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.     

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。     

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.     

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.     

Presence of a lethal protease in the extracellular products of Vibrio splendidus-Vibrio lentus related strains

The presence of a lethal extracellular 39-kDa protease, a virulence determinant of a Listonella pelagia strain which produces vibriosis in turbot, was determined in the extracellular products (ECP) of 33 Vibrionaceae strains. Both immunological and enzymatic techniques distinguished this specific protease from other Vibrionaceae proteins. It was detected in 15% (5/33) of the ECPs assayed belonging to strains of the Vibrio splendidus-V. lentus related group isolated in Galician aquaculture systems (NW Spain). As these strains were associated with diseased octopus and cultured turbot, were able to colonize the internal organs of fish and produced a lethal ECP for fish, they are a potential risk for the health of reared aquatic organisms.     

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.     

The effects of Vibrio anguillarum extracellular products on Japanese eels

To test the effect of Vibrio anguillarum extracellular products (ECP) on Japanese eels, test fish were injected intramuscularly with ECP at a dose of 1 mg protein/100 g body weight of fish. At 3, 6, 12, 24 and 36 h post-injection, blood samples were collected for haematocrit, haemoglobin, and serum protein determinations and tissues were fixed in Bouin's solution. Histopathological observations 24 h post-injection revealed that the ECP caused severe damage to muscle tissue, characterized by extensive muscle liquefaction and haemorrhaging. In addition, extensive haemosiderin deposits were observed in the spleen, with lesser deposits occurring in the kidney and liver. Haematocrit, haemoglobin, and serum protein values were lower in ECP-treated fish than in the untreated controls.     

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.     

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.     

嗜水气单胞菌源黄鳝出血病的组织病理学研究

应用组织学方法研究了分离自患出血病黄鳝(Monopterus albus)体内的嗜水气单胞菌(Aermonas hydrophila)人工感染健康黄鳝后引起的组织病理学变化。结果显示:健康黄鳝感染嗜水气单胞菌后发病呈急性过程,感染后15~33 h为发病死亡高峰期。黄鳝人工感染后出现的临床症状与自然发病病例相似,主要表现:体表出现出血斑、肛门红肿、腹腔充血以及各实质性器官出血、充血。人工感染黄鳝的肝脏、脾脏和肾脏的病理变化明显:肝脏出现广泛的充血、出血,肝细胞出现空泡变性、坏死;肾脏肾小管上皮的肿胀、结构的消失,肾小球出血、变性坏死;脾脏充血、出血;肠粘膜上皮脱落,基底膜排列紊乱,大量出血。结果表明,嗜水气单胞菌感染黄鳝后的病理变化主要表现为肝脏、肾脏等实质性器官充血、出血,及广泛的组织细胞变性和坏死。     

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.     

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.     

Putative virulence factors of atypical Aeromonas salmonicida isolated from Arctic charr,Salvelinus alpinus (L.), and European grayling,Thymallus thymallus (L.)

Atypical Aeromonas salmonicida (AAS) causes generalized lethal infections in farmed Arctic charr, Salvelinus alpinus (L.), and European grayling, Thymallus thymallus (L.), and is thus a serious threat for culture of these fish species. Virulence factors were studied among isolates of AAS from Arctic charr (n = 20), European grayling (n = 19) and other fish species (n = 20), of which 48 were of Finnish and 11 of Swedish origin. All isolates produced an A-layer. Extracellular products (ECP) of the AAS isolates did not produce detectable gelatinase and caseinase activity in test assays. Analysis of the same ECP preparations with substrate sodium dodecyl sulphate-polyacrylamide gel electrophoresis showed weak proteolytic activity, indicating the different sensitivity of the detection methods used. The ECP from AAS isolates showed low cytotoxic activity against cultured cells. However, the ECP did not induce mortality in challenged Arctic charr. The results suggest that toxic components, like ECP, secreted by the bacterium may not be the major virulence factor in AAS-infection in Arctic charr and European grayling, and hence the pathogenesis also differs from the pathogenesis of AAS-infection in Atlantic salmon, Salmo salar L.     

