In this study, a bacterial strain COFCAU_P1, isolated from the digestive tract of a freshwater teleost rohu (
Labeo rohita
), was identified as
Bacillus amyloliquefaciens
using 16S rRNA gene sequence ...analysis combined with amplification of species-specific
Bam
HI and barnase genes. The probiotic potential of the strain was evaluated using an array of in vitro tests along with safety and genetic analyses. The isolate showed potent antimicrobial response against several fish pathogenic bacteria, survived a wide pH range (2–9), and was resistant up to 10% bile salt concentration. With regard to the in vitro adhesion properties, the strain showed significantly high in vitro adhesion to mucus, auto and co-aggregation capacity, and cell surface hydrophobicity. The strain was non-haemolytic, able to produce extracellular enzymes, viz., proteinase, amylase, lipase, and cellulase, and showed significant free radical scavenging activity. A challenge study in rohu revealed the strain COFCAU_P1 as non-pathogenic. The presence of putative probiotic marker genes including 2, 3-bisphosphoglycerate-independent phosphoglycerate mutase, arginine/ornithine antiporter ArcD, choloylglycine hydrolase,
LuxS
, and E1 β-subunit of the pyruvate dehydrogenase complex was confirmed by PCR, suggesting the molecular basis of the probiotic-specific functional attributes of the isolate. In conclusion, the in vitro and genetic approaches enabled the identification of a potential probiotic from autochthonous source with a potential of its utilization in the aquaculture industry.
Motile
septicaemia (MAS), caused by
, is one of the most significant bacterial disease responsible for mortality in Indian catfish,
, 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 (
) and their response to bacterial pathogens remains unexplored in magur. Toll-like receptor 2 (
), 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,
gene of magur was characterized and downstream signaling was studied following challenge with
. The full-length cDNA of
(
) 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,
is closely related to pangasius and channel catfish. Highest basal expression of
,
and
in spleen,
in anterior kidney was observed. Lowest basal expression of
in skin and
88,
-
and
in muscle was detected. Significant up-regulation of
and downstream expression occurred at 3, 8, 24 h post infection to
in important immune organs such as liver, spleen, intestine and kidney. These findings highlight the vital role of
in eliciting innate immune defence against
infection.
Toll like receptor (TLR) signalling pathway plays a central role in inflammatory responses through the recognition of PAMPs and DAMPs. Toll-like receptor 4 (TLR4) is well known for the recognition of ...LPS of gram-negative bacteria leading to production of inflammatory cytokines. Modulation of a TLR pathway gene in different tissues indicates the functional importance of the tissues in innate immunity. The aim of this study was to clone and characterize TLR4 and evaluate its constitutive tissue distribution in Pangasianodon hypophthalmus, a commercially important fish species. The study was carried out at ICAR-CIFE, Mumbai during 2017. Partial nucleotide and amino acid sequences of TLR4 were identified in P. hypophthalmus and termed as phTLR4. Bio-informatics analysis of partial sequence revealed four putative LRR domains characteristic of TLRs. Nucleotide and amino acid sequences showed varying degree of identity with TLR4 sequences from other fish species in BLASTn and BLASTp analysis. Phylogenetic analysis placed the phTLR4 close to Ictalurus punctatus. TLR4 was found to be constitutively expressed in all the tissues studied. The significantly highest expression was recorded in intestine and spleen. The lowest expression was in muscle, heart and skin. House-keeping gene ß-actin was used as an internal control. The outcome of this research will be helpful for studying mRNA expression levels of phTLR4 under different conditions in different tissues of Pangasianodon hypophthalmus.
