Epizootics in farm-raised channel catfish, Ictalurus punctatus (Rafinesque), caused by the enteric redmouth bacterium Yersinia ruckeri

In the winters of 1995 and 1996, unusual disease outbreaks occurred on two separate channel catfish farms in Arkansas, USA. Affected fish exhibited extraordinary haemorrhaged rings around the eyes and raised haemorrhaged areas overlying the frontal foramens. Other signs included abnormal swimming, lethargy, loss of equilibrium, and exophthalmia. Bacterial isolates from the moribund fish were identified as Yersinia ruckeri by biochemical tests, no lysis by the Hafnia-specific bacteriophage 1672, and Y. ruckeri-specific growth patterns on Shotts-Waltman media. Fingerling catfish injected intraperitoneally with the bacterial isolate at 7.8 × 10⁶ bacteria fish^?1 developed lesions characteristic of the epizootics at 13, 18, and 22 °C and a biochemically identical isolate was recovered. Fingerling rainbow trout injected with the channel catfish isolate at 1 × 10⁵ bacteria fish^?1 and held at 20 °C developed signs typical of enteric redmouth by 4 days post-inoculation and were moribund by 5 days post-inoculation. Some differences of clinical signs occurred between experimentally infected rainbow trout and channel catfish. Clinical and biochemical similarities between infections of Y. ruckeri and many warmwater pathogens in affected catfish may lead to incorrect diagnosis of ERM infections.     

Antimicrobial peptide CAP18 and its effect on Yersinia ruckeri infections in rainbow trout Oncorhynchus mykiss (Walbaum): comparing administration by injection and oral routes

The antimicrobial peptide CAP18 has been demonstrated to have a strong in vitro bactericidal effect on Yersinia ruckeri, but its activity in vivo has not been described. In this work, we investigated whether CAP18 protects rainbow trout Oncorhynchus mykiss (Walbaum) against enteric red mouth disease caused by this pathogen either following i.p. injection or by oral administration (in feed). It was found that injection of CAP18 into juvenile rainbow trout before exposure to Y. ruckeri was associated with lowered mortality compared to non‐medicated fish although it was less effective than the conventional antibiotic oxolinic acid. Oral administration of CAP18 to trout did not prevent infection. The proteolytic effect of secretions on the peptide CAP18 in the fish gastrointestinal tract is suggested to account for the inferior effect of oral administration.     

Characterization of a novel Yersinia ruckeri serotype O1-specific bacteriophage with virulence-neutralizing activity

A lytic bacteriophage (φNC10) specific to serotype O1 Yersinia ruckeri has been identified and evaluated as a model to assess the potential use of bacteriophages and their products for disease control in aquaculture. Electron microscopy of purified φNC10 revealed a virion particle with a small (70 nm) polyhedral head and short tail. φNC10 infected only serotype O1 strains of Y. ruckeri and failed to bind a defined Y. ruckeri mutant strain lacking O1 lipopolysaccharides (O1-LPS), suggesting that φNC10 uses O1-LPS as its receptor. In addition, spontaneous φNC10-resistant mutants of Y. ruckeri exhibited defects in O1-LPS production and were sensitive to rainbow trout serum. Purified φNC10 displayed a polysaccharide depolymerase activity capable of degrading Y. ruckeri O1-LPS and thereby sensitizing Y. ruckeri to the bactericidal effects of rainbow trout serum. The φNC10-associated polysaccharide depolymerase activity also reduced the ability of Y. ruckeri cells to cause mortality following intraperitoneal injection into rainbow trout. These data demonstrate a potential utility of φNC10 and its associated polysaccharide depolymerase activity for Y. ruckeri disease prevention.     

