Microbiology and histopathology of 'saddleback' disease of underyearling Atlantic salmon, Salmo salar L.

Abstract. Heavy mortalities of underyearling Atlantic salmon parr have occurred at the Mactaquac Fish Culture Station in New Brunswick as the result of 'saddleback' disease. The organism causing this lesion was isolated and identified as Flexibacter columnaris. This bacterium was transmitted to other salmon parr, where it produced the typical saddle lesion from which it was re-isolated. The condition was more readily transmitted at 20°C than at 15°C. Transmission required entrance to the dermis through a break in the epidermal surface and was unsuccessful in intact fish. The morphology of the saddleback isolate and its histopathological effect on the fish tissues is described. F. columnaris appeared to be restricted to the dermis and underlying muscle of the saddle area when histological techniques were used, although it was also isolated from the kidneys of some affected fish. The gills, kidney and other organs of the fish did not seem to be affected by the presence of F. columnaris.     

Observations on the growth, survival and disease resistance of juvenile common sole, Solea solea (L.), fed Mytilus edulis L.

Abstract. The growth and survival of groups of juvenile sole, Solea solea (L.), fed exclusively fresh or heat-treated and frozen (stored) mussel, Mytilus edulis L., were compared and the effect of supplementing the stored mussel with one and two feeds per week of fresh mussel was assessed. Over the 9 weeks of the experiment the survival of the fish fed fresh mussel exceeded 90%. In contrast, less than 3% of those fed stored mussel alone survived. The inclusion of two feeds per week of fresh mussel increased survival to the same level as that of the groups fed only fresh mussel, though growth rates were significantly lower. The mortalities of the fish fed stored mussel were associated with an outbreak of the disease 'black patch necrosis' (BPN). The fish fed fresh mussel did not develop the symptoms of this disease despite their proximity to diseased fish and the lack of precautions to prevent its spread. The fish were not provided with a sand substrate, often considered necessary for sole ongrowing. It is suggested that a combination of adequate nutrition and attention to tank cleanliness is sufficient to avoid the occurrence of BPN and that the provision of a sand substrate is not a prerequisite for successful culture of juvenile sole.     

Effect of environmental conditions on the infectivity of Flexibacter columnaris to fish

Abstract. Columnaris disease, Flexibacter columnaris infection, was first described in North America. Recent publications indicate that its distribution is worldwide. Both warmwater and coldwater fish are infected with F. columnaris. All freshwater fish are probably susceptible to the disease under environmental conditions favourable to the bacterium and stressful to the fish. Flexibacter columnaris cannot tolerate sea water. Generally, outbreaks occur when the average water temperatures reach 15°C. The bacterium apparently enters the fish through the skin and gills. The swarms of bacteria involved give the lesions a characteristic yellow pigmentation. The external lesions destroy the skin, muscle and gill tissues. It is relatively easy to isolate the bacteria from the internal organs of the diseased fish, but there is no evidence of internal tissue damage. In this short review, emphasis is placed on several factors relative to water environment which play a role in the development of outbreaks of columnaris disease:

• 1 The optimum temperature for an outbreak of F. columnaris infection is thought to be between 20 and 30°C. Mortalities seldom occur at temperatures below 15°C, although highly virulent strains can kill fish at lower temperatures.
• 2 Flexibacter columnaris can survive longer in water of normal to high hardness, as well as in water containing small amounts of sodium, potassium, calcium or magnesium salts, but it cannot live in sea water.
• 3 Flexibacter columnaris grows well on paniculate fish feeds so that such deposits in the water may act as reservoirs of infection.
• 4 An important source of infection is feral fish. In the Columbia River or the Fraser River, resident coarse fish such as suckers, Catostomus spp., are thought to transmit the infection to returning adult salmon.
• 5 Flexibacter columnaris fails to invade fish in the presence of competitive bacteria like Citrobacter freundii when its initial numbers are approximately one in a 100 less than those of the competitors.
• 6 Crowding seems to be not only a stressor for fish, but also a factor that increases the chance of adhesion of F. columnaris to the external tissues of fish.

