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Nocardia seriolae is the main pathogen causing fish nocardiosis, which can infect various aquatic freshwater and marine fish species. We have previously confirmed that the heme-binding protein of N. seriolae (NsHBP) is a secreted protein relating to bacterial virulence. In the present study, a ΔNsHBP mutant of N. seriolae was successfully generated by deleting NsHBP gene, and it was further used for attenuated vaccine development and evaluation in hybrid snakehead (Channa maculata ♀ × Channa argus ♂). The LD 50 of strain ΔNsHBP by intraperitoneal injection in hybrid snakehead was presented at 5.47 × 10 7  CFU/mL, significantly (p < 0.01) higher than that of the wild-type strain at 5.28 × 10 5  CFU/mL, indicating the virulent reduction of strain ΔNsHBP in comparison to wild-type strain. The live attenuated vaccine used strain ΔNsHBP at sub-clinical dosages (LD 15 , 1.26 × 10 7  CFU/mL) to immunise the fish by intraperitoneal injection for 56 days could provide a relative percentage survival (RPS) at 82.40% against artificial challenge with wild-type strain ZJ0503. Additionally, serum enzymes activities of lysozyme (LZM), peroxidase (POD), acid phosphatase (ACP), alkaline phosphatase (AKP), superoxide dismutase (SOD) as well as specific antibody (IgM) titers were induced in fish following ΔNsHBP vaccination. Furthermore, the expression of immune-related genes (TNFα, IL-1β, MHCIα, MHCIIα, CD4 and CD8α) was significantly increased in comparison to the control group during immunisation period with ΔNsHBP in fish. Taken together, it is revealed that the deleted strain ΔNsHBP could be a potential candidate for live attenuated vaccine development to control fish nocardiosis in aquaculture., (© 2025 John Wiley & Sons Ltd.)

Crocodilians are susceptible to a range of virus infection including influenza A virus (IAV). However, little is known about the ecology and epidemiology of IAV in crocodile species. This study aimed to investigate IAV infection in farmed Siamese crocodiles in central Thailand. We collected plasma samples and pharyngeal swab samples from Siamese crocodiles residing in 13 crocodile farms in 9 provinces of central Thailand during 2019. Additional archival plasma samples of Siamese crocodiles collected in 2012 and 2018 were also included in the study. Plasma samples were screened for influenza A antibodies by a hemagglutination inhibition (HI) assay and positive were evaluated by a cytopathic effect/hemagglutination based-microneutralization (MN) assay. Swab samples were tested for influenza viral RNA by a real-time RT-PCR assay targeting the influenza matrix (M) gene. Among 246 tested plasma samples, the overall seroprevalence of antibodies against IAV in farmed Siamese crocodiles was 17.5% (43/246). The most common hemagglutinin (HA) subtype was H2 (46.5%, 20/43) followed by H9 (39.5%, 17/43), human H1 (14%, 6/43) and H1 (7%, 3/43). Multiple HA subtypes were also detected in 7% (3/43) of infected crocodiles with combination of H1 and H2 subtypes. All 126 tested swab samples were negative for influenza viral RNA. In addition, we demonstrated the ability of wild-type IAV subtypes (H1, H2, H9 and human H1) to infect primary Siamese crocodile fibroblast cells. To our knowledge, this is the first report of serological evidences of avian and human IAV infection in Siamese crocodiles. Our findings highlighted the role of crocodile species in the ecology of IAV particularly the potential to serve as the reservoir or mixing vessel for the viruses that significantly threaten both human and animal health., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2025 Thongdee et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The Vibrio parahaemolyticus strain causing acute hepatopancreatic necrosis disease (AHPND) in shrimp secretes toxins A and B (PirA Vp /PirB Vp ). These toxins have been implicated in pathogenesis and are targets for developing anti-AHPND therapeutics or prophylactics that include passive immunization. We have previously reported that Ccombodies (recombinant hagfish variable lymphocyte receptor B antibodies; VLRB) targeting PirB Vp conferred protection against V. parahaemolyticus in shrimp when administered as a feed supplement. In this study, we screened a phage-displayed library of engineered VLRBs for PirA Vp -targeting Ccombodies that were mass-produced in a bacterial expression system. We then introduced these Ccombodies into the diet of Pacific white shrimp (Penaeus vannamei) over a seven-day period. Subsequently, the shrimp were exposed to a challenge with V. parahaemolyticus. Mortality rates were then observed and recorded over the following seven days. Administering shrimp feed supplemented with Ccombodies at a high dose (100 mg per 100 g feed) reduced mortality in recipient animals (2.96-5.19 %) statistically similar to mock-challenged control (1.48 %), but significantly different from the Ccombody-deficient control (74.81 %). This suggests that the Ccombodies provided strong protection against the