Outbreak of Ichthyophthirius multifiliis associated with Aeromonas hydrophila in Pangasianodon hypophthalmus: The role of turmeric oil in enhancing immunity and inducing resistance against co-infection.

Ichthyophthirius multifiliis, a ciliated parasite causing ichthyophthiriasis (white spot disease) in freshwater fishes, results in significant economic loss to the aquaculture sector. One of the important predisposing factors for ichthyophthiriasis is low water temperature (i.e., below 20°C), which affects the health and makes freshwater fishes more susceptible to parasitic infections. During ichthyophthiriasis, fishes are stressed and acute immune reactions are compromised, which enables the aquatic bacterial pathogens to simultaneously infect the host and increase the severity of disease. In the present work, we aimed to understand the parasite-bacteria co-infection mechanism in fish. Later, Curcuma longa (turmeric) essential oil was used as a promising management strategy to improve immunity and control co-infections in fish. A natural outbreak of I. multifiliis was reported (validated by 16S rRNA PCR and sequencing method) in Pangasianodon hypophthalmus from a culture facility of ICAR-CIFRI, India. The fish showed clinical signs including hemorrhage, ulcer, discoloration, and redness in the body surface. Further microbiological analysis revealed that Aeromonas hydrophila was associated (validated by 16S rRNA PCR and sequencing method) with the infection and mortality of P. hypophthalmus, confirmed by hemolysin and survival assay. This created a scenario of co-infections, where both infectious agents are active together, causing ichthyophthiriasis and motile Aeromonas septicemia (MAS) in P. hypophthalmus. Interestingly, turmeric oil supplementation induced protective immunity in P. hypophthalmus against the co-infection condition. The study showed that P. hypophthalmus fingerlings supplemented with turmeric oil, at an optimum concentration (10 ppm), exhibited significantly increased survival against co-infection. The optimum concentration induced anti-stress and antioxidative response in fingerlings, marked by a significant decrease in cortisol and elevated levels of superoxide dismutase (SOD) and catalase (CAT) in treated animals as compared with the controls. Furthermore, the study indicated that supplementation of turmeric oil increases both non-specific and specific immune response, and significantly higher values of immune genes (interleukin-1ß, transferrin, and C3), HSP70, HSP90, and IgM were observed in P. hypophthalmus treatment groups. Our findings suggest that C. longa (turmeric) oil modulates stress, antioxidant, and immunological responses, probably contributing to enhanced protection in P. hypophthalmus. Hence, the application of turmeric oil treatment in aquaculture might become a management strategy to control co-infections in fishes. However, this hypothesis needs further validation.

Genetic diversity and multiple antibiotic resistance index study of bacterial pathogen, Klebsiella pneumoniae strains isolated from diseased Indian major carps.

Intensive fish farming systems have led to increase in disease incidence, due to higher stocking density, high organic matter levels, and poor quality of the aquatic environment. Diseased fish samples showing hemorrhages and reddish lesions were collected from different freshwater fish farms located at three different districts of West Bengal, India (Burdwan, North 24 Parganas, and Nadia). The present study was conducted to evaluate the genetic diversity of ten different Klebsiella pneumoniae strains isolated from different infected freshwater fish samples based on 16S rRNA gene sequence analysis. Primarily, Klebsiella-specific media was used for the isolation and characterization of Klebsiella pneumoniae. Further, through a biochemical test, all the strains were confirmed as K. pneumoniae. PCR analysis of 16S-23S internal transcribed spacer (PCR ribotyping) was carried out to study the species variation within different Klebsiella pneumoniae isolates. For all the isolates, a conserved PCR ribotype pattern was observed while differing from other bacterial species. Phylogenetic study showed the high degree of homology with diverse source of other strains. The multiple antibiotic resistance (MAR) values of the present study for the isolates were found to be 0.468. MAR value above 0.2 indicates that the source of isolation was highly contaminated with antibiotics. Based on the 16S rRNA gene sequence analysis, the present study revealed the genetic diversity of Klebsiella pneumoniae isolated from the different diseased fish farms of West Bengal. All the strains were found to be hypermucoviscous and multidrug-resistant, thus making it pathogenic towards the host organisms. Further, the study revealed a high prevalence of K. pneumoniae in aquaculture farms, representing a risk towards successful aquaculture.

