Current knowledge of nocardiosis in teleost fish

Nocardia sp. is the causative agent of nocardiosis, a lethal granulomatous disease of the skin, muscle, and various inner tissues affecting various teleost and shellfish. Four species of Nocardia have been isolated from diseased fish and shellfish, namely Nocardia asteroides, Nocardia seriolae, Nocardia salmonicida and Nocardia crassostreae. Therefore, in fish aquaculture, nocardiosis has caused severe economic losses, especially in the Asian region. Considerable research has been performed, since the first report of identified Nocardia sp. in fish, to characterize Nocardia sp. and identify rapid detection techniques, immune response against infection and prophylactic approaches. In this review, the current state of knowledge about nocardiosis in fish has been presented, including the pathogenesis, diagnosis, host immune response and vaccine development.     

Mycobacteria in aquarium fish: results of a 3‐year survey indicate caution required in handling pet‐shop fish

Fish are commonly infected with non‐tuberculous mycobacteria (NTM), which should be regarded as potential pathogens when handling aquarium fish and equipment. This study examined 107 aquarium fish from pet shops. Cultivation of the fish samples using different selective media was conducted for identification of NTM. Isolates were identified using the GenoType Mycobacterium common mycobacteria and additional species assays, sequencing of the 16S rRNA and rpoB genes, and real‐time PCR assay for identification of Mycobacterium (M.) marinum. Among the investigated fish, 79.4% (85/107) were positive for mycobacteria, with 8.2% (7 of 85) having two mycobacterial species present. Among the positive fish, the common pathogens M. marinum, Mycobacterium fortuitum (M. fortuitum group) and Mycobacterium chelonae were identified in approx. 90% of fish and other NTM species in 10%, including Mycobacterium peregrinum/septicum, Mycobacterium gordonae, Mycobacterium arupense, Mycobacterium kansasii, Mycobacterium ulcerans and Mycobacterium setense. The well‐known human pathogen M. marinum was present in 10.6% of the positive fish (9 of 85). The species of mycobacteria identified in the study are not only recognized as aquarium fish pathogens, but can also cause pathology in humans. Microbiological and clinical communities should therefore be sensitized to the role of NTM in infections associated with exposure to aquarium fish.     

The efficacy of MS‐222 as anaesthetic agent in four freshwater aquarium fish species

The efficacy of anaesthetic tricaine methanesulfonate (MS‐222) was evaluated in four freshwater aquarium fish species, Zebrafish (Danio rerio), Guppy (Poecilia reticulata), Discu (Symphysodon discus) and Green swordtail (Xiphophorus helleri). The correct dose of anaesthetic should induce the plane 4 of anaesthesia in less than 180 s, recovery in less than 300 s and must survive when exposed during 30 min to anaesthetic. Fishes were exposed to six concentrations of anaesthetic (75, 100, 125, 150, 200 and 250 mg L^?1) and the time of fish reaching plane 4 of anaesthesia, post exposure recovery, and the percentage of survival when fish were subject to 30 min in the anaesthetic were recorded. The optimal doses varied according to the species: D. rerio – 75, 100 and 125 mg L^?1, P. reticulata – 125, 150 and 200 mg L^?1, S. discus – 75 and 100 mg L^?1 and X. helleri – 125 and 150 mg L^?1. The induction time generally decreased significantly with increasing concentration of MS‐222 for all of the species evaluated. The recovery time had a tendency to increase with the increase of the MS‐222 concentration for D. rerio, P. reticulata and S. discus. On the other hand, X. helleri recovery time decreased with the increase of MS‐222 concentration. MS‐222 proved to be effective in anaesthesia for all the freshwater ornamental species studied. The main results clearly show that the optimal dose to anesthetize is fish species dependent and it is completely wrong to extrapolate optimal anaesthetic concentrations between different species.     