Identification of Yersinia ruckeri from diseased salmonid fish by Fourier transform infrared spectroscopy

Yersinia ruckeri is the causative agent of enteric redmouth disease (ERM), which mainly affects salmonid fish. Isolates of Y. ruckeri from diseased salmonid fish were obtained over a 6‐year period from eight fish farms in the State of Baden‐Württemberg, Southwest Germany. The strains were characterized by biochemical methods and Fourier transform infrared spectroscopy (FT‐IR) combined with artificial neural network analysis. These methods were complemented by 16S rDNA sequencing for several isolates. The set of strains from these fish farms included sorbitol‐positive, gelatinase‐positive and non‐motile Y. ruckeri. These variants were differentiated with an advanced FT‐IR module, which is part of a higher‐ranking method including more than 200 well‐defined Yersinia strains against a background of more than 1000 other Gram‐negative isolates. Validation of the newly constructed method yielded 97.4% of Y. ruckeri identified correctly on the species level. Thus, the FT‐IR analysis enables distinction of all Y. ruckeri from other Yersinia species (e.g. fish‐borne Y. enterocolitica) and other Enterobacteriaceae typically misidentified because of similar biochemical reaction profiles, especially Hafnia alvei. The differentiation of sorbitol‐positive variants of Y. ruckeri using FT‐IR was demonstrated.     

Experimental infection by Yersinia ruckeri O1 biotype 2 induces brain lesions and neurological signs in rainbow trout (Oncorhynchus mykiss)

Pathological manifestations in rainbow trout (Oncorhynchus mykiss) following experimental waterborne infection with Yersinia ruckeri serotype O1 biotype 2 (strain 07111224) were investigated. Rainbow trout were exposed to 8 × 10⁷ CFU/ml of Y. ruckeri by bath for 6 hr, and mortality was then monitored for 22 days post‐infection (dpi). Organs were sampled at 3 dpi and also from moribund fish showing signs of severe systemic infection such as bleeding, exophthalmia or erratic swimming behaviour. Y. ruckeri was observed in the meninges and diencephalon of the brain, and lamina propria of olfactory organ at 3 dpi. At 12 dpi, Y. ruckeri had spread throughout the brain including cranial connective tissues and ventricles and the infection was associated with haemorrhages and an infiltration with leucocytes. Y. ruckeri infection and associated with leucocyte infiltration were observed at 13 dpi. In conclusion, Y. ruckeri strain 07111224 causes encephalitis in the acute phase of infection, which could explain why Y. ruckeri‐affected fish show exophthalmia and erratic swimming known as signs of ERM.     

Health status of two salmonid aquaculture facilities in North Portugal: characterization of the bacterial and viral pathogens causing notifiable diseases

A comparative bacteriological and virological survey was conducted in two fish farms in the North of Portugal. The fish species examined included cultured rainbow trout, Oncorhynchus mykiss (Walbaum), and brown trout, Salmo trutta L., as well as wild fish captured near both facilities. The microbial load in the internal organs of apparently healthy fish was nonitored over a year, an all the disease problems occurring during this period were investigated. Although both farms presented intermediate levels of infection(30–40% infected fish), farm B showed the poorest microbiological quality since constant but low mortalities were observed throughout the year. Flavobacterium and Psedomonas-Xanthomonas were the predominant bacterial groups, comprising around 40–50% of the isolates from each farm. In farm B, members of the Enterobacteriaceae and mortile Aeromonas also showed significant prevalence (about 20%). The only outbreak of a notifiable disease was an occurrence of furunculosis, caused by Aeromonas salmonicida subsp. salmoncida, in farm A. However, Yersinia ruckeri was isolated not only from diseased fish, but also from asymptomatic fish, usually in mixed infections with motile Aeromonas or infections with motile Aeromonas or infectious pancreatic necrosis virus (IPNV). While Y. ruckeri isolates associated with mortalities belonged to the serotype O1 (subgroup a), those isolated from asymtomatic fish corresponded to serotype O3. Two strains of IPNV (serotype Ab) were isolated in farm B, which represents the first viral agent detected in Portuguese aquaculture. Qualitative and quantitative differences in microbial load were observed between cultured and wild fish. No notifiable bacterial or viral pathogens were detected in any of the feral species studied.     