In Silico Structural Studies and Molecular Docking Analysis of Delta6-desaturase in HUFA Biosynthetic Pathway.

Fish are an important source of highly unsaturated fatty acids (HUFA) such as eicosapentaenoic acid EPA (20:5 n-3) and docosahexaenoic acid DHA (22:6 n-3) and play a significant role in human nutrition. The fatty acyl delta6-desaturase (Δ6 desaturase) is a rate-limiting enzyme in the biosynthetic pathway of highly unsaturated fatty acids (HUFA) that converts polyunsaturated fatty acids (PUFA) such as linoleic (18:2n-6) and α-linolenic (18:3n-3) acids into HUFA. In this study, fatty acyl Δ6 desaturase was identified from pangasius (Pangasianodon hypophthalmus) and further analyzed for sequenced-based characterization and 3D structural conformation. Sequenced-based analysis revealed some important secondary information such as physicochemical property. e.g., isoelectric point, extinction coefficient, aliphatic index, and grand average hydropathy, among others, and also post-translational modification sites were identified. An evolutionary-conserved stretch of amino acid residue and a functionally significant conserved structural ancestor, N-terminal cytochrome b5 and membrane FADS-like superfamily, were identified. Protein association analysis showed a high confidence score with acyl-CoA synthetase, elovl5, elovl2, and phospholipase A2. Herein, we report, for the first time, a 3D native structure of Δ6 desaturase protein by homology modeling approach; molecular docking analysis was performed with linoleic (18:2n-6) and α-linolenic (18:3n-3) acids, which are the two key substrates in the HUFA biosynthetic pathway. This work provides insight into the structural and functional characterization of Δ6 desaturase, which is involved in HUFA biosynthesis as a rate-limiting enzyme.

Acute Phase Proteins and their Potential Role as an Indicator for Fish Health and in Diagnosis of Fish Diseases.

The acute phase proteins are biochemically and functionally unrelated protein predominantly synthesized in the liver. The local inflammatory cells i.e. macrophages and neutrophils secretes various cytokines like IL-1, IL-6, IL-8 (interleukins) and TNF-α into bloodstream in response to injury and tissue damage, which stimulate hepatocytes to produce protein and release them into the circulation; these proteins are called as acute phase protein (e.g. C-reactive protein (CRP), serum amyloid A (SAA), metal binding protein, lysozyme, lectin, etc.). The acute phase proteins are involved in variety of defence related activities e.g., inactivation of proteolytic enzymes, preventing the distribution of infectious agents (i.e. either by destruction of microorganism or making microbial cell suitable for cell response by modifying surface targets) and restoration of damage tissue and healthy condition. A number of well-known acute phase proteins have disease prognosis importance and change in the APPs level reflects the presence and intensity of inflammation during infection or injury. Further studies are still necessary to develop our knowledge in diagnostic importance of different acute phase proteins in fish and more efforts are needed to differentiate the APPs levels in case of viral, bacterial and parasitic diseases.

Relationship between endometritis and oxidative stress in the follicular fluid and luteal function in the buffalo.

In this study, alteration in the follicular fluid composition and luteal function was investigated in the buffalo with endometritis. Genitalia were classified into cytological and purulent endometritis on the basis of polymorphonuclear cell cut off while non-endometritis served as control (n = 10/group). In the follicular phase, the number of surface follicles was counted, diameter of the largest follicle was measured and the follicular fluid was assayed for total protein, cholesterol, malondialdehyde (MDA), total antioxidant capacity (TAC), oestradiol (E2 ) and progesterone (P4 ). The P4 content of corpus luteum during mid-luteal phase was estimated by radioimmunoassay. Ovaries from the follicular phase of oestrous cycle showed no significant difference in the total number of surface follicles, size of the largest follicle and volume of follicular fluid in the buffaloes with and without endometritis (p > .05). However, the antral fluid of the largest follicle from the genitalia of buffalo with cytological and purulent endometritis showed a significant decrease in the concentration of total protein, cholesterol, TAC and E2 and a significant increase in the concentration of MDA and P4 (p < .05). The results indicated that there is an association between endometritis and decreased ovarian function.