Impact of Pseudomonas H6 surfactant on all external life cycle stages of the fish parasitic ciliate Ichthyophthirius multifiliis

A bacterial biosurfactant isolated from Pseudomonas (strain H6) has previously been shown to have a lethal effect on the oomycete Saprolegnia diclina infecting fish eggs. The present work demonstrates that the same biosurfactant has a strong in vitro antiparasitic effect on the fish pathogenic ciliate Ichthyophthirius multifiliis. Three life cycle stages (the infective theront stage, the tomont and the tomocyst containing tomites) were all susceptible to the surfactant. Theronts were the most sensitive showing 100% mortality in as low concentrations as 10 and 13 μg/ml within 30 min. Tomonts were the most resistant but were killed in concentrations of 100 μg/ml. Tomocysts, which generally are considered resistant to chemical and medical treatment, due to the surrounding protective cyst wall, were also sensitive. The surfactant, in concentrations of 10 and 13 μg/ml, penetrated the cyst wall and killed the enclosed tomites within 60 min. Rainbow trout fingerlings exposed to the biosurfactant showed no adverse immediate or late signs following several hours incubation in concentrations effective for killing the parasite. This bacterial surfactant may be further developed for application as an antiparasitic control agent in aquaculture.     

Comparison between the intestinal microbiome of healthy fish and fish experimentally infected with Streptococcus agalactiae

Nile tilapia (Oreochromis niloticus) farming is an economic activity that is soaring in the whole world. Septicemia due to Streptococcus agalactiae is the main disease impacting fish farming. The aim of this study was to compare the gut microbiome of healthy animals and animals experimentally infected with S. agalactiae strain 21171A. The microbiome was established with 16S ribosomal DNA next‐generation sequencing (NGS). One hundred Nile tilapias, with an average weight of 35 g, were distributed into two groups. Fifty fish from the challenged group were orally inoculated with 100 μl of a bacterial solution containing 1.98 × 10³ CFU/ml of S. agalactiae strain 21171A, while 50 controls were orally inoculated with sterile saline. After the experiment, 24 fish from the challenged group and 27 fish from the control group were analysed. For both groups, bacteria attached to the mucosa (M) and present in faeces (F) were analysed. The mean of the number of taxa identified in the infected group (M + F) (45.87 ± 30.13) was lower than in the control (M + F) (67.70 ± 21.10) (p < .01). Nineteen bacterial taxa were more abundant in faecal samples from the infected group when compared with the control group (p < .01). Thirty‐nine taxa were associated with mucosa samples from the challenged group when compared to the control samples (p < .01). No OTU was associated with healthy samples. The results demonstrate that the infection with S. agalactiae reduces the variability of the gut microbiota. Moreover, some bacteria proliferate during the infection.     

The role and mechanism of icmF in Aeromonas hydrophila survival in fish macrophages

Survival in host macrophages is an effective strategy for pathogenic bacteria to spread. Aeromonas hydrophila has been found to survive in fish macrophages, but the mechanisms remain unknown. In this paper, the roles and possible mechanisms of IcmF in bacterial survival in fish macrophages were investigated. First, a stable silencing strain icmF‐RNAi was constructed by shRNA and RT‐qPCR confirmed the expression of icmF was down‐regulated by 94.42%. The expression of Hcp, DotU and VgrG was also decreased in icmF‐RNAi. The intracellular survival rate of the wild‐type strain was 92.3%, while the survival rate of icmF‐RNAi was only 20.58%. The escape rate of the wild‐type strain was 20%, while that of the icmF‐RNAi was only 7.5%. Further studies indicated that the expression of icmF can significantly affect the adhesion, biofilm formation, motility and acid resistance of A. hydrophila, but has no significant effect on the growth of A. hydrophila even under the stress of H₂O₂. The results indicated that IcmF of A. hydrophila not only acts as a structural protein which participates in virulence‐related characteristics such as bacterial motility, adhesion and biofilm formation, but also acts as a key functional protein which participates in the interaction between bacteria and host macrophages.     

