Lack of relationship between virulence of Aeromonas salmonicida and the putative virulence factors: A-layer, extracellular proteases and extracellular haemolysins

Abstract The role of A-layer (A), protease (P) and haemolysin (H) as virulence factors of Aeromonas salmonicida, the aetiological agent of fish furunculosis, was a investigated using three strains of the bacterium. Strain MT004 lacked the A-Iayer (A⁻) and produced extracellular caseinase and gelatinase (P⁺) and haemolysin (T-lysin; H⁺). Strain MT028 was A⁻, P⁻ and H⁻, and strain MT048 was A⁺, P⁺ and H⁻. The pathology and LD50 produced in rainbow trout by cells or extracellular products (ECP) of each strain were determined. The ECP was produced by two different methods where the protease and haemolytic activities differed in relative levels, or when the protease of MT004 ECP was inhibited by the serine protease inhibitor PMSF. The results indicate that the presence of A-layer is not essential, at least for a moderate degree of virulence; that in vitro production of extracellular proteases is not a requisite of virulent strains; that presence of protease and haemolysin in ECP can be correlated with the development of certain lesions and a rapid time to death but cannot be correlated with the lethal toxicity of the ECP. The authors conclude that an as-yet unrecognized component of ECP is responsible for killing fish.     

The humoral immune response of rainbow trout, Salmo gairdneri Richardson, and rabbits to Aeromonas salmonicida extracellular products

Abstract. Controversy exists concerning the efficacy of vaccinating fish against furunculosis. Where success is claimed, there has been little attempt to characterize the protective antigens or confirm their immunogenicity. In this report, the immunogenicity of native extracellular products (ECP) of Aeromonas salmonicida and a formalin-inactivated toxoid of ECP (f-ECP) was studied in rainbow trout and rabbits, with particular attention to the putative bacterial virulence factors protease and haemolysin. Using crossed immunoelectrophoresis and Protein-A absorption, antibodies to seven ECP components were detected in the rabbit following immunization with native ECP; antihaemolysin antibodies were found but antibodies to the protease could not be detected. Antibodies to at least 14 components of ECP, including haemotysin and protease, were detected in the rabbit following immunization with f-ECP. In trout immunized either with native ECP or f-ECP, antibodies to only four ECP antigens were detected and no antibodies to haemolysin or protease were found. The results may explain previous reports that passive immunization with rabbit antisera gave superior protection against furunculosis compared with antisera raised in fish, and indicate that many extracellular antigens of A. salmonicida may require modification in order to improve their immunogenicity in fish.     

Extracellular products from Mycobacterium spp. in fish

Mycobacterium spp. isolated from food and ornamental fish in Thailand (TB1, TB40, TB267 and TB268), and the type strains Mycobacterium marinum (NCIMB 1298), M. fortuitum (NCIMB 1294) and M. chelonae (NCIMB 1474) were cultured in Long's medium, Eagle's minimum essential medium, Sauton's medium and modified Sauton's medium. The latter enabled excellent growth and production of extracellular products (ECP) from TB 40, TB267, TB268 and M. marinum NCIMB 1298 in particular, whereas growth and production of ECP for all strains was limited in Long's medium. SDS-PAGE protein profiles of ECPs from 14-day culture supernatants showed major bands at 65 and <14 kDa. After 2 days culture at the higher temperature of 37°C (heat shock), the production of ECP from all mycobacteria strains except M. marinum averaged approximately four- to 10-fold higher than from strains cultured for 14 days at 28°C. Enzyme testing for the type strains indicated only mucinase activity for M. marinum, while lipase and RNase activities were detected for M. chelonae and M. fortuitum . Protease and DNase activities could not be detected for any of the Mycobacterium spp. tested. The medium lethal dose (LD₅₀) of ECP to rainbow trout and Nile tilapia was greater than 400 μg protein fish^–1.     

