In vivo fish models for visualizing Aeromonas hydrophila invasion pathway using GFP as a biomarker

The invasion pathway of Aeromonas hydrophila in vivo was studied in Crucian carp (Carassius auratus gibelio) with a virulent strain A. hydrophila J-1 transformed with a plasmid encoding green fluorescent protein (pGFPuv) (A. h J-1^GFP), which had similar virulence characteristics (haemolysin, extracellular proteases production, toxicity for EPC cells, survival in fish serum and LD₅₀ value) as the parent strain. Fish were divided into four experimental groups: (1) normal fish; (2) bacteria bath challenged, unwounded fish; (3) skin artificially wounded by scalpel, bacterial bath challenged fish; and (4) skin mucus layer partially removed by paper towel, bacterial bath challenged fish. The number of bacteria from blood, gills, kidney, muscle, liver and intestine were detected at 2, 4, 8, 12, and 24 h post-challenge. High bacterial numbers were observed in the muscle of the artificial wound group, in the kidney of the mucus removed group and in the gills of all groups. In conclusion, the gills and damaged skin are likely to be the main routes of entry for A. hydrophila, and GFP can be used as a real-time biomarker to study intimate host–pathogen interaction in fish.     

In situ adherence of Vibrio spp. to cryosections of Atlantic salmon, Salmo salar L., tissue.

Pathogenic and presumed non-pathogenic bacteria isolated from fish were tested for their adhesion to cryosections from different mucosal surfaces of Atlantic salmon, Salmo salar L. Adhered bacteria were detected by immunohistochemistry. Mucus was stained and fixed with Alcian blue after incubation of bacteria. The majority of the bacteria tested, i.e. Vibrio anguillarum serotype O1 , Vibrio salmonicida , Vibrio viscosus, Flexibacter maritimus and 'gut vibrios', i.e. Vibrio iliopiscarius and intestinal isolates of V. salmonicida , all adhered to mucus on all salmon epithelial surfaces tested, including sections from the foregut, hindgut, pyloric caeca, gills and skin. In contrast, V. anguillarum serotype O2, including both serotypes O2a and O2b, did not adhere to mucus, but did adhere to all other components of the tissues. As a positive control for adhesion of bacteria on cryosections, Escherichia coli was bound to piglet ileal mucosal lining, and as a negative control for adhesion, Staphylococcus aureus was found not to bind to any of the tissues tested. The present study shows that adhesion to mucus was not restricted to pathogenic bacteria, and furthermore, that not all pathogenic bacteria studied adhered to mucus. Hence, on the basis of these findings, the present authors suggest that V. anguillarum O2 may have an invasion strategy which does not involve adhesion to mucus, and thus, differs from the other pathogenic bacteria in the present study, which all bound to salmon mucus.     

Translocation of viable Aeromonas salmonicida across the intestine of rainbow trout, Oncorhynchus mykiss (Walbaum)

The pathogenic bacterium Aeromonas salmonicida is the causative agent of the destructive disease furunculosis in salmonids. Horizontal transmission in salmonids has been suggested to occur via the skin, gills and/or intestine. Previous reports are contradictory regarding the role of the intestine as a route of infection. The present study therefore investigates the possibility of bacterial translocation across intestinal epithelia using Ussing chamber technology, in vitro. Intestinal segments were exposed for 90 min to fluorescein isothiocyanate-labelled pathogenic A. salmonicida. Sampling from the serosal side of the Ussing chambers showed that bacteria were able to translocate across the intestinal epithelium in both the proximal and distal regions. Plating and subsequent colony counting showed that the bacteria were viable after translocation. During the 90 min exposure to A. salmonicida, the intestinal segments maintained high viability as measured by electrical parameters. The distal region responded to bacterial exposure by increasing the electrical resistance, indicating an increased mucus secretion. This study thus demonstrates translocation of live A. salmonicida through the intestinal epithelium of rainbow trout, suggesting that the intestine is a possible route of infection in salmonids.     

