Morphology and distribution of granulomatous inflammation in freshwater ornamental fish infected with mycobacteria

Mycobacteriosis in fish is a chronic progressive ubiquitous disease caused by Mycobacterium marinum, M. gordonae and M. fortuitum in most cases. The aim of this study was to describe the morphology and distribution of lesions in 322 freshwater ornamental fish across 36 species. Granulomatous inflammation was diagnosed by gross examination and histopathology testing in 188 fish (58.4%); acid‐fast rods (AFR) were determined in only 96 (51.1%) fish from 19 species after Ziehl–Neelsen staining. The most often affected organs with AFR were the kidney (81.2%), digestive tract (54.1%), liver (48.2%), spleen (45.9%) and skin (21.2%); sporadically, AFR were found in the branchiae (9.4%) and gonads (4.7%). In 14 randomly selected fish originating from four different fish tanks, the distribution of mycobacterial infection was studied by culture examination of the skin, gills, muscle tissue, digestive tract, liver, spleen and kidney. In 12 fish, the species M. marinum, M. gordonae, M. fortuitum, M. triviale, and M. avium subsp. hominissuis (serotypes 6 and 8 and genotype IS901? and IS1245+) were detected; mixed infection caused by different mycobacterial species was documented in five of them.     

First report of Shewanella sp. and Listonella sp. infection in freshwater cultured loach,Misgurnus anguillicaudatus

A novel disease outbreak occurred among cultured loach, Misgurnus anguillicaudatus in China. The diseased fish displayed lethargy, loss of appetite, black discolouration, anasarca and ventral skin hyperaemia. Clinical signs were observed in most of the fish examined, which consisted of discoloured liver, swollen spleen and petechial haemorrhage in the intestine and peritoneal membranes. The causative agent was found to be two species of bacteria by experimental infection, identified as Shewanella sp. and Listonella sp. The result showed that these were likely to be Shewanella putrefaciens and Listonella anguillarum respectively. Bacterial identification consisted of physiological and biochemical tests as well as 16S rRNA sequence analysis. In addition, antibiotic susceptibility testing was performed, revealing that Shewanella putrefaciens and Listonella anguillarum were susceptible to enrofloxacin, ciprofloxacin, levofloxacin, pipemidicacid and norfloxacin. This is the first report of S. putrefaciens and L. anguillarum from cultured loach.     

Infection of Tilapia Oreochromis sp. by Vibrio vulnificus in Freshwater and Low-salinity Environments

The first isolation of Vibrio vulnificus in southern Taiwan from hybrid tilapia Oreochromis sp. raised in freshwater and brackish water environments is documented in this report. The infection was only found in fish in ponds where the salinity was less than 10 ppt. Tilapia raised in water of higher salinities in the same region were not affected. Grossly visible signs of infection included dark coloration, lethargy, and external hemorrhage and ulceration of the skin. The most prominent internal signs of infection included splenomegaly and severe hemorrhagic lesions in the liver. Septicemia was documented in moribund fish. All bacterial isolates from moribund fish were tested by polymerase chain reaction, using V. vulnificus hemolysin/cytolysin gene‐specific primers. Sequence data from the 16S ribosomal RNA gene suggest that these isolates were V. vulnificus. The isolates were indole and mannitol positive, characteristics shared by human clinical isolates and isolates from freshwater European eel, Anguilla anguilla. The isolates from tilapia were unique in that they were negative for ornithine decarboxylase and citrate.     

Genotypic diversity among Shewanella spp. collected from freshwater fish

Different Shewanella species are isolated both from healthy and from diseased fish. To date, contemporary methods do not provide sufficient insight to determine species and detail differentiation between tested strains. Bacteria isolated from cultured (n = 33), wild (n = 12) and ornamental (n = 6) fish, as well as several reference strains, were tested by 16S rRNA gene sequencing, ERIC‐PCR and pulsed‐field gel electrophoresis (PFGE) assays. Our study indicates that isolates collected from freshwater fish were genetically diverse. Based on 16S rRNA gene sequences, bacteria were clustered into groups S. putrefaciens, S. xiamenensis and S. oneidensis. Some isolates were classified only to genus Shewanella; thus, 16S rRNA gene analyses were not enough to determine the species. ERIC‐PCR revealed 49 different genotype profiles indicating that the method might be useful for differentiation of Shewanella isolates irrespectively to species identification, contrary to PFGE which is not suitable for Shewanella typing.     