Serratia marcescens: a potential pathogen for fish

Abstract. Characterization of a red pigmented enterobacterium isolated from natural population of white perch, Morone americanus (Gmelin), during the course of a bacteriological survey in the Back River (Baltimore, Maryland, USA) indicated that the bacterium belonged to the species Serratia marcescents. The virulence properties of this isolate (RB 469), studied in comparison with the reference strain of S. marcescens ATCC 1800 and S. plymuthica K1R, revealed that all the strains were highly pathogenic for fish with LD₅₀s raging from 5 × 10³ to 1 × 10⁵. Similarly, te extracellular products (ECP) from the three isolates were lethal for fish (LD₅₀ ranging from 0·22 to 4·8 μg protein g^-1 fish). However, only ECP from strains with strong proteolytic activity (the white perch isolate and S. plymuthica ) were cytotoxic for both in fish and homoiothermic cell cultures and both activities were completely destroyed by heating at 100°C for 10min. In contrast, only the two S. marcescens strains which possessed phospholipase active were pathogenic for mice and produced enterotoxins. None of the Serratia strains displayed dermonecrotic factor in rabbits. All these lindings indicate that a direct relationship between eytotoxicity and virulence cannot be established.     

Do extracellular products of Aeromonas salmonicida induce thrombosis by entering the fish coagulation system at factor X?

Abstract. Thrombosis of minute vessels is a common feature of acute furunculosis in Atlantic salmon. Crude extracellular products (F.CP) of Aeromans, salmonicida injected into the dorsal aorta of cannulated fish elicited an activation of the coagulations systems of the fish When screened with a number of chromogenic substrates, the ECP protease revealed a factor-X like activity. Thus, it may he hypothesized that the ECP induces thrombosis by entering the fish coagulation systems at factor X. and that thrombus formation is a way of creating a site of infection.     

Production of the lethal acetylcholinesterase toxin by different Aeromonas hydrophila strains

Abstract. Seven fish pathogenic isolates of Aeromonas hydrophila , one A. sobria and one A. caviae were investigated for production of the fish lethal acetylcholinesterase toxin (AcChE-toxin). Western blotting was used for screening the ECP of these strains with a rabbit antiserum prepared against the purified toxin of strain B₃₂ and all the isolates (except A. sobria ) gave positive results with different patterns of bands. The AcChE-toxin appears to be secreted as a protein of high molecular weight which is stable at −20°C, and in 90% of the strains tested, it appears to be split into lower molecular weight fragments by the action of other components present in the ECP. The smallest, stable and highly active fragment has a MW of 15kDa.     

The non-specific activation of rainbow trout, Salmo gairdneri Richardson, complement by Aeromonas salmonicida extracellular products and the correlation of complement activity with the inactivation of lethal toxicity products

Abstract The possible mechanism of inactivation of the toxicity of Aeromonas salmonicida extracellular products (ECP) by normal rainbow trout serum was investigated using juvenile rainbow trout. ECP was prepared from culture supernatant by an acetone precipitation method. The ECP was incubated with normal rainbow trout serum at 20°C for 2 h, and the interrelationship between ECP proteolytic activity and immune complex-initiating, haemolytic complement activity (CH₅₀) of normal serum against antibody-sensitized goldfish red blood cells was evaluated. When normal serum was incubated with increasing concentrations of ECP, the CH₅₀ activity of serum decreased. The CH₅₀ activity was completely abolished in serum treated with undiluted ECP. ECP treated with serum was administered to trout intraperitoneally to determine mortality. All the fish receiving untreated ECP (0.05 ml = 0.5 mg protein) alone died within 24 h. When ECP was treated with serum at 1:1 to 4:1 (serum: ECP) in volume a similar high mortality was produced. These inocula possessed high protease activity and no or low CH₅₀ activity. However, mortality decreased and finally no mortality was recorded as ECP was treated with large volumes of serum (9:1 to 19:1). These inocula had lower protease activity and considerably higher CH₅₀ activity. Fish receiving ECP treated with heat-inactivated serum at 19:1 showed 100% mortality. A serum: ECP inoculum derived from fish which had been administered lipopolysaccharide from Salmonella enteritidis and which possessed a low CH₅₀ activity also gave a high mortality when used at 19:1. These results suggest that rainbow trout complement is implicated in the inactivation of toxicity of A. salmonicida ECP.     

The role of the lethal extracellular cytolysin of Aeromonas salmonicida in the pathology of furunculosis

Abstract. The histopathological effects of intramuscular (i.m.) and intraperitoneal injections of the purified lethal cytolytic toxin of A. salmonicida in Atlantic salmon, Salmo salar L., were restricted to extensive degranulation of eosinophilic granular cells (EGCs) in the gills (both routes) and a limited coagulative necrosis of muscle fibres at the site of i.m. injection. The severity of these lesions did not apparently differ amongst fish that were moribund within 48 h and the surviving fish at 90 h. Previous studies indicate a maximum time to death caused by this toxin of 48 h. Thus, it is difficult to account for death of the fish from the toxin solely on the grounds of the histopathological features. It is suggested that a complex metabolic dysfunction results in death. The mechanism of 'furuncle' formation was demonstrated to result from a combined effect of the cytolysin and the purified 70-kDa protease present in the ECP. When i.m.-injected singly, the purified cytolysin produced a limited coagulative necrosis and the purified protease produced a limited liquefaction and haemorrhaging. In combination, they induced the extensive liquefactive haemorrhagic 'furuncle' characteristic of the whole ECP.     