Comparative challenge model of Flavobacterium columnare using abraded and unabraded channel catfish, Ictalurus punctatus (Rafinesque)

The early entry of the fish pathogen Flavobacterium columnare and enhancement by abrasion was studied in channel catfish, Ictalurus punctatus (Rafinesque), using the polymerase chain reaction and a species-specific primer set for a bacterial 16S rRNA gene product. Evaluations were conducted following an abrasion bath immersion challenge with F. columnare. Abrasion, a practice which has historically been used prior to bacterial challenge, had significant effects on the early entry of the pathogen and on cumulative percent survival (CPS). The FvpF1-FvpR1 primer set was useful in detecting the early entry of F. columnare in mucus, skin, gill, blood, liver and trunk kidney tissues in both abraded and unabraded fish following immersion challenge at 29 +/- 2 degrees C. Bacteria were detected earlier in all tissues in abraded fish, except in the trunk kidney. These differences were not significant, except in the case of blood. Mucus, skin and gill tissues were positive for F. columnare earliest regardless of treatment (after 5 min in abraded fish and after 15 min in unabraded fish). CPS following challenge with F. columnare was significantly affected by abrasion, which supports the use of abrasion for the F. columnare challenge model for channel catfish.     

Effect of three dietary oils on disease susceptibility in Arctic charr (Salvelinus alpinus L.) during cohabitant challenge with Aeromonas salmonicida ssp. salmonicida

Arctic charr ( Salvelinus alpinus L.) were fed diets based on a commercial recipe supplemented with either linseed, soybean or marine oil prior to cohabitant challenge with Aeromonas salmonicida ssp. s almonicida . Mortality varied significantly between the three dietary groups. Highest mortality (48%) was observed in fish fed the marine oil and the lowest mortality (20%) was in the group fed soybean oil. Transmission electron microscopy (TEM) examination of the digestive tract of uninfected fish demonstrated substantial numbers of bacterial cells between microvilli. However, only a few bacteria were recovered that were associated with the microvilli of infected fish. Immunocytochemical staining/labelling investigations using TEM and an immunogold method were performed on mid-gut segments of fish fed the marine oil diet and showed augmentation of goblet cells and the presence of A. salmonicida ssp. salmonicida in the gastrointestinal tract of diseased fish after challenge with the pathogen. It is suggested that the gastrointestinal tract could be an infection route of A. salmonicida ssp. salmonicida. The greater prevalence of goblet cells supports the suggestion that sloughing off mucus is a protective response against bacterial infections. These results make an important contribution to our understanding of how nutrition can affect the disease resistance of fish.     

Use of probiotics to control furunculosis in rainbow trout, Oncorhynchus mykiss (Walbaum)

Aerobic heterotrophic bacteria were isolated from the intestinal contents of Atlantic salmon, Salmo salar , rainbow trout, Oncorhynchus mykiss , and turbot, Scophthalmus maximus , on tryptone soya agar and De Man Rogosa and Sharpe agar, of which 11 of 177 (6% of the total) of the isolates were antagonistic to Aeromonas salmonicida . Four of these cultures, which were identified tentatively as A. hydrophila , Vibrio fluvialis , Carnobacterium sp. and an unidentified Gram-positive coccus, were beneficial to fish when fed singly or as an equi-mixture. Feed supplemented with the putative probiotics indicated survival of the organisms in the gastrointestinal tract for 7 days. Feeding with the probiotics for 7 and 14 days led to better survival following challenge with A. salmonicida . There was no indication of serum or mucus antibodies to A. salmonicida , but there was an increased number of erythrocytes, macrophages, lymphocytes and leucocytes, and enhanced lysozyme activity in the fish.     

Protection against carp erythrodermatitis following bath or subcutaneous exposure to sublethal numbers of virulent Aeromonas salmonicida subsp. nova

Abstract. A bath challenge system was used to infect carp. Cyprinus carpio L., with Aeromonas salmonicida subsp. nova , the causative agent of carp cruthrodermatitis. Bath-challenged fish became infected with the bacterium exihibitinng typical signs of the disease, Carp that were sublethally bath exposed became infected and exhibited some skin lesions, but after one week, these quickly healed and the animals fully recovered from the infection, Naive fish that had not been previously exposed to the bacterium had mortalities of 100% when infected by the subcutaneous route and 40–60% by the bath route of infection. Carp that received sublethal infections were able to withstand subsequent lethal infection and recover regardless of the route of infection. Sublethally bath-exposed carp were protected from subsequently lethal challenges of A. salmonicida subsp. nova for at least 5 months.     