     

A comparative study of the tissue response to invasion and encystment by Stephanochasmus baccatus (Nicoll, 1907) (Digenea: Acanthocolpidae) in four species of flatfish

Abstract. Four species of flatfish [plaice, Pleuronectes platessa L.; turbot, Scophthalmus maximus (L.); Dover sole, Solea solea (L.) and the common dab, Limanda limanda (L.)] were experimentally infected with cercariae of Stephanochasmus baccatus under similar conditions and examined at intervals up to 7 weeks post-infection- The tissue response was found to be qualitatively the same in all four hosts and was consistent with a chronic granulomatous inflammatory reaction induced by resistant particulate foreign bodies. The intensity and rate of the response varied between host species but the capsule formation was essentially similar in all. The mature capsule consisted of an inner layer composed of leucocytes which underwent epithelioid transformation. The cellular structure of this tissue gradually degenerated from the oldest layer to form a dense eosinophilic ground substance in which was embedded nuclear debris. The outer layer of the capsule consisted of fibroblasts and reticulin collagen fibres. Muscle and fin cysts differed, with muscle cysts tending to be less discrete and more fibrous. The rate and intensity of the response to the parasite varied between different host species. Plaice and turbot showed the most intensive response and in turbot the reaction was considerably accelerated. Mortality of encysted metacercariae was only seen in turbot. The overall effect on larger 1-group fish was minimal, but the smaller 0-group plaice were more severely affected by the relatively large cvsts that interfered with organ function.     

草鱼细菌性烂鳃病病原的研究

从患细菌性烂鳃病的草鱼病灶分离得滑动细菌M_(165),经多次水中感染(及反复分离、再感染),能使草鱼及多种池塘鱼类显症、死亡。M_(165)的生物学特性与鱼害粘球菌相似,但不能用Dworkin法快速诱生小孢子。该菌DNA碱基比为35.3％,与粘球菌有明显差异,采用柱状屈桡杆菌学名Fiexibacter columnaris (Davis)Leadbetter。     

Dominant pathogenic species of mesophilic aeromonads isolated from diseased and healthy fish cultured in Poland

Aeromonas isolates were collected from cultured fish, characterized phenotypically and identified to species using 16S rDNA. The pathogenicity of all isolates was assayed on the basis of haemolytic and proteolytic activity and challenge tests were performed for isolates from healthy fish. A total of 131 Aeromonas isolates were obtained and identified as follows: A. hydrophila (13), A. bestiarum (23), A. salmonicida (motile biogroup) (19), A. caviae (2), A. sobria (18), A. veronii bt. sobria (42), A. jandaei (1), A. encheleia (11) and A. allosaccharophila (2). All isolates of A. hydrophila and A. bestiarum and most isolates of A. salmonicida and A. veronii were classified as pathogenic. Aeromonas hydrophila was isolated only from diseased trout except for one isolate obtained from carp fry. The other potentially pathogenic Aeromonas species were present in diseased as well as healthy fish. The pathogenicity of isolates from healthy fish was correlated with their enzymatic activity and was also tested by challenge experiments. The dominant pathogenic species were A. veronii bt. sobria, A. bestiarum and A. salmonicida in common carp and A. hydrophila in rainbow trout.     

The influence of salmon surface mucus on the growth of Flavobacterium columnare

Flavobacterium columnare is the causative agent of columnaris disease. The presence of lesions on the gills, skin and fins of diseased fish suggests that F. columnare is able to utilize fish skin mucus as a substrate for growth and that exposure to this material would alter the expression of genes involved in the colonization of the outer surfaces of the fish. Growth, biofilm formation, extracellular protease production and changes in protein expression of F. columnare strain C#2 cultured in media supplemented with juvenile Atlantic salmon skin mucus were compared with the same media without mucus. C#2 was able to grow by using mucus as the sole nutrient source. Growth in mucus-containing media induced cells to grow as a biofilm and extracellular protease activity increased in mucus-containing cultures. SDS-PAGE protein profiles showed that expression of six extracellular proteins increased in mucus-containing media. These results demonstrate that salmon surface mucus promotes the growth of F. columnare and that exposure to mucus alters the growth characteristics of this bacterium with regard to protease production and biofilm formation. Further characterization of mucus-induced physiological changes will increase our understanding of the basis of virulence of this economically important fish pathogen.     

