Response of the intestinal mucosal barrier of carp (Cyprinus carpio) to a bacterial challenge by Aeromonas hydrophila intubation after feeding with β‐1,3/1,6‐glucan

The effect of dietary β‐glucan on the bacterial community in the gut of common carp (Cyprinus carpio) was examined after oral application of Aeromonas hydrophila. Carp received either feed supplemented with 1% MacroGard^®, a β‐1,3/1,6‐glucan, or a β‐glucan‐free diet. Fourteen days after feeding, half of the carp from each group were intubated with 10⁹ colony‐forming units (CFU) of a pathogenic strain of A. hydrophila. Gut samples were taken 12 hr to 7 days after application and analysed using microbiological and molecular biological techniques (NGS, RT‐PCR‐DGGE). The reaction of the mucosa and the microbiota to an A. hydrophila intubation differed in carp fed with β‐glucan compared to carp from the control group. In β‐glucan fed carp, the total bacterial amount was lower but the number of bacterial species was higher. Bacterial composition was different for carp from both treatment groups. The number of mucin filled goblet cells was reduced in carp fed the β‐glucan diet. Mucus was obviously released from the goblet cells and was probably washed out of the gut together with high numbers of bacteria. This might be protective against pathogenic bacteria and, therefore, feeding with β‐glucan may provide protection against infections of the gut in carp.     

Effects of juniper berry oil on growth performance and blood parameters in common carp (Cyprinus carpio)

In this study the effects of commercial juniper berry oil (JBO) as a feed additive in diets for common carp (Cyprinus carpio) was investigated in terms of growth performance, feed utilization and blood parameters. The experimental diets were prepared to contain JBO at rate of 0 (JBO‐0), 5 (JBO‐5) and 10 (JBO‐10) ml/kg. Common carps (3.07 ± 0.15 g) were fed with test diets for a period of 60 days in 40‐L aquariums of triplicate groups. The feeding experiment revealed that JBO groups showed significantly better growth performance and feed utilization compared with the control group (JBO‐0) (p < 0.05). No significant effect was observed on hemoglobin amount, haematocrit ratio and consequential erythrocyte indices (p > 0.05). In the JBO groups, serum cholesterol, triglyceride, glutamic–pyruvic transaminase and alkaline phosphatase levels decreased significantly (p < 0.05). In contrast, the total protein level showed a significant increase, while glucose, albumin and lactate dehydrogenase levels did not present any significant variation (p > 0.05). Therefore, dietary addition of 5 ml/kg JBO promoted fish growth and positively influenced the blood parameters in common carps.     

Anaemia and plasma lipid profile in common carp (Cyprinus carpio) exposed to ambient copper sulphate and nano‐scale copper oxide

The objective of this study was to determine the effects of copper sulphate (CS) and copper oxide nanoparticles (NCuO) on haematological characteristics and plasma lipid profile in Cyprinus carpio. Fish were exposed to CS (0.25 mg L^?1 Cu) and NCuO [0.25 (LNP) and 25 mg L^?1 (HNP) Cu] for 14 days. CS and LNP treatments showed hypochromic anaemia after 3 and 7 days, and macrocytosis and haemolytic anaemia after 14 days. HNP treatment showed hypochromic anaemia and polycythemia after 3 days, and macrocytosis and hypochromic anaemia after 7 days and haemolytic hypochromic anaemia after 14 days. All copper‐exposed fish showed leukopenia at all times, except for HNP after 7 days and CS after 3 days. CS treatment at all times, and NCuO treatments after 14 days showed elevated plasma bilirubin. CS (3rd and 7th days) and HNP (3rd day) treatments had significantly higher plasma triglyceride compared to the control. Significant increases in plasma cholesterol and lipoprotein levels were observed after 3 and 14 days in CS, and after 14 days in LNP treatment; whereas HNP treatment had significantly higher high density lipoprotein (HDL) and very low‐density lipoprotein (VLDL) levels after 3 days, and cholesterol, HDL and low‐density lipoprotein (LDL) after 14 days. In conclusion, CS and NCuO induce anaemia probably due to iron metabolism disorder and RBC destruction in carp. CS and NCuO (at high concentration) alter plasma lipid profile, which may be due to stress‐induced lipolysis and hepatobiliary damages. At similar concentrations, copper nanoparticles are less toxic to carp than copper ions.     