Methods for identification and differentiation of different Shewanella spp. isolates for diagnostic use

Shewanella spp. are Gram‐negative, rod‐shaped, motile bacteria that are widely distributed in marine and freshwater environments. The bacteria are present in the physiological microflora of fish from temperate waters and are known as fish spoilage species. From clinically healthy fish and from fish with skin ulcerations, Shewanella spp. is regularly isolated, indicating a possible role as fish pathogen. In this study, 74 isolates of Shewanella spp. were analysed. For species identification, biochemical techniques, 16S rRNA sequencing, MALDI‐TOF MS and the Sherlock Microbial Identification System (MIS) based on the composition of fatty acid ethyl esters were compared. The phylogenetic relationship, cytotoxicity in vitro and resistance against antibiotics were tested. The most reliable method for species identification was 16S rRNA sequencing. From diseased fish, clinically healthy fish and the aquatic environment, different Shewanella species were isolated. This indicates that Shewanella spp. is widespread in the aquatic milieu and acts as a secondary pathogen. The virulence of Shewanella spp. is probably not depending on the species but on the isolate itself. Many isolates of Shewanella spp. were showing multiresistances against antibiotic substances, especially in samples derived from retailers and in routine diagnostics, all Shewanella spp. should therefore be tested for resistances against antibiotic agents.     

Current status of parasitic ciliates Chilodonella spp. (Phyllopharyngea: Chilodonellidae) in freshwater fish aquaculture

Freshwater fish farming contributes to more than two‐thirds of global aquaculture production. Parasitic ciliates are one of the largest causes of production loss in freshwater farmed fishes, with species from the genus Chilodonella being particularly problematic. While Chilodonella spp. include ‘free‐living’ fauna, some species are involved in mortality events of fish, particularly in high‐density aquaculture. Indeed, chilodonellosis causes major productivity losses in over 16 species of farmed freshwater fishes in more than 14 countries. Traditionally, Chilodonella species are identified based on morphological features; however, the genus comprises yet uncharacterized cryptic species, which indicates the necessity for molecular diagnostic methods. This review synthesizes current knowledge on the biology, ecology and geographic distribution of harmful Chilodonella spp. and examines pathological signs, diagnostic methods and treatments. Recent advances in molecular diagnostics and the ability to culture Chilodonella spp. in vitro will enable the development of preventative management practices and sustained freshwater fish aquaculture production.     

Predation on native fish eggs by invasive round goby revealed by species‐specific gut content DNA analyses

1. Conservation of riverine fish often aims to improve access to spawning grounds and restore longitudinal connectivity by removing migration barriers, and involves substantial investments. However, these investments also enable non‐native predators to invade upstream into spawning areas and potentially adversely affect the recruitment of threatened freshwater fish through egg or fry predation.
2. Detecting egg predation is often challenging. Visual inspections of fish gut contents may underestimate predation of soft materials such as eggs and fry, which limits the discovery of predators preying upon these life‐stages. DNA‐based detection assays may offer a more sensitive tool to assess predation of soft materials.
3. A conservation issue was confirmed by developing and applying a species‐specific DNA‐based detection assay: invasive round goby (Neogobius melanostomus) prey on the eggs or fry of the threatened common nase (Chondrostoma nasus) in Switzerland.
4. DNA‐based detection assays were also developed for five other valuable native fish species, including endangered salmonid and cyprinid river spawners. The applicability of the assays was confirmed in a series of laboratory and field feeding experiments involving eggs and fish tissue. In addition, this work provides a guiding framework for conservation managers regarding the use and applicability of different DNA‐based detection approaches for gut content analysis.
5. The results of this study could inform local conservation measures – such as temporary reductions in the density of round goby at spawning sites prior to spawning – and demonstrate how targeted application of species‐specific molecular markers may advance freshwater fish management.

     

Betanodavirus ability to infect juvenile European sea bass,Dicentrarchus labrax,at different water salinity

Viral encephalopathy and retinopathy (VER) is one of the most devastating and economically relevant diseases for marine aquaculture. The presence of betanodavirus in freshwater fish is recorded, but very little is known about VER outbreaks in marine species reared in freshwater. Our study investigated the ability of betanodavirus to cause disease in European sea bass, Dicentrarchus labrax, reared at different salinity levels. Fish were challenged with RGNNV or mock infected by bath at different salinity levels (freshwater, 25‰ and 33‰). Fish were checked twice a day and the dead ones were examined by standard virological techniques, by rRT‐PCR and by histochemical and immunohistochemical analyses. All the infected groups showed a significant higher mortality rate than the one of the mock‐infected group. VERv presence was confirmed by rRT‐PCR. Histochemical and immunohistochemical analyses highlighted the typical lesions associated with VER. Our results highlight that salinity does not affect the ability of betanodavirus to induce clinical signs and mortality in European sea bass infected under experimental conditions. These results underline the great adaptation potential of VERv, which in combination with its already known high environmental resistance and broad host range, may explain the diffusion of this disease and the threat posed to aquaculture worldwide.     

