Journal of fish diseases最新文献

Lactococcosis is a common bacterial fish disease caused by Lactococcus garvieae, L. petauri and L. formosensis. Although there are different PCR-based techniques to identify the etiological agent, none of these can differentiate these two bacteria without sequencing PCR-amplified fragments. In the present study, we developed a multiplex PCR assay for simultaneous detection and differentiation of L. garvieae and L. petauri. The specificity of the primers was validated against the bacterial DNA of the targeted and non-targeted bacteria. The sizes of the PCR amplicons were obtained as 204 bp for the DUF1430 domain-containing protein gene of L. garvieae, 465 bp for the Lichenan permease IIC component gene of L. petauri, and 302 bp for the teichoic acid biosynthesis protein F gene of both L. garvieae and L. petauri. The PCR amplicons were clearly separated by agarose gel electrophoresis. The multiplex PCR assay did not produce any amplification products with the DNA of the non-targeted bacteria. The multiplex PCR detection limits for L. garvieae and L. petauri were 5 and 4 CFU in pure culture and 50 and 40 CFU/g in spiked tissue samples, respectively. It takes less than 2 h from plate-cultured bacteria and 3 h from tissue samples to get results. In conclusion, the developed multiplex PCR assay is a rapid, specific, accurate, and cost-effective method for the detection and differentiation of L. garvieae and L. petauri and is suitable to be used for routine laboratory diagnosis of L. garvieae and L. petauri.

Globally, streptococcal disease caused by Streptococcus agalactiae is known for its high mortality rate, which severely limits the development of the tilapia breeding industry. As a third-generation vaccine, DNA vaccines have shown great application prospects in the prevention and control of aquatic diseases, but their low immunogenicity limits their development. The combination of DNA vaccines and molecular adjuvants proved to be an effective method for inducing protective immunity. This study constructed recombinant plasmids encoding tilapia HSP70 and IL-1β genes (pcHSP70 and pcIL-1β) to verify their effectiveness as molecular adjuvants for S. agalactiae DNA vaccine (pcSIP) in the immunized tilapia model. The results revealed that serum-specific IgM production, enzyme activities, and immune-related gene expression in tilapia immunized with pcSIP plus pcHSP70 or pcIL-1β were significantly higher than those in tilapia immunized with pcSIP alone. It is worth noting that combination with molecular adjuvants improved the immune protection of DNA vaccines, with a relative percentage survival (RPS) of 51.72% (pcSIP plus pcHSP70) and 44.83% (pcSIP plus pcIL-1β), respectively, compared with that of pcSIP alone (24.14%). Thus, our study indicated that HSP70 and IL-1β in tilapia are promising molecular adjuvants of the DNA vaccine in controlling S. agalactiae infection.

This study investigated the in vitro antimicrobial and anthelmintic effect of copper nanoparticles (CuNPs) against the bacterium Aeromonas hydrophila, the monogeneans Dactylogyrus minutus, Dactylogyrus extensus, Gyrodactylus cyprini, and the cestode Schyzocotyle acheilognathi, as well as their toxicity to Cyprinus carpio Koi. In the antimicrobial in vitro test, the inhibition zone method and minimum inhibitory concentration (MIC) were performed. In order to determine the time and efficacy of monogenean parasite mortality, the parasites were exposed to CuNP concentrations of 20, 50, 100, 150, 200, and 300 mg L⁻¹, and a control group with tank water and one with copper sulphate pentahydrate (CuSO₄.₅H₂O) at a concentration of 0.3 mg L⁻¹, performed in triplicate. The parasites were observed every 10 min for 300 min, and mortality was recorded. For the cestodes, parasites were immersed in CuNP concentrations of 50, 100, 150, and 300 mg L⁻¹. At the end of the in vitro tests, the anthelmintic efficacy of each treatment was calculated. To assess the tolerance and toxicity in fish, they were exposed to CuNP concentrations of 0.6, 1.25, 2.5, 5, 10, 20, and 50 mg L⁻¹ for 12 h. The MIC demonstrated that CuNPs effectively inhibited the growth of A. hydrophila up to a dilution of 12,500 mg L⁻¹ and showed an inhibition zone of 14.0 ± 1.6 mm for CuNPs. The results of anthelmintic activity showed a dose-dependent effect of concentration for both groups of parasites, with the most effective concentration being 300 mg L⁻¹ in 120 min. In the toxicity test, the carps showed tolerance to lower concentrations. The study indicated that CuNPs were effective against the studied pathogens. However, it proved to be toxic to fish at high concentrations. The use of low concentrations is recommended still requires further investigation.