Are brown trout Salmo trutta fario and rainbow trout Oncorhynchus mykiss two of a kind? A comparative study of salmonids to temperature‐influenced Tetracapsuloides bryosalmonae infection

Proliferative kidney disease (PKD) of salmonids caused by Tetracapsuloides bryosalmonae causes high mortalities of wild brown trout (Salmo trutta fario) and farmed rainbow trout (Oncorhynchus mykiss) at elevated water temperatures. Here the aim was to compare the temperature‐dependent modulation of T. bryosalmonae in the two salmonid host species, which display different temperature optima. We used a novel experimental set‐up in which we exposed brown trout and rainbow trout to an identical quantified low concentration of T. bryosalmonae for a short time period (1 hr). We followed the development of the parasite in the fish hosts for 70 days. PKD prevalence and parasite kinetics were assessed using qPCR. Exposures were performed at temperatures (12°C and 15°C) that reflect an environmental scenario that may occur in the natural habitat of salmonids. T. bryosalmonae infection was confirmed earliest in brown trout kept at 15°C (day 7 post‐exposure) while, in all other groups, T. bryosalmonae was not confirmed until day 15 post‐exposure. Moreover, significantly greater infection prevalence and a faster increase of parasite intensity were observed in brown trout kept at 15°C than in all other groups. These results indicate that PKD is differentially modulated by water temperature in related host species.     

Yersiniosis in Atlantic cod,Gadus morhua (L.), characterization of the infective strain and host reactions

A disease outbreak in farmed Atlantic cod caused by Yersinia ruckeri is reported. Mortality started following vaccination of cod reared in two tanks (A and B). The accumulated mortality reached 1.9% in A and 4.8% in B in the following 30 days when treatment with oxytetracycline was applied. Biochemical and molecular analysis of Y. ruckeri isolates from the cod and other fish species from fresh and marine waters in Iceland revealed a high salinity‐tolerant subgroup of Y. ruckeri serotype O1. Infected fish showed clinical signs comparable with those of Y. ruckeri ‐infected salmonids, with the exception of granuloma formations in infected cod tissues, which is a known response of cod to bacterial infections. Immunohistological examination showed Y. ruckeri antigens in the core of granulomas and the involvement of immune parameters that indicates a strong association between complement and lysozyme killing of bacteria. Experimental infection of cod with a cod isolate induced disease, and the calculated LD₅₀ was 1.7 × 10⁴ CFU per fish. The results suggest that yersiniosis can be spread between populations of freshwater and marine fish. Treatment of infected cod with antibiotic did not eliminate the infection, which can be explained by the immune response of cod producing prolonged granulomatous infection.     

FTA® card tool for sampling and rapid diagnosis of bacterial diseases from rainbow trout (Oncorhynchus mykiss) tissue

The collection of farmed fish samples and subsequent diagnosis of microbial pathogens can be difficult and time-consuming procedure under field conditions. Correct diagnosis and identification of bacterial diseases agents (Yersinia ruckeri, Flavobacterium psychrophilum, Lactococcus garvieae, and Vagococcus salmoninarum) are crucial for the effective treatment. In this study, the feasibility of using FTA® card (Flinders Technology Associates filter papers) for sampling infected rainbow trout (Oncorhynchus mykiss) tissues and significantly accelerated the molecular diagnosis of enteric redmouth disease, rainbow trout fry syndrome, bacterial coldwater disease, lactococcosis associated with fatal hemorrhagic septicemia, and vagococcosis were evaluated. We also examined and compared a practical and reliable extraction obtained from immobilized DNA on FTA® card with one commonly used commercial kit to diagnose bacterial pathogens in fish tissues. Results proved that the active working time of extracting DNA from FTA® card is requiring only 30 min and the cost is less than USA $ 0.5 for each eluted sample. A total of hundred extracted DNA from tissues on FTA® card were tested and showed successful PCR amplification of the aroA, murG, pLG, and pSa1 genes. In conclusion, our finding indicates FTA® card as an appropriate tool for the simple collection, easy transfer, and stored at room temperature without contamination and retrieval of high-quality DNA for rapid molecular diagnosis of bacterial fish pathogens without isolation and culture.