Assessment of antimicrobial resistance (AMR) in freshwater fish farms has been rarely investigated. Aeromonas are considered as an indicator organism for monitoring AMR in aquatic environment. ...Identification of the geographical areas harbouring AMR have implications for developing AMR mitigation strategies. The present study investigated the prevalence of AMR in freshwater fish farms (n = 124) in four districts (A-D) of Uttar Pradesh, India. The study revealed significantly higher AMR in fish farms of district D for ceftazidime, cefotaxime and trimethoprim-sulfamethoxazole. Similarly, AMR to ciprofloxacin and tetracycline was significantly higher in district A, and cefoxitin (FOX) in district B. District D also showed the highest number of MDR strains, AmpC and ESBL producers. Overall, maximum AMR was seen to β-lactams (35.5%) and aminoglycosides (14.5%). Twenty-five AMR phenotypes and eighteen AMR genotypes were detected in the study. The farms of district D showed significantly higher number of resistant phenotypes (≥3 antimicrobials). Furthermore, the study identified the species of Aeromonas contributing to AMR. Two clusters were observed: cluster I- resistant to FOX only, and cluster II (comprising of A. caviae and A. veronii bv veronii)- resistant to multiple antimicrobials and included majority of AmpC producers. A. caviae had significantly higher MAR than other Aeromonas species. The ARGs shared among the cluster II species and its close relative A. veronii bv sobria were blaTEM,sul1, tet(E) and blaCIT. Overall, our study identified the geographical districts harbouring higher AMR in the freshwater fish farms. These findings suggest the need for spatial assessment of AMR in aquaculture.
•Significantly higher prevalence of AMR in fish farms of district D•Highest prevalence of MDR strains, AmpC and ESBL producers in district D•Twenty-five AMR phenotypes and eighteen genotypes observed, mostly in combination with β-lactams•Two clusters of Aeromonas species observed: cluster II- A. caviae and A. veronii bv veronii, major contributors of AMR
Flavobacterium columnare is currently one of the important bacterial pathogens causing columnaris disease in several farmed fish species across diverse geographies. A presumptive columnaris disease ...outbreak in farmed catla, Catla catla (Hamilton), was investigated with the aim of isolating and identifying the causative pathogen. F. columnare (strain RDC-1) was isolated from gills of infected fish and identified by conventional biochemical methods, and through species specific polymerase chain reaction and sequencing of the 16S rDNA for molecular identification. Strain RDC-1 belonged to genomovar II with 99% similarity to available 16S rDNA sequences of F. columnare, and also shared 70% DNA-DNA relatedness with known strains of F. columnare. Bath immersion studies of RDC-1 showed development of columnaris disease in catla fingerlings within 7 days, with a cumulative mortality of 83.3%. This is the first molecular confirmation of Flavobacterium columnare as a fish pathogen of farmed Catla catla in India.
Mortalities were observed in several Pangasianodon hypophthalmus farms (n = 52) of Uttar Pradesh, North India during December 2020–January 2021. The affected fish were lethargic, swimming near the ...water surface and had white or grayish cotton-like patches over the body surface. Wet mount examination of the patches revealed presence of non-septate hyphae suggestive of oomycete infection. However, no parasites were observed in skin and gill scrapings. Histopathological examination of the affected skin tissue indicated severe destruction of the epidermis, degenerative changes and penetration of the oomycete hyphae in the dermis and underlying musculature. From the affected skin lesions, oomycete could be isolated which was identified as Saprolegnia parasitica based on morphological characteristics, and amplification and sequencing of the internal transcribed spacer region. Importantly, from the diseased P. hypophthalmus, no bacteria and viruses could be isolated from kidney, and pooled kidney and spleen tissues, respectively. Further, following experimental infection with S. parasitica zoospores by immersion method, 100% mortality was observed in P. hypophthalmus, indicating that the isolate was virulent. From the experimentally-infected striped catfish, S. parasitica could be reisolated and the histopathological lesions were similar to those observed in naturally infected fish, thereby fulfilling Koch’s postulates. Based on the gross and histopathological lesions, isolation of oomycete, molecular identification and bioassay, it was concluded that S. parasitica was responsible for mortalities in P. hypophthalmus farms. This forms the first report of mortalities of striped catfish due to oomycete S. parasitica infection and the findings will pave way for developing control measures for reducing losses in striped catfish farms due to saprolegniasis.