Liver damage in brown trout, Salmo trutta L., and rainbow trout, Oncorhynchus mykiss (Walbaum), following administration of the cyanobacterial hepatotoxin microcystin-LR via the dorsal aorta

Purified microcystin-LR (MC-LR) was administered via the dorsal aorta to brown trout, Salmo trutta L., or rainbow trout, Oncorhynchus mykiss (Walbaum), and, within 24 h, a dose of 300 μ g MC-LR kg^–1 caused increased activities in the blood by enzymes originating mainly from the liver, i.e. lactate dehydrogenase (LDH) and alanine transaminase (ALT). A dose of 75 μ g MC-LR kg^–1 significantly increased liver enzyme activities in the blood of brown trout at 24 h, but was without effect on rainbow trout, whereas 25 μ g MC-LR kg^–1 had no effect on blood LDH or ALT activities in either species. However, histopathological analysis of liver from both species following administration of the lowest toxin dose showed hepatocyte swelling and necrosis. Liver damage was more severe in brown trout compared to rainbow trout following administration of 300 μ g MC-LR kg^–1, showing disruption of the parenchymal architecture. After 48 h, there was a dose-dependent increase in the hepatosomatic index in both species. It is concluded that brown trout are less tolerant to MC-LR than rainbow trout.     

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。     

Antigenicity of Streptococcus agalactiae extracellular products and vaccine efficacy

Streptococcus agalactiae is a major bacterial pathogen that is the cause of serious economic losses in many species of freshwater, marine and estuarine fish worldwide. A highly efficacious S. agalactiae vaccine was developed using extracellular products (ECP) and formalin-killed whole cells of S. agalactiae. The vaccine efficacy following storage of S. agalactiae ECP and formalin-killed S. agalactiae cells at 4 degrees C for 1 year was determined. The stored ECP containing S. agalactiae formalin-killed cells failed to prevent morbidity and mortality among the vaccinated fish, and the relative percentage survival was 29. Serum antibody responses of the stored ECP and freshly prepared ECP against soluble whole cell extract of S. agalactiae indicated that significantly less antibody was produced in fish immunized with stored ECP and S. agalactiae cells than in those fish immunized with freshly prepared ECP and S. agalactiae cells at day 31 post-vaccination. Silver staining of sodium dodecyl sulphate-polyacrylamide gels and immunostaining of Western blots with tilapia antiserum to S. agalactiae revealed that predominant 54 and 55 kDa bands were present in the freshly prepared ECP fraction. The 55 kDa band was absent from the stored ECP and new bands below 54 kDa appeared on the Western blot. The results of this study on S. agalactiae ECP provide evidence for a correlation between protection and antibody production to ECP and for the importance of the 55 kDa ECP antigen for vaccine efficacy.     

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.     

White spot syndrome virus (WSSV) infection in tiger shrimp <Emphasis Type="Italic">Penaeus monodon</Emphasis>: A non-lethal histopathological rapid diagnostic method using paraffin and frozen sections

White spot syndrome virus (WSSV) infection was induced in tiger shrimp, Penaeus monodon, under laboratory conditions, and histopathological changes in subcuticular epithelial cells of the eye stalk and pleopod were studied sequentially at different time post-challenge. Routine histological techniques using paraffin embedded tissues, as well as frozen tissues, were used to document WSSV infection. Histological manifestations such as cellular hypertrophy in the subcuticular epithelial cells of the eyestalk and pleopod could be detected as early as 18 h post-infection (p.i.) before the manifestation of clinical signs of the disease. However, no histopathological changes could be detected before 18 h p.i.. Hypertrophy of the nuclei in the epithelial cells was pronounced after 24 h p.i. Marked necrosis, and eosinophilic intranuclear inclusions, characteristic of early stages of WSSV infection were observed between 24–36 h p.i. Clinical signs of the disease appeared at 48 h p.i. The presence of WSSV at early asymptomatic stages of p.i. has been tested in parallel samples using polymerase chain reaction, for further confirmation of WSSV. This paper discusses the potential of a non-lethal and rapid histopathological diagnostic method to document WSSV infection, using the eyestalk or pleopod, when expensive DNA based diagnostics are not available or affordable.     

Effect of fish density and number of infectious fish on the survival of rainbow trout fry, Oncorhynchus mykiss (Walbaum), during epidemics of infectious pancreatic necrosis

Two laboratory studies compared the effect of fish density and number of infectious fish on characteristics of survival of rainbow trout fry during controlled epidemics of infectious pancreatic necrosis (IPN). Analyses of hazard functions and survivor functions were used to determine whether peak death rate, time at which the peak death rate occurred and probability of survival to the end of the experiment were associated with fish density and number of infectious fish added (i.e. pathogen concentration). When number of infectious fish was low and fish density increased, the peak death rate increased, time of the peak death rate decreased and the probability of survival to the end of the experiment decreased. When number of infectious fish was high, the effect of density diminished. Loglogistic regression of survival data revealed that fish density, number of infectious fish and interaction between these two variables significantly affected time to death from IPN (P < 0.01).