Seasonal intestinal coccidiosis in wild bluegill Lepomis macrochirus is associated with a spring bacterial epizootic

Wild bluegill, Lepomis macrochirus Rafinesque, succumb to seasonal mortality in the early spring during cool water temperatures, shown previously to be related to bacteraemia caused by a psychrotrophic bacterium, Pseudomonas mandelii. In the study herein, intestinal coccidiosis in wild bluegill had seasonal prevalence causing heavy intestinal infections and sloughing of intestinal epithelium occurring in late winter/early spring. Infections were predominantly related to two different species, Goussia washuti n. sp., an epicellular coccidium, and a coccidium closely resembling Goussia desseri Molnár 1996, previously only described in percid fish in Europe. In 2019, co‐infections of bacteraemia and intestinal coccidiosis occurred in bluegills. Evaluating coccidium infection intensity by fresh parasite examination and histology, an association was observed in which fish with moderate‐to‐heavy intestinal coccidiosis were 8–12 times more likely to have bacteraemia compared to fish with no or light coccidiosis. The association of these co‐infections suggests that intestinal coccidiosis could contribute to seasonal bacterial epizootics of wild bluegill.     

Isolation and evaluation of putative probiotic strains from different teleost to prevent Pseudomonas aeruginosa infection in Cyprinus carpio

Probiotics renowned as valuable microbes serve as a potential alternative to control diseases in aquaculture and are considered as an efficient and environment‐friendly approach to reduce the use of antibiotics. The present study aims at the isolation of putative probiotic bacteria from the intestinal tract of different fish species from the Doaba region of Punjab, India. In this study, isolated bacterial strains were characterized based on their morphological, biochemical and molecular characterization by 16S rRNA gene sequencing, followed by in vitro evaluation of different selection parameters described in FAO/WHO guidelines. A total of 169 different bacterial strains were isolated from the gastrointestinal tract of 52 different fish species. After in vitro evaluation, out of 169 bacterial strains only five bacteria (S3, S7, BDK2', BDK7 and BDK9) identified as Enterococcus and Bacillus species showed antagonistic activity against the fish pathogen Pseudomonas aeruginosa (MTCC 4 673). These isolates were screened based on their response towards bile tolerance, pH tolerance, adhesion and drug susceptibility to different antibiotic discs. And, the in vivo results indicated improved growth and survival against the infection (P. aeruginosa) after oral administration of the probiotics. The observations of in vitro and in vivo evaluation indicate that these isolated probiotic strains serve as effective probiotics and can be used as a novel and safe treatment to cure current issues in aquaculture.     

Swimming performance of brown trout and grayling show species‐specific responses to changes in temperature

Fishways have historically been constructed to restore and preserve the ecological connectivity for fish in fragmented rivers. However, the fishways are often selective on species due to different size and swimming capacity. As the proportion of dammed rivers is still increasing, there is a growing need for more information on wild fish and their migration potential. In this study, we compared the swimming capacity of wild caught brown trout (Salmo trutta) and European grayling (Thymallus thymallus) until the fish were exhausted in a critical swimming speed (U_crit) test, under three different naturally occurring stream temperatures in Norway: 1.7, 5.5 and 10°C. The results indicated that trout swim better at the warmer temperatures than at colder temperatures. The grayling showed consistent swimming patterns with little variation across all tested temperatures. The results therefore signify the need to have operational fishways already early in the spring when the grayling migration starts and highlight the need for more studies on fish migration abilities across a wider range of species and seasons.     

