Immunological response of the Indian major carps to Aeromonas hydrophila vaccine

Abstract. Fingerlings of the three major types of Indian carp, i.e. Catla catla (Hamilton), Labeo rohita (Hamilton) and Cirrhunas mrigala (Hamilton), were immunized using a haemolysin-negative mutant of Aeromonas hydrophila . Very high titres of antibodies were induced in C. catla , followed by C. mrigala and L. rohita . Immunized fish showed good protection against homologous challenge. Moderate protection against heterologous challenge was observed in C. mrigala and L. rohita .     

A comparative study on innate immunity parameters in the epidermal mucus of Indian major carps

The innate immune system, particularly the external body surface, plays a frontier role in protecting fish from relevant infections. The present study is aimed at understanding and comparing the mucosal immunity of Indian major carp, that is, Labeo rohita, Catla catla and Cirrhinus mrigala, by evaluating different immune parameters such as protein content, lysozyme, myeloperoxidase, proteases and alkaline phosphatase activity in the skin mucus. The protein content of mucus sample of these species was compared, and the highest protein content was found in C. catla among the three Indian major carp species. The levels of proteases (40 ± 0.211 units/ml) and myeloperoxidase OD450 nm (1.525 ± 0.108) were found to be highest in the skin mucus of C. catla. However, lysozyme levels were highest in the skin mucus of L. rohita (10.95 ± 0.330 μg/ml), and C. mrigala had the highest alkaline phosphatase activity (30.74111 ± 0.680 U/l). Besides the enzyme activities, the epidermal mucus samples of three Indian major carp species were also tested and compared for the antibacterial activity against seven bacterial strains. Skin mucus of C. catla showed highest antibacterial activity among the three Indian major carps against all the seven bacterial strain, except that Micrococcus lysodeikticus and Vibrio cholerae, however, showed highest activity against mucus of C. mrigala. Also, the epidermal mucus from all the three species successfully agglutinated three freshwater pathogens, viz. Aeromonas hydrophila, Pseudomonas fluorescens and Edwardsiella tarda, with agglutination titres being the highest for Labeo rohita for all the pathogens. The epidermal mucus samples from the Indian major carp species showed haemagglutination activity and successfully lysed human, chicken and goat RBCs showing highest activity in C. catla. These results provide information for a better understanding of the role of epidermal mucus and its components in the innate immune system of Indian major carps.     

Larval fish movement in the River Lohajang,Tangail, Bangladesh

Larval fish drift and distribution patterns were studied in the River Lohajang, a tributary of the River Jamuna, Bangladesh, during the monsoon seasons (June - October) of 1992, 1993 and 1994. Larval fish drift peaks with the water levels of the River Jamuna. In all three years, the Indian carp, Catla catla, Labeo rohita L. and Cirrhinus mrigala, were associated with the first pulses, but were absent from drift samples after September. Hilsa species and other non-identified fish species were found throughout the monsoon season. The highest larval fish densities were found in the upper strata of the river, near the shore. The drift densities of these fish became evenly distributed through time. Water turbidity probably influenced the vertical and horizontal distribution, and the absence of diel patterns. The most important species in the larval drift were Hilsa spp., followed by Labeo rohita L., Glossogobius giuris L. and Rasbora daniconius. Among the major carp, Labeo rohita was the dominant species, contributing 80-90%. The impact of flood control programmes on the reproduction of riverine and larval fish drift is discussed.     

Growth Rate, Feed Conversion, and Body Composition of Catla catla, Labeo rohita, and Cirrhinus mrigala Fry Fed Diets of Various Carbohydrate-to-Lipid Ratios