A study of the pathological effect of isolated Aeromonas salmonidda extracellular protease on Atlantic salmon, Salmo salar L.

Abstract. A comparison was made between the effects of Aeromonas salmonidda extracellular protease and total extracellular products (ECP) following intramuscular injection into juvenile Atlantic salmon, Salmo salar L. Thus, 20, 10, 5, 2.5 and 1.5 units of salt-free protease in 0.2 ml water were compared with ECP preparations with the same levels of proteolytic activity. The highest concentration of ECP produced a gross pathology with a large furuncular lesion 36 h after injection. The corresponding protease preparation had a lesser effect, although a furuncle was formed and tissue liquefaction was produced. These effects were less marked with reduced concentrations. At the lowest level studied, no significant effect was observed with protease alone but ECP (0.8 μg of protein) produced a small, characteristic lesion similar to that achieved with 5 units of isolated protease.     

Virulence properties of Moritella viscosa extracellular products

Moritella viscosa is the causative agent of winter ulcer disease of marine fish. Knowledge of its pathogenicity is limited and there are no reports comparing the virulence properties of a collection of bacterial isolates. The in vivo and in vitro virulence of the extracellular products (ECP) of 22 M. viscosa isolates was screened. Two non-virulent Canadian isolates and a Norwegian isolate with reduced virulence produced non-lethal ECP. Correlation was obtained between cytotoxin and haemolysin production of M. viscosa. Isolates from salmon produced ECP with lower cytotoxic and haemolytic activities than ECP of isolates originating from other hosts. Correlation was not found between lethality of ECPs in salmon and cytotoxic or haemolytic activities. All isolates secreted esterases and a metallopeptidase (MvP1), degraded starch and produced siderophores. Variable levels of ECP protein concentration, different enzymatic activities and siderophore production could not explain differences in virulence. The results show that virulent M. viscosa isolates secrete a lethal toxic factor of unknown nature and that cytotoxin production may reflect host adaptation. Cell-culture models may not be optimal for determining the virulence of M. viscosa, as no association between cytotoxicity and bacterial virulence was obtained. Non-virulent strains may be useful in future research on M. viscosa virulence, as construction of mutants has not been successful.     

高致病性维氏气单胞菌胞外产物对斑点鲖的致病性

为了研究维氏气单胞菌及其胞外产物的致病机制对预防和治疗斑点鲖维氏气单胞菌病的作用。通过对一株高致病性斑点鲖源维氏气单胞菌胞外产物(extracellular product,ECP)的提取并结合体内外实验研究其酶活性与致病性,以期为维氏气单胞菌的致病机制研究提供新思路。采用饱和硫酸铵沉淀法,对一株高致病性的斑点鲖源维氏气单胞菌的ECP进行了提取,将其进行浓度检测、SDS-PAGE分析和酶活测定;并将提取的ECP攻毒斑点鲖,通过病理学分析研究其致病机制。提取的ECP经考马斯亮蓝试剂盒确定其浓度为0.743 mg/m L;SDS-PAGE分析发现该ECP类型丰富,分子大小主要集中在20.0~33.0 ku。运用琼脂平板扩散法对ECP中主要毒力和代谢相关酶活性进行体外测定,发现ECP具有脂酶、蛋白酶、卵磷脂酶、DNA酶和溶血活性,不具有淀粉酶、明胶酶、脲酶活性;并对与毒力密切相关的溶血活性进行了进一步的溶血谱绘制,发现ECP对其他多种动物红细胞都有溶血活性,对鱼类红细胞溶血性较强,但对鸡、鸭红细胞无溶血活性。ECP攻毒健康斑点鲖后,发现其具有明显致病性,死亡率高达100%。病鱼大体病理变化为体表黏液增多,出现褪色斑以及不同程度的出血斑;眼球充血;肛门红肿外凸;剖检病变表现:鳃丝充血肿胀、肝脏肿大、散在少量出血点,脾脏肿大暗红,肠道扩张、肠壁变薄、肠腔内有大量黄色黏液。组织学病变主要表现为肝细胞变性、坏死;脾脏淋巴细胞减少,大量结缔组织增生;胃、肠道黏膜上皮坏死脱落。该株维氏气单胞菌的胞外产物具有多种酶活性,且对斑点鲖具有明显的致病性,推测该胞外产物在维氏气单胞菌入侵、感染甚至致死斑点鲖过程中都发挥了重要作用。     