Effect of temperature on growth performance of greater amberjack (SERIOLA DUMERILI Risso 1810) Juveniles

In order to successfully diversify Mediterranean aquaculture, it is necessary to determine optimum culture conditions of potential candidate species such as greater amberjack (Seriola dumerili). Among culture conditions, rearing temperature is a key factor for achieving optimum growth and maintaining fish welfare. However, little is known about the optimum culture conditions of greater amberjack (Seriola dumerili). Thus, the aim of this study was to determine the effect of three different rearing temperatures (17, 22 and 26°C) during 120 days on growth performance, body morphometry, biochemical composition, gut transit and liver morphology of greater amberjack (Seriola dumerili) juveniles. After 120 days of rearing, fish raised at 26°C showed higher (p < .05) body weight and specific growth rate than fish held at lower temperatures, as well as improved feed utilization, protein efficiency and nutrient retention percentages. Fish stomach emptying was faster (p < .05) in fish raised at 26°C than in fish held at 22°C and 17°C. Similar results were obtained for gut transit time, being gut emptying faster (p < .05) in fish reared at 26°C than in fish cultured at lower temperatures. Rearing temperature also induced changes in fish morphology which resulted in a higher (p < .05) caudal propulsion efficiency index for fish reared at 26°C. Based on these results, we conclude that greater amberjack fingerlings perform better at 26°C than at 22°C or 17°C.     

A metabolomic investigation into the temperature‐dependent virulence of Pseudomonas plecoglossicida from large yellow croaker (Pseudosciaena crocea)

Pseudomonas plecoglossicida is associated with multiple fish diseases, and temperature is one of the most important environmental factors related to its outbreak. To elucidate the influence of temperature variation on the pathogen, the global metabolomics of P. plecoglossicida (NZBD9) were analysed at the virulent (18°C) and avirulent (12°C and 28°C) temperatures. The result showed that the levels of Phosphoric acid, Tyrosine, Spermidine and Sucrose were significantly reduced，while Itaconic acid, Glucaric acid and Isomaltose were increased in P. plecoglossicida at 18°C. These metabolic adjustments assist P. plecoglossicida to survive in adverse environments, proliferate in the host, colonize and resist host immune clearance during the initial steps of infection. The results suggested that L321_03626 and L321_18122 genes played a key role in the regulation of these metabolic adaptions and thus regulated P. plecoglossicida virulence at virulent temperature, which was proved by further gene silencing and artificial infection. The present study, for the first time, determines the P. plecoglossicida metabolomic responses to temperature variation, which is helpful to explore its pathogenic mechanism and provides reference for disease control.     

Visceral granulomas in farmed large yellow croaker,Larimichthys crocea (Richardson), caused by a bacterial pathogen,Pseudomonas plecoglossicida

An enzootic disease characterized by granulomas in internal organs occurred in cage‐farmed large yellow croaker, Larimichthys crocea (Richardson), in April and November 2010, in Ningbo, Zhejiang Province. One bacterial strain, named XSDHY‐P, was isolated from the diseased fish and identified by biochemical characterization, fatty acid methyl ester (FAME) analysis and multilocus sequence analysis (MLSA). According to the results obtained from the biochemical tests, FAME analysis and phylogenetic analysis derived from 16S ribosomal RNA, gyrB, oprF, oprI, oprL and rpoD gene sequencing, the bacterial isolate, XSDHY‐P, was identified as Pseudomonas plecoglossicida. Moreover, lethal dose, 50% trials were carried out to demonstrate the virulence of XSDHY‐P in large yellow croaker when administered at 2.13 × 10⁵ colony‐forming units per fish. Visceral granulomas were found in the experimentally infected fish as well as in the naturally infected fish, indicating that P. plecoglossicida is another bacterial pathogen that causes granulomatosis in L. crocea.     

Does non-native pumpkinseed Lepomis gibbosus affect endemic algae-scraping Capoeta aydinensis in case of introduction to a small stream? An ex situ growth experiment

The survival and establishment of alien fishes in novel environments can result in resource partitioning with native fishes. This can cause ecological impact and suppression of native populations. However, quantifying the impact of novel interactions between alien and native species remains highly challenging in the wild. Consequently, to determine the ecological risk of Lepomis gibbosus in case of introduction to a small stream, experimental approach was used to predict its competitive interaction with a poorly studied endemic fish, Capoeta aydinensis. The aim was to test whether L. gibbosus has an adverse effect on native species using an experimental design under allopatric and sympatric context with temperature regimes of 15 and 24ºC. The results indicated that temperature and fish proportion had effects on the growth of C. aydinensis while these factors were not important for L. gibbosus. These results provided little evidence of L. gibbosus presence being detrimental for endemic C. aydinensis, but nevertheless steps should be taken to avoid their further co-habitation in the wild.     