Effects of low salinities on Flavobacterium columnare infection of euryhaline and freshwater stenohaline fish

Channel catfish, Ictalurus punctatus (Rafinesque), goldfish, Carassius auratus (L.), striped bass, Morone saxatilis (Walbaum), and Gulf sturgeon, Acipenser oxyrinchus desotoi Vladykov, were acclimatized to fresh water or salinities of 9.0‰ or less and then exposed to Flavobacterium columnare (formerly known as Flexibacter columnaris ), the bacterial pathogen that causes columnaris disease. None of the fish acclimatized to 3.0 or 9.0‰ salinity died, and all deaths in lower salinities occurred between 1 and 5 days after exposure to F. columnare . Mortality was 97.7% in fresh water and 67.1% in 1.0‰ salinity for channel catfish (model SE, 1.8) and 66.5% in fresh water and 40.8% in 1.0‰ salinity for goldfish (model SE, 1.2); and 96.9% in fresh water and 61.7% in 1.0‰ salinity for striped bass (model SE, 1.8). After exposure to F. columnare , none of the Gulf sturgeon died. Flavobacterium columnare was isolated from the skin and gills of all fish dying during the experiments, but was not isolated from survivors in fresh water and 1.0‰ salinity 21 days after bacterial exposure. In vitro growth of bacteria was significantly higher in 1.0 or 3.0‰ salinity than in control medium (0.3‰ salinity). However, in vitro adhesion of bacteria was reduced with increasing salinity, which could explain the lower mortality of fish at higher salinities.     

Bacteria associated with early life stages of halibut, Hippoglossus hippoglossus L., inhibit growth of a pathogenic Vibrio sp.

Abstract Bacteria capable of inhibiting growth of a pathogenic Vibrio sp. were isolated from the gastrointestinal tract of cultured halibut larvae during the first feeding and weaning stages. No such bacteria were found among isolates from the surface of eggs or the gastrointestinal tract of yolk sac larvae. The fraction of pathogen-inhibitors among the total number of isolates ranged between 0–100% (first feeding) and 0–66% (weaning). All pathogen-inhibitors were Gram-negative rods, and 95% were oxidase and catalase positive fermentative isolates, capable of producing acid aerobically from a varying range of carbohydrates. These isolates possessed the characteristics of the Vibrio/Aeromonas -group, but only 19% were sensitive to the vibriostatic agent 0/129. Isolates from eggs and yolk sac larvae were dominated by bacteria belonging to the Cytophaga/Flexibacter/Flavobacterium - group. The high fraction of isolates with the ability to inhibit growth of the pathogenic Vibrio sp. among the total number of isolates indicates that pathogen inhibition may be an important mutualistic role of the intestinal flora of early life stages of halibut.     

Adhesion to sole, Solea senegalensis Kaup, mucus of microorganisms isolated from farmed fish, and their interaction with Photobacterium damselae subsp. piscicida

Abstract Most studies carried out to select microorganisms as candidate probiotics have focused on in vitro antagonism tests, such as the production of inhibitory compounds against pathogenic microorganisms. However, attachment to mucous surfaces could be another criterion to be considered when selecting potential probiotics for aquaculture. Nineteen isolates obtained from farmed Senegalese sole, Solea senegalensis Kaup, and gilthead sea bream, Sparus aurata L., have been evaluated for their capacity to adhere to skin and intestinal mucus of Senegalese sole, and their antagonistic effect against Photobacterium damselae subsp. piscicida, an important pathogen for farmed sole. The isolates from gilthead sea bream showed the highest percentage of adhesion to sole mucus, whilst the pathogenic microorganisms assayed and the isolates from sole showed, in general, a lower ability to adhere to sole mucus. The results suggest that the adhesion to fish mucus was more dependent on the isolate tested than on the host mucus. The isolates from gilthead sea bream also showed a higher antagonistic activity against P. damselae subsp. piscicida than those from Senegalese sole. Four isolates were selected, on the basis of their adhesive ability and antagonistic effect on P. damselae subsp. piscicida, to study their interactions with the pathogen in respect of adhesion to skin and intestinal mucus under exclusion, competition and displacement conditions. The results obtained show the ability of three isolates to reduce the adhesion of P. damselae subsp. piscicida to sole mucus under displacement and competition conditions. The adhesion of the pathogen to sole intestinal mucus was also significantly reduced when three isolates were assayed under exclusion conditions.     