bacterium. Feeding shrimp with a median dose (10 mg/100 g feed) gave statistically comparable low mortality (5.93-6.67 %) as the high dose. Reducing the Ccombody dose to 1 mg/100 g feed showed variable effects. Ccombody A2 showed mortality (11.85 %) significantly lower than that of the Ccombody-deficient group (74.81 %), suggesting that it can effectively protect against the bacterial challenge at a low dose. Our results demonstrate the ability of the phage-displayed VLRB library to generate antigen-specific Ccombodies rapidly and simply, with the expression of high protein levels in bacteria. The protective effect provided by these Ccombodies aligns with our earlier results, strongly supporting the use of VLRB antibodies as a substitute for IgY in passive immunoprophylaxis against AHPND in shrimp., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Fish nocardiosis is a chronic disease mainly caused by Nocardia seriolae, which occurs in a variety of economically cultured freshwater and marine fish. Studies have shown that DNA vaccine is an effective treatment to protect fish from bacterial infection. In our previous experiment, an in vivo-induced gene of N. seriolae, encoding phosphoketolase (PK) family protein, was identified by in vivo-induced antigen technology. In the present study, the antigenic gene encoding PK family protein was analyzed by bioinformatics and further inserted into the eukaryotic expression vector pcDNA3.1-myc-his-A for DNA vaccine development. The immunological effects of pcDNA-PK DNA vaccine were assessed in hybrid snakehead (Channa maculata ♀ × Channa argus ♂), showing induction in several serum enzyme activity parameters (including LZM, SOD, ACP and AKP), increasing in specific-antibody IgM levels, as well as up-regulation in six immune-related genes (CD4, CD8α, TNFα, IL-1β, MHCIα and MHCIIα). Moreover, an immune-protection with a relative survival rate was provided at 53.82 % following artificial challenge with N. seriolae in vaccinated fish in comparison to the control group. In summary, these results indicate that pcDNA-PK DNA vaccine could boost strong immune responses in hybrid snakehead and show preferably protective efficacy against N. seriolae, which may be applied in aquaculture to control fish nocardiosis., (Copyright © 2024. Published by Elsevier Ltd.)

Group B Streptococcus (GBS) is a major human and animal pathogen that threatens public health and food security. Spill-over and spill-back between host species is possible due to adaptation and amplification of GBS in new niches but the evolutionary and functional mechanisms underpinning those phenomena are poorly known. Based on analysis of 1,254 curated genomes from all major GBS host species and six continents, we found that the global GBS population comprises host-generalist, host-adapted and host-restricted sublineages, which are found across host groups, preferentially within one host group, or exclusively within one host group, respectively, and show distinct levels of recombination. Strikingly, the association of GBS genomes with the three major host groups (humans, cattle, fish) is driven by a single accessory gene cluster per host, regardless of sublineage or the breadth of host spectrum. Moreover, those gene clusters are shared with other streptococcal species occupying the same niche and are functionally relevant for host tropism. Our findings demonstrate (1) the heterogeneity of genome plasticity within a bacterial species of public health importance, enabling the identification of high-risk clones; (2) the contribution of inter-species gene transmission to the evolution of GBS; and (3) the importance of considering the role of animal hosts, and the accessory gene pool associated with their microbiota, in the evolution of multi-host bacterial pathogens. Collectively, these phenomena may explain the adaptation and clonal expansion of GBS in animal reservoirs and the risk of spill-over and spill-back between animals and humans., Competing Interests: A patent for Group B Streptococcus (GBS) antigens associated with strains virulent in fish has been filed by the Moredun Research Institute. MF and RZ are named inventors on this application. The International Patent Application number is WO 2016/034879 Al. This application covers GBS genes required for virulence in fish, i.e. Locus 3 as described in this manuscript., (Copyright: © 2024 Crestani et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Chlamydiosis, an infection caused by several Chlamydia species, has been reported in Nile, saltwater, and Siamese crocodiles. Despite its widespread reports in various countries, including Thailand, genetic information on Chlamydia species remains limited. This study presents a whole-genome-based characterization of Siamese crocodile-isolated Chlamydia. The results showed that Siamese crocodile Chlamydia contained a single circular chromosome with a size of 1.22-1.23 Mbp and a plasmid with a size of 7.7-8.0 kbp. A plasmid containing eight coding sequences (CDSs) was grouped in a β lineage. A chromosome