Population genetic structure of Indian shad, Tenualosa ilisha inferred from variation in mitochondrial DNA sequences.

Indian shad, Tenualosa ilisha, is a commercially important anadromous fish representing major catch in Indo-pacific region. The present study evaluated partial Cytochrome b (Cyt b) gene sequence of mtDNA in T. ilisha for determining genetic variation from Bay of Bengal and Arabian Sea origins. The genomic DNA extracted from T. ilisha samples representing two distant rivers in the Indian subcontinent, the Bhagirathi (lower stretch of Ganges) and the Tapi was analyzed. Sequencing of 307 bp mtDNA Cytochrome b gene fragment revealed the presence of 5 haplotypes, with high haplotype diversity (Hd) of 0.9048 with variance 0.103 and low nucleotide diversity (π) of 0.14301. Three population specific haplotypes were observed in river Ganga and two haplotypes in river Tapi. Neighbour-joining tree based on Cytochrome b gene sequences of T. ilisha showed that population from Bay of Bengal and Arabian Sea origins belonged to two distinct clusters.

Probiotics in fish and shellfish culture: immunomodulatory and ecophysiological responses.

Aquaculture is emerging as one of the most viable and promising enterprises for keeping pace with the surging need for animal protein, providing nutritional and food security to humans, particularly those residing in regions where livestock is relatively scarce. With every step toward intensification of aquaculture practices, there is an increase in the stress level in the animal as well as the environment. Hence, disease outbreak is being increasingly recognized as one of the most important constraints to aquaculture production in many countries, including India. Conventionally, the disease control in aquaculture has relied on the use of chemical compounds and antibiotics. The development of non-antibiotic and environmentally friendly agents is one of the key factors for health management in aquaculture. Consequently, with the emerging need for environmentally friendly aquaculture, the use of alternatives to antibiotic growth promoters in fish nutrition is now widely accepted. In recent years, probiotics have taken center stage and are being used as an unconventional approach that has numerous beneficial effects in fish and shellfish culture: improved activity of gastrointestinal microbiota and enhanced immune status, disease resistance, survival, feed utilization and growth performance. As natural products, probiotics have much potential to increase the efficiency and sustainability of aquaculture production. Therefore, comprehensive research to fully characterize the intestinal microbiota of prominent fish species, mechanisms of action of probiotics and their effects on the intestinal ecosystem, immunity, fish health and performance is reasonable. This review highlights the classifications and applications of probiotics in aquaculture. The review also summarizes the advancement and research highlights of the probiotic status and mode of action, which are of great significance from an ecofriendly, sustainable, intensive aquaculture point of view.

Beta-glucan: an ideal immunostimulant in aquaculture (a review).

The major hindrance in the development and sustainability of aquaculture industry is the occurrence of various diseases in the farming systems. Today, preventive and management measures are central concern to overcome such outbreak of diseases. Immunostimulants are considered as an effective tool for enhancing immune status of cultured organisms. Among different immunostimulants used in aquaculture practices, ß-glucan is one of the promising immunostimulant, which is a homopolysaccharide of glucose molecule linked by the glycoside bond. It forms the major constituents of cell wall of some plants, fungi, bacteria, mushroom, yeast, and seaweeds. Major attention on ß-glucan was captivated with the gain in knowledge on its receptors and the mechanism of action. The receptor present inside the animal body recognizes and binds to ß-glucan, which in turn renders the animal with high resistance and enhanced immune response. This review highlights ß-glucan as an immunostimulant, its effective dosages, and route of administration and furthermore provides an outline on role of ß-glucan in enhancing growth, survival, and protection against infectious pathogens pertaining to fishes and shellfishes. Study also summarizes the effect of ß-glucan on its receptors, recognition of proteins, immune-related enzymes, immune-related gene expression and their mechanisms of action.

Statistical optimization of process variables for the production of an anticancer drug (colchicine derivatives) through fermentation: at scale-up level.

