Exploring the protective role of Bacillus velezensis BV1704-Y in zebrafish health and disease resistance against Aeromonas hydrophila infection.

Bacillus genus, particularly Bacillus velezensis, is increasingly considered as viable alternatives to antibiotics in aquaculture due to their safety and probiotic potential. However, the specific mechanisms through which probiotic B. velezensis confers protection against Aeromonas hydrophila infection in fish remain poorly understood. This study delved into the multifaceted impacts of B. velezensis BV1704-Y on diverse facets of zebrafish health, including gut barrier function, immune response, oxidative stress, gut environment, microbiome composition, and disease resistance. Our findings demonstrate that supplementation with B. velezensis BV1704-Y significantly alleviated symptoms and reduced mortality in zebrafish infected with A. hydrophila. Furthermore, a notable reduction in the expression of pivotal immune-related genes, such as IL-1ß, IL6, and TNF-α, was evident in the gut and head kidney of zebrafish upon infection. Moreover, B. velezensis BV1704-Y supplementation resulted in elevated activity levels of essential antioxidant enzymes, including SOD, CAT, and GSH, in gut tissue. Notably, B. velezensis BV1704-Y positively modulated the structure and function of the intestinal microbiome, potentially enhancing immune response and resilience in zebrafish. Specifically, supplementation with B. velezensis BV1704-Y promoted the relative abundance of beneficial bacteria, such as Cetobacterium, which showed a noteworthy negative correlation with the expression of pro-inflammatory genes and a positive correlation with gut barrier-related genes. Altogether, our study suggests that B. velezensis BV1704-Y holds promise as an effective probiotic for protecting zebrafish against A. hydrophila infection, offering potential benefits for the aquaculture industry.

Vitamin B12 produced by Cetobacterium somerae improves host resistance against pathogen infection through strengthening the interactions within gut microbiota.

BACKGROUND: Pathogen infections seriously affect host health, and the use of antibiotics increases the risk of the emergence of drug-resistant bacteria and also increases environmental and health safety risks. Probiotics have received much attention for their excellent ability to prevent pathogen infections. Particularly, explaining mechanism of action of probiotics against pathogen infections is important for more efficient and rational use of probiotics and the maintenance of host health. RESULTS: Here, we describe the impacts of probiotic on host resistance to pathogen infections. Our findings revealed that (I) the protective effect of oral supplementation with B. velezensis against Aeromonas hydrophila infection was dependent on gut microbiota, specially the anaerobic indigenous gut microbe Cetobacterium; (II) Cetobacterium was a sensor of health, especially for fish infected with pathogenic bacteria; (III) the genome resolved the ability of Cetobacterium somerae CS2105-BJ to synthesize vitamin B12 de novo, while in vivo and in vitro metabolism assays also showed the ability of Cetobacterium somerae CS2105-BJ to produce vitamin B12; (IV) the addition of vitamin B12 significantly altered the gut redox status and the gut microbiome structure and function, and then improved the stability of the gut microbial ecological network, and enhanced the gut barrier tight junctions to prevent the pathogen infection. CONCLUSION: Collectively, this study found that the effect of probiotics in enhancing host resistance to pathogen infections depended on function of B12 produced by an anaerobic indigenous gut microbe, Cetobacterium. Furthermore, as a gut microbial regulator, B12 exhibited the ability to strengthen the interactions within gut microbiota and gut barrier tight junctions, thereby improving host resistance against pathogen infection. Video Abstract.

Effects of dietary Cetobacterium somerae on the intestinal health, immune parameters and resistance against Nocardia seriolae of largemouth bass, Micropterus salmoides.

