In-silico insights of ESBL variants and tracking the probable sources of ESBL-producing Escherichia coli in a small-scale poultry farm.

Commercial broiler farms face challenges of extended spectrum beta-lactamase (ESBL)-producing Escherichia coli transmitted from both vertical and horizontal routes. Understanding the dynamics of ESBL-E. coli transmission in compromised biosecurity settings of small-scale rural poultry farms is essential. This study aimed to elucidate the probable transmission pathways of ESBL-E. coli in such settings, employing phylogenetic analysis and molecular docking simulations to explore the catalytic properties of ß-lactamase variants. Sampling was conducted on a small-scale poultry farm in West Bengal, India, collecting 120 samples at three intervals during the broiler production cycle. E. coli isolates underwent resistance testing against eight antimicrobials, with confirmation of ESBL production. Genotypic analysis of ESBL genes and sequencing were performed, alongside molecular docking analyses and phylogenetic comparisons with publicly available sequences. Among 173 E. coli isolates, varying resistance profiles were observed, with complete resistance to cefixime and high resistance to amoxicillin and tetracycline. The incidence of ESBL-E. coli fluctuated over the production cycle, with dynamic changes in the prevalence of blaCTX-M-type and blaSHV-type genes. Phylogenetic analysis indicated partial clonal relationships with human clinical strains and poultry strains from the Indian subcontinent. Molecular docking confirmed the catalytic efficiencies of these ESBL variants. The study highlights probable vertical transmission of ESBL-E. coli and emphasizes drinking water as a potential source of horizontal transmission in small-scale poultry farms. Strict biosecurity measures could prevent the spread of antimicrobial-resistant bacteria in birds and their products in a small scale poultry farm.

Phytochemical Analysis and Demonstration of Antioxidant, Antibacterial, and Antibiofilm Activities of Ethnomedicinal Plants of North East India.

Ethnomedicinal plants are a rich reservoir of active compounds with potent pharmacological properties. Therefore, plants could serve as a source for the discovery of active antimicrobial and antioxidant agents and are focused because of their low toxicity, economic viability, easy availability, etc. In this regard, phytochemical analyses, viz. ß-carotene, total sugar, reducing sugar, vitamin C, total carotenoids, protein, total phenolic content (TPC), and total flavonoid content (TFC) of 20 ethnomedicinal plants of North East India (NEI) were evaluated in this study. The antibacterial activity against human pathogens and antioxidant potential of plant extracts was also demonstrated. The minimum inhibitory concentration (MIC80), minimum bactericidal concentration (MBC), and total antibacterial activity (TAA) of the active extracts were evaluated against Pseudomonas aeruginosa and Chromobacterium violaceum. The active extracts were also examined for antibiofilm as well as anti-pyocyanin activities against P. aeruginosa and anti-QS activity against C. violaceum at sub-MICs. The study demonstrated variable concentration of phytochemicals of the extracts, viz. ß-carotene (0.29-8.91 mg g-1), total sugar (2.92-30.6 mM), reducing sugar (0.44-14.5 mM), vitamin C (8.41-31.3 mg g-1), total carotenoids (14.9-267.0 mg g-1), protein (5.65-283 mg g-1), TPC (5.32-31.0 mg GAE/g DW), and TFC (1.74-68.2 mg QE/g DW). The plant extracts also exhibited potent antioxidant and antibacterial activities against both Gram-positive and Gram-negative bacteria. Some of the extracts also demonstrated significant biofilm inhibition and eradication, anti-pyocyanin, and anti-QS activities at sub-MICs. The selected ethnomedicinal plants are rich in phytochemicals and demonstrated potent antioxidant, antibacterial, and antibiofilm activities, thus could serve as the important source of novel antioxidant and antimicrobial agents.

Immune responses and protective immunity in Pangasius pangasius (Hamilton, 1822) as induced by outer membrane proteins of Edwardsiella tarda and aluminium hydroxide adjuvant complex.