First detection of spring viraemia of carp virus in common carp (Cyprinus carpio L.) affected by a septicaemic disease in Mexico

Spring viraemia of carp (SVC) is an infectious disease responsible for severe economic losses for various cyprinid species, particularly common carp (Cyprinus carpio carpio). The causative agent is the SVC virus (SVCV), a member of the Sprivivirus genus, Rhabdoviridae family, and a List 1 pathogen notifiable by the World Organization for Animal Health. This study describes the diagnosis of an SVCV pathogen isolated in October 2015 from wild common carp inhabiting a natural lagoon in central Mexico. While neither an epidemic nor fish mortalities were reported, the collected killed specimens exhibited clinical signs of disease (e.g., exopthalmia, moderate abdominal distension and haemorrhaging, as well as internal haemorrhages and adhesions). Histological results of injuries were consistent with the pathology caused by SVCV. This finding was supported by the isolation of a virus in EPC and BF‐2 cells and subsequent RT‐PCR confirmation of SVCV. The phylogenetic analyses of partial SVCV glycoprotein gene sequences classified the isolates into the Ia genogroup. These findings make this the first report of SVCV detection in Mexico, extending the southern geographical range of SVCV within North America. However, since this pathogen was detected in fish inhabiting a natural body of water without tributaries or effluents, it is difficult to estimate the risk of SVCV for other wild/feral cohabitating cyprinid species in the lagoon. The status of this virus is also unknown for other bodies of water within this region.     

Protective effects of basil (Ocimum basilicum) ethanolic extract supplementation diets against experimental Aeromonas hydrophila infection in common carp (Cyprinus carpio)

This study evaluated the effects of basil (Ocimum basilicum) leaves extract supplement on growth, blood parameters and resistance to Aeromonas hydrophila in Cyprinus carpio fingerlings. Basil leaves were mixed thoroughly with feed at 0, 100, 200, 400, 800 and 1600 mg kg^?1 of diet and were fed in triplicate group for 2 months. At the end of this period, growth performance and survival were determined. Then fish were challenged intraperitoneally with A. hydrophila and mortalities were recorded up to 10 days post challenge. Results indicated that WBC, RBC, Ht, Hb, total protein, albumin and globulin were higher (P < 0.05) in fish fed diets containing O. basilicum compared with the control. Highest specific growth rate values were observed in 400 mg kg^?1 group. Feed conversion ratio was significantly lower (P < 0.05) in fingerlings fed 400 and 800 mg O. basilicum kg^?1 dry diet. After 10 days post challenge, total protein, WBC, RBC and Hb were significantly higher in 400 mg kg^?1 compared with other groups. These results reveal that a dietary O. basilicum leaves extract of 400 mg kg^?1 fed for 60 days leads to increased growth performance and survival rate as well as improved feeding efficiency in common carp fingerlings rendering them more resistant against infection by A. hydrophila.     

Investigation on protective effect of recombinant protein (OmpTS) of Aeromonas hydrophila in Common carp (Cyprinus carpio)

The outer membrane protein of Aeromonas hydrophila is a potential candidate for vaccine development. In this study, after cloning and expression of ompTS, 270 common carp, weighing 44 ± 5.7 g divided into five groups, were injected intraperitoneally twice with 3‐week intervals. Groups included the following: PBS, PBS plus Freund's adjuvant, recombinant protein, recombinant protein plus Freund's adjuvant and 20 fish as negative control. Two weeks after the second injection, 30 fish of each group were challenged with a dose of 2 × LD₅₀ of Aeromonas hydrophila and RPS was measured. The antibody level was measured using ELISA test. The protection of recombinant protein in the immunized fish with and without adjuvant, respectively, was about 82.61% and 78.26% (the protection of recombinant protein electroeluted from an SDS–PAGE with and without adjuvant, respectively, was about 78.62% and 69.57%). The average of antibody level in recombinant protein with and without adjuvant was significantly higher than the PBS group (p < .05). The ability of recombinant ompTS to increase the antibody level and to protect the fish from challenge by A. hydrophila demonstrated that recombinant ompTS protein injection can be used to immunize common carp against A. hydrophila infection.     

The effects of dietary inulin on growth performances,survival and digestive enzyme activities of common carp (Cyprinus carpio) fry

This study was conducted to investigate the effects of dietary inulin on growth performance, diet utilization, survival rate, carcass composition and digestive enzymes activities (amylase, lipase and protease) of carp (Cyprinus carpio) fry (0.55 ± 0.02 g). After acclimation, fish were allocated into 9 tanks (40 fish per tank) and triplicate fish groups were fed, control diet (0 g) or diets containing 5 g and 10 g inulin kg^?1 for 7 weeks. No significant effect on growth performance and diet utilization of fish fed inulin compared with the control group was observed. However, supplementation of inulin significantly increased survival rate and carcass lipid content, while carcass protein content significantly decreased. Dietary inulin had no significant effects on digestive lipase, protease and amylase activities.     