sequence had approximately 1,018-1,031 CDSs. Chlamydial factors involving virulence were documented in terms of the presence of cytotoxins and several virulence factors in the chromosomes of Siamese crocodile Chlamydia. The analysis of antimicrobial resistance genes in the Chlamydia genome revealed that the most common resistance genes were associated with aminoglycosides, fluoroquinolones, macrolides, tetracyclines, and cephalosporins, with loose matching (identities between 21.12 % and 74.65 %). Phylogenetic analyses, encompassing the assessments of both whole proteome and nine taxonomic markers, revealed that Siamese crocodile Chlamydia was separated into three lineages (lineages I-III) with high bootstrapping statistic support. Interestingly, isolate 12-01 differed genetically from the others, suggesting that it is a new member of Chlamydia. The study findings indicate that Siamese crocodiles are susceptible hosts to Chlamydia, involving more than one species. This study is the first employing the highest number of whole-genome data on Siamese crocodile Chlamydia and provides better insights into pathogen genetics., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Nocardia seriolae has been identified as the causative agent of fish nocardiosis, resulting in serious economic losses in aquaculture. With an aim to screen potential candidates for vaccine development against N. seriolae, the in vivo-induced genes of N. seriolae in hybrid snakehead (Channa maculate ♀ × Channa argus ♂) model were profiled via in vivo-induced antigen technology (IVIAT) in the present study, and 6 in vivo-induced genes were identified as follows: IS701 family transposase (is701), membrane protein insertase YidC (yidC), ergothioneine biosynthesis glutamate-cysteine ligase (egtA), molybdopterin respectively-dependent oxidoreductase (mol), phosphoketolase family protein (Ppl), hypothetical protein 6747 (hp6747). Additionally, the yidC was inserted into eukaryotic expression vector pcDNA3.1-myc-his-A to construct a DNA vaccine named as pcDNA-YidC to evaluate immunoprotection in hybrid snakehead after artificial challenge with N. serioale. Results showed that the transcription of yidC was detected in spleen, trunk kidney, muscle and liver in vaccinated fish, suggesting that this antigenic gene can be recombinantly expressed in fish. Meanwhile, indexes of humoral immunity were evaluated in the vaccinated fish through assessing specific-antibody IgM and serum enzyme activities, including lysozyme (LZM), superoxide dismutase (SOD), acid phosphatase (ACP) and alkaline phosphatase (AKP). Quantitative real-time PCR analysis indicated that pcDNA-YidC DNA vaccine could notably enhance the expression of immune-related genes (CD4、CD8α、MHCIIα、TNFα、IL-1β and MHCIα) in 4 tissues (spleen, trunk kidney, muscle and liver) of the vaccinated fish. Finally, an immuno-protection with a relative survival rate of 65.71 % was displayed in vaccinated fish in comparison to the control groups. Taken together, these results indicate that pcDNA-YidC DNA vaccine could boost strong immune responses in hybrid snakehead and show preferably protective efficacy against N. seriolae, indicating that IVIAT is a helpful strategy to screen the highly immunogenic antigens for vaccine development against fish nocardiosis., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Background: Although Chlamydia sp. causes widespread disease outbreaks in juvenile crocodiles in Thailand, data regarding the epidemiology, and risk factors of such infections are limited. The aim of this study was to investigate the prevalence and possible risk factors associated with Chlamydia sp. infections on Siamese crocodile (Crocodylus siamensis) farms in Thailand. A cross-sectional study was conducted from July to December 2019. Samples were collected from 40 farms across six regions in Thailand. Conjunctival, pharyngeal, and cloacal swab samples were analyzed for Chlamydiaceae nucleic acids using semi-nested PCR followed by phylogenetic analysis based on the ompA gene fragment. Risk factors of infection were analyzed using chi-square and univariate regression to calculate odds ratios., Results: The prevalence of Chlamydia sp. infection across all regions was 65%. The ompA phylogenetic analysis showed that Chlamydia sp. detected in this study was genetically closely related to Chlamydia crocodili and Chlamydia caviae. The risk factors for infection were water source, reusing treated wastewater from the treatment pond, not disposing of leftover food, low frequency of water replacement in the enclosure of juvenile crocodiles, and lack of water replacement after the death of a crocodile., Conclusion: The prevalence of Chlamydia sp. infection in farmed crocodiles in Thailand was 65% during the study period. Cloacal swabs were superior to conjunctival and pharyngeal swabs due to their higher sensitivity in detecting Chlamydia sp., as well as their lower invasiveness. Good management and biosecurity in crocodile farming can reduce the risk of Chlamydia sp., (© 2023. The Author(s).)