Edwardsiella tarda is considered one of the important bacterial fish pathogens. The outer membrane proteins (OMPs) of E. tarda are structurally and functionally conserved, and immunogenic. This study assessed the effects of the OMPs of E. tarda CGH9 as a vaccine without aluminium hydroxide [AH] (T1) and with AH adjuvant (T2) on the respiratory burst (ROB) activity, lymphocyte proliferation of head kidney (HK) leukocytes, and serum antibody production in pangas catfish Pangasius pangasius. The ROB activity and lymphocyte proliferation of HK leukocytes increased in both vaccinated groups compared to the control. Nonetheless, the T2 group showed a gradual increase in ROB activity and lymphocyte proliferation of HK leukocytes up to 3-weeks post-vaccination (wpv). The serum antibody production in the T1 group decreased initially for up to 2-wpv and increased from 3-wpv; whereas, in the T2 group, the serum-specific antibody levels were significantly high from 1-wpv compared to control. Simultaneously, the protective efficacy in terms of relative percentage survival in the T2 group after injecting with a lethal dose of E. tarda CGH9 was high (89.00±15.56) compared to the T1 group (78.00±0.00). Furthermore, the catfish administered with a booster dose of E. tarda OMPs with or without AH adjuvant showed no additional increase in immune response or protective immunity. These results suggested that E. tarda OMPs and AH adjuvant complex has a higher potential to induce protective immunity, which may be a good choice as a vaccine to combat E. tarda infection in catfish.

Docking analysis of circulating CTX-M variants in multi-drug resistant, beta-lactamase and biofilm-producing E. coli isolated from pet animals and backyard livestock.

The generation of antimicrobial-resistant bacteria largely depends on the use of antimicrobials not only in humans but also in pet animals and livestock. The present study was conducted to detect the occurrence of beta-lactamase and biofilm-producing- E.coli in healthy pet and backyard livestock. The study also intended on molecular docking experiments to confirm the nature of the catalytic mechanism in ß-lactamase enzymes, encoded by the various blaCTX-M genotypes and phylogenetic analysis to reveal clonal relationship of the animal origin E. coli isolates with human clinical strains. The rectal swabs were collected from healthy dogs (n = 254), cats (n = 108), sheep (n = 119) and goats (n = 143) in India. In total 247 (76.47%) E. coli strains were identified as ESBL producers. The possession of ESBL-producers was significantly more (p < 0.05) in pets than in the backyard livestock. Most of the strains possessed blaCTX-M-15 like clones. E. coli strains possessing blaCTX-M-15.2, blaCTX-M-157, blaCTX-M-181 and blaCTX-M-218 like clones, isolated from pets were not reported earlier. The study detected 56.65% of E. coli strains as moderate or strong biofilm producers possessing biofilm-associated genes (csgA, rcsA, rpoS, sdiA). ESBL-producing E. coli showed phenotypical resistance to tetracycline (93.1%), azithromycin (89.8%), ampicillin (84.2%), cefotaxime (80.9%), doxycycline (82.5%), co-trimoxazole (80.9%), ampicillin/cloxacillin (76.9%). The CTX-M variants obtained in this study were modelled by the SWISS-MODEL and verified. Ligand having minimum binding energy, show the highest affinity of ß-lactamases for cefotaxime and cefpodoxime. The Gibbs free energy release for all 14 different complex ranges between -6.9 (CTX-M-15.2+cefpodoxime) to -5.3 (CTX-M-218+cefpodoxime) Kcal/mol. Phylogenetic analysis of the animal origin ESBL-E. coli strains revealed a partial clonal relationship with the clinical isolates of local human patients. The present study described the significant presence of biofilm and ß-lactamase producing, multi-drug resistant E. coli in pet animals having public health importance.

Ecofriendly phytofabrication of silver nanoparticles using aqueous extract of Cuphea carthagenensis and their antioxidant potential and antibacterial activity against clinically important human pathogens.

The green synthesis of nanoparticles (NPs) is the safest, ecofriendly, cost-effective, and non-hazardous approach of nanotechnology. In the current study, we described the green synthesis of silver nanoparticles (AgNPs) using Cuphea carthagenensis aqueous leaf extract as a reducing, capping, and stabilizing agent. The study aims at the synthesis, characterization, optimization, and determination of the antibacterial activity of Cc-AgNPs against clinically important human pathogens. Coating of cotton fabrics with Cc-AgNPs and their efficacy against skin infection causing organisms was also evaluated. Furthermore, antioxidant activity, growth assay and time kill assay of Cc-AgNPs were also performed in the study. The biosynthesized Cc-AgNPs were characterized by UV-visible spectrometry, energy-dispersive X-ray spectroscopy (EDX), X-ray diffraction (XRD), transmission electron microscopy (TEM), and Fourier transform infrared spectroscopy (FTIR). The spectroscopic and microscopic analysis demonstrated biosynthesis of face-centered cubic (fcc) crystalline spherical Cc-AgNPs with an average particle size of 10.65 ± 0.1 nm. Optimized peak synthesis of Cc-AgNPs was reported at pH7, 55 °C, 4 mM silver nitrate, and 5:45 (plant extract: silver nitrate). Cc-AgNPs exhibited potent antioxidant effect and antibacterial activity against both Gram-positive and Gram-negative bacteria. The lowest MIC (15 µg/ml) and MBC (25 µg/ml) values were reported against S. typhimurium. The Cc-AgNPs coated fabrics demonstrated potent antibacterial activity against tested strains. This application could be helpful in wound healing management. Furthermore, the hemolytic analysis demonstrated that Cc-AgNPs exhibit non-toxic nature against Red Blood Cells (RBCs) at the tested concentrations. In conclusion, the investigation demonstrated a fast, stable, and eco-friendly approach to the biosynthesis of Cc-AgNPs along with their antibacterial and antioxidant properties.