•Mortalities were observed in striped catfish farms in Uttar Pradesh, India during winter months.•Oomycete was isolated from skin lesions showing cotton wool-like growth.•The oomycete was identified as Saprolegnia parasitica on basis of amplification and sequencing of ITS region.•The disease was successfully reproduced with zoospores of S. parasitica by immersion challenge.
Toll-like receptor (TLR) 22 is a non-mammalian TLR, which is identified initially as a functional substitute of mammalian TLR3 in recognizing cell surface long dsRNA in teleosts. To understand the ...pathogen surveillance role played by TLR22 in an air-breathing catfish model the full-length cDNA of TLR22 was identified in Clarias magur and found to be consisted of 3597 nucleotides encoding for 966 amino acids. In the deduced amino acid sequence of C. magur TLR22 (CmTLR22) key signature domains such as one signal peptide, 13 LRRs, one transmembrane domain, one LRR_CT domain and an intracellular TIR domain could be identified. The CmTLR22 formed a separate cluster with other catfish TLR22 genes and situated within the TLR22 cluster in the phylogenetic analysis of teleost TLR groups. The CmTLR22 was constitutively expressed in all the 12 tested tissues of healthy C. magur juveniles with the highest transcript abundance in spleen followed by brain, intestine and head kidney. Following induction with the dsRNA viral analogue, poly (I:C), the level of expression of CmTLR22 was up-regulated in tissues such as kidney, spleen and gills. Whereas, in Aeromonas hydrophila-challenged C. magur, the expression levels of CmTLR22 was found to be up-regulated in gills, kidney and spleen, and down-regulated in liver. The findings of the current study suggest that the specific function of TLR22 is evolutionarily conserved in C. magur and might play a key role in mounting immune response by recognizing Gram-negative fish pathogen such as A. hydrophila and aquatic viruses in air-breathing amphibious catfishes.
Aims
To investigate the antibacterial activity of three (palmarosa, basil and rosemary) essential oils (EOs) on Aeromonas veronii and Aeromonas caviae, and determine minimum inhibitory concentration ...(MIC) of potent EO against tetracycline and sulfonamide resistant strains.
Methods and Results
Palmarosa oil (PMO) showed significantly (p < 0.05) higher inhibition zones against both A. veronii and A. caviae (n = 30) than basil and rosemary in the disk diffusion assay. The MIC (% v/v) of PMO ranged from 0.008% to 1.00%. The mean MIC was significantly higher for A. caviae (0.48 ± 0.24%) than A. veronii (0.21 ± 0.15%). Further, the MIC of PMO was compared in six groups: Group 1: Tetracycline Resistant A. veronii (TRV); Group 2: Tetracycline Resistant A. caviae (TRC); Group 3: Sulfonamide Resistant A. veronii (SRV); Group 4: Sulfonamide Resistant A. caviae (SRC); Group 5: Susceptible A. veronii (SV) and Group 6: Susceptible A. caviae (SC). No significant differences were observed between overall resistant (TRV+ SRV) and susceptible A. veronii (SV). However, in A. caviae, the resistant group had a lower MIC than the susceptible group. Moreover, the MIC was significantly lower for TRC (0.31 ± 0.11%) as compared to SRC (0.46 ± 0.10%). The time of kill of PMO for both the species of Aeromonas was 20–30 min.
Conclusion
Palmarosa oil exhibited significantly higher activity on A. veronii than A. caviae. The resistant strains of A. caviae were inhibited at a lower concentration than susceptible strains.
Significance and Impact of the Study
Palmarosa oil could be explored as an alternative antimicrobial agent for mitigating antimicrobial resistance and managing Aeromonas infection in fish and their risks to public health.
PDF
