Emergence of Aeromonas salmonicida subsp. masoucida MHJM250: unveiling pathological characteristics and antimicrobial susceptibility in golden mahseer, Tor putitora (Hamilton, 1822) in India.

Aeromonas salmonicida subsp. masoucida, designated as laboratory strain MHJM250, was characterized from a naturally infected farmed golden mahseer, Tor putitora. The infected fish exhibited clinical signs of erosion at the caudal fin and hemorrhage onx the ventral body surface. Molecular identification through 16 S rDNA and phylogenetic analysis revealed 100% similarity with a known strain A. salmonicida subsp. masoucida (MT122821.1). MHJM250 exhibited positive reactions for oxidase, catalase, esculin, MR-VP, O/F and utilized arginine and lysine. It also demonstrated siderophore activity, thrived at various NaCl concentrations, hydrolyzed gelatinase, skimmed milk and casinase. In vitro studies exhibited its hemolytic nature, significant biofilm production in glucose-rich tryptone soya broth and beta-hemolysis. MHJM250 didn't produce slime and was non-precipitated upon boiling. It showed crystal violet binding characteristics and auto-agglutination with relatively weak hydrophobicity (25%). In the challenge assay, intraperitoneal administration of MHJM250 to T. pitutora fingerlings at 108 CFU mL-1 resulted in pathogenicity with 3% mortality and mild hemorrhagic symptoms. Histopathological analysis revealed degenerative changes in gill, kidney, liver, muscle, and intestine samples. The bacterium displayed resistance to several antibiotics (µg/disc); ampicillin (10 µg), ampicillin/ sulbactam (10/10 µg), clindamycin (2 µg), linezolid (30 µg), penicillin G (10 µg) and rifampicin (5 µg) and varied minimum inhibitory concentrations against oxytetracycline, erythromycin and florfenicol. Transmission electron microscopy showed its rod-shaped structure with single polar flagellum and lophotrichous flagella. An investigation on the molecular basis for virulence factors of A. salmonicida subsp. masoucida MHJM250 may offer crucial understandings to formulate disease prevention and control strategies in aquaculture.

Identification and antifungal sensitivity of Fusarium species isolated from piscine hosts.

Fusarium is a huge genus of filamentous fungi that has the potential to cause emerging diseases. Members of this genus can cause infections in plants, animals and humans. Here, we report the isolation of F. oxysporum and F. equiseti from 2 important fish species, Oncorhynchus mykiss (rainbow trout) and Tor putitora (golden mahseer), respectively. F. oxysporum has emerged as a significant fungal pathogen causing infection in many fish. However, F. equiseti has been isolated mainly from plants. As far as the available literatures are concerned, this is the first report on the isolation of F. oxysporum and F. equiseti from these hosts. The isolates were identified based on growth morphology and microscopic observation. F. oxysporum produced violet pigmentation on potato dextrose agar, while F. equiseti had yellow colouration. F. oxysporum produced 1- to 2-celled microconidia along with straight or curved macroconidia having 3 to 4 septa. F. equiseti produced abundant macroconidia with 4 or more septa. Species were further confirmed based on the nucleotide sequences of the internal transcribed spacer region. In a molecular phylogeny analysis, F. oxysporum and F. equiseti formed 2 different clades. In an antifungal sensitivity assay, F. oxysporum was found to be susceptible to clotrimazole with a minimum inhibitory concentration of 1.0 µg ml-1, whereas F. equiseti was susceptible to clotrimazole, ketoconazole and fluconazole. Overall, the main findings of this study are the infection of new hosts by Fusarium species and the limited activity of many antifungal drugs against these pathogens.

Assessment of Single-Dose Pharmacokinetics of Oxolinic Acid in Rainbow Trout and Determination of In Vitro Antibacterial Activity Against Pathogenic Bacteria From Diseased Fish.