     

Bacterial pathogens in rainbow trout, Oncorhynchus mykiss (Walbaum), reared at Danish freshwater farms

During a 2-year period, bacterial fish pathogens were monitored on five rainbow trout, Oncorhynchus mykiss (Walbaum), freshwater farms in Denmark. A total of 1206 fish were examined and 361 bacterial isolates were identified phenotypically. Enteric redmouth disease, furunculosis and rainbow trout fry syndrome/coldwater disease were recorded. Infections caused by Flavobacterium psychrophilum occurred most frequently, but only one outbreak of enteric redmouth disease caused by Yersinia ruckeri serotype O1 and one of furunculosis caused by Aeromonas salmonicida were recorded during the monitoring period. Flavobacterium psychrophilum was isolated on all farms, both during disease outbreaks and from fish without any signs of disease. Serological investigations of F. psychrophilum showed that serotype Th was the dominant serotype found. The serotypes Th and Fd were involved in disease outbreaks of fry and larger fish. All isolates of F. psychrophilum showed proteolytic activities; however, a few isolates, belonging to serotype FpT did not degrade elastin and were not associated with mortality. Increasing resistance problems to oxytetracycline were demonstrated. More than half of the F. psychrophilum isolates showed resistance to oxolinic acid and oxytetracycline. No antibiotic resistant isolates were found among Y. ruckeri and A. salmonicida .     

Secondary immune response of rainbow trout following repeated immersion vaccination

Teleosts are able to raise a protective immune response, comprising both innate and adaptive elements, against various pathogens. This is the basis for a widespread use of vaccines, administered as injection or immersion, in the aquaculture industry. It has been described that repeated injection vaccination of fish raises a secondary immune response, consisting of rapid, accelerated and increased antibody reaction. This study reports how rainbow trout responds to repeated immersion vaccination against yersiniosis (ERM) caused by the bacterial pathogen Yersinia ruckeri. It was found that rainbow trout does not raise a classical secondary response following repeated immersion vaccination. Serum antibody titres were merely slightly increased even after three immunizations, using 30‐s immersion into a bacterin consisting of formalin‐inactivated Y. ruckeri (serotype O1, biotypes 1 and 2), performed over a 3‐month period. The densities of IgM‐positive lymphocytes in spleen of fish immunized three times were increased compared to control fish, but no general trend for an increase with the number of immunizations was noted. The lack of a classical secondary response following repeated immersion vaccination may partly be explained by limited uptake of antigen by immersion compared to injection.     

Effects of dietary humic acid on growth performance,haemato‐immunological and physiological responses and resistance of Rainbow trout,Oncorhynchus mykiss to Yersinia ruckeri

This study investigated the effects of dietary humic acid sodium salt on growth performance, haemato‐immunological and physiological responses, and resistance of rainbow trout, Oncorhynchus mykiss to Yersinia ruckeri. The experimental fish were divided into four groups; three of them were fed with humic acid incorporated diets (0.3% H3, 0.6% H6, 1.2% H12) and an additive free basal diet served as the control. Growth performance and haematological parameters of rainbow trout were not affected by humic acid supplemented diets (p > 0.05). However, dietary humic acid especially with 0.6% incorporation significantly increased stomach pepsin, intestinal trypsin and lipase activities p < 0.05. Following 60 days of feeding trial, fish were challenged with Yersinia ruckeri for 20 days. At the end of the challenge period, significantly higher (p < 0.05) survival rates were found in the 6% humic acid group compared to all other experimental treatment. Thus humic acid might replace antibiotics in diets for rainbow trout to control yersiniosis.     