Efficacy of bath vaccination with a live attenuated Vibrio harveyi against vibriosis in Asian seabass fingerling,Lates calcarifer

Vibrio harveyi causes vibriosis in various marine aquaculture fish species, especially when they are young. The infection subsequently leads to significant economic losses for aquaculture farms. Vaccination is recommended to control this disease. This study describes the efficacy of a live attenuated V. harveyi strain MVh_vhs (LAVh) as a vaccine candidate in controlling infection by wild‐type V. harveyi (WTVh) in Lates calcarifer. A total of 240 fingerlings were divided into four groups. Group 1 was not vaccinated and was not challenged, Group 2 was vaccinated with a formalin‐killed V. harveyi (FKVh), Group 3 was vaccinated with the LAVh before challenge and Group 4 was not vaccinated and was challenged. Bath vaccination was employed for one hour before the LAVh distribution was determined in the fish mucus, gill, liver, gut, kidney and spleen. The gills, livers, kidneys and skins were also sampled for gene expression analysis. To challenge the fish, skin abrasion was conducted before the fish were challenged by immersion with WTVh. The results revealed an extensive distribution of the LAVh in the liver and kidneys of the fish in Group 3 for the first 12 hr, resulting in mild lesions compared with Group 1. Similarly, there were significantly (p < .05) higher expressions of the Chemokine ligand 4 and major histocompatibility complex I genes in the skin and liver of the fish in Group 3 in comparison with other groups. Vaccination with LAVh resulted in a significantly high rate of survival (68%) of the fingerlings after being challenged with WTVh.     

用绿色荧光蛋白标记的细菌研究鱼体吸收颗粒抗原的部位

用绿色荧光蛋白标记的嗜水气单胞菌４３３２株（Ａｈ４３３２＾ＧＦＰ）菌液高渗浸泡和直接浸泡异育银鲫,研究鱼体对颗粒抗原的吸收。经两种方法处理的鱼,在整个实验过程中显示,鱼鳃的标记菌检出量均高于皮肤和肠道中的标记菌,证实鳃是抗原吸收的主要部位。另外,鱼体经高渗处理,鳃和皮肤可检出的标记菌明显增加。在肠道中,除浸泡６０ｍｉｎ,浸泡１０ｍｉｎ、３０ｍｉｎ及１２０ｍｉｎ的细菌检出量也明显增加。显示高渗可促进抗原的吸收。     

Improved recapture rate of vaccinated sea-ranched Atlantic salmon, Salmo salar L.

Vaccination of sea-ranched Atlantic salmon was conducted in order to investigate if immuno-prophylactic measures could improve their survival. Fish were either vaccinated by bath or injection. A total of 66 000 fish were reared in fresh water at a hatchery on the island of Bornholm and at the presmolt stage were separated in three groups each comprising of 22 000 fish. One group was vaccinated intraperitoneally with a polyvalent vaccine (containing killed Vibrio anguillarum serotype O1 and O2, Yersinia ruckeri and Aeromonas salmonicida ). A second group was bath vaccinated with the corresponding vaccine-components and the third group was used as a non-vaccinated control. One month after vaccination these groups were allocated to three separate net-pens located 500 m from the coastline of the island. After 4 months in the net-pens, 1000 fish from each cage were tagged with Carlin-tags below the dorsal fin. The fish were then released for a migration period in the Baltic Sea. Following a sea period of 40 months (45 months post-vaccination), the recapture rates of the groups were calculated from the returned tags from fishermen. Recapture of the injection vaccinated group was significantly higher (25%) compared with the bath vaccinated fish (14.7%) and the control group (16.8%).     

