A comparative molecular study of the presence of “Candidatus arthromitus” in the digestive system of rainbow trout,Oncorhynchus mykiss (Walbaum), healthy and affected with rainbow trout gastroenteritis

Observations were made using histopathological techniques in conjunction with a nested polymerase chain reaction (PCR) protocol for the specific detection of “Candidatus arthromitus” on DNA extracted from wax‐embedded tissues and fresh digestive contents of rainbow trout. Samples positive for “Candidatus arthromitus” DNA included fish with rainbow trout gastroenteritis (RTGE), clinically normal cohabiting fish, and apparently healthy controls from RTGE positive and RTGE negative sites. The results obtained from the PCR were confirmed by nucleotide sequencing. “Candidatus arthromitus” DNA was found in distal intestine as well as in sections of pyloric caeca, suggesting that both these locations are appropriate for molecular detection of “Candidatus arthromitus” DNA in trout. Furthermore, rainbow trout fry distal intestinal samples from two different hatcheries where RTGE had not been reported were also positive. Differences in “Candidatus arthromitus” DNA detection between paraffin wax‐embedded and fresh digestive content samples from the same fish suggested that it may be predominantly epithelium‐associated in healthy trout. Parallel histopathological observations indicated that pyloric caeca are the preferred site for visualizing segmented filamentous bacteria (SFB) in trout with RTGE. The results of this study showed that the presence of SFB was not invariably associated with clinical disease and that more information is required to understand the role of these organisms.     

A study of the sequential clinical and pathological changes during proliferative kidney disease in rainbow trout, Salmo gairdneri Richardson

Abstract. A histological study of 0+ rainbow trout, exposed to proliferative kidney disease (PKD) on an infected farm, showed evidence of infection from the beginning of June, gross pathological changes from the beginning of July, and clinical signs from mid-July. Evidence of healing was present in some samples from the second week of August. Kidney samples were divided into five grades (0–4) representing increasing severity of pathology. A sixth grade (H) was applied to kidneys showing signs of healing. The most frequent clinical signs were abdominal distension and exophthalmos. Melanosis, corneal cloudiness and ecchymoses were observed occasionally. Apart from renal enlargement, other internal gross pathological changes included ascites, gill and liver pallor, oedema, petechiation, and splenic enlargement. The majority of clinical signs and internal changes were only seen in fish with advanced renal swelling (grades 3 and 4). Histopathology was most marked in the kidney and was of three types: haemopoietic hyperplasia, vascular pathology and diffuse inflammatory change. Vascular changes, including occlusion of blood vessels in several organs and apparent haemoglobin crystallization, and destruction of renal excretory structures within areas of chronic diffuse inflammation were marked features of the histopathology in fish with clinical signs. The pathogenesis of PKD is discussed with reference to the histopathological changes.     

虹鳟肠炎红嘴病病理模型的构建

为探讨鲁氏耶尔森菌侵染虹鳟的致病机制,本实验建立了鲁氏耶尔森菌感染虹鳟引起的肠炎红嘴病的病理模型,制定相应的临床症状及组织病理学评分系统,并对该模型进行研究。将43尾平均体质量约为12 g的健康虹鳟随机分成5组:3个实验组(n=30)、对照组(n=10)和哨兵组(n=3)。3个实验组分别采用2.0×106、2.0×107和2.0×108 CFU/m L的鲁氏耶尔森菌感染浓度,通过腹腔注射方式进行人工感染试验。对感染鲁氏耶尔森菌的虹鳟肠、肝脏、脾脏和肾脏组织进行镜检及临床症状、剖检病变判断,结合细菌学检测,按制定的评分系统评价各组肠炎红嘴病造模效果,确定最佳造模方案。结果显示,各攻毒组虹鳟感染后72 h均出现不同程度死亡,临床症状表现为红嘴、肛门红肿、鳍(胸鳍、腹鳍、臀鳍)等出现不同程度充血,下颌部、腹部出现出血点等。组织病理学可见肝脏、脾脏、肾脏及肠组织均有炎性细胞浸润现象出现,肝细胞、肠上皮细胞和肾小管上皮细胞等实质细胞变性、坏死,脾脏部位淋巴细胞减少、红细胞死亡堆积。综合各组得分发现,2.0×107 CFU/m L组的鲁氏耶尔森菌感染虹鳟造模效果最佳,患病虹鳟的临床症状显著且组内差异较小,病程迁延较长,便于研究。研究表明,对体质量约12 g的虹鳟幼鱼腹腔注射0.1 m L浓度为2.0×107 CFU/m L的鲁氏耶尔森菌可成功构建肠炎红嘴病病理模型。     