Asian seabass (Lates calcarifer) is an economically important fish species that is widely cultivated in Thailand. However, aquaculture of Asian seabass is limited by infectious diseases. One of the most serious diseases is photobacteriosis, caused by Photobacterium damselae. Vaccination is recognized as an efficient disease prevention and pathogen control method for strengthening the aquaculture industry. To promote vaccine development, the characterization of pathogenic bacteria and their pathogenesis is required. In this study, isolates of P. damselae were obtained from commercial aquaculture farms in Thailand during 2019–2021. Analyses of 16S rRNA and the urease subunit alpha genes identified the isolates as P. damselae subsp. damselae (Phdd). Antibiotic susceptibility analyses showed that all Phdd isolates were resistant to amoxicillin (10 μg). Haemolysis and phospholipase activities were used to categorize P. damselae into three groups based on their biological activities. The pathogenicity of four candidates (SK136, PD001, PD002 and T11L) was tested in Asian seabass. Isolate SK136 showed the highest virulence, with a lethal dose (LD₅₀) of 1.47 × 10⁵ CFU/fish, whereas isolate PD001 did not show any virulence. Genotypic characterization, based on multi-locus sequence typing analysis, demonstrated that all candidates were novel strains with new sequence types (64, 65, 66 and 67). Preliminary vaccination using formalin-killed cells (FKCs) protected Asian seabass from artificial challenges. Taken together, these results provide fundamental knowledge for vaccine development against Phdd infection in Asian seabass.

Studying inflammatory responses induced by vaccination can contribute to a more detailed understanding of underlying immune mechanisms in lumpfish (Cyclopterus lumpus). Tissue samples from lumpfish intraperitoneally immunized with a divalent oil-adjuvanted vaccine (Aeromonas salmonicida and Vibrio salmonicida) at water temperatures of 5, 10, and 15°C were collected at 630 day degrees and 18 weeks post injection. The relative amount of secretory and membrane-bound immunoglobulin M (IgM) gene transcripts in the head kidney was determined by qPCR. Vaccine-induced inflammatory lesions were assessed on histological sections of abdominal pancreatic/intestinal tissue from vaccinated fish in all three temperature groups. Inflammatory cells forming dense aggregations in lesions showed proliferative activity, many of which were identified as eosinophilic-granulocyte-like cells. IgM+ cells were scattered in inflammatory tissue dominated by connective tissue, showing no difference in numbers between lesions from fish vaccinated at 5, 10, and 15°C. Relative gene expression analysis of secretory and membrane-bound IgM revealed low overall expression in the head kidney of vaccinated fish at both 630 day-degrees and 18 weeks post injection. The results of this study indicate that the vaccine stimulated prolonged local inflammatory responses at the injection site, which were not influenced by temperature.

The Strep Easy Kit, a bio-enrichment dual ICG-strip test, is a diagnostic tool designed for the detection of Streptococcus agalactiae, an important pathogenic bacterium in tilapia farming. The kit can simultaneously identify two different serotypes of S. agalactiae, serotype Ia and serotype III. This capability is crucial for the collection of valuable epidemiological data and facilitates strategic planning for effective vaccine development and deployment. The Strep Easy Kit consists of two main steps: pathogen enrichment and pathogen detection. The enrichment step increases the concentration of bacteria so that the bacterial load is raised to the level reliably detectable by the subsequent ICG strip test. This is achieved by incubating the fish samples in a suitable liquid medium under specified temperature and time conditions. The second step involves the use of the dual-ICG strip test. This strip test consists of two monoclonal antibodies and one polyclonal antibody that are specific to S. agalactiae and can distinguish between S. agalactiae serotype Ia and S. agalactiae serotype III. This dual capability enables the ICG strip test to simultaneously detect both serotypes of S. agalactiae in a single test kit. The detection limit of the test kit, which consists of a dual ICG-Strip test combined with an enrichment step, is 100 CFU/mL. The kit can be used to detect S. agalactiae in both live and dead fish samples, making it versatile for various testing scenarios. The test results obtained using the Strep Easy Kit have shown a 94.4% correlation with the standard method (Thai Agricultural Standard; TAS 10453-2010), with 90.2% sensitivity and 100% specificity. Significant advantages of the Strep Easy Kit lie in its simplicity and portability, allowing farmers to perform the test by themselves and on-site. This makes it a practical and accessible tool for the tilapia farming industry.