发病鲆鲽类分离菌株的16S rRNA基因测序分析

细菌性疾病是中国海水养殖鲆鲽类的主要病害,为全面了解病原菌种类,本研究对1999~2012年从山东、江苏、河北、天津等沿海地区养殖场发病鲆鲽鱼类中分离得到的124株优势菌株进行了16S rRNA基因测序和系统发育学分析。将基因序列与GenBank核酸序列数据库进行相似度比对分析,结果显示,有83株与弧菌属(Vibriosp.)细菌相似度最高,11株与气单胞菌属(Aeromonas sp.)细菌相似度最高,4株与爱德华氏菌属(Edwardsiella sp.)细菌相似度最高,26株为其他15种属的细菌。根据系统发育学分析结果,进一步将66株菌鉴定为16个种,优势种为溶藻弧菌(V.alginolyticus)、哈氏弧菌(V.harveyi)、鳗弧菌(V.anguillarum)、杀鲑气单胞菌(A.salmonicida)和迟缓爱德华氏菌(E.tarda)。选择其中的9株鳗弧菌和4株迟缓爱德华氏菌进行人工感染实验,结果显示,其中7株鳗弧菌和3株迟缓爱德华氏菌对大菱鲆(Scophthalmus maximus)有较强的致病性。研究结果可为阐明中国海水养殖鲆鲽类的流行病发生历史、病原种类、病原监测及疾病控制提供重要参考。     

草鱼和银鲫肠道产消化酶细菌的研究

检测了分别从草鱼(Ctenopharyngodon idellus)和银鲫(Carassius auratus gibelio)肠道中分离的180株细菌的蛋白酶、脂肪酶、淀粉酶和纤维素酶的产酶能力。结果显示,两种鱼肠道内可分泌胞外消化酶的细菌包括Aero-monas(气单胞菌属,Aer.)、Vibrio(弧菌属,Vib.)、Bacillus(芽孢杆菌属,Bac.)、Pseudomonas(假单胞菌属,Pse.)四个种属的细菌,Aer.在其中占主要优势,45.71%的Aer.可分泌胞外消化酶。草鱼可分泌上述四种胞外消化酶的菌株共有33株,占肠道菌总数的36.67%;银鲫43株,占47.78%。产酶菌的分布上,草鱼中肠内产消化酶细菌数量显著多于前肠和后肠(P<0.05),前、中、后肠分别是6株、20株和7株;银鲫中肠和后肠数量差异不显著,前肠分布最少。草鱼分泌蛋白酶、脂肪酶、淀粉酶和纤维素酶的菌株分别有21株(23.33%)、10株(11.11%)、30株(33.33%)和16株(17.78%)。银鲫肠道内未检测到可分泌纤维素酶的细菌,蛋白酶、脂肪酶和淀粉酶菌株的数量分别是21株、37株和17株。可见鱼类肠道细菌对食饵消化有重要作用。     

Transmission of protease genes into a protease-deficient mutant of Aeromonas salmonicida in river sediments