Largemouth bass (Micropterus salmoides), one of the most important freshwater commercial fish species has been widely cultivated in China. In recent years, the nocardiosis caused by Nocardia seriolae has greatly damaged the M. salmoides industry and there is no effective treatment at present. Currently, Cetobacterium somerae, the predominant bacteria in the gut of many freshwater fishes has been reported to be associated with fish health. However, whether the native C. somerae could protect the host from N. seriolae is unclear. In this study, M. salmoides were fed with three different diets, including control diet (CD), low C. somerae diet (106 CFU/g as LD) and high C. somerae diet (108 CFU/g as HD). After 8-week feeding, growth performance, gut health index, serum enzyme activities and the expression of inflammation-related genes were tested. Results showed that the LD and HD diets had no adverse effects on the growth performance. Moreover, dietary HD enhanced gut barrier and reduced intestinal ROS and ORP, as well as increased serum enzyme activities including ACP, AKP, SOD and LZM compared to the CD group. In addition, the HD diet significantly up-regulated the expression of TNF-α, IL8, IL-1ß and IL15, while down-regulating the expression of TGF-ß1 and IL10 in kidney. Moreover, the expression of antibacterial genes was significantly increased in HD group after being challenged by N. seriolae. And the fish fed HD diet exhibited higher survival rate (57.5%) than that in CD (37.5%) and LD groups (42.5%). To summarize, our study demonstrates that dietary HD can enhance gut health, improve immune response and strengthen pathogen resistance, suggesting that C. somerae is a potential probiotic for defending against N. seriolae infection in M. salmoides.

Dietary supplementation of Bacillus velezensis B8 enhances immune response and resistance against Aeromonas veronii in grass carp.

The heavy use of prophylactic antibiotics in aquaculture leads to elevated antibiotic residues, posing a huge hidden danger in aquaculture products and other natural aquatic environments. Therefore, this study aims to isolate probiotics that can replace antibiotics from the gut of grass carp for disease control. Bacillus velezensis B8 was isolated from the gut of grass carp and showed broad-spectrum antimicrobial activity against several fish pathogenic bacteria, including Aeromonas hydrophilis, Aeromonas veronii, Vibrio parahaemolyticus, Escherichia coli, Edwardsiella tarda and Vibrio mimicus. The safety evaluation showed that the strain B8 was non-toxic to grass carp, had no hemolytic activity, and was sensitive to most antibiotics. In vitro study indicated that strain B8 was viable at pH 2-7, had weak tolerance to 0.1% (w/v) bile salt, and could grow at 10°C-40 °C. The grass carps were fed with diets containing 0 (control), 107, and 109 cfu/g of strain B8 for 4 weeks. Various immune parameters were measured at 1, 2, 3, and 4 weeks of post-feeding. The results of non-specific immunoassay showed that diets supplemented with B8 significantly increased alkaline phosphatase (AKP) and superoxide dismutase (SOD) activity in serum samples (p < 0.05). The expression levels of immune-related genes in the kidney and spleen of grass carp were measured. Among them, the expression levels of IgM and TNF-α both in spleen and kidney were significantly increased after 3 and 4 weeks of post-feeding (p < 0.05). The expression of IgD and MHCI in kidney was significantly upregulated in high-dose groups after 2 and 3 weeks of feeding, respectively (p < 0.05). In addition, after 7 days of challenging with A. veronii, the high-dose group and low-dose group had 48% and 53% survival compared to 25% survival for the control group. These results suggest that B. velezensis B8 has the potential to be developed into a microecological preparation for the alternatives of antibiotics in aquaculture.

Dietary supplementation of salidroside increases immune response and disease resistance of crucian carp (Carassius auratus) against Aeromonas hydrophila.

Some medicinal plants have been known as immunostimulants, and the medicinal plants extract has been used to control the outbreak of the disease in aquaculture for many years. In this study, a total of 270 crucian carp (30 ± 5 g) were randomly distributed in 9 aquaria (55 cm l × 40 cm w × 50 cm h) and divided into three feeding groups including 0 (Control), 50 mg kg-1 (Diet A) and 100 mg kg-1 (Diet B) of salidroside. The expression of immune-related genes (IL-1ß, TNFα, MYD88, CXCL-8, TGF-ß, and IL-11) in the kidney had a significant increase when the crucian carp fed with Diet B for 4 weeks (P < 0.05). Meanwhile, the expression of IL-1ß, TNFα, and CXCL-8 in the spleen was significantly up-regulated when the fish fed with Diet B (P < 0.05). Higher serum alkaline phosphatase (AKP) activity, catalase (CAT) activity, superoxide dismutase (SOD) activity, and complement C3 content were found in the fish which fed with salidroside-supplemented diet. Our results also proved that fish fed with salidroside-supplemented diet for four weeks, especially at a concentration of 100 mg kg-1 diet, improved the protection of crucian carp against A. hydrophila. The amount of A. hydrophila in the kidney and spleen was significantly decreased in salidroside-supplemented diet groups (P < 0.05). In conclusion, the present results demonstrate that the addition of salidroside for four weeks can improve the immune response of crucian carp and increase the protection against the pathogen, especially at the concentration of 100 mg kg-1 diet. The protective effect of the salidroside to the crucian carp could be used as alternatives to antibiotics for controlling fish diseases in aquaculture.