In response to the heightened risk of bacterial diseases in fish farms caused by increased demand for fish consumption and subsequent overcrowding, researchers are currently investigating the efficacy and residue management of oxolinic acid (OA) as a treatment for bacterial infections in fish. This research is crucial for gaining a comprehensive understanding of the pharmacokinetics of OA. The present study investigates pharmacokinetics of OA in juvenile rainbow trout. The fish were given a 12 mg kg-1 dose of OA through their feed, and tissue samples were collected of the liver, kidney, gill, intestine, muscle, and plasma for analysis using LC-MS/MS. The highest concentrations of the drug were found in the gill (4096.55 µg kg-1) and intestine (11592.98 µg kg-1), with significant absorption also seen in the liver (0.36 L/h) and gill (0.07 L/h) (p < 0.05). The liver (0.21 L/h) and kidney (0.03 L/h) were found to be the most efficient (p < 0.05) at eliminating the drug. The study also confirmed the drug antimicrobial effectiveness against several bacterial pathogens, including Shewanella xiamenensis (0.25 µg mL-1), Lactococcus garvieae (1 µg mL-1), and Chryseobacterium aquaticum (4 µg mL-1). The study concludes significant variations among different fish tissues, with higher concentrations and longer half-lives observed in the kidney and intestine. The lowest MIC value recorded against major bacterial pathogens demonstrated its therapeutic potential in aquaculture. It also emphasizes the importance of understanding OA pharmacokinetics to optimize antimicrobial therapy in aquaculture.

Denaturing Gradient Gel Electrophoresis Approach for Microbial Shift Analysis in Thermophilic and Mesophilic Anaerobic Digestions.

To determine the evolution of microbial community and microbial shift under anaerobic processes, this study investigates the use of denaturing gradient gel electrophoresis (DGGE). In the DGGE, short- and medium-sized DNA fragments are separated based on their melting characteristics, and this technique is used in this study to understand the dominant bacterial community in mesophilic and thermophilic anaerobic digestion processes. Dairy manure is known for emitting greenhouse gases (GHGs) such as methane, and GHG emissions from manure is a biological process that is largely dependent on the manure conditions, microbial community presence in manure, and their functions. Additional efforts are needed to understand the GHG emissions from manure and develop control strategies to minimize the biological GHG emissions from manure. To study the microbial shift during anaerobic processes responsible for GHG emission, we conducted a series of manure anaerobic digestion experiments, and these experiments were conducted in lab-scale reactors operated under various temperature conditions (28 °C, 36 °C, 44 °C, and 52 °C). We examined the third variable region (V3) of the 16S rRNA gene fingerprints of bacterial presence in anaerobic environment by PCR amplification and DGGE separation. Results showed that bacterial community was affected by the temperature conditions and anaerobic incubation time of manure. The microbial community structure of the original manure changed over time during anaerobic processes, and the community composition changed substantially with the temperature of the anaerobic process. At Day 0, the sequence similarity confirmed that most of the bacteria were similar (>95%) to Acinetobacter sp. (strain: ATCC 31012), a Gram-negative bacteria, regardless of temperature conditions. At day 7, the sequence similarity of DNA fragments of reactors (28 °C) was similar to Acinetobacter sp.; however, the DNA fragments of effluent of reactors at 44 °C and 52 °C were similar to Coprothermobacter proteolyticus (strain: DSM 5265) (similarity: 97%) and Tepidimicrobium ferriphilum (strain: DSM 16624) (similarity: 100%), respectively. At day 60, the analysis showed that DNA fragments of effluent of 28 °C reactor were similar to Galbibacter mesophilus (strain: NBRC 10162) (similarity: 87%), and DNA fragments of effluent of 36 °C reactors were similar to Syntrophomonas curvata (strain: GB8-1) (similarity: 91%). In reactors with a relatively higher temperature, the DNA fragments of effluent of 44 °C reactor were similar to Dielma fastidiosa (strain: JC13) (similarity: 86%), and the DNA fragments of effluent of 52 °C reactor were similar to Coprothermobacter proteolyticus (strain: DSM 5265) (similarity: 99%). To authors' knowledge, this is one of the few studies where DGGE-based approach is utilized to study and compare microbial shifts under mesophilic and thermophilic anaerobic digestions of manure simultaneously. While there were challenges in identifying the bands during gradient gel electrophoresis, the joint use of DGGE and sequencing tool can be potentially useful for illustrating and comparing the change in microbial community structure under complex anaerobic processes and functionality of microbes for understanding the consequential GHG emissions from manure.

Unveiling the molecular mechanisms of pigmentation control in Queen Loach, Botia dario (Hamilton, 1822): Insights from sesame seed and marigold-induced antityrosinase effects.