Abstract. The transformation of Aeromonas salmonicida with DNA fragments from bacterial cell-free sonicates was investigated with intraspecific, interspecific band intergeneric fish pathogenic bacteria including Aeromonas salmonicida, Aeromonas hydrophila, Pseiidomonas fluorescens and Vibrio anguillarum strains as donor bacteria. A phenotypic marker for transformation was extracellular protease production since a protease-deficient mutant NTG-1 induced from pathogenic A. salmonicida strain A-7301 by mutagenesis was used as a recipient. This mutant was non-pathogenic to rainbow trout. The mutant was incubated with each sonicate at 20°C for 20 days with a nutrient-poor medium containing a trace (5 μg/ml each) of both humic acid and tryptone in the presence of clean river sand (100 g/100 ml medium) corresponding with an environment of rivers. During the incubation, the survival of mutant NTG-1 cells was observed and protease positive NTG-1 cells were isolated from each culture. The protease production of the isolates was due to the transmission of protease genes of the donor strains. The activity of proteases produced by the transformants extra-cellularly was determined. These transformants induced with the sonicates of the parent strain, intraspecific strain and with the sonicates of the interspecific A. hydrophila strain were pathogenic to rainbow trout, whereas the transformants derived with the sonicates of the intergeneric strains P. fluorescens and V. anguiUarum showed non-pathogenicity, although all the donor strains, with the exception of the P. fluorescens strain, were pathogenic. These findings are interesting since they demonstrate that trausformation in A. salmonicida occurs with considerable ease even intergenencally and interspecifically, as well as intraspecifically in river environments, and that there is a large difference in the lethal toxicity of extracellular protease produced by these bacteria.     

Non-specific immune response of Nile tilapia,Oreochromis nilotica,to the extracellular products of Mycobacterium spp. and to various adjuvants

Nile tilapia were immunized by injecting extracellular products (ECP) of Mycobacterium spp. (strain TB40, TB267 or the type strain Mycobacterium marinum) into their swim bladders. A variety of adjuvants – Freund's complete adjuvant (FCA), Freund's incomplete adjuvant (FIA) and Titremax – were similarly injected into additional groups of tilapia. Phosphate-buffered saline (PBS) was used as a control. The number of nitroblue tetrazolium (NBT)-positive cells observed in the swim bladder of the immunized fish had significantly increased by the fourth day post-immunization. By day 8, the number of NBT-positive cells in fish immunized with ECP from mycobacteria strains TB40 or TB267 were fewer than in fish immunized with ECP from M. marinum or fish injected with FCA or FIA. The level of lysozyme activity detected in the serum of fish 4 days after being immunized with ECP from various Mycobacterium spp. was also significantly higher than that found in the serum of the control fish. Head kidney macrophages showed an enhanced reduction of NBT when cultured in vitro with 1 μg ml^–1 of ECP. Concentrations greater than this (10 or 100 μg ml^–1) were found to suppress the reduction of NBT by the macrophages.     

Experimental infection of turbot, Psetta maxima (L.), with strains of viral haemorrhagic septicaemia virus isolated from wild and farmed marine fish

The susceptibility of turbot, Psetta maxima, to infection with two strains of viral haemorrhagic septicaemia virus (VHSV) obtained from wild Greenland halibut, Reinhardtius hippoglossoides, and from farmed turbot was examined. A marine VHSV strain known to be highly pathogenic for turbot was also utilized for comparative purposes. Fish were infected by intra-peritoneal (i.p.), immersion or cohabitation, and maintained at two different temperatures (8 and 15 degrees C). Infection trials showed that the three VHSV isolates were pathogenic for turbot fingerlings by i.p. injection at both temperatures, with high levels of mortality. Virus was recovered from most pools of dead fish i.p. challenged, but not from surviving fish. Although clinical signs were not induced following waterborne exposure, viral growth was obtained from some pools of surviving fish challenged by immersion with strain GH40 from Greenland halibut, which indicates that the virus can survive in sea water and infect other fish via horizontal transmission. Furthermore, although low, the clinical signs and mortality observed in fish cohabitating with turbot challenged with strain GH40 confirms horizontal transmission and indicates that the passage through fish increases the virulence of this strain for turbot. These findings indicate that Greenland halibut, as other wild fish, may play an important role in the epizootiology of VHSV and suggest a potential risk for the turbot farming industry.     

南方鲇源豚鼠气单胞菌胞外产物活性与致病性研究

采用琼脂扩散法测定了南方鲇源豚鼠气单胞菌胞外产物酶活性和溶血活性,同时对胞外产物的细胞毒性和其致病性进行了研究。结果显示:南方鲇源豚鼠气单胞菌胞外产物具有蛋白酶、脂酶、明胶酶和脲酶活性,但不具有淀粉酶和卵磷脂酶活性,具有很强的溶血活性和细胞毒性。肌肉注射感染发现,其对南方鲇有强致病性,其LD50为每千克鱼体重0.802 mg;注射后的南方鲇肌肉、心、肝、肾、脾、肠和胃等组织发生了严重组织病理变化,骨骼肌和心肌坏死断裂,炎症细胞浸润;肝脏严重空泡变性;肾小管上皮细胞变性、坏死、间质内大量炎症细胞浸润;脾充血、出血,淋巴细胞减少,胃肠黏膜上皮细胞变性、坏死、脱落。     