Habitat partitioning among three predatory fish in a temperate reservoir

The diversity of predatory species plays a key role in ecosystem functioning but our understanding of the mechanisms underlying their coexistence is limited, particularly in freshwater ecosystems. Northern pike Esox lucius, European perch Perca fluviatilis and pikeperch Sander lucioperca are three widespread predatory species in European lakes, where they often coexist. As potential competitors, we hypothesised that partitioning habitat is a determinant of species coexistence. This was tested by quantifying the variability of their habitat use in tracking adult individuals in the Bariousses reservoir (France, 86.6 ha, mean depth 7.1 m). Specifically, we investigated their distribution along the littoral–pelagic and depth axes along the daily cycle and across seasons. From littoral to pelagic waters were first found pike, then perch and finally pikeperch. Pike was the closest to the surface while pikeperch was the deepest. This general pattern was, however, variable across seasons with the three species located in the upper layer in summer during reservoir stratification. Individuals were more evenly distributed along the littoral–pelagic axis and closer to the bottom when water was mixing (autumn, winter). In summer, perch used more intensively in the pelagic zone during the daytime. Other species did not show any diel change of habitats. Our results highlighted that species coexistence is associated with habitat partitioning among these three predators, with perch showing a more variable behaviour regarding habitat characteristics. Now more than ever, in the context of global change which modifies habitats, it is of crucial importance to understand the coexistence mechanisms of species that shape ecosystems.     

Larval feeding behaviour of angel fish Pterophyllum scalare (Cichlidae) fed copepod Eucyclops serrulatus and cladoceran Ceriodaphnia quadrangula

Three different live diets, Eucyclops serrulatus, Ceriodaphnia quadrangula and equal combination of E. serrulatus copepodid and C. quadrangula, were offered to angelfish (Pterophyllum scalare) larvae viz 1‐week, 2‐week and 3‐week old at prey densities of 2, 5 and 10 individuals mL^?1. Results showed that 1‐week‐old P. scalare larvae consumed E. serrulatus copepodid at a rate of 31.3–56.7 ind. h^?1, C. quadrangula at 8.0–12.0 ind. h^?1, and mixture of E. serrulatus and C. quadrangula at 20.7–40.7 ind. h^?1. For 2‐week‐ and 3‐week‐old larvae, consumption rate increased accordingly. The electivity indices (E) of P. scalare (1‐week‐old larvae) for E. serrulatus copepodid were +0.18, +0.23 and +0.22 at prey densities of 2, 5 and 10 ind. mL^?1 respectively. Tendency towards E. serrulatus copepodid consumption reduced by aging P. scalare as indicated by the E values for 2‐ and 3‐week‐old larvae. However, growth and survival of P. scalare larvae was greatest when fed on combination of copepod E. serrulatus and C. quadrangula.     

Evidence of fish and human pathogens associated with doctor fish (Garra rufa,Heckel, 1843) used for cosmetic treatment

Doctor fish (Garra rufa, Heckel, 1843) are increasingly used for cosmetic treatment raising particular concerns regarding the potential transmission of infections to clients. Investigations of microbial causes undertaken in two outbreaks of mortality among G. rufa used for cosmetic treatment revealed the presence of multiple bacteria, including both fish and human pathogens such as Aeromonas veronii, A. hydrophila, Vibrio cholerae, Shewanella putrefaciens, Mycobacterium marinum and M. goodii. This range of bacteria indicates an intense microbial proliferation involving multiple pathogens, most likely induced by the poor health condition of the fish. Most of the detected pathogens are well‐known agents of zoonosis. Indeed, M. goodii is an emerging nosocomial human pathogen that has never been detected in fish to date, nor in other animals. This first detection of M. goodii associated with fish infection points out a new zoonotic potential for this pathogen. These findings point out that handling, poor environmental conditions and the presence of fish pathogens, that can compromise the immune system of fish, can result in a mixed microbial proliferation and increase the spread of waterborne bacteria, including zoonosis agents. Accordingly, the microbiological surveillance of fish used for cosmetic treatment is extremely important, particularly in association with mortality outbreaks.     