Development of a selective-differential medium for the isolation of Yersinia ruckeri and its application in epidemiological studies

Abstract. The development of a selective-differential medium (ROD) for the isolation of Yersinia ruckeri from carrier and clinically infected rainbow trout, Oncorhynchus mykiss (Walbaum), is described. This medium was a useful aid for the detection of Y. ruckeri in faecal material and allowed the epidemiology of enteric redmouth disease to be studied in greater detail. Regular use of ROD at two fish farms in southern England has demonstrated that isolation of Y. ruckeri from faeces was possible under field conditions up to 4–6 weeks before acute clinical kidney infection occurred.     

In vitro markers for virulence in Yersinia ruckeri

In this study, different traits that have been associated with bacterial virulence were studied in Yersinia ruckeri. Two isolates that had been shown to cause disease and mortality in experimentally infected rainbow trout were compared with five avirulent isolates. Both virulent isolates showed high adhesion to gill and intestinal mucus of rainbow trout, whereas the majority of non‐virulent strains demonstrated significantly lower adhesion. A decrease in adherence capability following bacterial treatment with sodium metaperiodate and proteolytic enzymes suggested the involvement of carbohydrates and proteins. All strains were able to adhere to and invade chinook salmon embryo cell line (CHSE‐214), fathead minnow epithelial cell line (FHM) and rainbow trout liver cell line (R1). One non‐virulent strain was highly adhesive and invasive in the three cell lines, whereas the virulent strains showed moderate adhesive and invasive capacity. The internalization of several isolates was inhibited by colchicine and cytochalasin‐D, suggesting that microtubules and microfilaments play a role. For all strains, intracellular survival assays showed a decrease of viable bacteria in the cells 6 h after inoculation, suggesting that Y. ruckeri is not able to multiply or survive inside cultured cells. Analysis of the susceptibility to the bactericidal effect of rainbow trout serum demonstrated that virulent Y. ruckeri strains were serum resistant, whereas non‐virulent strains were generally serum sensitive.     

Effect of combination of dietary Bacillus subtilis and trans‐cinnamic acid on innate immune responses and resistance of rainbow trout,Oncorhynchus mykiss to Yersinia ruckeri

The present study investigated the effects of combination of dietary Bacillus subtilis and trans‐cinnamic acid on serum biochemical parameters, innate immune responses and resistance of rainbow trout, Oncorhynchus mykiss to Yersinia ruckeri. Six experimental groups of fish with mean weights of 20.58 ± 0.35 g were used in the study. Five experimental groups of fish were fed diets containing Bacillus subtilis (10⁷ per gram) or a mix of the Bacillus subtilis (BS) and trans‐cinnamic acid (25 mg/kg‐25trcBS, 50 mg/kg‐50trcBS, 75 mg/kg‐75 trcBS, 150 mg/kg‐150 trcBS), whereas an additive‐free basal diet served as the control (Cont). In this study, an increase was observed in granulocyte percentage, respiratory burst activity, phagocytic activity, phagocytic index, myeloperoxidase activity and total antiprotease activity especially in fish fed with mix of the BS and trans‐cinnamic acid‐supplemented diets (p < .05). Moreover, at the end of the 20‐day challenge period the survival rates and antibody titre (p < .05), and relative per cent survival were higher in the BS group and all trcBS groups compared with control group. As a conclusion, the results in the present study show that feeding rainbow trout with diets containing a mix of B. subtilis and trans‐cinnamic acid over a 60‐day period might be sufficient for improving fish immune responses and disease resistance against Y. ruckeri.     