Action of selected antibiotics on four common bacteria associated with diseases of fish

Abstract. Four bacteria commonly associated with diseases of fish ( Aeromonas hydrophila complex, Aeromonas salmonicida, Flexibacter columnaris -like organisms and Pseudomonas fluorescens ) were evaluated in a serial dilution system for susceptibility to four antibiotics commonly used in the treatment of bacterial fish diseases (chloramphenicol, erythromycin, furpyrinol and oxytetracycline). Findings were expressed as minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). Furpyrinol was found to have the lowest MIC and MBC against A. hydrophila complex, A. salmonicida and F. columnaris-like organisms. MIC and MBC's for oxytetracycline were complicated by the presenceof R-plasmids, but were less than 4.0 mcg/ml for those without plasmids. Pseudomonas fluorescens was susceptible only to oxytetracycline. MIC for all groups of organisms using chloramphenicol was generally less than 16.0 mcg/ml. Susceptibility to erythromycin by these groups of organisms was greater than 16 mcg/ml.     

采用通用引物PCR配合SSCP和RFLP技术检测鱼病病原菌

采用通用引物PCR（UPPCR）、PCR－RFLP、PCR－SSCP技术,研究快速鉴别鱼病病原菌的分子生物学诊断技术。结果发现,采用细菌16S rRNA基因保守区特异性引物,以嗜水气单胞菌、鲁克氏耶尔森菌、鳗弧菌、柱状曲挠杆菌、乙型链球菌、荧光假单胞菌等部分常见鱼病病原菌为对象,可以建立一种UPPCR技术。该技术能在保证实验条件不变的基础上,检出上述所有细菌,并还可检出大肠杆菌和双歧杆菌等非鱼病病原菌。并且认为,该法与SSCP配合即采用UPPCR－SSCP技术能较好地鉴别被检菌而用于鱼病病原菌的快速诊断。     

Chemotherapy of Cytophaga/Flexibacter-like bacteria (CFLB) infections in fish: studies validating clinical efficacies of selected antimicrobials

Abstract. In vitro and in vivo efficacies of commonly used chemotherapeutants were determined for Cytophaga johnsonae, Cytophaga psychrophila, Flexibacter colutnnaris and Flexibacter maritimus. Treatmeat of barramundi, Lutes calcarifer (Bloch), with oxolinic acid (OA) as a bath (50 ppm) or by mouth (10 mg kg⁻¹ body weight) resulted in serum levels above the minimum inhibitory concentration (MIC) for F. columnaris and produced significant clinical efficacy ( P < 0.05). Amoxycillin (AM) was found to produce adequate serum levels against F. maritimus when used as a bath (200 ppm) or given orally (80 mg kg⁻¹ body weight) to Atlantic salmon, Salmo salar L. and rainbow trout, Oncorhynchus mykiss (Walbaum), respectively. At these dose rates, this antibiotic was also clinically efficacious against F. maritimus ( P <0.05). Trimethoprim produced more than adequate serum levels for the control of F. maritimus when given as a bath (50 ppm) or orally (10 mg kg⁻¹ body weight). Trimethoprim was significantly more protective than AM when tested in vivo ( P < 0.05). For C. johnsonae and C. psychrophila , the MIC values for OA and oxytetracycline were low, whereas that for TMP was high. Also, based on MIC values, C. psychrophila strains were more sensitive to AM and norfloxacin than C. johnsonae.     