Exploring broilers and native fowls of Andaman and Nicobar Islands as a source of ß-lactamase-producing Enterobacteriaceae even with limited anthropogenic activities and docking-based identification of catalytic domains in novel ß-lactamase variants.

Objectives: The present study was conducted to detect the occurrence of ß-lactamase and biofilm-producing Escherichia coli, Salmonella, and Klebsiella in broilers and native fowl reared in the Andaman and Nicobar Islands, India. The study also included molecular docking experiments to confirm the nature of the catalytic domains found in the ß-lactamase variants obtained and to reveal the clonal relationship of the isolates with human clinical strains from the database. Materials and methods: A total of 199 cloacal swabs were collected from five poultry breeds/varieties (broiler, Vanraja, Desi, Nicobari, and layer) in three districts of the Andaman and Nicobar Islands. E. coli, Salmonella enterica, and Klebsiella pneumoniae were isolated by standard techniques and confirmed by PCR. Phenotypical ß-lactamase producers were identified by a double-disc test. The genes (bla CTX, bla SHV, bla TEM , and bla AmpC) were screened, and selected sequences of ß-lactamase variants were submitted to DDBJ. Homology modeling, model validation, and active site identification of different ß-lactamase variants were done by the SWISS-MODEL. Molecular docking was performed to identify the catalytic domains of the ß-lactamase variants. The selected ß-lactamase sequences were compared with the Indian ESBL sequences from human clinical strains in NCBI-GenBank. Results: In total, 425 Enterobacteriaceae strains were isolated from the collected samples. Klebsiella pneumoniae (42.58%) was found to be the most prevalent, followed by Salmonella enterica (30.82%) and E. coli (26.58%). The phenotypical antibiogram of all 425 isolates showed the highest resistance against oxytetracycline (61-76%) and the lowest against gentamicin (15-20%). Phenotypical production of ß-lactamase enzymes was observed in 141 (33.38%) isolates. The isolation rate of ß-lactamase producing E. coli, Salmonella enterica, and Klebsiella pneumoniae was significantly higher (p < 0.05) in the birds reared in the South Andaman district (25.6, 17.5, and 18.7%, respectively) than in Nicobar (11.5, 7.6, 7.1%, respectively). Genotyping of the ß-lactamase-producing isolates revealed the maximum possession of bla TEM, followed by bla SHV and bla CTX - M. The nucleotide sequences were found to be similar with bla CTX - M-15, bla SHV - 11, bla SHV - 27, bla SHV - 228, bla TEM - 1, and bla AmpC in BLAST search. Distribution of studied biofilm-associated genes in Enterobacteriaceae strains from different varieties of the birds revealed that the layer birds had the maximum possession, followed by Vanraja, Desi, broilers, and Nicobari fowls. The phylogenetic analysis of selected sequences revealed a partial clonal relationship with human clinical strains of the Indian subcontinent. Molecular docking depicted the Gibbs free energy release for 10 different macromolecules (proteins) and ligand (antibiotic) complexes, ranging from -8.1 (SHV-27 + cefotaxime) to -7 (TEM-1 + cefotaxime) kcal/mol. Conclusion and relevance: The study revealed ß-lactamase variants circulating in the fowl population of the Andaman and Nicobar Islands (India), even in remote places with low anthropogenic activity. Most of the strains possessed bla TEM - 1, followed by bla CTX - M-15. Possession of bla SHV - 11, bla SHV - 27, and bla SHV - 228 in poultry Enterobacteriaceae strains was not reported earlier from any part of the world. The phylogenetic analysis revealed a partial clonal relationship of ß-lactamase sequences with the human clinical strains isolated from the Indian subcontinent.

