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.

Assessing the effect of therapeutic level of oxytetracycline dihydrate on pharmacokinetics and biosafety in Oncorhynchus mykiss (Walbaum, 1792).

The aim of the experiment was to investigate the pharmacokinetics of oxytetracycline dihydrate after a single oral administration of 80 mg kg-1 day-1 in rainbow trout and assess its biosafety at concentration of 80, 240, 400, and 800 mg kg-1 day-1 over 30 days, focusing on various aspects such as effective feed consumption, physiological responses, drug tolerance, and detection of low drug concentrations in rainbow trout. The pharmacokinetics study spanned a duration of 5 days, while the assessment of biosafety extended for a 30-day safety margin, followed by a subsequent 10-day residual analysis. Pharmacokinetic analysis revealed slow absorption with low-rate constant in tissues. Absorption rates vary among tissues, with the gill showing the highest rate (0.011 h-1) and plasma exhibiting the slowest (0.0002 h-1). According to pharmacokinetic analysis, the highest concentration, Cmax (µg kg-1) was observed in the kidney (9380 µg kg-1) and gill (8710 µg kg-1), and lowest in muscle (2460 µg kg-1). The time (Tmax) to reach peak concentration (Cmax) varied among tissues, ranging from 3 h in the gill to 32 h in the muscle, with 24 h in plasma, 32 h in the kidney, and 16 h in both the liver and skin. The liver and kidney had the highest area under the concentration-time curve (AUC(0-128)), indicating widespread drug distribution. Prolonged elimination occurred at varying rates across tissues, with the gill showing the highest rate. The study found that OTC concentrations exceeded the LOD and LOQ values. Biosafety evaluation showed effective feed consumption, physiological responses, and low drug concentrations in muscle at the recommended dosage of 80 mg kg-1 fish day-1.

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.

Emerging bacterial fish pathogen Lactococcus garvieae RTCLI04, isolated from rainbow trout (Oncorhynchus mykiss): Genomic features and comparative genomics.

Lactococcus garvieae is one of the emerging zoonotic bacterial pathogen, causes fatal hemorrhagic septicemia in cultured fish species, animals and humans, worldwide. Here, we report the genomic features of whole-genome sequence (WGS) of L. garvieae strain RTCLI04, recovered from lower intestine of farmed rainbow trout, Oncorhynchus mykiss in the northwest Himalayan region India. The genome of L. garvieae RTCLI04 is a single circular chromosome of 2,054,885 base pairs (bp), which encodes 1993 proteins and has G + C content of 39%. The bioinformatics analysis of WGS of RTCLI04, confirmed the presence of 51 tRNAs genes (including two pseudogenes), six rRNAs genes (four genes for 5S rRNA; one gene for 16S rRNA and one gene for 23S rRNA), five virulent domains, and twenty eight different genetic pathways. A Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) finder tool indicates that three different CRISPR and one cas system with common spacer was present in the genome of L. garvieae RTCLI04. Pan-genome analysis of RTCLI04 and all the other reference L. garvieae strains shows that pan-genome of this bacterium consisted of 2239 putative protein-coding genes in which 1850 genes are core gene, 389 genes are dispensable gene, and 221 genes are unique to RTCLI04. L. garvieae RTCLI04 lacks genomic island of 16.5 Kb capsule gene cluster. In addition, 39 virulence-associated genes (VAGs) including hly1,-2,-3; PavA, PsaA; eno; LPxTG containing surface proteins 1, 2, 3 and 4; pgm, sod and 29 antimicrobial resistant genes (ARGs) including mefE (clindamycin), srmB (lincomycin), dfrA26 (trimethoprim), gyrB (nalidixic acid), arr-3 (rifampin), otrB (tetracycline), aac(6)-Ic (tobramycin), IrgB (penicillin), mecA (oxacillin), vanRB (vancomycin) and mfpA (fluoroquinolone) were also predicted in the genome of L. garvieae RTCLI04. Our study provides new insight into understanding the virulence mechanism, antimicrobial resistance, and development of effective therapeutic measures against L. garvieae during a disease outbreak in aquaculture.

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.

Molecular cloning, characterization and expression profile of kisspeptin1 and kisspeptin1 receptor at brain-pituitary-gonad (BPG) axis of golden mahseer, Tor putitora (Hamilton, 1822) during gonadal development.