Proteome interrogation using gold nanoprobes to identify targets of arctigenin in fish parasites.

Gold nanoparticles (GNPs) are one of the most widely used nanomaterials in various fields. Especially, the unique chemical and physical properties make them as the promising candidates in drug target identification, unfortunately, little is known about their application in parasites. In this paper, GNPs were employed as new solid support to identify drug targets of natural bioactive compound arctigenin (ARG) against fish monogenean parasite Gyrodactylus kobayashi. Before target identification, GNPs with ARG on the surface showed the ability to enter the live parasites even the nucleus or mitochondria, which made the bound compounds capable of contacting directly with target proteins located anywhere of the parasites. At the same time, chemically modified compound remained the anthelminthic efficacy against G. kobayashii. The above results both provide assurance on the reliability of using GNPs for drug target-binding specificity. Subsequently, by interrogating the cellular proteome in parasite lysate, myosin-2 and UNC-89 were identified as the potential direct target proteins of ARG in G. kobayashii. Moreover, results of RNA-seq transcriptomics and iTRAQ proteomics indicated that myosin-2 expressions were down-regulated after ARG bath treatment both in transcript and protein levels, but for UNC-89, only in mRNA level. Myosin-2 is an important structural muscle protein expressed in helminth tegument and its identification as our target will enable further inhibitor optimization towards future drug discovery. Furthermore, our findings demonstrate the power of GNPs to be readily applied to other parasite drugs of unknown targets, facilitating more broadly therapeutic drug design in any pathogen or disease model.

The immunoprotective effect of whole-cell lysed inactivated vaccine with SWCNT as a carrier against Aeromonas hydrophila infection in grass carp.

Aeromonas hydrophila is a strong gram-negative bacterium that can cause a mass death of grass carp, and result in the huge economic loss. Development of practical vaccines is the best way to control the outbreak of this bacterial disease. In this study, a whole-cell inactivated vaccine was obtained via sonication, and then single-walled carbon nanotubes (SWCNTs) was used to link to the bacterial lysate (BL) for a novel vaccine (SWCNTs-BL). A total of 400 fish were vaccinated with BL and SWCNTs-BL via immersion (5, 10 mg L-1) or injection (5, 10 µg/fish) before challenge with live A. hydrophila at the 28 days post immunization (d.p.i.). The results showed that the antibody titer, enzymatic activity, expression of some immune-related genes (especially IgM and TNF-α) and RPS of fish in the injection groups were significantly increased compared to the control group after 28 d.p.i. For the immersion groups, immunological parameters were increased compared to the control group. Furthermore, the immuno-protective effects of SWCNTs-BL were better than BL. The above results indicated that BL of A. hydrophila can effectively induce specific immune response of grass carp, and BL linked with functionalized SWCNTs could enhance the protective effect of immersion immunization. Our results may provide a practical vaccine, with a simple production, to fight against bacterial diseases in aquaculture industry.

Effects of potential probiotic Bacillus velezensis K2 on growth, immunity and resistance to Vibrio harveyi infection of hybrid grouper (Epinephelus lanceolatus♂ × E. fuscoguttatus♀).

Nowadays, there is no suitable treatment for vibriosis in groupers. So an eco-efficient and environmentally friendly treatment is necessary for the grouper industry. Probiotic-feeding has been a promising strategy to control the bacterial pathogens in aquaculture. A new Bacillus velezensis strain named K2 was isolated from the intestinal tract of healthy grouper, and exhibited wide antimicrobial spectrum of against fish pathogens, including Vibrio harveyi, Vibrio alginolyticus, Aeromonas hydrophila, Aeromonas veronii, Aeromonas caviae, Enterococcus casseliflavus and Lactococcus garvieae. Moreover, results of the safety of B. velezensis K2 showed that intraperitoneal injection of K2 in healthy grouper did not cause any pathological abnormality or death, indicating this bacteria could be considered as a candidate probiotic in aquaculture. Groupers were fed with the diets containing 1 × 107 cfu/g of B. velezensis K2 for 4 weeks. Various immune parameters were examined at 1, 2, 3, and 4 weeks of post-feeding. Results showed that diets supplemented with K2 significantly increased serum acid phosphatase (ACP) activity (P < 0.05). Results of the mRNA expression of immune-related genes in the head kidney of hybrid grouper showed that the expression of lysozyme gene was significantly upregulated after 1 and 2 weeks of feeding (P < 0.05). A significant up-regulation of the expression of piscidin, IgM and MyD88 were detected at day 21, whereas the TLR3 and TLR5 showed lower expression compared to the controls during 21 days, and a significant decrease of TLR3 gene was found at day 28 (P < 0.05). After challenge with V. harveyi, the survival rate of fish administrated with the strain K2 for 28 days was signifiacantly higher than the controls without this strain (P < 0.05). These results collectively suggest that B. velezensis K2 is a potential probiotic species to improve health status and disease resistance and can be developed as a probiotic agent in grouper industry.