Fish pigmentation study can reveal understandings in dermatological research based on functional genomics. Cultured ornamental fish becomes dull coloured and antityrosinase activity through sesame seed may enhance skin colour, which has not been studied. Botia dario is an indigenous fish, having ornamental and aesthetic value and can be studied as a model for fish pigmentation genetics. In this study, fish specimens were fed with 15% marigold petal meal along with 5, 10 and 15% w/w sesame seed in diet. Pigmentation genes, that is, tyr, tyrp1a, asip1, gnaq, kitlga, mc1r, mitf, pax7a, rab38, slc7a11, sox9a, sox10, csf1r, bcdo2 and gsta2 in skin and immunogens, that is, il20, nramp, tlr9 and trail in kidney were studied. Gene expression in tissues revealed enhanced pigmentation and immunity as well as the role of tyr, tyrp1a and asip1 in pigmentation. Immunogenes and blood parameters confirmed the best pigmentation diet. Colorimetric analysis also showed the enhancement of pigmentation. Insights from sesame seed and marigold-induced antityrosinase effects will be applied in aquaculture to develop natural, dietary formulations that will enhance pigmentation in ornamental fish, leading to improved skin colour and market value.

Quantitative Analysis of Genomic DNA Degradation of E. coli Using Automated Gel Electrophoresis under Various Levels of Microwave Exposure.

The problem that this study addresses is to understand how microwave radiation is able to degrade genomic DNA of E. coli. In addition, a comparative study was made to evaluate the suitability of a high-throughput automated electrophoresis platform for quantifying the DNA degradation under microwave radiation. Overall, this study investigated the genomic DNA degradation of E. coli under microwave radiation using automated gel electrophoresis. To examine the viable organisms and degradation of genomic DNA under microwave exposure, we used three methods: (1) post-microwave exposure, where E. coli was enumerated using modified mTEC agar method using membrane filtration technique; (2) extracted genomic DNA of microwaved sample was quantified using the Qubit method; and (3) automated gel electrophoresis, the TapeStation 4200, was used to examine the bands of extracted DNA of microwaved samples. In addition, to examine the impacts of microwaves, E. coli colonies were isolated from a fecal sample (dairy cow manure), these colonies were grown overnight to prepare fresh E. coli culture, and this culture was exposed to microwave radiation for three durations: (1) 2 min; (2) 5 min; and (3) 8 min. In general, Qubit values (ng/µL) were proportional to the results of automated gel electrophoresis, TapeStation 4200, DNA integrity numbers (DINs). Samples from exposure studies (2 min, 5 min, and 8 min) showed no viable E. coli. Initial E. coli levels (at 0 min microwave exposure) were 5 × 108 CFU/mL, and the E. coli level was reduced to a non-detectable level within 2 min of microwave exposure. The relationships between Qubit and TapeStation measurements was linear, except for when the DNA level was lower than 2 ng/µL. In 8 min of microwave exposure, E. coli DNA integrity was reduced by 61.7%, and DNA concentration was reduced by 81.6%. The overall conclusion of this study is that microwave radiation had a significant impact on the genomic DNA of E. coli, and prolonged exposure of E. coli to microwaves can thus lead to a loss of genomic DNA integrity and DNA concentrations.

Antibacterial activity of a short de novo designed peptide against fish bacterial pathogens.

In the face of increasing antimicrobial resistance in aquaculture, researchers are exploring novel substitutes to customary antibiotics. One potential solution is the use of antimicrobial peptides (AMPs). We aimed to design and evaluate a novel, short, and compositionally simple AMP with potent activity against various bacterial pathogens in aquaculture. The resulting peptide, KK12YW, has an amphipathic nature and net charge of + 7. Molecular docking experiments disclosed that KK12YW has a strong affinity for aerolysin, a virulence protein produced by the bacterial pathogen Aeromonas sobria. KK12YW was synthesized using Fmoc chemistry and tested against a range of bacterial pathogens, including A. sobria, A. salmonicida, A. hydrophila, Edwardsiella tarda, Vibrio parahaemolyticus, Pseudomonas aeruginosa, Escherichia coli, Staphylococcus epidermidis, and methicillin-resistant S. aureus. The AMP showed promising antibacterial activity, with MIC and MBC values ranging from 0.89 to 917.1 µgmL-1 and 3.67 to 1100.52 µgmL-1, respectively. In addition, KK12YW exhibited resistance to high temperatures and remained effective even in the presence of serum and salt, indicating its stability. The peptide also demonstrated minimal hemolysis toward fish RBCs, even at higher concentrations. Taken together, these findings indicate that KK12YW could be a highly promising and viable substitute for conventional antibiotics to combat microbial infections in aquaculture.