Fish–habitat relationships and fish conservation in small coastal streams in southern Spain

• 1. Studies dealing with the fish fauna of coastal streams are scarce in the scientific literature, particularly those from Mediterranean climates. Owing to their small size, these systems suffer extreme seasonal fluctuations, following the typical Mediterranean flood–drought cycle and leading to a high risk of extinction to freshwater fish.
• 2. This work analyses fish distribution in 14 stream stretches belonging to eight basins in the northern sector of the Strait of Gibraltar (southern Spain). Fish–habitat relationships were studied through multivariate ordination techniques at two scales: basin and stretch.
• 3. A principal components analysis clearly discriminated larger and more sinuous basins from smaller and steeper ones. This ordination was related to the non‐migratory freshwater fish species richness and to the total number of fish species present in the middle reaches of each basin.
• 4. The main sources of variation in community composition and habitat characteristics in the different stretches were related to a clear upstream–downstream gradient, along which total species richness increased.
• 5. These small coastal basins are inhabited by two highly endangered species, Andalusian toothcarp (Aphanius baeticus) and Iberian chub (Squalius pyrenaicus), and have similar or higher overall freshwater species richness than larger adjacent basins. The near absences of flow regulation and introduced species make these streams one of the few types of Iberian aquatic system where unaltered fish–habitat relationships can be studied.

Copyright © 2005 John Wiley & Sons, Ltd.     

Characterization of Mycobacterium salmoniphilum as causal agent of mycobacteriosis in Atlantic salmon,Salmo salar L., from a freshwater recirculation system

Thirty Atlantic salmon, Salmo salar L., with low corporal condition relative to other fish present in the culture system, were sampled from a freshwater recirculation pisciculture located in Chile. The most characteristic signs and lesions were cachexia and presence of multiple greyish‐white granulomas within internal organs. The external and internal lesions, along with the microscopic, histologic and biochemical findings, were consistent with mycobacteriosis. The identification of Mycobacterium salmoniphilum as the causal agent of the lesions was possible through the use of molecular analyses. This study represents the first report of Mycobacterium salmoniphilum in a freshwater recirculation system and the first case of fish mycobacteriosis described in Chile.     

Lumpfish (Cyclopterus lumpus,Linnaeus) is susceptible to viral nervous necrosis: Result of an experimental infection with different genotypes of Betanodavirus

In recent years, the use of cleaner fish for biological control of sea lice has increased considerably. Along with this, a number of infectious diseases have emerged. The aim of this study was to investigate the susceptibility of lumpfish (Cyclopterus lumpus) to Betanodavirus since it was detected in asymptomatic wild wrasses in Norway and Sweden. Three betanodaviruses were used to challenge lumpfish: one RGNNV genotype and two BFNNV genotypes. Fish were injected and monitored for 4 weeks. Brain samples from clinically affected specimens, from weekly randomly selected fish and survivors were subjected to molecular testing, viral isolation, histopathology and immunohistochemistry. Reduced survival was observed but was attributed to tail‐biting behaviour, since no nervous signs were observed throughout the study. Betanodavirus RNA was detected in all samples, additionally suggesting an active replication of the virus in the brain. Viral isolation confirmed molecular biology results and revealed a high viral titre in BFNNV‐infected groups associated with typical lesions in brains and eyes of survivor fish. We concluded that lumpfish are susceptible to Betanodavirus, as proven by the high viral titre and brain lesions detected, but further studies are necessary to understand if Betanodavirus can cause clinical disease in this species.     