Interactions of microorganisms isolated from gilthead sea bream, Sparus aurata L., on Vibrio harveyi, a pathogen of farmed Senegalese sole, Solea senegalensis (Kaup)

Four bacterial isolates from farmed gilthead sea bream, Sparus aurata, included in a previous study as members of the Vibrionaceae and Pseudomonodaceae and the genus Micrococcus, have been evaluated for their adhesive ability to skin and intestinal mucus of farmed Senegalese sole, Solea senegalensis, and their antagonistic effect on Vibrio harveyi, a pathogen of sole. These isolates showed higher adhesion to sole mucus than the pathogenic strains of V. harveyi assayed. Only two of the isolates showed antagonistic activity to V. harveyi. Interactions of the four isolates with V. harveyi in respect of adhesion to skin and intestinal mucus under exclusion, competition and displacement conditions were studied. Three isolates were able to reduce the attachment to skin and intestinal sole mucus of a pathogenic strain of V. harveyi under displacement and exclusion conditions, but not under competition conditions. The in vivo probiotic potential of isolate Pdp11 was assessed by oral administration followed by challenge with the pathogenic V. harveyi strain Lg14/00. A group of 50 Senegalese sole received a commercial diet supplemented with 10(8) cfu g(-1) of lyophilized Lg14/00 for 15 days. A second group of fish received a non-supplemented commercial diet. After challenge the mortality of the fish receiving the diet supplemented with the potential probiotic isolate was significantly lower than that in the fish receiving the non-supplemented commercial diet. This study has shown that the ability to interfere with attachment of pathogens, as well as the adhesion to host surfaces, are suitable criteria for selection of candidate probiotics for use in the culture of Senegalese sole.     

Influence of water quality and temperature on adhesion of high and low virulence Flavobacterium columnare strains to isolated gill arches

The ability of Flavobacterium columnare ( Flexibacter columnaris ) to attach to the gills of common carp, Cyprinus carpio L., was evaluated using a gill perfusion model. A comparison between a high and a low virulence strain of F. columnare was made and evaluated in comparison to results obtained previously with an in vivo model. The ion composition of the water of the organ bath in which the gills were suspended was varied and the influence on adhesion processes assessed. Experiments were carried out to examine the influence of water quality (i.e. nitrite and organic matter) and temperature on the capacity of the bacteria to adhere. It was found that the high virulence strain adhered more readily than the low virulence strain, as was found during the in vivo experiments. Moreover, it was observed that adhesion of the high virulence strain was enhanced by a number of factors. These were immersion of the gill in bivalent, ion-rich water, the presence of nitrite or organic matter, and high temperatures.     

Identification of pathogenic bacteria associated with the occurrence of 'red spot' in sea mullet, Mugil cephalus L., in south-eastern Queensland

Abstract. An investigation was undertaken to determine the bacterial species associated with the fish disease known as 'red spot' which affected wild fish stocks in south-eastern Queensland. Outbreaks of the disease in the Noosa River were examined with particular reference to the sea mullet, Mugil cephalus. The bacterial flora of the skin of healthy fish as well as those exhibiting early and advanced lesions was determined. In the lower Noosa River estuary, Vibrio anguillarum was the sole organism associated with very early lesions. It was later shown that V. alginolyticus was also associated with V. anguillarum in early lesions, but was considered to be a secondary invader. Aeromonas hydrophila was isolated from advanced lesions on fish taken in freshwater reaches of the river. This organism was also regarded as a secondary invader.     

Genome-wide comparison of Carnobacterium maltaromaticum derived from diseased fish harbouring important virulence-related genes

Although Carnobacterium maltaromaticum has been used as a probiotic in fish, it was reported to cause disease for the first time in Korea. The objective of this study was to understand the differences between pathogenic and non-pathogenic strains. Pathogenicity was tested by challenging rainbow trout with C. maltaromaticum ATCC35586 and 18ISCm isolated from diseased fish, and DSM20342 isolated from a dairy product. We also compared 24 genomes of C. maltaromaticum strains plus the genome of our isolate 18ISCm sequenced in this study. Only the strains from diseased fish caused high mortality with severe histopathological changes. Although all strains shared more than 90% of Ko_id, wecC and xtmA were found only in strains from diseased fish. Interestingly, only strains from diseased fish harboured two wecC paralogs involved in the production of D-mannosaminuronic acid which is a major component of a well-known virulence factor, teichuronic acid. Two wecC paralogs of 18ISCm were increased when they were co-cultured with trout blood cells, suggesting that wecC genes might play a role in virulence. The results of this study show that strains isolated from diseased fish are different from strains derived from food in terms of pathogenicity to fish and the presence of virulence-related genes.     