Determining the prevalence of Escherichia coli, Salmonella, and shiga toxin-producing Escherichia coli in manure of dairy lagoons.

AIM: The aim of this study was to determine the prevalence of microbial pathogens in manure of dairy lagoons in California. METHODS AND RESULTS: To determine pathogens in dairy manure stored in anaerobic lagoons of dairy farm, an extensive field study was conducted across California to sample manure from 20 dairy farms. Samples were analyzed to determine the prevalence of indicator Escherichia coli, Shiga toxin producing E. coli (STEC), Salmonella, and E. coli O157: H7. To test the E. coli, STEC, and Salmonella, we used agar culture-based method followed by polymerase chain reaction (PCR) method. In addition, a real- time PCR based method was used to determine the presence of E coli O157: H7. Study demonstrated that the prevalence of Salmonella in manure sample is lower than E. coli. The presence of Salmonella was found in 2.26% of the samples, and both the culture-based and PCR methods yielded comparable outcomes in detecting Salmonella. Moreover, ∼11.30% of the total samples out of the 177 were identified as positive for STEC by qPCR. CONCLUSION: These findings demonstrate that indicator E. coli are abundantly present in anaerobic lagoons. However, the presence of STEC, and Salmonella is substantially low.

Assessing safety, efficacy and residue depletion in golden mahseer, Tor putitora (Hamilton, 1822): biochemical and physiological responses to graded concentrations of oxytetracycline dietary supplementation.

The safety and effectiveness of oxytetracycline can potentially manage bacterial infections in fish. This, in turn, might reduce the concerns related to its use in aquaculture and human consumption, such as toxicity, antimicrobial resistance, and other associated risks. The primary objective of this study was to assess how adding oxytetracycline dihydrate to the diet affects its effectiveness, safety, and the presence of residues in T. putitora. T. putitora fingerlings, subjected to experimental infection with Aeromonas hydrophila at a concentration of 108 CFU mL- 1, received an oral administration of oxytetracycline dihydrate. The oxytetracycline dihydrate was added to the feed (corresponding to 2% of the fish body weight) at concentrations of 44.1, 88.2, 132.3 and 176.4 mg Kg- 1 fish body weight per day. This treatment was carried out for 10 consecutive days. The biochemical and physiological responses of T. putitora and efficacy of oxytetracycline dihydrate were determined through estimation of microbial load (CFU mL- 1), haematogram, serum biomarkers, behavioral characteristics, non-specific immunity and residue depletion. Experimentally infected fish showed disease progression and induced histopathological conditions with highest microbial load (CFU mL- 1) in the muscle of both control and treated fish. The fish haematogram showed increased leucocyte and haemoglobin content, influenced by dietary oxytetracycline dihydrate. The fish demonstrated adaptive physiological response to oxytetracycline dihydrate at 44.1 to 88.2 mg and resulted in increased albumin and globulin content. The serum-enzyme assay showed significant increase in aspartate aminotransferase (AST), alanine aminotransferase (ALT) and plasma alkaline phosphatase (ALP) activities in the test fish (< 0.05). Oxytetracycline dihydrate at 88.2 to 132.3 mg Kg- 1 fish body weight per day recorded higher feed intake (75%), significant survivability (66-68%) and histopathological recovery. The suppressed immune response was manifested with decreased respiratory burst and lysozyme activity. The palatability, treatment of bacterial infection, histopathological changes and survivability by fingerlings of golden mahseer determined the safety and optimized the therapeutic potential of the oxytetracycline dihydrate at 88.2 mg Kg- 1 fish body weight per day for 10 days to contain the infection by A. hydrophila. A withdrawal period of 8-d was recommended as oxytetracycline dihydrate concentration depleted below the legal maximum residue limit (MRL 2.0 mg g- 1) in the edible muscle of the golden mahseer reared at an average water temperature of 20 °C. This is considered safe for human consumption.

Microbial diversity in dairy manure environment under liquid-solid separation systems.