Role of dietary ascorbic acid in vitellogenesis in rainbow trout (Salmo gairdneri)

Adult rainbow trout which were fed a diet devoid of ascorbic acid for 21 months, including the stages of gonadal development, showed no macroscopic signs of avitaminosis C and no increased mortality compared to a control group of fish with dietary supplementation of this vitamin. Fish without dietary supplementation of ascorbic acid contained more lipid in the liver and less in the ovaries than the reference fish. The lipid metabolism of the maturing fish is thus affected when the dietary intake of ascorbic acid is very low or absent. Fish without dietary ascorbic acid became anaemic (reduced levels of haemoglobin and haematocrit) towards the end of the feeding period. The serum levels of oestradiol-17β and vitellogenin were also lower in these fish. Possible modes of action of ascorbic acid in maturing rainbow trout are discussed on the basis of the presented analytical data on ascorbic acid, gross composition, blood serum levels of oestradiol-17β, vitellogenin, total protein, albumin, cholesterol, calcium, phosphorus and haematological parameters.     

Infectious pancreatic necrosis virus isolated from farmed rainbow trout and tilapia in Kenya is identical to European isolates

Infectious pancreatic necrosis virus (IPNV) is an aquabirnavirus that causes serious diseases in a variety of fish species worldwide. It has been isolated from a large number of healthy fresh and marine water fish. Prior to this study, there was no record of the presence of IPNV infection in Kenya. Here, the presence of IPNV in farmed rainbow trout and tilapia was examined in Nyeri County of central Kenya. Head kidney samples taken from five rainbow trout and three tilapia farms and stored in RNALater^® were processed by PCR followed by sequencing of a segment A fragment covering nucleotide positions 2,120–2,343 bp. IPNV was detected in all the farms sampled with infection ratios ranging from 0.3 to 0.78 although the infections were not associated with any specific clinical signs of disease. These findings were supported by immunohistochemistry staining of the virus in the kidney and exocrine pancreas of rainbow trout. Sequence alignment and phylogenetic analysis revealed that the Kenyan isolates were identical to European isolates, suggesting a common origin. These findings highlight the need for better biosecurity procedures with more stringent surveillance programmes and control for fish diseases, especially focusing on imported breeding materials to Kenya.     

Oral transmission as a route of infection for viral haemorrhagic septicaemia virus in rainbow trout, Oncorhynchus mykiss (Walbaum)

Surveys among wild marine fish have revealed occurrence of viral haemorrhagic septicaemia virus (VHSV) infections in a high number of diverse fish species. In marine aquaculture of rainbow trout, preying on invading wild fish might thus be a risk factor for introduction and adaptation of VHSV and subsequent disease outbreaks. Our objective was to determine whether an oral transmission route for VHSV in rainbow trout exists. Juvenile trout were infected through oral, waterborne and cohabitation transmission routes, using a recombinant virus strain harbouring Renilla luciferase as reporter gene. Viral replication in stomach and kidney tissue was detected through bioluminescence activity of luciferase and qRT-PCR. Replication was detected in both tissues, irrespective of transmission route. Replication patterns, however, differed among transmission routes. In trout infected through oral transmission, replication was detected in the stomach prior to kidney tissue. In trout infected through waterborne or cohabitation transmission, replication was detected in kidney prior to stomach or in both tissues simultaneously. We demonstrate the existence of an oral transmission route for VHSV in rainbow trout. This implies that preying on invading infected wild fish is a risk factor for introduction of VHSV into marine cultures of rainbow trout.     

Pseudomonas putida infection in rainbow trout

A disease outbreak occurred during the Spring of 2006 among rainbow trout, Oncorhynchus mykiss, kept in growout tanks. During the outbreak 35% of the rainbow trout died. Typical clinical signs were ulceration on the dorsal surface of the fish. Based on biochemical characteristics and sequence of 16S rRNA, the causative bacteria isolated from diseased fish were identified as Pseudomonas putida. Histologically, skin ulcer initially was characterized by sloughed off epithelia and epithelial necrosis. In the advanced form of the disease, almost all layers of the skin down to the epidermis were lost. Outside of Japan, this study reports the first P. putida infection in fish.     