Effect of various nutritional components and fermentation parameters on biotransformation of colchicine using Bacillus megaterium ACBT03 to produce 3-demethylated colchicine (3-DMC) has been investigated. Initially, Plackett-Burman (PB) design was used to evaluate the 11 nutritional variables to screen medium component that significantly influences the 3-demethylated colchicine (3-DMC) production. Three most statistically significant nutrients that is starch, glycerol and yeast extract were identified. Response surface methodology was used to optimize fermenter operational factors pH, temperature, impeller tip velocity and aeration rate for high 3-DMC production. The predicted results of 3-DMC yield (7689.13mg/l) were obtained at 5-l-jar fermenter after optimization of pH 6.8, temperature 28°C, impeller tip velocity 4650cm/min, and aeration rate 2.0vvm. The validity of model was confirmed; and the optimum medium designed led a maximum 3-DMC yield of 7335mg/l, which had 3-4% variation with predicted results. Furthermore, the experiment was scaled up in the 70-l fermenter.

Regulation of tacrolimus production by altering primary source of carbons and amino acids.

AIMS: In the present communication, attempts have been made to regulate the tacrolimus production by supplementing commercial source of carbons and amino acids timely. METHODS AND RESULTS: Tacrolimus production was regulated by supplying vegetable oils and amino acids, individually and in combination. Tacrolimus quantification was done by HPLC. Streptomyces spp. MA6858 B3178 was found to produce 115.3 mg l(-1) of tacrolimus. The rotation speed of shake flask, pH of the broth and supply of air were maintained at 7.1, 230 rev min(-1) and 2.0 vv m air respectively. CONCLUSIONS: The effect of carbons on tacrolimus production was noticed to be of diphasic manner. During the first 24 h of culture, monosaccharide is used for the growth of microbe. However, after the lapse of 36 h, addition of soya oil and l-lysine in combination enhanced the tacrolimus production to 115.3 mg l(-1). Besides this, pH of broth was also noticed as a critical factor in monitoring tacrolimus biosynthesis. SIGNIFICANCE AND IMPACT OF THE STUDY: The newly isolated mutant Streptomyces spp. MA6858 B3178 having high potential for tacrolimus production as compared to existing data can be well used for the commercialization of tacrolimus.

Patient-controlled epidural analgesia with fentanyl and bupivacaine provides better analgesia than intravenous morphine patient-controlled analgesia for early thoracotomy pain.

BACKGROUND: Intravenous patient-controlled analgesia (IVPCA) and patient-controlled epidural analgesia (PCEA) were studied in terms of analgesic efficacy, respiratory function and side effects after thoracic surgery for 24h. PCEA using fentanyl and bupivacaine as compared to IVPCA using morphine provides better pain relief both at rest and during coughing and is associated with fewer side effects. AIMS: To compare IVPCA and PCEA in terms of analgesic efficacy, respiratory function and side effects after thoracic surgery. SETTINGS AND DESIGN: Tertiary care teaching hospital. Prospective, randomized and open study. MATERIALS AND METHODS: Thirty ASA-I or II patients undergoing thoracotomy were assigned randomly to receive either IVPCA using morphine or PCEA using fentanyl and bupivacaine combination postoperatively. No background infusion was administered in either group. Postoperative evaluation included pain intensity both at rest and during coughing, degree of sedation, arterial blood gas, forced vital capacity (FVC), peak expiratory flow rate (PEFR), presence of side effects such as nausea/vomiting and pruritus at 0, 2, 8, 12 and 24h. The primary outcome of the study was the percentage of patients with analgesia failure defined as VAS>30 despite three consecutive PCA boluses requiring rescue analgesia with intravenous fentanyl. STATISTICAL ANALYSIS: Data were analyzed using t -test, chi2 test and Mann-Whitney test. RESULTS: Significantly less number of patients required rescue analgesia in PCEA group ( P< 0.05). Pain relief was better both at rest and during coughing ( P< 0.05) in PCEA group as compared to IVPCA. Patients in the PCEA group were less sedated and had fewer incidences of side effects, i.e. nausea/vomiting and pruritus. Postoperative FVC and PEFR were reduced significantly compared to baseline only in IVPCA group ( P< 0.05). CONCLUSION: After thoracic surgery, PCEA using fentanyl and bupivacaine as compared to IVPCA using morphine provides better pain relief both at rest and during coughing and associated with fewer side effects.

Sustained release of antimicrobial drugs from polyvinylalcohol and gum arabica blend matrix.

