic approach and the proteomic data also contribute to a better understanding of the molecular basis for the antagonism of B. amyloliquefaciens.

Lactococcus lactis anchoring avian infectious bronchitis virus multi-epitope peptide EpiC induced specific immune responses in chickens.

Mucosal immunity is critical in preventing infectious bronchitis virus (IBV) infection. To deliver viral antigens to the mucosal immune system of chickens safely and effectively, we constructed a Lactococcus lactis strain carrying IBV multi-epitope gene EpiC fused with the gene of the cell-wall anchoring domain of Staphylococcus aureus protein A. SDS-PAGE and Western blot results indicated that the fused peptide was located partially on the cell surface. Oral and nasal inoculation with the recombinant L. lactis of chickens elicited significantly high humoral and mucosal immune responses, especially in the nasally immunized group. Eighty percent chickens of the nasally immunized group with recombinant L. lactis did not show any clinical signs after a lethal dose challenge with IBV SAIBk strain, while all the non-recombinant L. lactis immunized chickens exhibited obvious and typical symptoms. These results indicate that needle-free recombinant lactococci anchoring the IBV antigen makes a promising vaccine candidate against the spread of IB.

Biofilm electrode reactor coupled manganese ore substrate up-flow microbial fuel cell-constructed wetland system: High removal efficiencies of antibiotic, zinc (II), and the corresponding antibiotic resistance genes.

A coupled system comprised of a biofilm electrode reactor (BER) and a manganese ore substrate microbial fuel cell-constructed wetland (MFC-CW) system was used to remove co-exposed antibiotic and Zn (II), as well as simultaneously reduce copies of antibiotic resistance genes (ARGs) in the current study. In this system, BER primarily reduced the concentrations of antibiotics and Zn (II), and the effluent was used as the input to the MFC-CW, thereby providing electricity to BER. Co-exposure to a high concentration of Zn (II) decreased the relative abundances (RAs) of ARGs in the BER effluent, whereas the remaining sub-lethal concentration of Zn (II) increased the RAs of ARGs in the MFC-CW effluent. Even though the absolute copies of ARGs in the effluents increased during co-exposure, the total number of target ARG copies in the effluent of MFC-CW was significantly lower than that of BER. Moreover, BER pre-treatment eliminated most of Zn (II), which improved the electrical power generation characteristic of the MFC-CW unit. Correspondingly, the bacterial community and the ARGs hosts were analyzed to demonstrate the mechanism. In conclusion, the coupled system demonstrates significant potential to reduce antibiotics, Zn (II) and environmental risks posed by ARGs.

Expression of avian infectious bronchitis virus multi-epitope based peptide EpiC in Lactococcus lactis for oral immunization of chickens.

The avian infectious bronchitis virus (IBV) multi-epitope based peptide EpiC was found to be effective in inducing strong humoral and cellular responses against IBV. In this study, the gene EpiC was introduced into Lactococcus lactis NZ3900, and three recombinant strains expressing EpiC in intracellular and extracellular forms were constructed. SDS-PAGE and Western blot results indicated that EpiC was successfully expressed and had good immunoreactivity with chicken anti-IBV serum. Fusion of the signal pepitide gene SPusp45 and the nine-peptide LEISSTCDA encoding oligonucleotide to EpiC increased the secretion of EpiC, but reduced the total yields of EpiC. Oral immunization to specific-pathogen-free (SPF) chickens with recombinant strains induced significantly higher levels of humoral immune responses, and provided protection against lethal dose challenge by the IBV SAIBk strain. These results indicate that it is feasible to use L. lactis as an antigen delivery vehicle in developing oral vaccines against IBV infection.

Phenotypic and genomic characterization of pathogenic Providencia rettgeri from kuruma shrimp Marsupenaeus japonicus.

Providencia rettgeri has been recognized as a zoonotic pathogen of humans and aquaculture animals and has become a global public health concern. However, scarce information is available on the characterization of pathogenic P. rettgeri from kuruma shrimp Marsupenaeus japonicus. In the present study, a P. rettgeri isolate (KM4) was confirmed as a causative agent of red leg disease in cultured M. japonicus, which showed a median lethal dose (LD50 ) value of 5.01 × 105 CFU·ml-1 and had multiple resistances to aminoglycosides, sulfonamides, and tetracycline antimicrobials used in aquaculture. In addition, the whole genome of isolate KM4 was sequenced and found to consist of a single circular chromosome of 4,378,712 bp and a circular plasmid of 171,394 bp. The genome sequence analysis further revealed the presence of potential virulence and antibiotic resistance genes in isolate KM4, which probably rendered this isolate particularly virulent. To our knowledge, this is the first study to characterize P. rettgeri pathogens from kuruma shrimp infected with red leg disease. The findings of this study can provide novel insights into the presence and distribution of pathogenicity-associated genes in shrimp-pathogenic P. rettgeri.