Histopathology of head kidney tissues in challenged rohu, Labeo rohita Hamilton after vaccinating with Aeromonas hydrophila antigens.

The study was conducted to evaluate the vaccination effects on rohu, Labeo rohita head kidney tissues while assessing the vaccine efficacy of Aeromonas hydrophila antigens. Six acclimatized rohu groups were immunized with three antigenic formulations (outer membrane proteins, somatic and whole-cell antigen) @ 200 µg/fish and also with equal volume of Freund's incomplete adjuvant (FIA), separately. Simultaneously, two non-vaccinated groups, i.e., injected with FIA (100µl), normal saline solutions (0.85%) and one control without injection were maintained for 28 days. All rohu were challenged with median lethal dose of A. hydrophila (2.85 × 106 cells/rohu) intraperitoneally. After 7 days, highest cumulative mortality (%) of ˃88% was found for all non-vaccinated groups. During histopathological observations in head kidney tissues of all treatment and control groups, numerous histopathological changes in the nephritic cells like mild loss of typical tubular epithelial lining, necrosis, thickening of renal epithelial lining, haemorrhages, inflammation, distorted and widening of the lumen with vacuolated surrounding and the constricted lumen of nephritic tubules were noticed for vaccinated rohu in contrast to non vaccinated groups before A. hydrophila challenge. In case of all non-vaccinated fish, including control, extensive degenerated and necrotized head kidney tissues were observed, whereas it was least observed in vaccinated rohu after 7 days A. hydrophila challenge. Results suggest that OMPs antigen along with FIA was the premier vaccine approach for improving resistance to Aeromonas disease and reduce mortality in rohu. Similarly, vaccination with all three antigenic formulations, preferably when applied along with FIA, can effectively protect the head kidney against A. hydrophila infection.

Efficacy evaluation of ethanolic extract of Tamarindus indica L. leaves as possible alternate therapy in septic arthritis model of rabbit.

BACKGROUND: Our previous study exhibited free radicals scavenging and antioxidant activities of ethanolic and aqueous extracts of Tamarindus indica L. leaves in chronic sodium fluoride poisoning in rats. Tamarindus indica L. seed extract was also reported to have anti-arthritic efficacy by inhibiting cartilage and bone degrading factors. Therefore, an attempt was made to evaluate the effects of ethanolic extract of Tamarindus indica L. leaves in septic arthritis. METHODS: The safety study was performed by oral dosing of ethanolic extract of the plant leaves at 2 g kg- 1 for consecutive 28 days in rabbits. Septic arthritis was induced in rabbits by single intra-articular inoculation of 104 c.f.u. of Staphylococcus aureus to the left stifle joint and was monitored by bacterial colony count, some relevant biochemical parameters and histopathological interpretation of the affected joint. For efficacy evaluation in septic arthritis, linezolid at 75 mg kg- 1 twice daily for 10 days and the ethanolic extract of Tamarindus indica L. at 500 and 1000 mg kg- 1 for consecutive 14 days were administered orally to the rabbits after 48 h of induction of arthritis. RESULTS: In sub-acute toxicity study of Tamarindus indica L. leaves ethanolic extract, no significant change between days was found for aspertate aminotransferase, alanine transaminase, alkaline phosphatase, blood urea nitrogen and creatinine compared to day 0 values of the same group. The bacterial colony count of synovial fluid following Staphylococcus aureus inoculation to left stifle joint was found to be 1.08 ± 0.47 and 1.19 ± 0.29 c.f.u. mL- 1 in ethanolic extract low dose and high dose groups respectively, on day 2 which was reduced to 0.057 ± 0.036 c.f.u. mL- 1 and nil on day 16. The test extract was also found to markedly reduce simultaneous glucose difference, total protein ratio of serum and synovial fluid, joint radius and joint narrowing. CONCLUSION: Ethanolic extract of Tamarindus indica L. leaves at 500 mg kg- 1 and 1000 mg kg- 1 produced anti-arthritic effects against S. aureus induced septic arthritis in rabbits. However, the ethanolic extract at 1000 mg kg- 1 orally for consecutive 14 days showed better effects in septic arthritis.

Decellularized Caprine Conchal Cartilage toward Repair and Regeneration of Damaged Cartilage.

