Aeromonas hydrophila is considered an important pathogen of fish causing substantial economic loss in aquaculture worldwide. The objective of the current study was to assess the virulence potential of A. hydrophila strains isolated from gills of apparently healthy fish using phenotypic assays, PCR based genotypic tests, and in-vivo pathogenicity assays in Indian catfish, Clarias magur. For this, motile aeromonads (n = 50) were isolated from gills of apparently healthy freshwater food fish (n = 50) collected from domestic fish market. Gill swabs were incubated for primary enrichment in alkaline peptone water supplemented with cephalothin (10 mg/mL) for 12 hours followed by plating on Starch Ampicillin Agar. A single big yellow colony was selected from each sample, purified, and presumptively identified as Aeromonas with the help of Aerokey-II. Out of the fifty isolates of Aeromonas, five isolates of diverse origin (9C, 10G1, 7C, 10C and 4P) were confirmed to be A. hydrophila by automated bacterial identification system VITEK 2 with confidence interval of 90-98%. In the phenotypic assays, strains 9C and 10G1 showed high serum resistance, swimming and swarming activity and low biofilm producing capabilities, which were indicative of putative virulence. Both the strains belonged to genotype aah + act + alt + ascV + eno + lip + ast + on the basis of detection of virulence genes by PCR, i.e. extracellular haemolysin (aah), cytolytic enterotoxin (act), heat-labile cytotonic enterotoxin (alt), type-III secretion system (ascV), enolase (eno), lipase (lip) and heat-stable cytotonic enterotoxin (ast). In-vivo pathogenicity assays confirmed that both the strains were pathogenic to magur fish. The LD 50 for 9C strain was 6.81 × 10 4 CFU/mL and 7.62 × 10 5 CFU/mL for 10G1strain. In conclusion, our phenotypic and genotypic findings showed that A. hydrophila isolated from apparently healthy fish harbour number of important virulence genes/factors and could have important implications in triggering disease in farmed fish under stress.

Motile Aeromonas septicaemia (MAS), caused by Aeromonas hydrophila, is one of the most significant bacterial disease responsible for mortality in Indian catfish, Clarias magur, a potential aquaculture species in the Indian subcontinent. In fish, innate immunity elicited by pathogen recognition receptors (PRRs) plays an important role in providing protection against bacterial infection. Information on PRRs including Toll-like receptors (tlrs) and their response to bacterial pathogens remains unexplored in magur. Toll-like receptor 2 (tlr2), a phylogenetically conserved germ-line encoded PRR recognizes specific microbial structure and trigger MyD88-dependent signaling pathway to induce release of various cytokines responsible for innate immune response. In the present study, tlr2 gene of magur was characterized and downstream signaling was studied following challenge with A. hydrophila. The full-length cDNA of magur tlr2 (mtlr2) comprised of 3,066 bp with a single open reading frame of 2,373 bp encoding 790 amino acids having a theoretical pI value of 6.11 and molecular weight of 90 kDa. Structurally, it comprised of signal peptide (1–42aa), one leucine-rich repeat region (LRR) at N-terminal (LRR1-NT: 50–73 aa) and C-terminal (LRR-CT: 588–608 aa), twenty LRRs in between, one trans-membrane (Tm) domain (609–631aa) followed by cytoplasmic TIR domain (670–783aa). Phylogenetically, mtlr2 is closely related to pangasius and channel catfish. Highest basal expression of mtlr2, myd88 and il-1β in spleen, nf-kb in anterior kidney was observed. Lowest basal expression of mtlr2 in skin and myd88, nf-kb and il-1β in muscle was detected. Significant up-regulation of mtlr2 and downstream expression occurred at 3, 8, 24 h post infection to A. hydrophila in important immune organs such as liver, spleen, intestine and kidney. These findings highlight the vital role of tlr2 in eliciting innate immune defence against A. hydrophila infection.

Motile Aeromonas septicaemia (MAS), caused by Aeromonas hydrophila, is one of the most significant problem among the bacterial diseases responsible for causing mortality in catfishes. The objective this study was to understand the modulation of innate immune parameters and histopathological alteration in Indian catfish Clarias magur (Hamilton, 1822), following intraperitoneal injection with A. hydrophila. At 3 to 8 h post-infection (hpi), respiratory burst activity, myeloperoxidase, protease, total anti-protease and α2 macroglobulin increased significantly (p<0.05) indicating immune response of host against pathogen invasion at an early stage of infection. The total protein, albumin, albumin/globulin ratio and bactericidal activity were significantly decreased indicating the response of pathogen to suppress host immune response to establish infection. Serum bactericidal activity, bacterial agglutination titre and lysozyme activity increased significantly at 24 hpi. Histopathological examination of infected fish revealed the accumulation of melanomacrophage centres, congestion, hepatocyte degeneration and vacuolated hepatocytes of liver; hyaline droplet accumulation, lymphocytes infiltration and diffuse necrosis of renal tubule in kidney; unilateral fusion of secondary gill lamellae, leukocytic infiltration as well as dilated central venous sinus in gills. Knowledge regarding the immune response in magur could be useful for developing strategies for improving disease resistance against A. hydrophila infection.

No abstract

The reverse transcription quantitative real‐time PCR (RT‐qPCR) is widely preferred tool for expression analysis of target gene at mRNA level. However, in order to obtain significant biological comparison, data normalization to a stable endogenous internal control gene also called reference gene (RG) is of utmost importance. In the present study, we evaluated the expression stability of four commonly used housekeeping genes, that is beta actin, β‐actin; glyceraldehyde‐3‐phosphate dehydrogenase, GAPDH; elongation factor‐1 alpha, EF‐1α and 18S ribosomal RNA, 18S for qPCR data normalization. This study was conducted for two varying physiological (Group I: fed vs. non‐fed and Group II: earthen pond vs. fibre reinforced plastic tank reared) and one pathological condition (Group III: Aeromonas hydrophila injected vs. PBS injected) in seven different tissues of Indian catfish, Clarias magur, an economically valuable food fish of South‐East Asia. Tissue wise expression stability analysis using web‐based comprehensive tool Reffinder revealed that EF‐1α was the most stable RG in kidney, liver, spleen and gills of group I; spleen, intestine, muscle and brain in group II; and kidney, liver, intestine, muscle and brain in group III. β‐actin was the most stable RG in intestine of group I; liver and brain in group II; spleen and gill in group III. 18S was the most stable RG in muscle and brain of group I; kidney and gill in group II. GAPDH was the least stable RG among four selected genes under all the experimental conditions. These findings on selection of RGs will lead to precise interpretation of relative gene expression in different tissues by RT‐qPCR in C. magur in the context of physiological studies and bacterial infection.

No abstract

No abstract