Pancreas disease in farmed Atlantic salmon, Salmo salar L., and rainbow trout, Oncorhynchus mykiss (Walbaum), in Norway

The present paper describes, for the first time, clinical signs and pathological findings of pancreas disease (PD) in farmed Atlantic salmon, Salmo salar L., and rainbow trout, Oncorhynchus mykiss (Walbaum), in sea water in Norway. Similarities and differences with reports of PD from Ireland and Scotland are discussed. Samples of 68 rainbow trout from disease outbreaks on 14 farms and from 155 Atlantic salmon from outbreaks on 20 farms collected from 1996 to 2004 were included in the present study. The histopathological findings of PD in Atlantic salmon and rainbow trout in sea water were similar. Acute PD, characterized by acute necrosis of exocrine pancreatic tissues, was detected in nine Atlantic salmon and three rainbow trout. Salmonid alphavirus (SAV) was identified in acute pancreatic necroses by immunohistochemistry. Most fish showed severe loss of exocrine pancreatic tissue combined with chronic myositis. Myocarditis was often but not consistently found. Kidneys from 40% and 64% of the rainbow trout and Atlantic salmon, respectively, had cells along the sinusoids that were packed with cytoplasmic eosinophilic granules. These cells resembled hypertrophied endothelial cells or elongated mast cell analogues. Histochemical staining properties and electron microscopy of these cells are presented. SAV was identified by RT-PCR and neutralizing antibodies against SAV were detected in blood samples.     

Comparative sensitivity of carp, Cyprinus carpio L. and rainbow trout, Salmo gairdneri Richardson, to Renibacterium salmoninarum

Abstract. The pathogenicity of Renibacterium salmoninarum to carp, Cyprinus carpio L., and rainbow trout, Salmo gairdneri Richardson, was investigated. All carp injected with 4·8 × 10⁸ cells/fish, or 4·8 × 10⁷ cells/fish survived for 38 days. R. salmoninarum was isolated from all moribund fish, but not from the kidney of surviving fish, although R. salmoninarum antigen was detected in several of these fish by the dot blot assay. On the other hand, mortality in rainbow trout was 95% in the fish injected with 4·8 × 10⁸ cells/fish, and 15% in those which received 4·8 × 10⁷ cells/fish. R. salmoninarum antigen was detected by the dot blot assay in all surviving rainbow trout. The number of R. salmoninarum cells was immediately decreased by carp or rainbow trout serum, and the serum bactericidal activity of carp was higher than that of rainbow trout. Carp blood leucocytes had higher phagocytic activity than those of rainbow trout.     

Lysozyme from rainbow trout, Salmo gairdneri Richardson, as an antibacterial agent against fish pathogens

Abstract. The antibacterial effect of two lysozyme variants purified from rainbow trout, Salmo gairdneri Richardson, kidney was investigated as part of a project directed towards increasing the disease resistance of fish by the transgenic technique. Seven bacterial strains from five Gram-negative species, of which one was considered non-pathogenic, were examined. One of the rainbow trout lysozymes was surprisingly potent, having substantial antibacterial activity on all strains tested. Hen egg-white lysozyme was bactericidal only against the one species considered non-pathogenic. The data suggest that lysozyme does play a role in the disease defence of rainbow trout and that the gene for the most active lysozyme may be suitable for testing the transgenic strategy.     

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

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

Further consideration of the haematology of proliferative kidney disease (PKD) in rainbow trout, Salmo gairdneri Richardson

Abstract. A study of selected haematological parameters in rainbow trout, Salmo gairdneri Richardson, exposed to proliferative kidney disease (PKD) under field conditions is described. Changes in the blood picture are related to kidney pathology at different stages of the disease. As extensive renal vascular pathology, haemoglobin crystallization and inflammatory lesions developed, there were progressive decreases in haemoglobin and packed cell volume values, erythrocyte numbers declined to less than 50% of those in control fish, and numbers of misshapen erythrocytes increased. Leucopenia, neutrophilia and thrombocytopenia developed, especially in oedematous fish. With recovery, most values were similar to those of control fish. In recovered, re-challenged fish, erythrocyte counts, packed cell volume and haemoglobin values decreased in infected fish compared with uninfected fish. The results of the study are compared with those of other authors and the type of anaemia in PKD is discussed.     