Evaluation of an experimental Aeromonas salmonicida epidemic in chinook salmon, Oncorhynchus tshawytscha (Walbaum)

To determine the dynamics of the transmission of Aeromonas salmonicida ssp. salmonicida infection, chinook salmon, Oncorhynchus tshawytscha, were exposed to bacteria by cohabitation. The latent period (time between exposure and infectivity) was determined by exposing a group of chinook salmonid fingerlings to A. salmonicida by bath, then, at daily intervals, by holding five exposed (donor) fish with approximately 50 naive fish for 24 h. The latent period was 3 days post-infection and the time period between the initial exposure to bacteria and the beginning of bacterial shedding was 4.5 days for the same animals. The prevalence and intensity of infection in the donor fish, to which recipient fish were exposed, i.e. the level of exposure, was highly correlated with the development of disease in recipient (susceptible) chinook salmon (r2 = 0.57). An experiment was conducted to determine the daily progress of infection and development of a furunculosis epidemic among recipient fish by cohabiting a single exposed fish with 43 unexposed salmon. At daily intervals, all fish (in seven treatment tanks and one control tank daily) were sacrificed and tested for the presence of A. salmonicida in the kidney (n = 3520). Over 10 days, mean prevalence among recipient fish reached 75% and disease related mortality exceeded 50%. Bacterial concentrations in the water continued to increase over the duration of the experiment in concert with the number of infected animals present in the population.     

Plasma ladderlectin concentration following sterile inflammation and Aeromonas salmonicida subsp. salmonicida infection

Plasma samples obtained from rainbow trout either experimentally infected with Aeromonas salmonicida or injected with either A. salmonicida lipopolysaccharide (LPS) or a commercial A. salmonicida vaccine (Lipogen) were analysed by enzyme immunoassay to evaluate changes in rainbow trout ladderlectin (RTLL) concentrations during the acute phase response (APR). Plasma RTLL concentrations in fish injected with A. salmonicida LPS, vaccine or live A. salmonicida varied over a 10 day period, but did not significantly increase. In contrast, fish experimentally infected with A. salmonicida exhibited a modest, but statistically significant ( P  <   0.05), decrease in RTLL concentration. These studies demonstrate that RTLL is not detectably induced during the trout APR to sterile inflammation or A. salmonicida infection, but plasma concentration of this protein may be reduced during bacterial infection.     

Pathogenicity of Pasteurella sp. in lumpsuckers (Cyclopterus lumpus L.)

The incidence of disease caused by Pasteurella sp. in farmed lumpsuckers in Norway has been steadily increasing in recent years, causing significant economic losses and fish welfare issues. The disease affects all life stages, both in hatcheries and after release into salmon cages. Therefore, it is important to establish robust challenge models, to be used for vaccine development. Exposure experiments via intramuscular and intraperitoneal injection underlined the high virulence of the bacteria, whereas the cohabitation and bath models allowed the chronic symptoms of the disease to be studied more accurately. Skin lesions and haemorrhage at the base of fins were observed in the more acute cases of the disease. Symptoms including white spots over the skin, especially around the eyes, characterized the chronic cases. The latter were most prominent from the bath challenge model. Histopathology indicated a systemic pattern of disease, whereas qPCR analysis from head kidney showed that bacteria may be present in survivor fish at the end of the challenges. In all the challenge models investigated, Pasteurella sp. was re‐isolated from the fish, thus fulfilling Koch's postulates. These findings highlight the importance of screening of lumpsuckers prior to transfer to minimize the risks of carrying over asymptomatic carriers.     

Influence of dietary carbohydrate on blood chemistry, immunity and disease resistance in Atlantic salmon, Salmo salar L.

Abstract. The influence of dietary carbohydrate (CHO) on blood chemistry, immunity and disease resistance was studied in two experiments with Atlantic salmon, Salmo salar L. Moist diets with increasing amounts of digestible CHO ranging from 0 to 30% (dry weight) were used. In the first experiment with adult (0.5 kg) fish, blood haemoglobin concentration was negatively correlated with increasing dietary CHO level, while serum glucose and protein did not differ between the groups. Serum cortisol increased linearly in fish fed from 5 to 30% CHO. Serum haemolytic activity was negatively correlated with dietary levels of CHO. Humoral immune responses elicited after vaccination by intraperitoneal injection or by dip immersion with Vibrio salmonicida showed no differences according to diet 10 and 17 weeks post-vaccination. Mortality after challenge with live Aeromonas salmonicida by intraperitoneal injection was lowest in fish fed 10% CHO. In the second experiment with juvenile Atlantic salmon (3g), there were minor differences in body and organ weights. Plasma glucose, protein and cholesterol were elevated in fish fed the highest CHO levels. Fish exposed to immersion challenge with different water concentrations of Vibrio anguillarum showed no statistical differences in mortality. The studies indicate that varying dietary levels of CHO affected immunity and resistance to bacterial infections to a minor extent in Atlantic salmon at low water temperatures during freshwater and seawater stages.     