Bluegill Picornavirus isolated from a mortality event involving Bluegill (Lepomis macrochirus) in the upper Mississippi River

A mortality event involving an estimated 1,000 adult bluegills (Lepomis macrochirus) was observed in an ice‐covered backwater lake of the upper Mississippi River near Alma, Wisconsin, in December of 2017. Macroscopic signs of disease included abdominal distension due to fluid accumulation within the internal organs as well as external and internal haemorrhaging. Histological evaluation revealed chronic peritonitis with peritoneal adhesions in all fish examined. Kidney, spleen and ascites fluid samples were collected from diseased bluegills and examined for the presence of pathogens. Bluegill picornavirus (BGPV) was isolated using tissue cell culture methods utilizing a recently developed, uncharacterized bluegill fry cell line (BF‐4), and the presence of this virus was confirmed through molecular identification. The current geographic range, known susceptible hosts as well as historical epizootics associated with BPGV is discussed. The ability of BGPV to cause significant mortality in wild fish further emphasizes the importance of monitoring both wild and hatchery populations for this pathogen.     

Food consumption and digestive enzyme activity of Clarias batrachus exposed to various temperatures

The effect of temperature on the food consumption rate and the digestive enzyme activities of Clarias batrachus (80.60 ± 5.34 g) were evaluated. Fish were exposed to six different temperatures of 10, 15, 20, 25, 30 and 35 °C following an acclimation temperature of 25 °C. The rate of temperature change was 2 °C day^?1. Highest food consumption was recorded at 25 °C. It gradually reduced with decreasing water temperature. Food consumption rate was significantly (P < 0.05) lower at 10 °C compared with other treatments. Hence, 46.67, 8.20–23.58 and 1.02–6.15% reduced food consumptions were recorded in groups exposed at 10, 15 and 20 °C temperatures, respectively, compared with the 25 °C. The consumption rate was not affected in fish exposed at 30 and 35 °C. Total protease, trypsin and chymotrypsin activities were significantly (P < 0.05) higher in fish exposed at 25 °C compared with others. Lipase activity was significantly (P < 0.05) higher in fish exposed at 30 °C compared with others. Lowest enzyme activities were recorded at 10 °C. Water temperature below 25 °C affected the food consumption and digestive enzyme activities in fish that served as indicators of stress in fish.     

Thermal ecology of subadult and adult muskellunge in a thermally enriched reservoir

The movement of adult muskellunge, Esox masquinongy Mitchill, has been investigated in a variety of systems, but temperature selection by muskellunge has not been examined where well‐oxygenated waters were available over a range of temperatures for much of the year. Thirty subadult and adult muskellunge tagged internally with temperature‐sensing radio tags were tracked from March 2010 to March 2011 in a Tennessee reservoir. Mean tag temperatures were 18.9 °C in spring (March to May), 22.1 °C in summer (June to August), 16.5 °C in autumn and 9.8 °C in winter (December to February). When the greatest range in water temperatures was available (7.1–33.3 °C; May to early August 2010), their realised thermal niche (mean ± 1 SD) was 22.3 °C ± 1.8; the realised thermal niche was affected by fish size (smaller fish selected slightly warmer temperatures) but not sex. An electric generating steam plant discharging warm water resumed operation in January 2011, and most (86%) tagged fish occupied the plume where temperatures were ≈10 °C warmer than ambient water temperatures. No mortalities were observed 15 days later when plant operations ceased. Their affinity for the heated plume prompted concerns that muskellunge will be too easily exploited when the plant operates during winter.     