Exposure to temperatures outside of a fish's optimal range results in suppression of the immune system, ultimately leaving aquaculture stocks susceptible to disease outbreaks. This effect is exacerbated in triploid fishes, which demonstrate greater susceptibility to stress than their diploid counterparts. This study investigates the impacts of acute heat stress on the abundance of immune transcripts and proteins in diploid and triploid Chinook salmon (Oncorhynchus tshawytscha), an important finfish crop. This study also demonstrates that acute heat stress induces significant increases in the abundance hsp70, hsp90 and il1b transcripts in the head kidneys, gills and heart ventricles of both diploid and triploid Chinook salmon. Widespread dysregulation of antigen-presentation transcripts was also observed in fish of both ploidies. These results suggest that acute heat stress activates acute-phase responses in Chinook salmon and dysregulates antigen presentation, potentially leaving fish more susceptible to infection. At the protein level, IL-1β was differentially expressed in the head kidney and ventricles of diploid and triploid salmon following heat shock. Differential expression of two tapasin-like proteins in diploid and triploid salmon subjected to heat shock was also observed. Altogether, these data indicate that diploid and triploid Chinook salmon respond differently to acute thermal stressors.

Displaying antigens on yeast surface as an oral vaccine has been widely explored, while its potential as an immersion vaccine has not been evaluated. Here, an immersion vaccine was prepared by displaying ORF25 of Cyprinid herpesvirus 2 (CyHV-2) on the surface of Saccharomyces cerevisiae. Carassius auratus gibelio was immersion immunized by 2 × 10⁷ CFU/mL yeast for 2 h, and reinforce the immunity using the same method 14 days after the first immunization. The results showed that ORF25 specific antibody in immunized crucian carp serum was detected at a high level, and the mRNA expression level of IgM, IgT, IL-1β, and IFN-1 in vaccinated head-kidney and spleen tissues were higher than the control group, indicating that innate and adaptive immunity were induced. Moreover, the immersion vaccination provided effective protection for fish against CyHV-2, leading to a relative percent survival of 50.2%. Meanwhile, immersion vaccination restrained virus replication and histological damage in CyHV-2 infected crucian carp. Our data suggested that immersion immunization of S. cerevisiae-displayed ORF25 could be served as a candidate vaccine to prevent CyHV-2 infection.

With the emergence of diseases, the U.S. catfish industry is under challenge. Current trends prefer autochthonous bacteria as potential probiotic candidates owing to their adaptability and capacity to effectively colonize the host's intestine, which can enhance production performance and bolster disease resistance. The objective of this study was to isolate an autochthonous bacterium as probiotic for hybrid catfish. Initially, an analysis of the intestinal microbiota of hybrid catfish reared in earthen ponds was conducted for subsequent probiotic development. Twenty lactic acid bacteria were isolated from the digesta of overperforming catfish, and most of the candidates demonstrated probiotic traits, including proteolytic and lipolytic abilities; antagonistic inhibition of catfish enteric bacterial pathogens, negative haemolytic activity and antibiotic susceptibility. Subsequent to this screening process, an isolate of Lactococcus lactis (MA5) was deemed the most promising probiotic candidate. In silico analyses were conducted, and several potential probiotic functions were predicted, including essential amino acids and vitamin synthesis. Moreover, genes for three bacteriocins, lactococcin A, enterolysin A and sactipeptide BmbF, were identified. Lastly, various protectant media for lyophilization of MA5 were assessed. These findings suggest that Lactococcus lactis MA5 can be an autochthonous probiotic from hybrid catfish, holding promise to be further tested in feeding trials.