Effects of dietary carbohydrate-to-lipid (CHO : L) ratios on the growth rate, conversion efficiencies, and body composition were studied in the Indian major carp fry Catla catla, Labeo rohita , and Cirrhinus mrigala. Six isonitrogenous (40% crude protein) and isocaloric (3.46 kcal metabolizable energy/g) semi-purified diets, with CHO: L ratios of 0.02, 0.60, 1.54, 3.38, 8.93, and 43.00 were fed to triplicate groups of fish in 70-L flow-through (1.5–2.0 L/min) indoor circular troughs. Fish were fed to apparent satiation (about 10% body weight), 6 d/wk, twice daily at 0800 and 1600 h for 6 wk. Fish growth rates differed significantly (P < 0.05) with CHO: L ratio in the diets. Maximum weight gain (%) and specific growth rate (SGR %) were observed in C. catla and L. rohita fed a diet with 36% carbohydrate and 4% lipid, corresponding to a CHO: L ratio of 8.93. In C. mrigala , highest weight gain (%) and SGR (%) were noted in fish fed 27% carbohydrate and 8% lipid, corresponding to a CHO: L ratio of 3.38. In all the species, fish fed either diet with the lowest (0.02) or the highest (43.00) CHO: L ratio tended to have significantly lower (P < 0.05) growth and conversion efficiencies. Percentage of dry matter and whole-body lipid of fish significantly (P < 0.05) increased as CHO: L ratio decreased. However, whole-body crude protein content of fish significantly (P < 0.05) increased as CHO : L ratio increased. The results of this study indicate that the Indian major carp fry efficiently utilize carbohydrate for energy, and excess dietary lipid resulted in increased lipid accumulation in the body.     

The immune response of turbot, Scophthalmus maximus (L.), skin to high water temperature

The immune responses of mucus from the skin of turbot, Scophthalmus maximus, were studied in relation to changing water temperature. Groups of fish were exposed to a programmed increase of 3 °C per 48 h, until the experimental water temperatures of 16, 20, 23, 25, 27 or 28 °C were reached. After 48 h at the relevant temperature, the expressions of immune‐related factors were determined, including immunoglobulin M (IgM), IL‐1β, hepcidin, transferrin, lysozyme, acid/alkaline phosphatase and superoxide dismutase using RT–PCR and spectrophotometric methods. Significant changes in mucus immunity were observed, which paralleled with those previously reported for serum in other fish species. Hence, it is suggested that the serum and mucus immune system of turbot have a similar regulatory system. This information could be useful in better understanding the role of the mucus as a component of the innate immune system.     

Identification and characterization of proteases from skin mucus of tambacu,a Neotropical hybrid fish

Skin secretions of fishes constitute a rich source of proteins with a broad spectrum of antimicrobial properties. We report here the characterization of proteases from skin mucus of tambacu, an economically important Neotropical hybrid fish. The effects of pH on the proteolytic activities of the mucus acting on various substracts – hemoglobin, casein, bovine serum albumin (BSA) and ovalbumin (OVA) – were tested. Optimal pH values for protease activity on hemoglobin were 4.5 and 8.5, on casein, 8.5, on BSA, 5.0 and 7.5, and on ovalbumin, 4.5 and 6.5. The proteolytic activity was inhibited on all of these substrates in the presence of specific inhibitors: caseinolytic activity was inhibited by inhibitors of serine and metalloproteases; hemoglobinolytic activity was inhibited by serine, aspartic and metalloproteases inhibitors; albuminolytic activity on BSA was inhibited by serine and aspartic proteases inhibitors, and on ovalbumin, by cysteine and aspartic proteases inhibitors. Gelatin zymography revealed that the skin mucus of tambacu consisted primarily of serine and metalloproteases. Hemoglobin zymography showed one proteolytic band inhibited by EDTA, whereas casein zymography showed two proteases inhibited by serine proteases inhibitors. We were able to identify all classes of proteases in the mucus from the skin of tambacu. These, and these results suggest that the proteolytic activities of the skin mucus of fish may play an important role in the defense against microorganisms and ectoparasites.     

Purification and characterization of a 630 kDa bacterial killing metalloprotease (KilC) isolated from plaice Pleuronectes platessa (L.), epidermal mucus

Antibacterial chemicals in the mucus of fish such as lysozyme, lectins, peptides and proteases provide an efficient first line of defence against pathogens. This study shows that there are at least three antibacterial proteins in plaice skin mucus in addition to lysozyme. One of these proteins is responsible for approximately 74% of the antibacterial activity and is a 630 kDa protease complex designated KilC (bacterial killing metalloprotease C). Purified KilC kills the bacteria Staphylococcus aureus, Escherichia coli, Bacillus subtilis and Pseudomonas aeruginosa efficiently. The protease activity of KilC is dependent upon the divalent cation Mg(2+) and shows pH dual optima of 5.0 and 8.0. The enzyme has a temperature optimum of 25 degrees C and is made up of at least five different sized peptides. Studies with protease inhibitors show that the catalytic site of KilC may be cysteine- or serine protease-like. KilC may kill bacterial cells by acting directly upon the bacteria or by producing low molecular weight bioactive compounds such as peptides.     