Edwardsiella ictaluri bacteraemia elicits shedding of Aeromonas hydrophila complex in latently infected channel catfish, Ictalurus punctatus (Rafinesque)

Edwardsiella ictaluri is a primary bacterial pathogen of channel catfish, Ictalurus punctatus, and the causative agent of enteric septicaemia of catfish . Edwardsiella ictaluri is known to gain entry to the host by infection of the nares, gastrointestinal tract, and gills, and to disseminate to organs via an as yet uncharacterized acute bacteraemia. In this study, fluorescent microscopy showed E. ictaluri on the gill within 5 min of immersion challenge and E. ictaluri could also be isolated from the circulation within 5 min. When removed to clean water, catfish cleared circulating bacteria within 15 min and the blood remained free of E. ictaluri until its reappearance at the 12 h post-infection sampling. However, Aeromonas hydrophila , the aetiological agent of motile aeromonad septicaemia, appeared within the circulation 7 h post-challenge with E. ictaluri and was detected in all fish at 12 h post-infection. Only 20% of fish carried A. hydrophila in the trunk kidney that could be detected by plate culture on Rimler–Shotts agar; however, 100% of challenged and stress-control fish were A. hydrophila complex positive at 24 h post-challenge. These results suggest that although the catfish is capable of clearing its circulation of E. ictaluri , superinfection with latent A. hydrophila may enhance clinical signs of edwardsiellosis. This is the first report of a bacterial superinfection appearing in fish.     

鲤浮肿病毒人工感染锦鲤的试验研究

为研究鲤浮肿病毒的感染方式及在锦鲤各组织器官中的动态分布,在水温20～22℃,溶解氧7～8mg/L试验条件下,采用注射、浸泡、划伤后浸泡3种方式人工感染锦鲤,观察了试验鱼的发病和死亡情况;检测了鲤浮肿病毒在感染鱼体内各组织器官中的存在情况,探讨了主要被感染组织的病理变化。试验结果显示,采用注射、浸泡、划伤后浸泡3种方式均可导致健康锦鲤感染该病毒,病鱼出现昏睡、烂鳃、凹眼等症状,累积死亡率35%～88%。锦鲤感染该病毒后至少有3d的潜伏期,随后病毒在鳃组织中大量增殖,至7d达到高峰,病毒量314.8个/ng,之后病毒量开始逐渐下降,至12d在鳃组织中检测不出病毒。鲤浮肿病毒在肾、肝、脑、肌肉和脾中存在的时间较短且病毒量较低,在血液和肠道中未检出。结合鳃组织病理切片可知,鳃为该病毒感染的主要靶器官。本研究结果为鲤浮肿病的诊断和防控提供了一定的科学依据。     

Virulence of Flavobacterium branchiophilum in experimentally infected salmonids

Abstract. The ability of selected strains of Flavobacterium branchiophilum to attach to and colonize the gills of five species of teleosts (four salmonid and one cyprinid) and cause mortality was investigated. In virulence studies with F. branchiophilum strain LAB4a, cumulative mortality was dose-dependent in rainbow trout, Oncorhynchus mykiss (Walbaum), and ranged from 0 to 75%. However, regardless of dose, the relative amount of gill-associated F. branchiophilum antigen increased 1 h after challenge. The gill-associated F. branchiophilum antigen in fish which survived (moribund fish) increased by four to six times compared to that detected on the gills 1 h after challenge. The gill-associated antigen on moribund fish was not significantly different from that on the gills of dead fish. Flavobacterium branchiophilum strain LAB4a also attached to the gills of brook trout, Salvelinus fontinalis (Mitchell), rainbow trout, chinook salmon, Oncorhynchus tshawytscha (Walbaum), Arctic charr, Salvelinus alpinus (L.), and common shiners, Luxilus cornutus (Mitchell), following a 1-h bath exposure, and caused mortality in all species. The virulence of eight strains of F. branchiophilum for rainbow trout was examined. Both virulent and avirulent strains adhered to the gills following bath exposure (fimbriae were observed on all strains), but only virulent strains had the capacity to further colonize the gills and cause mortality.     