In dairy manure, a wide array of microorganisms, including many pathogens, survive and grow under suitable conditions. This microbial community offers a tremendous opportunity for studying animal health, the transport of microbes into the soil, air, and water, and consequential impacts on public health. The aim of this study was to assess the impacts of manure management practices on the microbial community of manure. The key novelty of this work is to identify the impacts of various stages of manure management on microbes living in dairy manure. In general, the majority of dairy farms in California use a flush system to manage dairy manure, which involves liquid-solid separations. To separate liquid and solid in manure, Multi-stage Alternate Dairy Effluent Management Systems (ADEMS) that use mechanical separation systems (MSS) or weeping wall separation systems (WWSS) are used. Thus, this study was conducted to understand how these manure management systems affect the microbial community. We studied the microbial communities in the WWSS and MSS separation systems, as well as in the four stages of the ADEMS. The 16S rRNA gene from the extracted genomic DNA of dairy manure was amplified using the NovoSeq Illumina next-generation sequencing platform. The sequencing data were used to perform the analysis of similarity (ANOSIM) and multi-response permutation procedure (MRRP) statistical tests, and the results showed that microbial communities among WWSS and MSS were significantly different (p < 0.05). These findings have significant practical implications for the design and implementation of manure management practices in dairy farms.

Physico-chemical assessment of on-farm bioconversion of organic waste in dairy farms in context to sustainability and circular bioeconomy.

On a milk-producing dairy farm, milk production is correlated with manure production and the number of cattle, and manure is widely used as a soil fertilizer. However, excessive dairy manure production is linked with greenhouse gas emissions and water quality issues. On-farm planning of manure storage and application to enhance soil nutrients are essential in a circular economy to reduce environmental impact, where manure is not landfilled and incinerated. Instead, it creates a nutrient resource for crops and soil. Dairy manure, which is rich in nutrients, is a valuable fertilizer that contains many nutrients such as nitrogen (N), organic matter (OM), phosphorous (P), Potassium (K) and micronutrients. In this work, a pilot field research was conducted between 2016 and 2018 in various parts of California, USA (San Joaquin Valley, Sacramento Valley, Shasta Cascade, and the North Coast of California) to assess physio-chemical characteristics of solid fractions of dairy manure among various dairy farms. A total of 156 samples were collected from the gut (n = 107) and toe (n = 49) of the manure piles across California for determining total solid (TS), volatile solid (VS), temperature, moisture content and carbon-nitrogen ratio (C: N). Here, using the observations of field study and analysis, we show that C: N, OM and MC of solid fractions of dairy manure vary significantly among dairy farms. The average C: N ratio of manure (26-32) among various regions was close to an ideal C: N value of 24:1 for soil microbes to stimulate nutrient release to crops. Manure pH ranged between 7.0 and 8.0, which was close to an optimal pH range for common crops (6.0-8.0). Moreover, considering less cost and surplus availability, manure will likely continue providing a cost-effective organic fertilizer resource compared to commercial chemical fertilizers.

A novel scalable polycationic melamine sponge-based filtration matrix for continuous ultrafast adsorption of anionic pollutants.

Effective capturing of anionic pollutants from wastewater under industrial operating conditions, which requires high processing flux and fast adsorption rate remains a challenge. Here, a commercially available melamine sponge (MS) with reticulated 3D macroporous structures was covalently modified with positively charged moieties using a single step functionalization under mild conditions. The developed novel polycationic melamine sponge (MS+) was formed by a nucleophilic addition reaction between glycidyltrimethylammonium chloride (GMTA) and MS, followed by a self-propagation of GMTA. The produced MS+ possessed strong electrostatic interactions with different anions such as Rose Bengal (RB) and phosphates (P) under a wide pH range (3-11). The MS+ exhibited promoted static adsorption efficiencies of 400 mg g-1 (P) and 600 mg g-1 (RB), within 5 min and 60 s, respectively. Furthermore, the MS+ showed high stability and recyclability for up to 15 cycles of uses, and the recycling process was environmentally friendly by using 1 M NaCl as a releasing solution. Benefiting from fast flow through the macroporous MS+ and highly positive charged skeleton, the MS+ was applied for rapid dynamic enrichment process of P from real manure wastewater with an enrichment factor of 4.4. Utilization of the MS+ as the substrate brings additional advantages such as low cost, availability, and flexibility to fit into existing filtration devices. The developed MS+ could be expanded for enrichments of other anionic species from various polluted water sources.

Effect of glyphosate-based herbicide roundup on hemato-biochemistry of Labeo rohita (Hamilton, 1822) and susceptibility to Aeromonas hydrophila infection.