Recovery of Bacillus mycoides,B. pseudomycoides and Aeromonas hydrophila from common carp (Cyprinus carpio) and rainbow trout (Oncorhynchus mykiss) with gill disease

During a 3‐month period from June to the end of August 2016, ~5% mortalities were observed in a farm with rainbow trout (Oncorhynchus mykiss Walbaum) and one farm of common carp (Cyprinus carpio L.) in Bulgaria. The disease was manifested by gill ulcers/rot, asphyxiation and bloody ascites. Aeromonas hydrophila was isolated from the internal organs of all the diseased fish. Bacillus mycoides or B. pseudomycoides were recovered from the gill lesions on diseased carp and rainbow trout, respectively, with identification achieved by conventional phenotyping and by sequencing of the 16S rRNA gene. In vivo experiments confirmed that all three organisms were pathogenic to rainbow trout.     

Effect of dietary soybean lecithin on growth parameters,digestive enzyme activity,antioxidative status and mucosal immune responses of common carp (Cyprinus carpio)

Common carp, Cyprinus carpio (13.54 ± 0.48 g), were fed soybean lecithin‐incorporated diets for 8 weeks. After the feeding trials, the different groups of fishes were assessed by survival, growth parameters, digestive enzymes, antioxidant enzyme activities, mucosal immune parameters, etc. The lecithin‐incorporated diets (10–30 g kg^–1) helped to increase the weight gain significantly (p < .05) compared with control groups; activity of digestive enzymes was also significantly (p < .05) higher compared with experimental groups. The antioxidant enzymes including catalase and serum superoxide dismutase were significantly (p < .05) higher in the C. carpio fed lecithin‐incorporated feed than the ones fed the control feed. The serum thiobarbituric acid‐reactive substances and serum glutathione peroxidase activity did not differ significantly (p > .05) between control and experimental groups. The positive correlation supported that antibacterial activity was significantly (p < .05) increased with increasing concentration of lecithin in the diets. Among the different mucosal immune parameters, lysozyme, protease and esterase activities were significantly (p < .05) increased with lecithin‐incorporated diets. The results of this study confirm that 20 and 30 g kg^–1 lecithin inclusion in diets helped to enhance the activities of digestive enzymes, antioxidant enzymes, antibacterial factors and mucosal immune parameters in C. carpio.     

Molecular cloning and expression of kidney‐type glutaminase from common carp (Cyprinus carpio) and its up‐regulation by glutamine in primary culture enterocyte

Glutaminase (GLS) is the key enzyme of glutamine (Gln) metabolism and utilization. In this study, a cDNA encoding GLS protein was identified from common carp Cyprinus carpio intestine. The open reading frame of GLS cDNA encodes a polypeptide of 595 amino acids, which shows a high similarity with its zebrafish Danio rerio counterpart. Bioinformatic analysis showed the protein belongs to kidney‐type GLS. The putative protein has glutaminase domain and ankyrin repeats domain, which are highly conserved among vertebrate orthologues. Real‐time quantitative PCR analysis revealed that the abundance of GLS mRNA was the highest in the white muscle, followed by the brain, eyeball and pituitary. Glutaminase was ubiquitously expressed in all intestinal segments of common carp. The activity of GLS did not distribute uniformly along the entire length of the intestine. In primary culture enterocyte, and the expression of GLS mRNA is up‐regulated quickly and effectively by Gln.     

Modulation of growth performances,survival, digestive enzyme activities and intestinal microbiota in common carp (Cyprinus carpio) larvae using short chain fructooligosaccharide

This study investigates the effects of inclusion of low levels of dietary short chain fructooligosacchairde (sc‐FOS) on physiological response and intestinal microbiota of carp (Cyprinus carpio) larvae. After acclimation, fish (550 ± 20 mg) were allocated into nine tanks (40 fish per tank) and triplicate groups were fed a control diet (0%) or diets containing 0.5% and 1% sc‐FOS for 7 weeks. At the end of the experiment, the growth performance parameters (final weight, weight gain, specific growth rate (SGR), food conversion ratio (FCR) and condition factor (CF), survival rate as well as digestive enzyme activities (amylase, lipase and protease), total viable counts of heterotrophic aerobic bacteria (TVC) and lactic acid bacteria (LAB) level in intestinal microbiota were measured. Our results revealed no significant (P > 0.05) effects of sc‐FOS on growth performance and TVC when compared with the control group. However, administration of low levels of dietary sc‐FOS significantly increased digestive enzyme activities (lipase and amylase) and LAB levels (P < 0.05). Also, survival rate was significantly elevated in sc‐FOS fed carp. These results revealed that administration of low levels of sc‐FOS can be considered as a beneficial dietary supplement for larval stage of common carp.     