Integrated Duck-cum-Fish Farming in Bangladesh

An experiment on integrated duck-cum-fish farming was conducted in 11 ponds of 0.1 ha each at the Fisheries Research Institute, Mymensingh, Bangladesh. Khaki Campbell layer ducks Anas platyrhychos were housed directly over the pond water surface at 200, 400 and 500 dncks/ha. Each density had three replicate ponds and two ponds had no ducks. Fish fingerlings were stocked at a total density of 8,850/ha. The species composition was silver carp Hypophthalmicthys molitrix 33.9%; catla Catla catla 12.4%; rohu Labeo rohita 18.1%; mrigal Cirrhina mrigala 28.8%. grass carp Ctenopharyngodon idella 1.4% and sor puti Puntius gonlonotus 5.4%. The fish were not given any supplemental feed, and the ponds were not fertilized except for the split duck feed and duck manure falling directly into the ponds. Ducks were fed with a feed formulated from locally available ingredients.
After one year the fish were harvested. The yield of fish increased with an increase in duck density. Highest net fish production of 4,250 kg/ha/yr was obtained from ponds with 500 ducks/ ha, compared to 490 kg/ha/yr from the control ponds. Manure of each duck contributed a net fish yield of 6.9–7.5/yr. Average egg production was 237 eggs per female duck per year. Economic analysis of the technology showed a net profit of about 100% of the total costs, indicating the economic viability of the technology.     

Biochemical and Serological Characterization of Flavobacterium columnare from Freshwater Fishes of Eastern India

Flavobacterium columnare is an important pathogen of freshwater fishes often causing high mortality. Seven strains of F. columnare have been isolated from gill necrosis, skin lesions and internal organs of Catla catla, Labeo rohita, Cirrhinus mrigala, Carassius auratus, Anabas testudineus and Clarias batrachus and characterized by biochemical reactions and serological tests viz indirect enzyme‐linked immunosorbent assay (ELISA), Dot‐ELISA, agglutination test. All the strains showed binding to Congo red dye as well as hemolysis. All the seven strains were susceptible to amikacin, gentamycin and ofloxacin. In vivo LD₅₀ dose of virulent strain MS2 was found to be 6 × 10⁴ CFU/mL after experimental infection to L. rohita. Serologically all the seven strains showed a positive result to agglutination, Dot‐ELISA and indirect ELISA. The agglutination titer was found to be in the range of 256‐131,072. The lysozyme activity of hyperimmune sera was found to be 39.37 ± 0.80 units/mL. This is the first extensive study that report about the various strains of F. columnare from freshwater fishes of Eastern India and their differentiation by biochemical and serological methods.     

Evaluation of antibacterial activity and innate immune components in skin mucus of Indian major carp,Cirrhinus mrigala

The present work has been undertaken to analyse the antibacterial activity and innate immune components in the skin mucus of Indian major carp, Cirrhinus mrigala. Skin mucus was extracted separately in triple‐distilled water (TDW), 3% acetic acid (3% AA) or 0.1% trifluoroacetic acid (1% TFA). All mucus extracts exhibited different spectrum of the antibacterial activity against different groups of pathogenic bacteria. Protein profiling by polyacrylamide gel electrophoresis revealed a series of protein bands in the TDW extract, four major protein bands in the AA extract and two protein bands in the TFA extract. Tandem mass spectrometry analysis of distinct protein bands identified potential innate immune factors – histone H2A, histone H3, histone H4, haemoglobin, cofilin and nucleoside diphosphate kinase in the TDW extract, and ubiquitin and histone H2B isoforms in acidic extracts of skin mucus of C. mrigala. The presence of these innate immune molecules suggests that skin mucus play an important role in the protection of the fish against microbial invasion.     