Biocontrol of saprolegniosis in rainbow trout (Oncorhynchus mykiss Walbaum) using two bacterial isolates (LE89 and LE141) of Pseudomonas fluorescens

The probiotic activity of 15 bacterial isolates that inhibit Saprolegnia parasitica in vitro was tested for the biocontrol of saprolegniosis in rainbow trout (Oncorhynchus mykiss Walbaum), adding the bacteria to tank water for 14 days at a concentration of 10⁶ bacteria ml^?1 water. Pseudomonas fluorescens LE89 and Pseudomonas fluorescens LE141 were effective in controlling experimental infection with S. parasitica since of the fish treated with LE89, 24.5% ± 16.27% (p < 0.05) became infected, as did 42.8% ± 8.41% (p < 0.05) of those treated with LE141. Given their protective effect when administered in water, their effect was also studied when administered in feed before and after experimental infection. Both bacterial isolates survived low pH levels and the action of bile, grew in skin and intestinal mucus, were resistant to several antibiotics and survived in feed; however, neither of the two isolates prevented S. parasitica infection when administered in feed.     

Evidence of exotoxin secretion of Piscirickettsia salmonis,the causative agent of piscirickettsiosis

Piscirickettsia salmonis is the aetiological agent of piscirickettsiosis, a disease which affects a variety of teleost species and that is particularly severe in salmonid fish. Bacterial‐free supernatants, obtained from cultures of three isolates of Piscirickettsia salmonis, were inoculated in Atlantic salmon, Salmo salar L., and in three continuous cell lines in an effort to determine the presence of secretion of extracellular products (ECPs) by this microorganism. Although steatosis was found in some liver samples, no mortalities or clinical signs occurred in the inoculated fish. Clear cytotoxicity was observed after inoculation in the cell lines CHSE‐214 and ASK, derived from salmonid tissues, but not in MDBK, which is of mammalian origin. The degree of cytotoxicity of the ECPs was different among the P. salmonis isolates tested. The isolate that evidenced the highest cytotoxicity in its ECPs exhibited only an intermediate virulence level after challenging fish with bacterial suspensions of the three P. salmonis isolates. Almost complete inhibition of the cytotoxic activity of ECPs was seen after proteinase K treatment, indicating their peptidic nature, and a total preclusion of the cytotoxicity was shown after their incubation at 50 °C for 30 min. Results show that P. salmonis can produce ECPs and at least some of them are thermolabile exotoxins that probably play a role in the pathogenesis of piscirickettsiosis.     

Development and characterization of a fibroblastic‐like cell line from caudal fin of the red‐line torpedo,Puntius denisonii (Day) (Teleostei: Cyprinidae)

A fibroblastic‐like cell line was established from the ornamental fish, red‐line torpedo (Puntius denisonii). The red‐line torpedo fin (RTF) cell line is being maintained in Leibovitz's L‐15 medium supplemented with 10% fetal bovine serum (FBS) for over 1 year at 28 °C on a continuous basis in normal atmosphere. The growth rate of RTF cells increased as the FBS proportion increased from 5% to 20% at 28 °C with optimum growth at the concentrations of 10% FBS. The morphology of RTF cell was predominantly fibroblastic like. Propagation of these cell lines was serum dependent, with a low plating efficiency (<15%). Karyotyping analysis of RTF cells at the 25th passage indicated that the modal chromosome number was 2n=50. The cell line was cryopreserved in liquid nitrogen at ?196 °C and could be recovered from storage after 6 months with good cell viability. Polymerase chain reaction amplification of a fragment of two mitochondrial genes, 16S rRNA and CO1, confirmed the identity of these cell lines with those reported from this animal species, confirming that the cell lines originated from P. denisonii. The bacterial extracellular products from Vibrio cholerae MTCC3904 and Aeromonas hydrophila were found to be toxic to RTF. The cell lines were not susceptible to viral nervous necrosis virus, a marine fish virus.     