冬夏两季五种经济鱼类组织脂肪酸含量及组成分析

为了解不同季节不同鱼类不同组织中的不同脂肪酸含量,科学地指导鱼类膳食消费,本实验研究了冬夏两季,采集自上海市场常见的5种经济鱼类:大黄鱼(海洋肉食性),银鲳(海洋杂食性),日本鳗鲡(淡水肉食性),莫桑比克罗非鱼(淡水杂食性),草鱼(淡水草食性),分别检测鱼背部肌肉、腹部肌肉、尾部肌肉、肝脏和腹腔脂肪组织的脂肪含量和脂肪酸绝对含量。结果显示,5种鱼肌肉脂肪酸谱存在显著差异,并与各自的生活环境及食性均有关系;在鱼的腹腔脂肪或肝脏中,饱和脂肪酸(SFAs)和单不饱和脂肪酸(MUFAs)含量较高,且与组织脂肪含量密切相关;而多不饱和脂肪酸(PUFAs)、n-3系多不饱和脂肪酸(n-3 PUFAs)和n-6系多不饱和脂肪酸(n-6 PUFAs)含量较低,且与组织脂肪含量关系不大;大黄鱼和银鲳各肌肉组织中的n-3 PUFAs、二十碳五烯酸(EPA)和二十二碳六烯酸(DHA)含量以及n-3/n-6值高于莫桑比克罗非鱼和草鱼,并与组织脂肪含量呈正相关;冬季草鱼腹部肌肉、莫桑比克罗非鱼尾部肌肉以及日本鳗鲡和银鲳的肝脏中的n-3 PUFAs含量较夏季高。研究表明,脂肪酸组成与物种、食性、水域环境以及季节温度和组织部位均有关系。从补充n-3 PUFAs摄入的角度分析,日本鳗鲡、大黄鱼和银鲳营养价值高于莫桑比克罗非鱼和草鱼,冬季鱼类的营养价值高于夏季。     

Fish fauna in Iberian Mediterranean river basins: biodiversity,introduced species and damming impacts

• 1. A basin approximation was used to analyse distribution patterns of different components of biodiversity (taxonomic richness, endemicity, taxonomic singularity, rarity) and conservation status of freshwater fish fauna in 27 Mediterranean Iberian rivers.
• 2. Basin area alone explained more than 80% of variation in native species richness. Larger basins featured not only a higher number of native species, but also more endemic and rare species and fewer diversified genera than smaller basins.
• 3. In contrast, smaller basins scored higher community conservation values, owing to their lower degree of invasion by introduced species.
• 4. The presence of dams was the most important factor determining the conservation status of fish communities, and it was also positively associated with the number of introduced species.
• 5. While the most important components of Iberian freshwater fish biodiversity are located in large basins, small unregulated basins feature better conserved fish communities.

Copyright © 2004 John Wiley & Sons, Ltd.     

Detection and molecular characterization of infectious spleen and kidney necrosis virus from major ornamental fish breeding states in Peninsular Malaysia

‘Gold standard’ OIE reference PCR assay was utilized to detect the presence of infectious spleen and kidney necrosis virus (ISKNV) in freshwater ornamental fish from Malaysia. From total of 210 ornamental fish samples representing 14 species, ISKNV was detected in 36 samples representing 5 fish species. All positive cases did not show any clinical signs of ISKNV. Three restriction enzymes analyses showed that the fish were infected by identical strains of the same virus species within Megalocytivirus genus. Major capsid protein (MCP) genes of 10 ISKNV strains were sequenced and compared with 9 other reference nucleotide sequences acquired from GenBank. Sequence analysis of MCP gene showed that all strains detected in this study were closely related to the reference ISKNV with nucleotide sequence identity that was ranging from 99.8% to 100%. In addition, phylogenetic analysis of MCP gene revealed that viruses from genus Megalocytivirus can be divided into three genotypes: genotype 1 include reference ISKNV and all other strains that were detected in this study, genotype 2 include viruses closely related to red sea bream iridovirus (RSIV), and genotype 3 include viruses closely related turbot reddish body iridovirus (TRBIV).     

IUCN Red List of freshwater fishes and lampreys in Flanders (north Belgium)

Red Lists are used to assess the extinction risk of species. Regional IUCN criteria were applied to categorise 42 indigenous freshwater fish species in Flanders into Red List categories. As such, three species are assessed as Regionally Extinct, eight as Critically Endangered, two as Endangered and eight as Vulnerable. A further five species are considered Near Threatened, 15 species as Least Concern and one species is Data Deficient. Five species disappeared from the Regionally Extinct category compared with the previous Red List (1988) as a consequence of improved water quality of estuaries and reintroduction programmes. In total, 62% of the freshwater fish species in Flanders was considered extinct or threatened. This is higher than neighbouring regions and Europe as a whole (44%). This new Red List of freshwater fishes in Flanders can be used to inform conservation actions.     