Bacillus amyloliquefaciens G1: A Potential Antagonistic Bacterium against Eel-Pathogenic Aeromonas hydrophila.

Recent studies have revealed that the use of probiotics is an alternative to control marine aeromonas. However, few probiotics are available against Aeromonas hydrophila infections in eels. In the present study, a potential antagonistic strain G1 against the eel-pathogenic A. hydrophila was isolated from sediment underlying brackish water. Its extracellular products with antibacterial activities were shown to be stable under wide range of pH, temperature, and proteinase K. It was initially identified as Bacillus amyloliquefaciens using API identification kits and confirmed to be B. amyloliquefaciens strain (GenBank accession number DQ422953) by phylogenetic analysis. In addition, it was shown to be safe for mammalians, had a wide anti-A. hydrophila spectrum, and exhibited significant effects on inhibiting the growth of the eel-pathogenic A. hydrophila both in vitro and in vivo. To the best of our knowledge, this is the first report on a promising antagonistic Bacillus amyloliquefaciens strain from brackish water sediment against eel-pathogenic A. hydrophila.

Encapsulated Bdellovibrio Powder as a Potential Bio-Disinfectant against Whiteleg Shrimp-Pathogenic Vibrios.

Liquid preparations of bdellovibrios are currently commercialized as water quality improvers to control bacterial pathogens in whiteleg shrimp Penaeus vannamei. However, the efficacy of these liquid preparations is significantly impaired due to a dramatic loss of viable cells during long-term room temperature storage. Thus, new formulations of bdellovibrios are greatly needed for high-stablility room-temperature storage. In the present study, the encapsulated powder of Bdellovibrio sp. strain F16 was prepared using spray drying with 20 g L-1 gelatin as the coating material under a spray flow of 750 L h-1, a feed rate of 12 mL min-1, and an air inlet temperature of 140 °C. It was found to have a cell density of 5.4 × 107 PFU g-1 and to have spherical microparticles with a wrinkled surface and a diameter of 3 µm to 12 µm. In addition, the encapsulated Bdellovibrio powder presented good storage stability with its cell density still remaining at 3.5 × 107 PFU g-1 after 120 days of room-temperature storage; it was safe for freshwater-farmed whiteleg shrimp with an LD50 over 1200 mg L-1, and it exhibited significant antibacterial and protective effects at 0.8 mg L-1 against shrimp-pathogenic vibrios. To our knowledge, this is the first report on a promising Bdellovibrio powder against shrimp vibrios with high stable room-temperature storage.

Aeromonas hydrophila, an Emerging Causative Agent of Freshwater-Farmed Whiteleg shrimp Litopenaeus vannamei.

Aeromonas hydrophila is a well-known bacterial pathogen associated with mass mortalities in aquaculture. Yet, few reports are available on whiteleg shrimp-pathogenic A. hydrophila. In the present study, a virulent isolate WS05 was confirmed as a causative agent of diseased freshwater-cultured whiteleg shrimp and showed a mean lethal dose (LD50) value of 4.8 × 104 CFU mL-1. It was identified phenotypically and molecularly as an A. hydrophila strain, and exhibited susceptibility to several veterinary antibiotics extensively used in aquaculture, including cotrimoxazole, doxycycline, florfenicol, neomycin, and tetracycline. In view of the strongest inhibition zone of florfenicol against isolate WS05, the synergistic effect of the combinations of florfenicol and herb extracts was further evaluated, and the result indicated that Punica granatum extract was a potential synergist of florfenicol against isolate WS05 and the fractional inhibitory concentration index (FICI) for the florfenicol-P. granatum extract was calculated as 0.31. When combined with 7.81 mg mL-1 P. granatum extract, the minimum inhibitory concentration (MIC) of florfenicol against isolate WS05 was reduced from 0.50 to 0.03 mg L-1, and its activity against isolate WS05 was also enhanced with a significant reduction of ≥3.61 log in cell density after 24 h of treatment compared with that in the single drug treatment. In addition, the protective effect was potentiated by the combination of florfenicol and P. granatum extract, with a cumulative mortality of 36.66% (p < 0.05) and 33.33% (p < 0.05) lower than that in the single treatment with florfenicol and P. granatum extract after the challenge with isolate WS05 for seven days. As far as we know, this is the first study to describe whiteleg shrimp-pathogenic A. hydrophila and suggest P. granatum extract as a potential synergist of florfenicol against the A. hydrophila pathogen.