Synthetic polymers are widely used in biomedical applications. Polymer blends have recently paved their way in this field. An attempt to prepare blend of synthetic polymer polyvinylalcohol and natural macromolecule gum arabica is made in this paper. Characterization of these blends by NMR, DSC and viscoelastic studies reveal preparation of a blend composition with synergistic properties. The blend composition with synergistic properties was used to release various antimicrobial drugs. The duration and release of the drug depends on the amount of drug loaded in the matrix and solubility of the drug in the matrix and release medium. The advantage of this system is that the release kinetics of the drug from the system can be tailored by adjusting plasticizer, homopolymer and crosslinker composition depending on the drug to be released.

Sustained release of ferrous sulfate from polymer-coated gum arabica pellets.

Sustained release of ferrous sulfate (FeSO4) from gum arabica pellets has been achieved for 7 h. Release is further sustained for 12 to 600 h by coating the pellets with polyvinyl acetate and ethylene vinyl acetate, respectively. An increase in the amount of gum arabica in the pellets decreases the rate of release due to the gelling property of gum arabica. The gel layer acts as a barrier and retards the rate of diffusion of FeSO4 through the pellet. In coated pellets, an increase in thickness of membrane helps to sustain the release of FeSO4 for a longer duration. Thus, the release of FeSO4 can be sustained for the required duration by adjusting the amount of gum arabica in the pellet and the composition and thickness of polymeric coat. The mechanism of release accordingly varies from Fickian to zero-order and super case II transport.

Effect of sugar mill effluent on oxygen uptake and carbon dioxide output of rice (Oryza sativa L.c.v. Mushoori) seedlings.

The effects of a sugar mill effluent on respiration of rice (Oryza sativa L.c.v. Mushoori) seedlings have been investigated. Each experiment consisted of two parts, the effect of various concentrations of the effluent and the time-dependent changes induced by the undiluted effluent. A marked increase in the rate of respiration was noticed upon treatment with various concentrations of the effluent. The time-dependent changes in the respiratory rate were about threefold with the 12 hr of treatment, and thereafter a rapid loss was noticed. Additionally, the respiratory quotient was studied to elucidate the nature of the metabolism of seedlings as influenced by the effluent.

The effect of sugar mill effluent on enzyme activities of rice seedlings.

The effect of sugar mill effluent on the activities of peroxidase, amylase, and nitrate reductase of rice (Oryza Sativa L. C.V. Mushoori) seedlings have been investigated. In addition, an attempt was also made to investigate effluent-induced changes in the activities of mitochondrial enzymes, such as succinate dehydrogenase, during germination of rice seedlings. The effects were studied in relation to both concentration of effluent and time of exposure. The activity of all enzymes tested, except succinate dehydrogenase, decreased during effluent treatment. A marked increase in succinate dehydrogenase activity was noticed at various concentrations of effluent treatment. When time-dependent changes in succinate dehydrogenase activity were followed a nearly threefold increase was found with 12 hr of treatment after which a rapid loss was noticed. A correlation was established between the retardation of growth and development of effluent-treated rice seedlings with the activities of some key enzymes regulating growth and development.

Analysis of the effect of industrial effluent on pigments, proteins, nucleic acids, and the 2,6-dichlorophenol indophenol Hill reaction of rice seedlings.

The effect of an industrial effluent on cellular macromolecular composition and indophenol photochemical activity of isolated chloroplasts of rice (Orvza sativa L. C. V. Mushoori) seedlings have been investigated. Each experiment consisted of two parts. One was the effect of various concentrations and the other was the time-dependent changes induced by the undiluted effluent. The total pigments, proteins, and nucleic acids of rice seedlings declined with an increase in effluent concentration and the time of incubation. The loss in contents of macromolecules like deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and protein was relatively more marked in the root than in the shoot. RNA and chlorophyll (Chl) contents of the seedlings were found most susceptible to effluent stress. Loss in Hill reaction activity measured as photoreduction of 2,6-dichlorophenol indophenol (DCPIP) of isolated chloroplasts could be correlated in a general way with the loss of pigments, proteins and nucleic acids. Diphenyl carbazide- and Mn2+-induced restoration of loss in DCPIP photoreduction suggests that the damage of oxygen evolving systems is the initial site of action of the effluent.