In the present study, comprehensive research was executed to investigate the salient toxic effects of glyphosate herbicide in static water system by evaluating the haemato-biochemical profiles of Labio rohita. A challenge study against Aeromonas hydrophila was conducted to determine disease susceptibility of the fish, treated to varying concentrations of commercial-grade glyphosate herbicide. A static range finding bioassay and definitive test revealed that the 96-h LC50 value of glyphosate was 10.16 mg L-1. The experimental fish were subjected to three sub-lethal concentrations of 2.06, 1.03, and 0.63 mg l-1 for 28 days and changes were documented bi-fortnightly to study haemato-biochemical alterationsin the fish. Significantly (p < 0.05) low values in red blood corpuscles (RBC), hemoglobin (Hb), and hematocrit value (Hct) were documented. In contrast, a significant (p < 0.05) escalation in white blood corpuscles (WBC) was documented in comparison to the control. Biochemical and stress markers such as blood glucose, total protein, and alkaline phosphatase (ALP) were significantly (p < 0.05) low, whereas serum glutamate pyruvate transaminase (SGPT) and serum glutamate oxaloacetate transaminase (SGOT) escalated significantly (p < 0.05). Chronic exposure to glyphosate, on the other hand, had the least effect on the Na+ and K+ ions. Further, a challenge assay against A. hydrophila at three sub-lethal glyphosate concentrations demonstrated a synergistic impact that reduced the fish survivability. The findings conclude that persistent low glyphosate concentrations in aquatic ecosystems show significant pathophysiological changes in L. rohita, with increased vulnerability to infections. Altogether, our findings indicate the need to further study the possible assessment for a sustainable bio-remediation technique, mitigation of the detrimental effects of glyphosate exposure in fish, and recommendation of an acceptable residue concentration of the glyphosate in aquatic ecosystem.

Pathological analysis and antimicrobial susceptibility of Chryseobacterium balustinum RTFCP 298 isolated from diseased rainbow trout, Oncorhynchus mykiss.

In this study, six isolates of Chryseobacterium balustinum were characterized from diseased rainbow trout fingerlings. The virulence characteristics, pathogenicity, and antimicrobial susceptibility pattern of these isolates were investigated. The bacterium showed positive results for catalase, cytochrome oxidase, and aesculin hydrolysis, while negative results were obtained for DNase, gelatinase, methyl red, Voges-Proskauer's reaction, Simon citrate, Hydrogen sulphide, and starch hydrolysis. Amino acid metabolism analysis revealed the inability to metabolize arginine, lysine, and ornithine decarboxylase. Molecular characterization (16S rRNA) and phylogenetic analysis revealed the test isolates as C. balustinum, closely related to strain WLT (99.85% similarity) and C. balustinum P-27 (99.77%). Virulence assay indicated haemolytic activity and biofilm formation by the test bacterium. The challenge test confirmed moderate pathogenicity in rainbow trout and established Koch's postulates. The clinical manifestations of infection included fin erosion, eye and body surface haemorrhage, exophthalmia, and organ liquefaction. The minimum inhibitory concentrations of various antimicrobials ranged from 1 to > 256 µg mL-1. The novel synthetic antimicrobial peptides exhibited MICs of 8 to > 256 µg mL-1, suggesting a potential control method. These findings suggest that C. balustinum is an opportunistic pathogen with moderate pathogenicity in rainbow trout. Further research on the host-pathogen relationship is necessary to understand virulence characteristics and pathogenicity in aquaculture.

First report of Achlya bisexualis infection in captive-reared Endangered golden mahseer Tor putitora.

Achlya bisexualis is a notorious oomycete pathogen with the potential to cause emerging disease in fish farms. In this study, we report the first isolation of A. bisexualis from captive-reared golden mahseer Tor putitora, an Endangered fish species. The infected fish showed a cotton-like growth of mycelia at the site of infection. The mycelium when cultured on potato dextrose agar produced radially growing white hyphae. The hyphae were non-septate, and some of them carried matured zoosporangium with dense granular cytoplasmic contents. Spherical gemmae with stout stalks were also observed. All the isolates had 100% identity in internal transcribed spacer (ITS)-rDNA sequence and showed highest similarity to that of A. bisexualis. In molecular phylogeny, all the isolates formed a monophyletic group with A. bisexualis which was supported by a bootstrap value of 99%. Based on the molecular and morphological findings, all the isolates were confirmed as A. bisexualis. Further, the anti-oomycete effect of boric acid, a known antifungal agent, against the isolate was evaluated. The minimum inhibitory concentration and minimum fungicidal concentration were found to be 1.25 and >2.5 g l-1, respectively. Isolation of A. bisexualis from a new fish species indicates its possible occurrence in other unreported hosts. Considering its wide infectivity and the potential to cause disease in farmed fishes, its probable prevalence in a new environment and host needs to be closely monitored to prevent the spread of infection, if any, by adopting suitable control measures.