Characterization of β‐catenin 1 during the gonad development in the common carp (Cyprinus carpio)

β‐catenin gene is a pivotal gene for gonad development and maintenance of ovarian function in mammals. However, little is known about its expression and function in gonad development of fish. In this study, a complete cDNA (3342 bp) sequence of β‐catenin 1 was cloned from the common carp, Cyprinus carpio, by RACE PCR, which encodes a 780‐amino‐acid protein. Quantitative real‐time PCR demonstrated that β‐catenin 1 mRNA expressions were high in the testis and ovary tissue and the expression increased as the testes developed and the early stage ovaries developed. Western blot results revealed a single immunoreactive band with an estimated molecular weight of 90 kDa in testes. Immunohistochemistry analysis revealed that the β‐catenin 1 protein was concentrated mainly in the cytoplasm of early development stage of oocyte cells and in the cytomembrane of developing and mature sperm cells. 17β‐Ethinylestradiol injecting intraperitoneally into the fish decreased the relative β‐catenin 1 mRNA expression level except 1 μg/g 72 hr and 5 μg/g 48 hr of treatments in the ovary by real‐time PCR. These results suggest, for the first time, that β‐catenin 1 is an essential protein in gonad development and might be involved in ovarian early development of C. carpio.     

The induction of Hsp70 synthesis by non‐lethal heat shock confers thermotolerance and resistance to lethal ammonia stress in the common carp,Cyprinus carpio (Linn)

Exposure to heat‐shock protein (Hsp) stimulating factors induces Hsp accumulation and confers tolerance to lethal ammonia stress on the common carp Cyprinus carpio. This study investigated whether a non‐lethal heat shock bestowed similar protective effects against ammonia and induced thermotolerance, both thought to be rendered by increased amounts of Hsps. The 30‐min lethal temperature (30 min LHT) and 1‐h lethal ammonia concentration (1 h LCT) for this species occurred at 41°C and 14.2 mg/L NH₃ respectively. Heating juvenile carp (5 cm) from 28°C to 32, 34 and 38°C, with a subsequent 8‐h recovery period augmented tolerance to lethal heat and ammonia perturbation by two to threefold as compared with animals held at 28°C. Protection occurred in conjunction with Hsp70 accumulation in gills, substantiating the role of this Hsp in enhancing the stress tolerance of common carp.     

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.     

A novel 79‐bp insertion/deletion polymorphism in 3′‐flanking region of IGF‐I gene is associated with growth‐related traits in common carp (Cyprinus carpio L.)

Insulin‐like growth factor‐I (IGF‐I) participates in the modulation of the cellular development, differentiation and proliferation in various vertebrate tissues. Therefore, variations in IGF‐I gene are good candidates as growth regulators. In this study, a novel 79‐bp insertion/deletion (I/D) polymorphism was identified in 3′‐flanking region of IGF‐I gene by polymerase chain reaction (PCR) and sequencing approaches in common carp Cyprinus carpio. To gain insight into the association of the 79‐bp I/D polymorphism with growth‐related traits, a total of 747 individuals from Heilongjiang carp Cyprinus cario haematopterus (YL) (n = 263), selective line of German mirror carp Cyprinus carpio L. mirror (JL) (n = 229), and cold‐resistance line of Hebao red carp Cyprinus carpio var. wuyuanensis (HL) (n = 255) were genotyped. The DD genotype of the polymorphism was significantly associated with higher body length (135), body weight (325, 385, and 445 days), net weight (445 days) and lower overwintering body loss rate in JL. In addition, in pooled population (YL+JL+HL), fish with DD genotype also showed significantly higher body weight (445 days) and net weight (445 days) than those with II genotype. Taken together, these results suggest that the novel polymorphism in IGF‐I gene could be considered as a potential genetic marker for selection of growth performance in common carp.     