Atypical bacterial gill disease: a new form of bacterial gill disease affecting intensively reared salmonids

An unusual form of bacterial gill disease (BGD) was identified which affected five species of cultured salmonids from Canada (i.e. rainbow trout, chinook salmon and Atlantic salmon), Norway (i.e. brown trout) and Chile (i.e. coho salmon). All outbreaks occurred at low water temperatures (< 10 °C) and with clinical presentations distinct from classical BGD, which is caused by Flavobacterium branchiophilum. In contrast to classical BGD, fish did not show marked respiratory distress with flaring of the opercula, the animals did not orientate at the surface of the water column near inflow water or at the margins of the tanks, and the feed response of the fish was varied. While mortality was increased, it was not precipitous as in classical BGD. Eight outbreaks were examined in greater detail using histopathology, scanning electron microscopy, bacteriology and immunohistochemistry. Large numbers of small bacterial rods were seen adhering to the lamellar epithelium of affected gills from all outbreaks. Histologically, the lamellar epithelium appeared swollen, often with evidence of single cell degeneration and exfoliation. In more severe instances, the formation of lamellar synechiae was seen, usually associated with sequestration of bacteria between fused lamellae. By contrast with typical BGD, overt epithelial hyperplasia, lamellar fusion and filamental clubbing were not common sequelae to infection; instead, the end result was shortened and somewhat stubby lamellae covered with swollen epithelial cells. The predominant bacterium recovered from affected gills was a small, Gram-negative, motile, fluorescent pigment-producing rod that shared phenotypic characteristics with Pseudomonas fluorescens. Polyclonal antisera prepared against three representative isolates indicated a weak antigenic similarity among them. Immunohistochemistry corroborated this finding, in that the antisera reacted strongly with gill sections containing the homologous bacteria, but not against morphologically similar bacteria in heterologous sections. A Gram-negative, yellow pigmented bacterium (YPB), identified as Flavobacterium psychrophilum, was also recovered, but only from the gills in the Ontario outbreaks. Antiserum prepared against this YPB indicated an antigenic similarity among isolates recovered from the Ontario outbreaks, but immunohistochemistry failed to recognize antigenically related bacteria on the gills of fish from the other outbreaks. Based on the unusual clinical presentation and the histopathological appearance of the gills, in conjunction with the absence of filamentous bacteria associated with and recovered from affected gills, the present authors have called this condition ‘atypical bacterial gill disease’ or ABGD.     

Vaccination of Atlantic lumpfish (Cyclopterus lumpus L.) at a low temperature leads to a low antibody response against Aeromonas salmonicida

We present a study on the effect of water temperature on immunization of Atlantic lumpfish. In total, 360 fish were vaccinated with either 50 μl of an oil‐based injection vaccine (VAX), with Aeromonas salmonicida and Vibrio salmonicida antigens, or PBS. Fish were vaccinated at three different water temperatures, 5°C, 10°C and 15°C, and sorted into six groups (N = 60). Lumpfish were weighed every 3 weeks after vaccination, sampled at 3, 6, 9 and 18 weeks post‐immunization (wpi) and evaluated by modified Speilberg score, ELISA and immunoblotting. Vaccinated fish showed low antibody response against V. salmonicida. Fish vaccinated at 5°C showed significantly lower antibody response against A. salmonicida throughout the study. At higher temperatures, vaccinated fish showed significantly increased antibody responses, at 18 wpi for 10°C and at 6 and 18 wpi for 15°C. Immunoblotting demonstrated specific response against the LPS antigen of A. salmonicida in the 10°C and 15°C VAX groups. Mean body weight increased in all groups throughout the study. Vaccinated fish had low Speilberg scores with no melanization of abdominal tissue. Our results show that vaccinating lumpfish at a lower water temperature may lead to a low antibody response against A. salmonicida.     