Lysozyme: an important defence molecule of fish innate immune system

The innate immune system of fish is considered to be the first line of defence against a broad spectrum of pathogens and is more important for fish as compared with mammals. Lysozyme level or activity is an important index of innate immunity of fish and is ubiquitous in its distribution among living organisms. It is well documented that fish lysozyme possess lytic activity against both Gram‐positive bacteria and Gram‐negative bacteria. It is also known to be opsonic in nature and activates the complement system and phagocytes. It is present in mucus, lymphoid tissue, plasma and other body fluids of freshwater and marine fish. It is also expressed in a wide variety of tissues. Lysozyme activity has been shown to vary depending on the sex, age and size, season, water temperature, pH, toxicants, infections and degree of stressors. Here, we review our current understanding of different types of lysozyme and their expression and its role in fish innate immune system.     

The effects of dietary vitamin C on mucosal immune responses and growth performance in Caspian roach (Rutilus rutilus caspicus) fry

This study was conducted to investigate the effects of different levels of dietary vitamin C on some skin mucus immune parameters, mucus antimicrobial activity and growth performance of Caspian roach (Rutilus rutilus caspicus) fry. Six hundred sixty Caspian roach (1.4 ± 0.02 g) fry were allocated to 12 tanks (55 fish per tank), and triplicate groups were fed diets containing 0, 1,000, 1,500 and 2,000 mg kg^?1 vitamin C for 60 days. At the end of the trial, the epidermal mucus protein level, alkaline phosphatase and antimicrobial activity against two gram-positive bacteria (Streptococcus faecium and Micrococcus luteus) and gram-negative bacteria (Escherichia coli and Serratia marcescens) as well as growth performance were measured. The results demonstrated that feeding on vitamin C significantly elevated skin mucus alkaline phosphatase and protein levels compared to the control group (P < 0.05). However, lysozyme activity was undetectable in both the vitamin C-fed roach fry and the control group. Skin mucus antimicrobial activity was increased following vitamin C administration, and the bacterial growth inhibition zones were significantly elevated in vitamin C-fed roach (P < 0.05). Similar results were obtained in case of the minimum inhibitory concentration of skin mucus. Also fish fed the control diet had a significantly lower weight gain, specific growth rate and condition factor compared to the other treatments (P < 0.05). These results revealed that dietary vitamin C beneficially affects the skin mucus immune parameters and growth performance of Caspian roach fry.     

Development and characterization of cell lines derived from rohu, Labeo rohita (Hamilton), and catla, Catla catla (Hamilton)

Two new cell lines, designated RE and CB, were derived from the eye of rohu, Labeo rohita , and the brain of catla, Catla catla , respectively. The cell lines were maintained in Leibovitz's L-15 supplemented with 20% foetal bovine serum. The RE cell line was sub-cultured for more than 70 passages and the CB cell line for more than 35 passages. The RE cells are rounded and consist predominantly of epithelial cells. The CB cell line consists of predominantly fibroblastic-like cells. Both cell lines are able to grow at temperatures between 25 and 32 °C with an optimum of 28 °C. The growth rate of the cells increased as the foetal bovine serum concentration increased from 2% to 20% at 28 °C, with optimum growth at concentrations of 15% or 20% foetal bovine serum. The cells were successfully cryopreserved and revived at different passage levels. The cell lines were not susceptible to four marine fish viruses. Extracellular products from Aeromonas sp . were toxic to the cell lines. When the cells were transfected with plasmid eukaryotic green fluorescent protein (pEGFP [Clontech, Carlsbad, CA, USA]) vector DNA, a significant fluorescent signal was observed suggesting that these cell lines could be a useful tool for transgenic and genetic manipulation studies. Polymerase chain reaction amplification of mitochondrial 12S rRNA from rohu and catla confirmed that the cell lines originated from these fish species. The cell lines were further characterized by immunocytochemistry using confocal laser scanning microscopy.     

