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.

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.

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.

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.

Characterization and pathogenicity of Aeromonas veronii associated with mortality in cage farmed grass carp, Ctenopharyngodon idella (Valenciennes, 1844) from the Central Himalayan region of India.

In the study, Aeromonas strains (n = 12) were isolated from moribund grass carp fry reared in the cage culture unit from the Central Himalayan region of India. They were identified as Aeromonas veronii, by biochemically and 16S rRNA analysis. The experimental bath infection of grass carp fry was performed using A. veronii GCAFBLC 228, one of the 12 isolates at cell concentrations 106 and 108 CFU mL-1. The infected fry showed varied behavioural characteristics followed by tail rot, black pigmentation and hemorrhage in the body 48-96 h post infection. The post bath challenged demonstrated maximum mortality (23%) at cell concentration 108 CFU mL-1 during 10th and 12th day. Histopathology revealed hypertrophy, hyperplasia, fusion of gill lamellae, detachment and epithelial cell detachment in gill, swelling of hepatocytes, granular deposition in liver and tubular degeneration and yellow pigmented macrophage aggregates in the kidney. The in vitro assays for virulence traits recorded that A. veronii GCAFBLC 228 was ß-haemolytic having strong cell surface hydrophobicity (CHS) characteristic (> 50%), precipitated after boiling, produced slime, non-suicidal and bound to crystal violet. The antibiogram showed that the strain was susceptible to ciprofloxacin (5 µg), cefotaxime (30 µg), ceftazidime (30 µg), cefoxitin (30 µg), ceftriaxone (30 µg), chloramphenicol (30 µg) and tetracycline (30 µg). Negative staining transmission electron microscopy revealed presence of the lateral flagellum-like structure and cell adherence possibly could be correlated with the pathogenicity of A. veronii GCAFBLC 228. The further investigation is warranted to study the transmission, pathogenesis and epidemiology of A. veronii GCAFBLC 228 to develop the best health management practice for cage farmed fish.

The emergence of zoonotic Fusarium oxysporum infection in captive-reared fingerlings of golden mahseer, Tor putitora (Hamilton, 1822) from the central Himalayan region of India.

Zoonotic Fusarium oxysporum infection was identified in captive-reared fingerlings of golden mahseer, Tor putitora (Hamilton, 1822) from the central Himalayan regions, India. Initially, fingerlings of T. putitora (mean length 10.8 ± 0.002 and weight 18.58 ± 0.054 g) were observed with cottony mass like growth completely covering the dorsal and caudal fins. The infected fingerlings were showing clinical signs such as sluggish, erratic movement, gasping, flared operculum and settling at one corner of the rearing tanks. The microscopic observation of 8-day old culture of cottony mass like growth showed the presence of septate macroconidia, randomly spread microconidia and chlamydospores in short-chain. From sequence analysis of ITS amplified fragment, the isolate was identified as Fusarium oxysporum, TPFCF 214 (MH464266.1) and clustered with F. oxysporum, strain NRRL 43504 (EF453107.1) and F. oxysporum, strain 20736 (JX 270150.1) isolated from the human in phylogenetic tree. An experimental infection of healthy golden mahseer fingerlings with 20 µl of F. oxysporum spore suspension (2.5 × 109 spore ml-1 ) showed the development of lesion 6-dpi at the site of injection. Experimental trial on EPC-2 cell culture recorded detachment in the monolayer, clumping and shrinking of the cell line 6-8 dpi with a spore suspension of F. oxysporum, TPFCF 214 (5.68 × 102 cell/ml). From the severity of its infection, there is a chance that F. oxysporum may emerge as pathogenically and pose a significant health risk on captive-reared golden mahseer in other Asian countries and world. As Fusarium solani and F. oxysporum are known to cause invasive fusariosis in human especially in immunocompromised patients, localized infection in immunocompetent individuals as well as osteomyelitis, arthritis, otitis, sinusitis and brain abscess, the global fish farmers, handlers and aquaculturist need to be aware of possible health hazards caused by Fusarium spp. and should adopt proper fish health management and animal husbandry practice to control the infection of Fusarium in culture environment.