Influence of temperature on growth and survival of juvenile winter flounder,Pseudopleuronectes americanus (Walbaum) reared under continuous light

Winter flounder, Pseudopleuronectes americanus, has emerged as a promising candidate flatfish for cold‐water aquaculture and restocking. Here, juveniles were reared for 8 weeks at three temperatures: 10, 15, and 20°C under 24‐hr light. All fish were imaged at stocking and at 2‐week intervals, where growth was measured as changes in standard length (S_L) and body area (B_A). By week 2, fish reared at 15 and 20°C were larger than those grown at 10°C. At weeks 4 to 6, fish at 15°C were larger than fish at 20°C. Linear regressions were used to model growth dynamics over time at each temperature. Highly significant linear growth trajectories were detected over time for S_L and B_A. S_L and B_A regressions also showed a significant difference among the slopes across temperatures, where comparing slopes showed the best temperature to rear the flounder was 15°C. Weights of fish held at 15°C and 20°C were greater than at 10°C at the termination of the experiment. Within each temperature, the growth rate of malpigmented fish was not different from that of the normally pigmented fish. Overall, growth of winter flounder was comparable to that of other commercially produced flatfish species, providing strong evidence for this flatfish species as a potential species for aquaculture.     

Bacterial community of pond's water,sediment and in the guts of tilapia (Oreochromis niloticus) juveniles characterized by fluorescent in situ hybridization technique

Information about bacterial community structure and functioning in fish farming ponds remains scarce, mainly due to methodological difficulties in counting and identifying uncultured bacteria. The main objective of this study was to evaluate the degree of similarity between the bacterial community of the digestive tract of tilapia (Oreochromis niloticus) juveniles and that of the test pond's water and sediment, using the Fluorescent In Situ Hybridization (FISH) technique. Samples of water, sediment and gut content of 30 tilapia juveniles from a single nursery ground were collected in January 2010. Potentially probiotic and pathogenic bacteria of the species Bacillus, Lactobacillus brevis, Lactobacillus collinoides, Lactobacillus coryniformis, Lactobacillus farciminis, Vibrio and Pseudomonas fluorescens were found in different samples using specific fluorescent probes. The similarity between bacterial community environments and gastrointestinal tracts was determined using the Morisita‐Horn index. The fish guts presented higher abundances of Pseudomonas fluorescens, Bacillus, Lactobacillus brevis and Lactobacillus collinoides. The bacterial community composition of tilapia gastrointestinal tract was more similar to the water than the sediment of the pond. The results of this study showed that the FISH technique can be easily used for monitoring of probiotics and pathogen detection in aquaculture systems.     

Isolation,identification and pathogenicity of Vibrio harveyi,the causal agent of skin ulcer disease in juvenile hybrid groupers Epinephelus fuscoguttatus × Epinephelus lanceolatus

The hybrid grouper, Epinephelus fuscoguttatus (♀) × Epinephelus lanceolatus (♂), is a newly bred cultivated marine fish species of high economic value. However, a skin ulcer disease with high mortality has occurred, and the responsible pathogen remains unknown. In this study, we summarized the epidemic status and external signs of this disease. We screened potential pathogens and finally isolated one bacterial strain ML01 from affected fish. We subjected healthy juvenile hybrid groupers to bacterial challenge tests with the isolate by immersion, immersion after dermal abrasion and intraperitoneal injection, respectively. Within 14 days post‐infection, the isolate ML01 caused mass mortality of juveniles infected via immersion after dermal abrasion or intraperitoneal injection. Diseased juveniles displayed obvious signs of skin ulcers. The median lethal dose of ML01 by intraperitoneal injection was 1.10 × 10⁵ colony‐forming units. ML01 was identified as Vibrio harveyi by bacterial morphology, analytical profile index identification, 16S rDNA sequencing and multilocus sequence analysis. Antibiotic susceptibility tests showed that ML01 was sensitive to ceftriaxone, doxycycline and minocycline. The results of this study suggest that V. harveyi is the causal agent of skin ulcer disease in juvenile hybrid groupers, thus providing a basis for effective control and prevention of this disease.