Protective Immunity of Goldfish Against Ichthyophthirius multifiliis Infection Induced by Different Trophont Vaccine Preparations

Ichthyophthirius multifiliis, commonly called “Ich,” is a protozoan parasite that infects the epidermis and gills of freshwater fish. Here, we used goldfish (Carassius auratus) to determine whether the four vaccine preparations (live trophonts, formalin‐fixed trophonts, freeze‐thawed trophont lysates, and trophont cilial and cell cortical fractions) of I. multifiliis (Fouquet) can elicit resistance of immunized fish against subsequent theront challenge, and whether a relationship exists between in vitro immobilization of theronts in sera or mucus from immunized fish and host protective immunity against the parasite. Experimental goldfish were randomly divided into five groups with five parallel controls, with each group or subgroup consisting of 20 individuals. Each test goldfish was immunized with one of the four Ich vaccine preparations administered by one of the three routes: live trophonts by gavage (Group 1), formalin‐fixed trophonts by injection (Group 2) or by immersion (Group 3), freeze‐thawed trophont lysates by injection (Group 4), and trophont cilial and cell cortical fractions by injection (Group 5). The fish were then challenged by a standard Ich theront challenge on Day 21 postimmunization. For every 10 d following immunization, we tested the in vitro immobilization of Ich theronts by sera or mucus from immunized and control fish. We found that although all control (nonimmunized) and Group 3‐immunized goldfish died following theront challenge, more than half (55–65%) of the immunized fish from Groups 1, 2, 4, and 5 survived. Furthermore, except for the fish from Groups 3 and 4, the number of mean days to death in each test group was significantly higher than that in the respective control. These results indicate that goldfish immunized by injection or by gavage with any of the four vaccine preparations gained resistance to Ich infection. Further analysis indicated that this protective immunity was closely associated with the in vitro immobilization of Ich theronts by fish sera or mucus.     

An experimental model for fish furunculosis caused by Aeromonas salmonicida

Abstract. Development of an experimental bath challenge method for fish furunculosis caused by Aeromonas salmonicida is described. The influence of certain important experimental variables was investigated and standardized. Possible application of the model is discussed.     

Feeding Atlantic salmon Salmo salar L. soybean products: effects on disease resistance (furunculosis), and lysozyme and IgM levels in the intestinal mucosa

Two trials were initiated to investigate the consequences of including various soybean products in diets for Atlantic salmon Salmo salar L. on (1) mortality following infection by Aeromonas salmonicida ssp. salmonicida during a cohabitation challenge, and (2) the lysozyme and IgM content of the intestinal mucosa. Groups of salmon were fed control diets containing fishmeal as the sole protein source (Contr1 and Contr2, respectively), soy concentrate-containing diets (SoyConc1 and SoyConc2, respectively), or diets containing either solvent-extracted soybean meal (SoyMeal, trial 1) or soybean molasses (SoyMol, trial 2), an alcohol extract of soybean meal. Both SoyMeal and SoyMol caused enteritis-like changes in the distal intestine, which were not observed in fish fed the Contr1, Contr2, SoyConc1, or SoyConc2 diets. There were significant differences ( P  < 0.05) in mortality between feeding groups following the A.s. salmonicida challenge: these differences were greatest in fish fed SoyMeal (65.6%), least in fish fed SoyConc1 (60.5%), and intermediate in the fish fed the Contr1 diet (62.9%). The SoyMol diet caused significantly ( P  < 0.0001) increased levels of both lysozyme and IgM in the mid and distal intestinal mucosa. It is concluded that components of soybean meal and soybean molasses cause an inflammatory response in the distal intestine that may lead to increased susceptibility to furunculosis.