大弹涂鱼皮肤黏液的抑菌活性和蛋白质组学

皮肤黏液是鱼类免疫防御的第一道防线。大弹涂鱼皮肤黏液对其免疫防御、渗透压维持以及适应水陆生活等方面具有重要意义。为深入了解大弹涂鱼皮肤黏液的蛋白质分子组成,对其皮肤黏液开展了抑菌活性分析和蛋白质组学分析。通过电刺激法收集大弹涂鱼皮肤黏液,采用打孔法比较了皮肤黏液和血清的抑菌活性差异;进一步利用鳗弧菌对大弹涂鱼进行诱导,采用生长曲线抑制法分析和比较了诱导前后皮肤黏液的抑菌活性差异。利用Shotgun质谱技术,结合大弹涂鱼皮肤转录组数据库,对大弹涂鱼皮肤黏液开展了蛋白质组学分析;进一步利用String软件对所鉴定的蛋白质开展了蛋白质相互作用预测。大弹涂鱼皮肤黏液具有广谱抑菌活性,鳗弧菌诱导后的大弹涂鱼皮肤黏液与诱导前相比,对部分革兰氏阴性菌的抑菌活性明显上升,但对其他菌株的抑菌活性差异不明显。从大弹涂鱼皮肤黏液中共鉴定各类蛋白质分子97种,基本分子组成与其他硬骨鱼类皮肤黏液蛋白相似,但也有少数蛋白如泛素蛋白、胸腺素蛋白等未在其他鱼类皮肤黏液中报道。此外,其他鱼类中所鉴定到的部分蛋白如热休克蛋白、抗菌肽等在大弹涂鱼皮肤黏液中未能鉴定到。大弹涂鱼皮肤黏液中已鉴定的蛋白多数具有与免疫功能相关的结构域,且存在一个以肌动蛋白为核心的相互作用网络。大弹涂鱼皮肤黏液具有明显的广谱抑菌活性,其蛋白质组成与其他鱼类皮肤黏液具有一定的相似性。本研究为深入了解大弹涂鱼皮肤黏液的分子多样性及功能机制奠定了基础。     

Food selection, electivity and dietary overlap among planktivorous Chinese and Indian major carp fry and fingerlings grown in extensively managed, rain-fed ponds in Bangladesh

Abstract. The fry of four species of planktivorous carp, silver carp, Hypophthalmichthys molitrix (Valenciennes), bighead carp, Aristichthys nobilis (Richardson), rohu, Labeo rohita (Hamilton), and catla, Catla catla (Hamilton), were grown in monoculture in ponds in Mymensingh, Bangladesh in order to determine electivity and the extent of dietary overlap. Other than an avoidance for Cyanobacteria, electivity was found to be low in most instances, although the low productivity of the ponds may have had some influence. Dietary overlap was greatest between catla and silver carp (0–52) and between catla and bighead carp (0–48).     

Characterization of carboxylesterase in skin mucus of Cirrhinus mrigala and its assessment as biomarker of organophosphate exposure

Presence of carboxylesterase (CbE) activity in the skin mucus of Cirrhinus mrigala was investigated. CbE activity in skin mucus showed higher substrate preference for α-naphthyl acetate over p-nitrophenyl acetate. Four CbE isozymes—CbE-1, CbE-2, CbE-3, and CbE-4 were observed in skin mucus during zymography. The isozyme CbE-4 was characterized as typical serine esterase, whereas CbE-1, CbE-2, and CbE-3 were identified as sulphhydryl group-dependent serine esterases. In vitro treatment of skin mucus with the organophosphorus insecticide, Nuvan^® showed strong inhibition of CbE activity. In vivo exposure of the fish to sublethal test concentrations (5 and 15 mg/l) of the insecticide also revealed significant inhibition of CbE activity in mucus. After the cessation of exposure, CbE activity recovered to its control level during the recovery periods. Thus, CbE activity in skin mucus could be considered a biomarker of the organophosphorus insecticide exposure to fish and a useful tool in monitoring environmental toxicity.     

3种野鲮亚科鱼类16SrRNA基因序列分析

野鲮亚科(Labeoninae)隶属鲤形目(Cypriniformes)中的鲤科(Cyprinidae,约有26个属近300个种。为研究其在鲤科鱼类中的系统发育关系,以16S rRNA基因作为遗传标记进行鲤科鱼类的系统发育分析。通过PCR方法, 扩增了长度为361-362 bp的3种野鲮亚科鱼类的16S rRNA基因部分序列,序列比对得到364个排列位点,其中94个为信息位点,占全部位点的26.8%。用Mega 2.1软件的NJ法构建分子系统树,结果表明:野鲮亚科鱼类没有形成单系类群,野鲮亚科的鲮、角鱼与鲤亚科的鲤、鲫形成姐妹群,然后再与野鲮亚科的麦鲮和露斯塔野鲮聚在一起,说明鲮与鲤亚科鱼类具有较近的亲缘关系。     