It is well known that the Asian water monitors or Varanus salvator are both scavengers and predators. They can live and survive in the place that exposed to harmful microorganisms. Most people believe that they have some protected mechanisms to confront those infections. The aim of this study is to determine the antibacterial activities of crude peptides and protein hydrolysates extracted from serum of the Varanus salvator. Ten types of bacteria were cultured with crude peptides and protein hydrolysates which were isolated from 21 Varanus salvator's serum. The crude peptides showed some interested inhibition percentages against Enterobacter aerogenes ATCC13048 = 25.6%, Acinetobacter baumannii ATCC19606 = 33.4%, Burkholderia cepacia ATCC25416 = 35.3% and Pseudomonas aeruginosa ATCC27853 = 25.8%, whereas the protein hydrolysates had some inhibition potential on Burkholderia cepacia ATCC25416 = 24.3%. For the rest results of other tests were below 20% of inhibition. In addition, the evidences show that crude peptides have better antibacterial performances significantly than protein hydrolysates on most tested bacteria. Furthermore, antimicrobial peptides prediction shows about 10 percent hit (41/432 sequences). The interpretation shows that the best hit sequence is highly hydrophobic. It may destroy outer membrane of Gram-negative hence prevents the invasion of those bacteria. Altogether, bioinformatics and experiments show similar trends of antimicrobial peptide efficacy from Varanus salvator. Further studies need to be conducted on peptide purification and antimicrobial peptide candidate should be identified., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Thanasak et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Group B Streptococcus (GBS) is a major pathogen of humans and aquatic species. Fish have recently been recognized as the source of severe invasive foodborne GBS disease, caused by sequence type (ST) 283, in otherwise healthy adults in Southeast Asia. Thailand and Vietnam are among the major aquaculture producers in Southeast Asia, with GBS disease reported in fish as well as frogs in both countries. Still, the distribution of potentially human-pathogenic GBS in aquaculture species is poorly known. Using 35 GBS isolates from aquatic species in Thailand collected from 2007 to 2019 and 43 isolates from tilapia collected in Vietnam in 2018 and 2019, we have demonstrated that the temporal, geographical, and host-species distribution of GBS ST283 is broader than previously known, whereas the distribution of ST7 and the poikilothermic lineage of GBS are geographically restricted. The gene encoding the human GBS virulence factor C5a peptidase, scp B, was detected in aquatic ST283 from Thailand but not in ST283 from Vietnam or in ST7 from either country, mirroring current reports of GBS strains associated with human sepsis. The observed distribution of strains and virulence genes is likely to reflect a combination of spill-over, host adaptation through the gain and loss of mobile genetic elements, and current biosecurity practices. The plastic nature of the GBS genome and its importance as a human, aquatic, and potentially foodborne pathogen suggests that active surveillance of GBS presence and its evolution in aquaculture systems may be justified.

Nile tilapia is one of the most consumed farmed fish in the world. The outbreak of pathogenic bacterial diseases causes high mortality rates and economic losses in Nile tilapia farming. Antibiotic administrations are commonly utilized to inhibit and prevent bacterial infections. However, antibiotics are expensive and cause serious concerns for antibiotic resistance in fish that can be potentially transferred to humans. As an alternative solution, probiotics can be used to prevent infection of pathogenic bacteria in fish. In this work, both bacteria and yeast were isolated from fish gastrointestinal tracts and their inhibitory activity against Nile tilapia pathogenic bacteria was evaluated, as well as other probiotic properties. In this study, 66 bacteria and 176 acid tolerant yeasts were isolated from fish gastrointestinal tracts. Of all isolated microorganisms, 39 bacterial and 15 yeast isolates with inhibitory effect against pathogens were then examined for their probiotic properties (acidic and bile salt resistance, adhesion potential, and biofilm formation), formation of antibacterial factor survival rate under simulated gastrointestinal fluid, and safety evaluation. AT8/5 bacterial isolate demonstrated probiotic properties and the highest inhibition against all 54 tested pathogens while YON3/2 yeast isolate outperformed the inhibitory effect among all yeast isolates. These two probiotic isolates were further identified by 16S rDNA and the D1/D2 domain of 26S rDNA sequence analysis for bacterial and yeast identification, respectively. AT8/5 and YON3/2 showed the highest similarity to Lactiplantibacillus argentoratensis and Candida tropicalis , respectively. This is the first report on isolated L. argentoratensis and C. tropicalis with antipathogenic bacteria of Nile tilapia properties. Collectively, AT8/5 and YON3/2 could be potentially used as promising alternatives to existing antibiotic methods to prevent pathogenic bacteria infection in Nile tilapia farming.