Antagonism against fish pathogens by cellular components and verification of probiotic properties in autochthonous bacteria isolated from the gut of an Indian major carp,Catla catla (Hamilton)

Probiotic potential of the autochthonous bacteria in catla, Catla catla has been evaluated through determination of antagonistic activity (in vitro) of the cellular components of gut bacteria against seven fish pathogens. Altogether 208 strains were isolated, inhibitory activity of the isolates was evaluated through cross‐streaking and 16 primarily selected antagonistic strains were confirmed using the double‐layer method. Four bacteria that showed antagonism against ≥4 pathogens were selected as putative probiotics. The intracellular, extracellular, whole‐cell and heat‐killed cell components exhibited bactericidal activity against the pathogens. In addition, the selected strains were capable of producing different extracellular enzymes, competent to grow in intestinal mucus and could tolerate diluted bile juice. Analysis of 16SrRNA partial gene sequence revealed that both the strains CC1FG2 and CC1FG4 were Bacillus methylotrophicus (KF559344 and KF559345), while the isolates CC1HG5 and CC2HG7 were Bacillus subtilis subsp. spizizenii (KF559346) and Enterobacter hormaechei (KF559347) respectively. Bio‐safety evaluation through intra‐peritoneal injection of the isolates did not induce any pathological signs or mortalities in C. catla. The study confirmed probiotic properties of autochthonous gut bacteria in C. catla and demonstrated potential for using them as bio‐control agents. However, in vivo studies are essential to explore their efficacy in the commercial aquaculture.     

Comparative assessment of Vibrio virulence in marine fish larvae

Vibrionaceae infections are a major obstacle for marine larviculture; however, little is known about virulence differences of Vibrio strains. The virulence of Vibrio strains, mostly isolated from vibriosis outbreaks in farmed fish, was tested in larval challenge trials with cod (Gadus morhua), turbot (Scophthalmus maximus) and halibut (Hippoglossus hippoglossus) using a multiwell dish assays with single‐egg/larvae cultures. The strains differed significantly in virulence as some caused a high mortality of larva reaching 100% mortality after a few days, while others had no or only marginal effects on survival. Some Vibrio strains were pathogenic in all of the larva species, while some caused disease only in one of the species. Twenty‐nine of the Vibrio anguillarum strains increased the mortality of larvae from at least one fish species; however, pathogenicity of the strains differed markedly. Other Vibrio species had no or less pronounced effects on larval mortalities. Iron uptake has been related to V. anguillarum virulence; however, the presence or absence of the plasmid pJM1 encoding anguibactin did not correlate with virulence. The genomes of V. anguillarum were compared (D. Castillo, P.W. D'Alvise, M. Middelboe & L. Gram, unpublished data) and most of the high‐virulent strains had acquired virulence genes from other pathogenic Vibrio.     

Effects of supplementary fennel (Foeniculum vulgare) essential oil in diet on growth and reproductive performance of the ornamental fish,Convict cichlid (Cichlasoma nigrofasciatum)

This study evaluated the effects of diet containing 0 (C: control), 75 (D₁), 100 (D₂), 125 (D₃) and 150 (D₄) mg kg^?1 F. vulgare essential oil on growth and reproductive performance of C. nigrofasciatum. A total of 225 Convict cichlids female with mean weight (1.65 ± 0.02 g) were distributed into 15 glass aquaria (15 fish tank^?1) in triplicate treatments. During 40 days of experimental period, fish fed at 3% of their body weight daily. At the end of experiment, growth indices, gonadosomatic index and reproductive indices were determined. The best feed conversion ratio was obtained in D₄ (1.19 ± 0.03, P < 0.05). The addition of F. vulgare essential oil did not have any statistical effects on other growth indices (specific growth rate, weight gain and condition factor). Total survival rates in all treatments and control were uniformly high, ranging from 93% to 97% (P > 0.05). F. vulgare essential oil has no negative effect on survival rate of C. nigrofasciatum. There were no significant differences among protein content of fish body (P > 0.05). The lowest value of fat content (26.5 ± 0.5%) was observed in D_2, and it had significant difference with control and other groups (P < 0.05). The ash percentage in treatments fed with F. vulgare essential oil were higher than control fish (P < 0.05). The highest moisture content was detected in D₄ (72.68 ± 0.19%) and D₂ (71.23 ± 2%) groups. Among the used dosage, D₄ was the most effective dosage that could significantly increase GSI (11.06 ± 1.55%), fecundity (340 ± 21) and hatching ratio (92.33 ± 1.63%). There were no significant differences in diameter of eggs among various treatments (P > 0.05).     