An outbreak of Shewanella putrefaciens group in wild eels Anguilla anguilla L. favoured by hypoxic aquatic environments

Microbiological analyses were conducted on wild eels from the L′Albufera Lake (Spain). A total of 174 individuals were collected in two surveys (i.e. year 2008 and autumn–winter 2014) among those caught by local fishermen into the lagoon. The prevalence of Shewanella putrefaciens group was 1.7% in 2008 and rose above 32% in 2014. It was due to an outbreak of shewanellosis that presented a morbidity rate of 64%. S. putrefaciens group strains were isolated as pure cultures from the sick eels that showed white ulcers surrounded by a reddish inflammation, damage of the mouth, extensive skin discoloration, exophthalmia, ascites and bad odour. The S. putrefaciens group was recovered from freshwater samples taken at the L′Albufera system, along autumn–winter 2015. Its counts significantly increased in freshwater parallel to hypoxia and temperature rising. Shewanellae strains were identified as S. putrefaciens and S. xiamenensis by 16S rRNA gene sequencing. These isolates recovered from sick eels or freshwater were virulent for European eel by IP challenge (LD₅₀ 10⁶ CFU g^?1 body weight). They also caused 30–38% cumulative mortality, in European eels challenged by a 2‐h bath (10⁷ CFU mL^?1). These results suggest that shewanellosis could be transmitted through water highlighting the fact that hypoxic conditions increase this bacterium levels in water.     

Fish resources of Dhofar khawrs (coastal lagoons) in the Sultanate of Oman

Abstract A fish resource monitoring programme was undertaken during 1992–1993 in nine Dhofar khawrs (coastal lagoons) of Oman. The khawrs contained a large number of fish comprising mostly milkfish, Chanos chanos (Forsskal), and mullet, Mugil cephalus L. A wide range of species were recorded (47) with Khawr Sawli supporting the highest number (26). Most fish use khawrs as nursery grounds and migrate to the sea for breeding. Mullets matured in winter, milkfish in early spring and the other species in spring-summer except the sleeper, Ophiocara porocephala (Valenciennes), which breeds all year round. Three species of invertebrates with commercial value were collected: Indian white shrimp, Penaeus indicus H. Milne Edwards, Mud crab, Scylla serrata (Forskal), and swimming crab, Portunus pelagicus (L.). Khawr water temperature followed a seasonal cycle and surface temperatures were higher than the deeper waters. Heavy rains in early spring caused extensive flooding and opening of khawrs to the sea. This had little effect on water salinity in some khawrs (Rawri and Mughsayl), while others displayed marked elevation in salinity (Taqah, Sawli and Jnawf) or reduced salinity by 50% (Qurm al-Kabir). Sedimentation and organic decomposition added during flooding produced drastic reduction in dissolved oxygen. Khawrs were again connected to the sea during monsoon (June–September) as water levels increased. Intrusion of sea water increased salinity and continued for several months in shallow khawrs (Jnawf); however, freshwater seepage reduced salinity in most khawrs. Massive fish mortality following heavy rains was the most significant seasonal occurrence. Extensive fish losses also occurred when water levels dropped after opening khawrs to the sea, and left many fish stranded in shallow areas. Other environmental perturbations existed in most khawrs (e.g. pollution and overgrazing). However, these khawrs have potential for small-scale fisheries and different types of aquaculture. Management problems are identified and options for solutions suggested.     

Susceptibility of freshwater rearing Asian seabass (Lates calcarifer) to pathogenic Streptococcus iniae

Asian seabass (Lates calcarifer) has been recognized as an economically important aquaculture species which can be adapted to and cultivated in wide range of salinities. The number of freshwater intensive seabass farms in Thailand is increasing annually. Here, we first describe the susceptibility of Asian seabass, which were cultured in freshwater, to Streptococcus inae (SI) and their pathological changes. Three isolates of putative SI were identified using a combination of standard biochemical assays and species‐specific PCR prior subjected to in vivo challenge. Accumulated mortalities of the fish which received 10⁷ CFU fish^?1 of either SI1J, SI SGSA or SI2J were 90%, 90% and 100% at 7 days‐post infection (dpi), respectively, and mortalities increased sharply between 3 and 5 dpi. Clinical signs such as erratic swimming and opaque eyes were identified from a few infected fish, while most died rapidly without any abnormal signs. Histopathological manifestations were observed in the multiple organs (kidney, liver and brain). Haemorrhage, hyperhemia, cellular degeneration and inflammatory cells infiltration were commonly found within the internal organs. Notably, the formation of numerous encyst‐like lesion aggregated by eosinophilic cells, resembling macrophages, were typically found in the brain of the infected fish. Summarily, this study first revealed that freshwater reared Asian seabass is highly susceptible to SI infection and haemorrhagic septicaemia was a major pathological change that could be found in the infected fish.