Comparative effect of Freund's adjuvant and Aloe vera L. gel on the expression of TNF‐α and IL‐1β in vaccinated Cyprinus carpio L. with Aeromonas hydrophila C. bacterin

The comparative effects of Freund's and Aloe vera gel as adjuvants on the expression of IL‐1β and TNF‐α genes were studied in vaccinated common carp (Cyprinus carpio) with Aeromonas hydrophila bacterin. Fishes were intraperitoneally immunized with A. hydrophila bacterin in combination with Aloe vera gel or Freund's and also without any adjuvant. At day 28 after immunization, all groups were challenged by lethal dose of A. hydrophila (10⁷ cells/fish). Changes in the expression of IL‐1β and TNF‐α genes were evaluated in anterior kidney before challenge and 12, 24, 72 and 7 days postchallenge using quantitative real‐time PCR. Higher expression levels of both genes were observed in all vaccinated groups compared with non‐immunized group. Fishes which received Aloe vera gel showed higher expression of IL‐1β and TNF‐α in relation to animals which vaccinated with or without Freund's adjuvant. We concluded that Aloe vera gel in compared with Freund's adjuvant had a more stimulatory effect on the expression of immune‐related genes in vaccinated common carp and it can use as a novel adjuvant in aquaculture.     

Dietary microbial levan ameliorates stress and augments immunity in Cyprinus carpio fry (Linnaeus, 1758) exposed to sublethal toxicity of fipronil

A 45‐day feeding trial was conducted to study the stress ameliorating and immunomodulatory role of microbial levan in Cyprinus carpio fry exposed to sublethal dose (1/10th LC₅₀) of fipronil [(±)‐5‐amino‐1‐(2,6‐dichloro‐α,α,α‐trifluoro‐p‐tolyl)‐4‐trifluoromethylsulfinylpyrazole‐3‐carbonitrile]. Two hundred and twenty‐five fry were randomly distributed in five treatments in triplicates. Four purified diets were prepared with graded levels of microbial levan. Five different treatment groups were levan control L₀P₀ (basalfeed + 0% levan without exposure to pesticide); pesticide control L₀P₁ (basalfeed + 0% levan with exposure to pesticide); L_0.25P₁ (basalfeed + 0.25% levan with exposure to pesticide); L_0.50P₁ (basalfeed + 0.50% levan with exposure to pesticide) and L_0.75P₁ (basalfeed + 0.75% levan with exposure to pesticide). Lactate dehydrogenase (LDH), malate dehydrogenase (MDH) and fructose‐1,6‐diphosphatase (FDPase) activites were significantly (P < 0.05) increased, whereas alkaline phosphatase (ALP), adenosine triphosphatase (ATPase) and acetyl choline esterase (AchE) activities were significantly (P < 0.05) reduced in higher levan‐fed groups. RBC, haemoglobin and WBC counts were significantly (P < 0.05) increased in the levan‐fed groups. Similar trends were also observed for the total serum protein, globulin, NBT and lysozyme activities. Blood glucose and serum cortisol exhibited a third order polynomial relationship with increasing level of dietary levan. Overall result showed stress ameliorating, immunostimulating and protective role of microbial levan against fipronil‐induced stress in C. carpio fry at 0.75% level of dietary levan supplementation.     

The protective effect of humic‐rich substances on atypical Aeromonas salmonicida subsp. salmonicida infection in common carp (Cyprinus carpio L.)

When challenged with atypical Aeromonas salmonicida subsp. salmonicida, exposure of the common carp (Cyprinus carpio L.) to different humic‐rich compounds resulted in a significant reduction in infection rates. Specifically, in fish exposed to (i) humic‐rich water and sludge from a recirculating system, (ii) a synthetic humic acid, and (iii) a Leonardite‐derived humic‐rich extract, infection rates were reduced to 14.9%, 17.0% and 18.8%, respectively, as compared to a 46.8% infection rate in the control treatment. An additional set of experiments was performed to examine the effect of humic‐rich components on the growth of the bacterial pathogen. Liquid culture medium supplemented with either humic‐rich water from the recirculating system, the synthetic humic acid or the Leonardite humic‐rich extract resulted in a growth reduction of 41.1%, 45.2% and 61.6%, respectively, as compared to the growth of the Aeromonas strain in medium devoid of humic substances. Finally, in a third set of experiments it was found that while the innate immune system of the carps was not affected by their exposure to humic‐rich substances, their acquired immune system was affected. Fish, immunized against bovine serum albumin, displayed elevated antibody titres as compared to immunized carps which were not exposed to the various sources of humic substances.