Augmenting fish health using Emblica officinalis against triarylmethane dye induced blood toxicity in Cyprinus carpio

Blood is a sensitive bioindicator of stress observed by organisms in response to toxicants. Erythrocytic morphological abnormalities serve as a reliable determinant of fish health. Malachite green, a multifunctional dye was assessed for toxicity in Cyprinus carpio. Dye‐induced blood toxicity has been determined through biochemical and ultra structural endpoints at 15, 30 and 60 days in the fish. Further, an attempt has been made to combat the adverse effects of the toxicant through supplementation with Emblica officinalis. Electron microscopic study depicted erythrocytic remodelling in form of surface blebbing, lobopodial projections and shrinkage; appearance of crenate, acanthocytes, echinocytes, dacrocytes, spherocytes and rhomboidal cells. Hepatic marker enzymes showed significantly (p < 0.05) elevated levels of biomarker enzymes, alkaline phosphatase, serum glutamate oxaloacetate transaminase and serum glutamate pyruvate transaminase. Contrarily feeding with E. officinalis extract (1,000 mg/kg feed) significantly attenuated biochemical and morphological alterations, indicating the protective efficacy of E. officinalis. Both toxicity and attenuation observed were in exposure‐dependent manner.     

Growth and economic analysis of freshwater prawn,Macrobrachium rosenbergii (de Man), produced with feeds substituting sunflower cake for fish meal,soya bean meal and mustard oil cake

A feeding trial with Macrobrachium rosenbergii was carried out to test the effectiveness of sunflower cake (SFC) as a replacement for conventional protein sources. Four isonitrogenous (~30% crude protein) and isocaloric (388–402 kcal) diets were formulated. Diet‐1 was a control made with conventional ingredients including fish meal, soya bean meal and mustard oil cake. Diet‐2, Diet‐3 and Diet‐4 were made with 30%, 40% and 50% SFC, respectively, replacing the usual protein sources used in Diet‐1. After 100 days, diets with SFC inclusion showed favourable growth and economic performance trends compared to control diet‐1, but without significant differences (p > .05). Better production (572.89 ± 62.99 kg/ha) was observed with Diet‐4 followed by Diet‐2, Diet‐3 and Diet‐1. Diet‐4 revealed the best feed conversion ratio (2.56 ± 0.24), specific growth rate (1.18 ± 0.05) and protein efficiency ratio (1.14 ± 0.10) among these diets. Whole carcass composition analysis revealed highest crude protein (18.19%) and lipid (4.43%) content in prawns fed Diet‐3 followed by Diet‐4. Highest total income, gross margin and benefit cost ratio (BCR) per hectare from prawns were US$5,759, US$2,623 and 1.84, respectively, found in Diet‐4. This study demonstrates that 30%–50% inclusion of SFC has favourable impacts on productivity and profitability compared with controls, with 50% SFC inclusion as the most cost‐effective diet for prawns without apparent compromise in growth, flesh quality or economics. The performances of the diets suggest that SFC may be a suitable substitute for fishmeal, soya bean meal and mustard oil cake in prawn feed. The effectiveness of higher SFC concentrations should also be investigated.     