Immunosuppression-induced alterations in fish gut microbiota may increase the susceptibility to pathogens.

Intestinal bacteria play an important role in the health and provide a variety of beneficial effects to host. Immunosuppressant can reduce the immunity of host and increase the susceptibility to pathogens. But it is not clear whether the increased susceptibility caused by immunosuppressant is related to changes of gut microbiota. In this study, we used crucian carp administrated with dexamethasone to explore the effects of immunosuppressants on gut microbial communities and further evaluate the potential association between changes in gut microbiota and susceptibility to pathogens. The results of MANOVA based on the top 10 PCoA axis scores from unweighed/weighted UniFrac distances showed that administration of dexamethasone (P = 0.021) and the administration time (P = 0.027) had a significant impact on the gut microbial composition, regardless of pathogens infection status (P = 0.35). After administration with dexamethasone, the fish had higher abundance of Cetobacterium and lower abundance of Bacillus and Lactococcus, and the abundance of genus Bacillus, Pseudomonas and Lactococcus decreased along with prolong administration time of dexamethasone. The results may help us understand the correlation between the host susceptibility to pathogenic bacteria and gut microbial community shift, and extend our knowledge regarding the role of gut microbiota in keeping the balance between pathogenic and symbiotic bacteria.

Cytokine gene expression profiles in goldfish (Carassius auratus) during Gyrodactylus kobayashii infection.

Monogeneans of the genus Gyrodactylus are well-known pathogens causing huge mortalities in wild and cultured fish. Cytokine expression is one of most important host defense mechanisms against parasite infections. In this study, the expression pattern of the key pro-inflammatory (IL-1ß, IL-8, IFN-γ, TNF-α, IL-12 and iNOS) and anti-inflammatory cytokine genes (IL-10, TGFß and IL-4) of Gyrodactylus kobayashii infected goldfish (Carassius auratus) were determined by real-time quantitative PCR analysis. Our results showed that G. kobayashii infection caused increased expression of the pro-inflammatory cytokines including IFN-γ, TNF-α and iNOS in all detected tissues throughout the infection period. Among these genes, iNOS has the highest transcript level accompanied with increased nitric oxide (NO) concentration in the serum of all infected goldfish. The mRNA level of IL-1ß in the liver, spleen and head kidney was significantly up-regulated during the early stage of infection (days 2-8). While high expression level of IL-8 and IL-12 was observed during the elimination phase of infection (days 10-14). As for anti-inflammatory cytokines, the expression profiles of IL-10 were distinct from those of TGF-ß and IL-4. Specifically, the mRNA level of IL-10 did not increase in the spleen and head kidney during the early stage of infection, while increased expression of TGF-ß and IL-4 were likewise seen. Besides, all infected fish had significantly higher complement C3 but lower IgM levels than the non-infected fish. The results provide insights into the interaction between gyrodactylids and the fish host, and indicate that systemic cytokine responses are critical for controlling parasite infection in fish.

Display of GCRV vp7 protein on the surface of Escherichia coli and its immunoprotective effects in grass carp (Ctenopharyngodon idella).