Immune response, growth and survival of Labeo rohita fingerlings fed with levamisole supplemented diets for longer duration

The study was to determine the effect of long-term administration of different dosages of levamisole on growth, immune response and disease resistance against Aeromonas hydrophila & Edwardsiella tarda in Labeo rohita fingerlings. Fish were fed with four different dosages of levamisole (0, 125, 250 and 500 mg kg⁻¹ diet) for 56 days. Different serum biochemical and haematological parameters such as serum total protein content, albumin content, globulin content, albumin/globulin ratio, glucose content, leucocytes count; cellular immune parameters including superoxide anion production, phagocytic activities, lymphokine production index; humoural immune parameters including lysozyme, complement and serum bactericidal activities were evaluated after 14 days interval. After 56 days, fish were divided into two subgroups under each treatment group for challenge with pathogens A. hydrophila and E. tarda . The cumulative mortality (%) and agglutinating antibody titre was recorded on 28th day postchallenge. WBC count, phagocytic ratio, lymphokine production index, lysozyme activity and serum bactericidal activity were increased upon administration of levamisole dosages for long term. However, the growth performance and survival against pathogens was not significantly changed over 56 days administration of levamisole. But incorporation of moderate dosage of levamisole for 42 days results better immune response without effect on growth and survival of L. rohita fingerlings.     

Characterization of Gelatinolytic Activity in Seminal Plasma of Some Teleost Fish

Gelatin-containing SDS-PAGE combined with the incubation of gels in buffers containing protease inhibitors was performed for the visualization and characterization of proteolytic activity in teleost fish seminal plasma. To demonstrate the class of detected enzymes we used serine protease inhibitor – benzamidine or EDTA which inhibits metalloproteases activity. Additionally the effects of calcium ions on protease activity were investigated. Multiple gelatinolytic activities in seminal plasma of 10 teleost fish species from three orders (Cypriniformes, Salmoniformes, Perciformes) were found. Most proteases were either stimulated by Ca²⁺ and inhibited by EDTA or inhibited by benzamidine. This suggests that metalloproteases and serine proteases are major gelatinolytic proteases of fish seminal plasma. In cyprinid species we found a common profile of two gelatinolytic activities; the first band (60–66 kDa) belonged to metalloproteases, and the second one (76–81 kDa) belonged to serine proteases. Other bands were also visible and they represented mostly serine protease activity. Species from the Salmoniformes order showed a similarity in metalloproteases with molecular weights of about 64 and 75 kDa. Salmonid species also had similar serine proteases with molecular weights of about 102 and 165 kDa. In European grayling seminal plasma we found metalloproteases with molecular weight of 51, 57, 64, 70 kDa and two serine proteases activities of 35 and 125 kDa. Percid species had metalloproteases activities of 53 and 63 kDa and serine protease activity of 100 kDa. Protease of other, presently unknown classes were also found in seminal plasma of asp, chub, European grayling and pikeperch. The physiological role of seminal plasma proteases is still unknown.     

In Vitro and Dietary Effects of Chitin on Cellular and Humoral Immune Parameters of Catla,Catla catla (Hamilton)

Immunostimulants increase the immunocompetency and disease resistance of fish by enhancing immune responses. This study determined the in vitro and dietary effects of chitin on immune responses of catla, Catla catla. Catla head‐kidney leukocytes were cultured in complete RPMI‐1640 medium containing 0.01, 0.1, and 1.0 mg/mL of chitin. Leucocyte viability and different cellular immune responses, namely, respiratory burst, nitric oxide production, and leucocyte peroxidase content were analyzed in vitro. In the dietary experiment, chitin‐supplemented diets (0.5, 1.0, and 2.0% of feed) were fed to catla for 2 wk, and respiratory burst, serum lysozyme, and total protein contents were measured after 1 and 2 wk of feeding. Leucocytes incubated in vitro with chitin for 72 h maintained their viability. Chitin significantly (P < 0.05) enhanced all the immune responses in vitro. The lowest dose showed the best response. Dietary chitin also significantly stimulated all the immune parameters in a dose‐dependent manner with the highest dose showing the maximum responses. All the responses were higher after 1 wk of feeding and declined thereafter. The results suggest that chitin stimulates catla cellular and humoral immune responses in vitro and in vivo.