Comparison of Culture Traits of Channel Catfish,Ictalurus punctatus,and Blue Catfish I. furcatus

Culture of channel catfish, "Ictalurus punctatus," accounts for virtually all catfish production. Recently, however, an increasing number of catfish farmers are growing or are considering the culture of blue catfish, "I. furcatus." Current and past research the compared the culture traits of channel and blue catfish are reviewed. Blue catfish could be potentially advantageous for certain farm environments. Strain effects in both species are important. In general, channel catfish grow faster to market size than do blue catfish. However, some strains of blue catfish grow faster than many strains of channel catfish. Blue catfish are more vulnerable to parasites such as "Icthyophthirius" and to bacterial infections such as those from "Flexibacter columnaris when compared to channel catfish. Blue catfish do not tolerate handling as well as channel catfish, and handling stress often leads to infections in blue catfish. However, blue catfish have high resistance to enteric septicemia of catfish caused by "Edwardsiella ictaluri" and to channel catfish virus when compared to channel catfish. Blue catfish are twice as vulnerable to harvest by seining than channel catfish, and have a higher dress-out percentage than most strains of channel carfish. Channel catfish tolerate lower dissolved oxygen levels than do blue catfish. Blue catfish require one more year to reach sexual maturity than do channel catfish.     

两种疾病发生对养殖半滑舌鳎肠道菌群结构的影响分析

为研究疾病发生情况下鱼类肠道菌群的结构变化,运用高通量测序方法分别对感染腹水病和皮肤溃疡病的工厂化养殖半滑舌鳎病鱼及同一养殖系统中健康鱼的肠道菌群结构进行了对比分析。结果发现,养殖半滑舌鳎肠道中的细菌种类要明显少于环境中的细菌种类,仅为养殖池进水和颗粒饲料中细菌种类总数的14.3%~44.7%。在疾病发生后,病鱼肠道中的细菌种类数量仅是同一养殖系统中健康鱼肠道细菌种类数量的45.8%和64.5%,病鱼肠道中细菌多样性下降非常明显。进一步分析肠道菌群结构的组成发现,虽然发病鱼和健康鱼肠道中超过60%的细菌种类不相同,但优势菌群的结构组成差异不明显,丰度最高的前10种细菌种类基本相同,其比例超过肠道相对总菌量的80%。不同疾病的发生对半滑舌鳎肠道菌群结构的影响也不同,腹水病会造成肠道中优势菌株的种类和相对丰度都发生比较明显的改变,而皮肤溃疡病发生前后肠道菌群结构组成几乎没有变化。研究表明,养殖半滑舌鳎的肠道菌群结构相对比较保守,而且并不是所有的疾病种类都会引起肠道菌群结构发生改变。     

Isolation and identification of Pseudoalteromonas piscicida strain Cura-d associated with diseased damselfish (Pomacentridae) eggs

Bacteria were isolated from the diseased eggs of two damselfish (Pomacentridae) species, Amphiprion clarkii and Amblyglyphidodon curacao . Healthy A. clarkii eggs were infected separately with four bacterial isolates from the diseased eggs, as well as with Vibrio parahaemolyticus and Listonella anguillarum. Amphiprion clarkii eggs exposed to Cura-d, an isolate from diseased A. curacao eggs, showed increased mortality compared to uninfected control eggs. The A. curacao eggs were infected separately with the same two isolates from diseased A. curacao eggs, and similarly, eggs exposed to isolate Cura-d also showed increased mortality compared to uninfected eggs. However, the increase in mortality associated with exposure to isolate Cura-d was most significant in A. clarkii eggs. A 16S rDNA comparative sequence analysis identified that isolate Cura-d was most similar to Pseudoalteromonas piscicida , a known fish pathogen. Both Cura-d and P. piscicida were found to be polarly flagellated, Gram-negative rods with an oxidative metabolism which grew on d-mannose, d-fructose, sucrose and maltose, but not on l-threonine. Based on these properties, it is proposed that Cura-d should be classified as P. piscicida strain Cura-d. It is concluded that the P. piscicida strain cura-d was associated with the increased mortality observed in A. clarkii eggs.