A survey on Tetracapsuloides bryosalmonae infections in Slovene fresh waters

Slovenia has no history of health problems related to proliferative kidney disease (PKD) either in farmed or in wild fish. However, due to the past molecular evidence for the presence of Tetracapsuloides bryosalmonae DNA in tissues of some fish from open waters, a survey was conducted on wild salmonids that were primarily sampled for other purposes. In winter 2010–2011, specimens from a total of 244 rainbow trout, Oncorhynchus mykiss (Walbaum), and brown trout, Salmo trutta L., from 30 bodies of fresh water were examined for T. bryosalmonae using a PCR method. The sampled fish showed no clinical signs or gross pathological lesions characteristic of PKD. Nineteen (7.8%) fish from seven (23.3%) fresh waters were positive for T. bryosalmonae. The identity of PCR amplicons was confirmed by sequencing. With one exception, all the positive fish were found in waters from the regions where the average yearly temperatures and the environmental pollution are higher. This preliminary countrywide survey provided the first insight into the situation regarding T. bryosalmonae infection of wild salmonids in Slovenia.     

Comparison of mortality and viral load in rainbow trout (Oncorhynchus mykiss) infected with infectious pancreatic necrosis virus (IPNV) genogroups 1 and 5

Infectious pancreatic necrosis virus (IPNV) is the aetiological agent of a highly contagious disease that affects farmed salmonids. IPNV isolates have been phylogenetically classified into eight genogroups, of which two are present in Chile, genogroups 1 and 5. Here, we compare the mortality rate caused by isolates from both genogroups in rainbow trout (Oncorhynchus mykiss) fry to determine if there is an association between host susceptibility and phylogenetic characterization of IPNV. Fish were challenged by immersion with one of four isolates (two for each genogroup), and mortality curves were assessed after 30 days. Viral load was measured in all mortalities and in live fish sampled at 1, 7 and 20 days post-infection. Although mortality was low throughout the challenge, differences were found between fish infected with different isolates. Both isolates from genogroup 1 caused greater cumulative mortalities than either of the isolates from genogroup 5. When combined, the overall mortality rate of fish challenged with genogroup 1 isolates was significantly higher than those infected with genogroup 5. However, viral load was lower on trout infected with genogroup 1 isolates. These results suggest that rainbow trout are more susceptible to IPNV isolates from genogroup 1 than genogroup 5.     

Effects of Panax ginseng extract in practical diets for rainbow trout (Oncorhynchus mykiss) on growth performance,immune response and resistance to Yersinia ruckeri

In a preliminary in vitro study, a Panax ginseng extract exhibited an evident antibacterial activity against Yersinia ruckeri and Lactococcus garvieae and affected the respiratory burst and proliferation of rainbow trout Oncorhynchus mykiss leukocytes. Subsequently, the effects of a dietary ginseng extract supplementation on growth, blood biochemical profile, innate immune response and resistance against Y. ruckeri infection were investigated in vivo in rainbow trout juveniles. Four experimental diets were obtained by adding 0.0%, 0.01%, 0.02%, 0.03% of ginseng ethanolic extract to a commercial feed. Each diet was fed to triplicate groups of fish (mean body weight 30.5 ± 0.15 g) at 1% of body weight day^?1 for 10 weeks. The dietary supplementation with ginseng extract did not affect growth performance, feed utilization, biometric traits and fish whole body composition (P > 0.05). No major changes due to graded levels of ginseng extract in the diet were observed in blood biochemical parameters except for increasing plasma triglycerides and non‐esterified fatty acids in fish fed diets including 0.01% and 0.02% of extract (P < 0.05). The innate immune response was barely modulated by the dietary addition of ginseng extract. Serum lysozyme and leukocytes respiratory burst activities were just slightly increased in fish fed all the ginseng extract‐supplemented diets compared with controls, whereas serum antiproteases and leukocyte MPO were not affected (P > 0.05). The dietary administration of ginseng extract induced a reduction in mortality of rainbow trout infected with Y. ruckeri, although no significant differences between treated and control groups were observed (P > 0.05).