Biosafety, histological alterations and residue depletion of feed administered anti-parasitic drug emamectin benzoate in golden mahseer, Tor putitora (Hamilton, 1822) as a model candidate fish for sport fishery and conservation in temperate waters.

In the present experiment, the attempt has been made to study the biosafety, toxicity, residue depletion and drug tolerance of graded doses of emamectin benzoate (EB) in juveniles of golden mahseer, Tor putitora as a model candidate fish for sport fishery and conservation in temperate waters through an extended medicated feeding. The graded doses of EB viz., 1× (50 µg/kg fish/day), 2 × (100 µg/kg fish/day), 5 × (250 µg/kg fish/day) and 10 × (500 µg/kg fish/day) were administered to golden mahseer juveniles through medicated diet for 21 days at water temperature of 18.6°C. The higher doses of EB did not cause any mortality during and 30 days after the end of medication period, but considerable variations in feeding and behavior were observed. Severe histological alterations observed after EB-diets (5 × and 10×) were vacuolation, pyknotic nuclei, melanomacrophage centre and necrosis in liver; Bowman's capsule dilation, degenerated renal tubules in kidney; myofibril disintegration, muscle oedema, splitting of muscle fibres, migration of inflammatory cells in muscle; and abundant goblet cells, dilated lamina propria and disarrangement of mucosa in intestine tissues. The residual concentrations of EB metabolites Emamectin B1a and B1b were analyzed using muscle extracts and were found to be peaked during medication period followed by gradual depletion in post-medication period. The outcome of this study showed that the Emamectin B1a residual concentration in fish muscle in 1×, 2×, 5×, and 10× EB treatment groups were 1.41 ± 0.49, 1.2 ± 0.7, 9.7 ± 3.3, and 37.4 ± 8.2 µg/kg at 30 days of post-medication period, respectively, which falls under the maximum residue limits (MRLs) of 100 µg/kg. The results support the biosafety of EB at recommended dose of 50 µg/kg fish/day for 7 days. As residue of EB is recorded falling within the MRL, no withdrawal period is recommended for golden mahseer.

Pharmacokinetics and biosafety evaluation of a veterinary drug florfenicol in rainbow trout, Oncorhynchus mykiss (Walbaum 1792) as a model cultivable fish species in temperate water.

In two experimental trials; florfenicol pharmacokinetics following a single dose oral administration at 15 mg kg-1 fish body weight and biosafety through extended medicated feeding were studied in the rainbow trout, Oncorhynchus mykiss. The pharmacokinetic trial was conducted for 5 days, whereas the biosafety experiment lasted for a 30-day safety margin followed by a 20-day residual period analysis at 3, 5 and 10 times greater than the therapeutic dose 10 mg kg-1 biomass day-1. C max µg kg-1 calculated for florfenicol were found to be 5,360 in intestine, 2,890 in gill, 2,250 in kidney, 973 in liver and 273 in plasma, obtained at T max of 16 h. Intestine had utmost area under the concentration-time curve (tissue/plasma) of 13.83 h µg kg-1 and a prolonged half life (t1/2ß) of 28.62 h. The highest apparent metabolic rate value in the kidney (0.327) showed a high level of biotransformation of florfenicol to its metabolite florfenicol amine. The apparent distribution rate of florfenicol amine in muscle, in comparison to the parent drug florfenicol, indicated elimination of the medication mostly in the form of florfenicol amine with t1/2 of 16.75 h. The biosafety of florfenicol orally administered to rainbow trout recorded effective feed consumption, physiological responses, drug tolerance and significantly low drug concentrations in muscle of rainbow trout, thus its usage at 10 mg kg-1 fish body weight is recommended. In the study, the rapid absorption, greater bioavailability, enhanced dispersion, slower elimination and biosafety of the drug form a significant basis for the florfenicol and its metabolite florfenicol amine as a useful antibacterial agent in aquaculture.