Multilocus variable number tandem repeat analysis of Edwardsiella piscicida isolates pathogenic to fish

This study describes a novel multilocus variable number tandem repeat analysis (MLVA) based on six variable number of tandem repeat (VNTR) loci for genotyping of 37 Edwardsiella piscicida (previously Edwardsiella tarda) isolates from multiple sources. The number of alleles identified for each of the six VNTR loci ranged from 3 to 5 with VNTR loci 1 (DI = 0.632) and 3 (DI = 0.644), displaying the highest degrees of polymorphism. MLVA typing of the 37 E. piscicida isolates resulted in the identification of five major clusters consistent with their geographical origins, and were designated as MLVA types I, II, III, IV and V. Types III and V were resolved further into subtypes largely consistent with outbreak source. An MLVA profile comprising a string of integers representing the number of tandem repeats for each allele provided a unique identification for each MLVA type and/or strain. The MLVA protocol described in the current study is robust, relatively simple, has a higher power of resolution than multilocus sequence analysis (MLSA) and is capable of discriminating closely related isolates.     

Underwater evaluation of habitat partitioning in a European river between a non‐native invader,the racer goby and a threatened native fish,the European bullhead

We conducted underwater surveys using SCUBA gear to examine habitat and microhabitat competition between the Ponto‐Caspian racer goby Babka gymnotrachelus and native European bullhead Cottus gobio to assess the potential for competitive displacement of the native species by the invading species. In summer, 88 surveys were made in a tributary of the River Vistula within defined benthic areas across the entire width of the river bed. The occurrence of fish by total length class (small: <6 cm; large: >6 cm) and environmental conditions (depth, water velocity, substratum type, plant cover, shelter type) was recorded. We found a substantial separation between the species and size classes in relation to substratum, shelter type and water velocity. European bullheads were limited to lotic areas with stony bottoms, whereas racer gobies also occupied lentic areas over sand or mud. European bullheads usually took refuge under stones in contrast to a wider range of shelters used by racer goby, including tree roots and rubbish. In general, the breadth of habitat used by the racer goby was wider than that of the European bullhead, although habitat overlap between the species was not statistically significant except for the type of shelter occupied by small fish, selecting smaller stones. An inverse relationship was observed between small European bullheads and all racer gobies in areas where they co‐occurred, suggesting that invader may be having an adverse effect on the distribution and habitat use of small native bullheads, particularly in areas of moderate water velocities over small stones and gravel.     

Efficacy of single and multiple doses of fenbendazole against gill parasites (Dactylogyrus sp.) of Labeo rohita (Hamilton, 1822) and its physio‐metabolic effects on the fish

The study was conducted to investigate the efficacy and physio‐metabolic responses of single and multiple doses of fenbendazole (FBZ) in Labeo rohita against gill parasite (Dacylogyrus sp.). Two Isoenergetic (395 Kcal DE/100 g) and isonitrogenous (35% CP) purified diets viz. control (without FBZ) and treatment (0.2% FBZ) were prepared. The efficacy of FBZ was measured after administration of single dose (20 mg/kg b. wt.) on 0th day and multiple doses of FBZ, that is, 20 mg/kg b. wt. on 0th, 3rd and 7th day through diet. Although oral administration of FBZ at single dose could significantly reduce the parasite in fish in comparison with the control group, 100% efficacy of the drug was not achieved up to 15th day of treatment. On the other hand, multiple doses of FBZ could show almost 100% efficacy on 15th day in terms of elimination of parasites from gill. The drug induced oxidative stress in examined fish. There was no significant change in activities of hepatic and muscle aspartate aminotransferase and alanine aminotransferase of L. rohita up to 15th day after oral administration of FBZ at single dose. But both single and multiple doses of FBZ could significantly enhance the hepatic superoxide dismutase, catalase and glutathione‐S‐transferase activities in fish up to 7th day of drug administration and significantly decreased thereafter up to 15th and/or 30th day. It is concluded that oral administration of FBZ at multiple doses (20 mg/kg b. wt.) on 1st, 3rd and 7th day could be effective against Dactylogyrus sp. infection in L. rohita.