Mucosal CD3ε+ cell proliferation and gut epithelial apoptosis: implications in rainbow trout gastroenteritis (RTGE)

Rainbow trout gastroenteritis (RTGE) is an emerging disease that has acquired new relevance in European rainbow trout, Oncorhynchus mykiss (Walbaum), culture, because of the economic losses it causes. Disease aetiology and pathogenesis remain unclear. The lesions appear restricted to the gastrointestinal tract where extensive mucosal detachment associated with high numbers of segmented filamentous bacteria (SFB) can be detected. In this study, an RTGE outbreak in north-western Spain was investigated, and findings observed in diseased trout were compared with control fish. PAS stain and immunohistochemical assays with anti-CD3ε and anti-active caspase-3 antibodies were performed. The results showed that CD3ε+ inflammatory infiltrates were present in the intestine of diseased trout both in the lamina propria-submucosa and within the epithelium. Moreover, an increased number of caspase-3+ cells in the intestinal mucosa and also strong anti-caspase-3 immunoreactivity in desquamated cells in the gut lumen were observed. Changes in the number of goblet cells were also found, resulting in an increase or depletion of mucous cells depending on the severity of the intestinal lesions. These findings suggest that T cells and apoptosis play an important role in the development and pathogenesis of RTGE.     

Changes in serum parameters of rainbow trout, Salmo gairdneri Richardson, and Atlantic salmon, Salmo salar L., infected with Renibacterium salmoninarum

Abstract. Bacterial kidney disease was induced experimentally in rainbow trout, Salmo gairdneri Richardson, and Atlantic salmon, S. salar L., and selected serum parameters measured over a 35-day period. In those fish which became infected a significant increase in the erythrocyte sedimentation rate and in the serum levels of bilirubin, blood urea nitrogen and potassium were recorded. A significant decrease in total serum protein, cholesterol and sodium also occurred in these fish. Electrophore-tic analysis of serum proteins showed the fast migrating fraction was reduced before the slower migrating material in infected fish.     

The thiamin deficiency signs and requirement of rainbow trout (Salmo gairdneri,Richardson)

Four growth studies were conducted to determine the signs, biochemical indices and histopathology of a thiamin deficiency and the thiamin requirement of young rainbow trout reared at 15°C on a semi-purified test diet. The major overt signs of a thiamin deficiency in rainbow trout are predominantly neurological: irritability and instability. Other signs include convulsions, feed refusal, dark pigmentation and finally mortalities. Growth reduction in the thiamin deficient trout appear to result from anorexia or feed refusal and not specifically to a thiamin deficiency. Although there were prominant neurological signs in the thiamin deficient trout, there were no histopathological signs in any tissues of the trout, including the brain and central nervous system, examined by light microscopic techniques. The tissue transketolase activity would appear to be a sensitive and specific indicator of the thiamin status in the trout. In addition, the levels of plasma lactate and serum pyruvate are also elevated in thiamin deficient trout. On the basis of the growth parameters, absence of deficiency signs and kidney and liver transketolase activity, the thiamin requirement of rainbow trout reared at 15°C on a semi-purified test diet is 1 mg/kg feed.     

Disease of rainbow trout caused by Pseudomonas luteola

Bacteria isolated from rainbow trout, Oncorhynchus mykiss, kept in a farm, in Turkey. During the outbreak, 40% of the rainbow trout (10-40 g) died. Typical clinical signs were exophthalmia, dark pigmentation, hemorrhage at the base of the pectoral, pelvic, anal fins and around the vent. Internal signs were enlarged spleen, pale liver and intestine filled with yellowish fluid. Liver, kidney and spleen of diseased fish, were aseptically streaked on Tryptic Soy Agar. After incubation, pure cultured colonies were observed and biochemically characterized with API 20 NE and other biochemical tests. Cultured bacterial 16 S rDNA gene was sequenced. Based on biochemical characteristics and sequence of 16 S rRNA, the causative bacteria were identified as Pseudomonas luteola. This study reports the first P. luteola infection in fish.     

Occurrence of viral haemorrhagic septicaemia in rainbow trout Salmo gairdneri Richardson reared in sea-water

Abstract. Viral haemorrhagic septicaemia (VHS) is reported for the first time in sea-water cultured rainbow trout. Heavy mortalities with typical signs and lesions A VHS virus (serotype 1) was isolated from the diseased fish. The mortalities were caused only by the VHS virus and 80 days post transfer of trout to sea-water the mortalities reached 85%, of the initial population.
The disease was experimentally transmitted to rainbow trout, both in sea-water 3·10⁴ pfu/ml of virus or by intramuscular injection of various doses of VHS₁ (7·10¹ 7·10⁴ or 7·10⁴ pfu per fish). Death occurred in all infected groups and started earlier in sea-water. Typical signs of VHS were observed in moribund fish. Viral multiplication was demonstrated to have occurred in fish organs.