Temperature alters the oxidative and metabolic biomarkers and expression of environmental stress-related genes in chocolate mahseer (Neolissochilus hexagonolepis).

Long-term acclimation temperature effects on biomarkers of oxidative stress, metabolic stress, expression of heat shock proteins (Hsps), and warm-temperature acclimation related 65-kDa protein (Wap65) were evaluated in the threatened chocolate mahseer (Neolissochilus hexagonolepis). Fifteen-day-old larvae were acclimated to different water temperatures (15, 19, 23-control group, 27, and 31 °C) for 60 days prior to the sampling for quantification of mRNA, enzyme, nitric oxide, and malondialdehyde (MDA) content. Acclimation to 31 °C increased the basal mRNA level of glutathione S-transferase alpha 1 (GSTa1), and activities of catalase (CAT), glutathione reductase (GR), and GST enzymes and but downregulated the expression of superoxide dismutase 1 (SOD1) in the whole-body homogenate. Other antioxidant genes, i.e., CAT and GPx1a, were unaffected at 31 °C, and nitric oxide (NO) concentration was significantly lower. In contrast, fish acclimated to 15 °C showed an upregulated transcript level of all the antioxidant genes and no significant difference in the CAT, GR, and GST enzymes. Activities of the metabolic enzymes, aspartate transaminase (AST) and alanine transaminase (ALT), were significantly lower at 15 °C. The expression of Hsp47 was upregulated at both 15 and 31 °C groups, whereas Hsp70 was elevated at 27 and 31 °C groups. Wap65-1 transcription did not show significant variation in treatment groups compared to control. Fish in the high (31 °C) and low-temperature (15 °C) acclimation groups were capable of maintaining oxidative stress by modulating their antioxidant transcripts, enzymes, and Hsps.

Long-Term Surgical Outcomes of Bilateral Symmetrical Superior Oblique Nasal Tenotomy in Patients of Large A-Pattern Exotropia.

Purpose: To report the long-term outcomes of bilateral symmetrical superior oblique (SO) nasal tenotomy in patients with large A-pattern exotropia (≥25 prism diopter [PD]). Methods: This retrospective study was conducted on 15 patients (aged: 4-28 years) of large A-pattern exotropia. An A-pattern was defined as >10 PD difference between up and down gaze at 6 m by use of the alternate prism cover test. Objective ocular torsion was assessed by fundus photography and subjective torsion by double Maddox rod test. All patients underwent horizontal muscle surgery according to the primary position horizontal deviation and bilateral symmetrical SO nasal tenotomy for A-pattern. Surgical success was defined as postoperative A-pattern of ≤10 PD, the absence of vertical and torsional diplopia, and the absence of V-pattern. The minimum follow-up period was 24 months. Results: A total of 15 patients of large A-pattern exotropia (7 males and 8 females) with a mean age of 17.09 ± 7.9 years were included in the study. All patients had bilateral SO overaction of grade +3 or +4 with a mean preoperative A-pattern of 30.3 ± 3.9 PD. At 24 months of follow-up, esotropia in down gaze (V-pattern) was present in four patients with a mean of 11.25 ± 2.5 PD, (range, 10-15 PD). The rest of the 11 patients maintained successful alignment with a mean A-pattern of 3.18 ± 1.17 PD, (range, 2-5 PD). There was significant A-pattern collapse with a mean of 31 ± 9.1 PD after 2 years of follow-up, which was significantly associated with preoperative A-pattern (Pearson correlation, r = 0.7; t[15] = 4.0; P = 0.002). The mean of pre- and postoperative objective ocular torsion was found to be -0.5 ± 4° and -4.8 ± 3.8° with a mean extorsion effect of 4.67 ± 3.85°. There was a statistically significant difference between pre- and postoperative ocular torsion (°) (t [30] = 5.42; P < 0.001), the change in ocular torsion was significantly associated with preoperative torsion (Pearson correlation, r = 0.5; t [30] = 7.2; P < 0.001). None of the patients had subjective torsion on the double Maddox rod test pre- and postoperatively. Conclusions: Bilateral symmetrical SO nasal tenotomy is effective in cases with large A-pattern (>25 PD). The reduction of A-pattern and postoperative change in fundus torsion have a positive correlation with preoperative A-pattern and preoperative torsion, respectively.