南方鲇源豚鼠气单胞茵胞外产物活性与致病性研究

采用琼脂扩散法测定了南方鲇源豚鼠气单胞菌胞外产物酶活性和溶血活性,同时对胞外产物的细胞毒性和其致病性进行了研究。结果显示:南方鲇源豚鼠气单胞菌胞外产物具有蛋白酶、脂酶、明胶酶和脲酶活性,但不具有淀粉酶和卵磷脂酶活性,具有很强的溶血活性和细胞毒性。肌肉注射感染发现,其对南方鲇有强致病性,其LD50为每千克鱼体重0.802 mg;注射后的南方鲇肌肉、心、肝、肾、脾、肠和胃等组织发生了严重组织病理变化,骨骼肌和心肌坏死断裂,炎症细胞浸润;肝脏严重空泡变性;肾小管上皮细胞变性、坏死、间质内大量炎症细胞浸润;脾充血、出血,淋巴细胞减少,胃肠黏膜上皮细胞变性、坏死、脱落。     

An appraisal of the extracellular toxins of Aeromonas salmonicida ssp. salmonicida

Abstract. Aeromonas salmonicida produces many extracellular enzymes, some of which are known to play an important role in pathogenesis and virulence, while the role of others is presently speculative. The latter group includes amylase, aryl-sulphatase, glucosidases, esterases and lysophospholipase. There are two enzymes which are known to be of prime importance in pathogenesis: a 70-kDa protease (caseinase) and a 25-kDa phospholipase (glycerophospholipid: cholesterol acyltransferase, GCAT). The protease causes extensive tissue liquefaction, activates the blood clotting system and is lethal for fish at 2·4 μg/g fish. It is inhibited by α2-macroglobulin but resistant to all the other serum protease inhibitors. Its role in vivo appears to be as a broad spectrum protease providing amino acids for in vivo growth. The GCAT is mainly present in a high molecular weight complex with LPS. The complex is extremely haemolytic for fish (but not mammalian) erythrocytes. It is the most lethal component of the exotoxins (lethal dose 45 ng/g fish). The complex with LPS confers enhanced toxicity to the GCAT and stability to heat and proteolytic degradation. In vitro , this toxin also has high leucocytolytic and cytolytic (RTG-2) activity. On injection into fish, it causes very little histopathology other than a marked degranulation of eosinophilic granular cells (EGCs) in the gills. Its precise mode of pathogenesis is uncertain and appears complex. The protease and the GCAT/LPS have an additive relationship in respect to lethal doses and mixtures of the two produce extensive liquefactive and haemorrhagic lesions typical of furuncles. The possible relationship of the GCAT/LPS to other less well characterized factors (cytotoxin, leucocytolysin, haemolysin, salmolysin) is discussed.     

The production of a lymphokine (macrophage activating factor) by rainbow trout, Oncorhynchus mykiss (Walbaum), leucocytes stimulated with the extracellular products of Mycobacterium sp.

The production of macrophage activation factor (MAF) by rainbow trout, Oncorhynchus mykiss (Walbaum) , head kidney leucocytes was examined after culturing in vitro with extracellular products (ECP) collected from Mycobacterium sp. Cultures of leucocytes were prepared from naive fish, or fish previously vaccinated with either the ECP or with formalin killed whole cell preparations (WC) of the bacterium. The cells were then incubated with the ECP in vitro and the ability of their supernatants to activate macrophages assessed. Macrophages from control fish were incubated with the supernatants, and their ability to reduce nitroblue tetrazolium (NBT) measured as an indicator of macrophage activation. Incubation of head kidney macrophages from naive fish directly with 1, 10 or 100 μg mL^–1 of ECP for 48 h significantly enhanced macrophage activation compared with control macrophages. Vaccination of fish with either ECP or WC had no significant effect on the respiratory burst of control macrophages 4 weeks post-vaccination. By the eighth week, however, absorbance levels of respiratory burst reflecting both the primary (cells from vaccinated fish cultured in vitro with PBS) and the secondary (cells cultured in vitro with ECP) MAF responses of fish vaccinated with ECP and WC, had peaked and these were significantly different from the non-vaccinated controls. This activity had fallen to levels similar to control fish by week 12 for fish vaccinated with WC.