Infection with Grass carp reovirus (GCRV) is becoming unprecedentedly widespread in grass carp (Ctenopharyngodon idella) aquaculture industry, yet the management of GCRV infection still remains a challenge. Therefore, it is of importance to develop effective means against GCRV. As a delivery system of viral antigens, surface displaying of heterologous proteins on bacteria using anchoring motifs has successfully been implemented in human and veterinary vaccines research. In this study, a novel vaccine (BL21/InpN/vp7) was developed based on surface displaying a major capsid protein (vp7) of GCRV using the anchoring motif of N-terminal unique domain of ice-nucleation protein (InpN) on Escherichia coli BL21 (DE3) vaccine. Then the grass carp were immunized by surface displaying BL21/InpN/vp7 vaccine against GCRV using both intraperitoneal injection and bath immunization and their immune responses were tested. The results revealed that some non-specific immune parameters (acid phosphatase (ACP), alkaline phosphatase (AKP) and total antioxidant capacity (T-AOC)) were strongly increased in grass carp post injection inoculation (vp7 dose ranged from 10 to 20 µg). The specific antibody levels against GCRV and the transcriptional of immune-related genes (TNF-α, IL-1ß, MHCI and IgM) were also significantly enhanced in grass carp by injection inoculation (vp7 dose ranged from 5 to 20 µg). On the other hand, only the highest dose of bath vaccination significantly induced the production of specific antibody and up-regulated transcriptions of several immune-related genes (IgM and MHCI) in grass carp. The lower cumulative mortality of grass carp in vaccinated groups after GCRV challenge clearly demonstrated that surface displayed vp7 vaccine could protect fish against GCRV infection. The relative percentage survival (RPS) value in injection vaccinated group (88.89%) was much higher compared to bath group (18.89%), which was in consistent with the production of specific serum antibodies, non-specific immune response and immune related genes expression. To sum up, our results indicated the surface display of heterologous antigenic proteins on E. coli BL21 (DE3) using the anchoring motif of ice-nucleation protein may provide a promising approach to the vaccine development of aquatic animals and suggested its potential to be used as vaccine to fight against GCRV infection.

Ammonia exposure alters the expression of immune-related and antioxidant enzymes-related genes and the gut microbial community of crucian carp (Carassius auratus).

Chronic exposure of ammonia in fish can affect the activities of antioxidant enzymes but few studies investigate the influence of ammonia exposure on the expression of immune-related and antioxidant enzymes-related genes. Also, there is no study demonstrates the effect of ammonia exposure on gut microbial community of fish. In this study, crucian carp (Carassius auratus) were exposed to the ammonia concentrations, 0 (control), 10 mg L-1 (low) or 50 mg L-1 (high) for consecutive 30 days at 25 ± 1 °C temperature, respectively, and after that, the fish from all exposure groups were maintained in control conditions for another 15 days. The results showed that low concentration ammonia increased the expression of immune-related genes and antioxidant enzymes-related genes, but high concentration ammonia inhibited the expression of immune-related genes and antioxidant enzymes-related genes. After a 15-day treatment without ammonia, the expression of antioxidant enzymes-related genes and immune-related genes showed no significant changes compared with control. The results of high-throughput sequencing showed that gut microbial communities were significantly differentiated following ammonia exposure. The abundance of Bacteroides and Cetobacterium (two kinds of potential probiotics) increased while fish exposed to 10 mg L-1 ammonia. The Flavobacterium (a potential fish pathogen) showed increasing trends when the exposure dose reached 50 mg L-1, while the Bacteroides and Cetobacterium showed almost no abundance. The results also revealed that ammonia exposure concentration or time can alter the intestinal microbial community. In conclusion, ammonia exposure could induce the immune response in crucian carp, and alter the gut microbial community. The results may help us understand the correlations of gut microbial community shift and ammonia exposure and extend our knowledge to comprehend the effects of environmental factors on intestinal microbial community.

Protective immunity of grass carp induced by DNA vaccine encoding capsid protein gene (vp7) of grass carp reovirus using bacterial ghost as delivery vehicles.