Complete cDNA sequences of kiss1 (gmkiss1) and its receptor kiss1r (gmkiss1r) were cloned and characterized from brain tissue of adult golden mahseer (Tor putitora). Thereafter, quantification of gmkiss1 and gmkiss1r mRNA expression in brain-pituitary-gonad (BPG) axis of male and female golden mahseer was carried out using quantitative real-time (qRT)-PCR assay during an annual reproductive cycle, at different gonadal development stages. The gmkiss1 cDNA was 508bp, with 330bp open reading frame (ORF), encoding a precursor protein of 109 amino acids, whereas gmkiss1r cDNA was 1383bp with an ORF of 1004bp, which encodes a 334 amino acid protein residue. The qRT-PCR study shows that gmkiss1 and gmkiss1r are expressed in brain, pituitary and gonads of both the sexes of golden mahseer. An apparent sexual dimorphism in transcript level of gmkiss1 and gmkiss1r in brain and gonads was evident during the reproductive cycle. Overall, in brain, testis and ovary, the gmkiss1 and gmkiss1r mRNA expression level was comparatively higher during the initial stages of gonadal development, than that of spermiation or ovulation stage. In pituitary of both the sexes, throughout the gonadal development, consistently low transcript level of gmkiss1 and gmkiss1r was observed. The gmkiss1 mRNA expression level in brain and ovary of female golden mahseer was several folds higher than the brain and testis of male fish. In conclusion, we confirm the presence of kiss1 and its receptor in golden mahseer, and results of our study strongly suggested the involvement of kisspeptin1 system in gonadal development and annual reproductive cycle of this species.

Impact of acid mine drainage on haematological, histopathological and genotoxic effects in golden mahaseer, Tor putitora.

The present study was carried out to evaluate sub-lethal mechanism of acid mine drainage toxicity in fingerlings (9.5 ± 2.4 cm) of golden mahseer, Tor putitora. Exposed fingerlings showed significant reduction (P < 0.01) in blood erythrocytes, neutrophils, thrombocytes, lymphocytes and leukocytes in contrast to increase in number of immature circulating cells. Hyperplasia, degeneration of glomeruli, presence of inflammatory cells and increased number of melanomacrophage aggregates, vacuolization of cell cytoplasm, hepatocyte swelling were marked in kidney and liver of fish. Ladder in, an increment of 180-200 bp of hepatic and kidney DNA, by electrophoresis were consistent with DNA damage. 10 day exposure to acid mine drainage resulted in reduction of double stranded DNA to 46.0 and 48.0 in hepatocytes and kidney cells respectively. Significant increase (P < 0.01) in tail length and percent tail DNA was evident by comet assay. The results suggest that exposure to acid mine drainage might cause irreversible damage to immune cells, tissue and DNA of fish, and this model of DNA damage may contribute in identifying novel molecular mechanism of interest for bioremediation application.

Spermatogenesis and related plasma androgen and progestin level in wild male golden mahseer, Tor putitora (Hamilton, 1822), during the spawning season.

Testicular development and plasma levels of sex steroid [11-ketotestosterone (11-KT), testosterone (T) and 17,20ß-dihydoxy-4-pregnen-3-one (17,20ß-P)] were studied for the first time in wild golden mahseer, Tor putitora. Testicular development was investigated by macroscopic observation and histology of the gonads, whereas steroids were measured by enzyme-linked immunosorbent assay. Based on macroscopic observation and germ cell types present in gonad histology, the testes of T. putitora were divided into five developmental stages: immature [stage I; spermatogonia (SPG)], early spermatogenesis [stage II; SPG and spermatocytes (SPC)], late spermatogenesis [stage III; SPG, SPC, spermatids (SPD) and spermatozoa (SPZ)], spermiation (stage IV; SPZ) and post-spawning (stage V; SPG, SPD and SPZ). During the stage I of the testes, the lowest levels of plasma sex steroid and gonadosomatic index (I G) were recorded. The highest plasma level of T was 0.89 ± 0.09 ng/mL and 11-KT was 4.23 ± 0.54 ng/mL, which was during the stage III and IV, respectively. The peak in 11-KT was coincident with the peak in I G (1.65 ± 0.12 %). The lowest T and 11-KT levels were 0.25 ± 0.02 ng/mL and 0.47 ± 0.09 ng/mL, respectively, which was at stage I. Plasma levels of 17,20ß-P increased significantly at stage III (1.04 ± 0.06 ng/mL) and stage IV testes (1.28 ± 0.03 ng/mL) and then declined in post-spawned fish. This indicates that 17,20ß-P could also be a possible maturation-inducing steroid in this fish. The condition factor (K) significantly decreased during the testicular development and was lowest at spermiation stage (0.53 ± 0.02 %). The proportion of running male peaked concomitantly with the appearance of stage IV testes. Presence of germ cells of different developmental stages indicates that T. putitora male is a multiple spawner, and the information generated here is important for developing a captive breeding, culture and conservation programs for this endangered coldwater Himalayan fish species.