Grass carp reovirus (GCRV) is one of the most pathogenic aquareovirus and can cause lethal hemorrhagic disease in grass carp (Ctenopharyngodon idella). However, management of GCRV infection remains a challenge. Therefore, it is necessary to find effective means for the control of its infection. The uses of bacterial ghost (BG, non-living bacteria) as carriers for DNA delivery have received considerable attentions in veterinary and human vaccines studies. Nevertheless, there is still no report about intramuscular administration of bacterial ghost-based DNA vaccines in fish. In the current study, a novel vaccine based on Escherichia coli DH5α bacterial ghost (DH5α-BG), delivering a major capsid protein gene (vp7) of grass carp reovirus encoded DNA vaccine was developed to enhance the efficacy of a vp7 DNA vaccine against GCRV in grass carp. The grass carp was injected intramuscularly by different treatments -i) naked pcDNA-vp7 (containing plasmid 1, 2.5 and 5 µg, respectively), ii) DH5α-BG/pcDNA-vp7 (containing plasmid 1, 2.5 and 5 µg, respectively) and iii) naked pcDNA, DH5α-BG or phosphate buffered saline. The immune responses and disease resistance of grass carp were assessed in different groups, and results indicated that the antibody levels, serum total antioxidant capacity (T-AOC), superoxide dismutase (SOD) activity, acid phosphatase (ACP) activity and alkaline phosphatase (AKP) activity and immune-related genes were significantly enhanced in fish immunized with DH5α-BG/pcDNA-vp7 vaccine (DNA dose ranged from 2.5 to 5 µg). In addition, the relative percentage survival were significantly enhanced in fish immunized with DH5α-BG/pcDNA-vp7 vaccine and the relative percentage survival reached to 90% in DH5α-BG/pcDNA-vp7 group than that of naked pcDNA-vp7 (42.22%) at the highest DNA dose (5 µg) after 14 days of post infection. Moreover, the level of pcDNA-vp7 plasmid was higher in DH5α-BG/pcDNA-vp7 groups than naked pcDNA-vp7 groups in muscle and kidneys tissues after 21 days. Overall, those results suggested that DH5α bacterial ghost based DNA vaccine might be used as a promising vaccine for aquatic animals to fight against GCRV infection.

Preliminary study on the relationship between dexamethasone and pathogen susceptibility on crucian carp (Carassius auratus).

Dexamethasone, a known immunosuppressant, can inhibit the immune response and increase the amount of pathogen in body, but the role of dexamethasone affecting susceptibility of crucian carp (Carassius auratus) to pathogen is unclear. The effects of dexamethasone on susceptibility of crucian carp to Aeromonas hydrophila were investigated in this study. The fish were divided into four groups randomly and injected intraperitoneally by dexamethasone for 0 day (group D), 3 days (group C), 6 days (group B), and 9 days (group A), respectively. The serum lysozyme activity was significantly declined in group A, B and C. Relative immune gene expression such as il-1ß, cxcl-8, tnfα and crp in kidney were down-regulation compared to group D. After that crucian carp were infected with A. hydrophila, crucian carp treated by dexamethasone had higher mortality (group A 95%, group B 76%, group C 31%) when compared to group D (4% mortality); the amount of pathogen in was significantly increased (P < 0.05) in liver, kidney and spleen of fish in group A-C compared to group D. These results implicated that higher susceptibility caused by dexamethasone may be induced by the decrease of lysozyme activity and the down-regulation of some immune genes.

Characterization of Toll-like receptor gene expression in goldfish (Carassius auratus) during Dactylogyrus intermedius infection.

Toll-like receptors (TLRs), the first and best understood innate immune receptors, play a notable role in the innate immune system by sensing pathogenic agents and initiating appropriate immune responses. However, studies about the roles of fish TLRs in response to the infection of the ectoparasitic monogenean Dactylogyrus intermedius have been surprisingly vacant. In the present study, cDNA fragments of five members of TLRs family in goldfish (Carassius auratus) were cloned and the expression patterns of nine TLRs in five tissues at different time points during D. intermedius infection were subsequently investigated. We found that the expressions of TLR4, TLR5, TLR20 and TLR22 were significantly elevated after infection at some time points, of which the transcription of TLR5 was progressively increased nearly in all tissues, whereas the mRNA levels of other TLRs (TLR2, 3, 7, 9 and 21) were down-regulated or showed no significant change compared with the control at most time points. Additionally, this paper was also conducted to explore the expression of above TLRs after re-infected with D. intermedius. The results showed a significant upregulation of TLR4, TLR5 and TLR22 in all tested tissues at these two time points, especially the levels of TLR4 and TLR22 expression, were even higher comparing with the first infection. Besides, tissue-specific expression analysis revealed that spleen featured the highest expressions of almost all the TLR-encoding genes among detected tissues. The informations obtained here could be helpful towards understanding the functions of TLRs in response to parasitic infection in goldfish and provide new insights for the development of preventive and therapeutic approaches against D. intermedius infection.