Repair and regeneration of nasal and auricular cartilage thrust significant challenges in reconstructive surgery. The burgeoning clinical requirement is yet to endorse a satisfactory cartilage replacement matrix. In this regard, we have bioengineered cross-linked decellularized caprine conchal cartilage (DC) as biocompatible, durable, and nontoxic matrices. The DC matrices exhibited reduced DNA and sulfated glycosaminoglycan (sGAG) with a minimal effect on the collagen content. Further, histology and scanning electron micrographs revealed a significant loss of cellular bodies and the presence of a compact matrix consisting of intricate collagen fibers, when compared to unprocessed matrices. An in vitro biological assessment of the matrices exhibited an increased chondrocyte proliferation and viability with a significantly higher DNA, sGAG, and total collagen content. The matrices showed a 3-fold increase in the expression of cartilage-specific genes, namely, aggrecan, collagen II, and sox-9, and exhibited a minimal in vitro immunogenicity. Further, an in vivo assessment was performed by xenografting these caprine matrices in a rabbit model. The retrieved matrices showed a well-organized structural and cellular orientation with extracellular matrix formation after 3 months of implantation. No significant infiltration of plasma cells, macrophages, lymphocytes, and immature fibroblasts was recorded. Therefore, these affordable, resourceful, xenocompatible matrices offer a potential alternate in the repair and regeneration of nasal and auricular cartilages.

Novel decellularized animal conchal cartilage graft for application in human patient.

Restoration of the external ear and nose in human patients, in either congenital deformity or acquired defects, is a challenge in reconstructive surgery. Optimization of the currently available materials is necessary for rhinoplasty and microtia correction to avoid intraoperative manoeuvring and early rejection. The aim of this study was to develop cross-linked decellularized caprine conchal cartilages as biocompatible, robust, and non-toxic matrix template. The characterization of the decellularized tissue encompasses in vitro lymphoproliferation assay, cytotoxicity test, agar gel precipitation test, in vivo immunocompatibility study, histology, and determination of pro-inflammatory cytokines in animal model. Decellularized cartilage was implanted in human volunteer at R. G. Kar Medical College and Hospital, Kolkata, India, and samples were assessed histologically by retrieving those after 4 months. The processed cartilages were implanted in rhinoplasty (nine) and microtia patients (six) keeping autogenous cartilage graft as control up to 18 months after surgery. Primary outcomes were viability and safety of the material, both in animal model and human pre-application in actual site. Secondary outcomes included self-assessed clinical findings on gross examination. This study is under the ethical approval no. RKC/14 dated January 27, 2012. The in vitro cellular reactivity was less in processed cartilage protein than control. Histology of retrieved tissues in animal model and human volunteer showed no adverse reactions. Production of IL-2, IL-6, and TNF-α cytokines was lower at 4 weeks. The rhinoplasty and microtia operation in clinical patients utilizing the processed cartilage showed satisfactory recovery with improved facial look. These low cost, easily available, biocompatible, safe xenocartilage biomatrices of caprine conchal cartilage origin are very flexible in shape and size, enabling them as potential bioimplant for repair of nasal and auricular structure without any rejection or diverse biomedical applications.

Detection, characterization, and antibiogram of extended-spectrum beta-lactamase Escherichia coli isolated from bovine milk samples in West Bengal, India.

BACKGROUND: Milk is considered as complete food and an important part of human diet throughout the world including India. Bacterial contamination of milk such as Escherichia coli due to unhygienic condition and poor udder health can cause infections, especially in infants and elders or in immunocompromised persons. Possession of antimicrobial resistance genes by commensal bacteria present in milk makes the issue more serious. AIM: The study was aimed to isolate and characterize extended-spectrum beta-lactamase (ESBL)-producing E. coli from milk samples collected from different parts of West Bengal, India, to assess the potential risk associated with the food. MATERIALS AND METHODS: Around 182 milk samples were collected from apparently healthy cows reared by organized dairy farms in West Bengal. E. coli was isolated from collected samples as per standard methods followed by serotyping. The detection of ESBL-producing E. coli was done both phenotypically and genotypically by detecting the presence of bla CTX-M gene. Antibiogram of the ESBL-positive isolates was done using common 12 antibiotics by disc diffusion method. RESULTS: A total of 22 (12.1%) samples were found to be positive for E. coli in this study. Different serotypes such as O11, O20, O22, O34, O35, O128, O149, and UT were isolated from the collected samples. 12 (54.5%) E. coli strains showed the capability of producing ESBL, both phenotypically and genotypically with the presence of bla CTX-M gene. Antibiogram of these ESBL-positive isolates revealed the drugs such as colistin (100%), levofloxacin (83.33%), and imipenem (66.67%) to be highly sensitive against this pathogen but drugs such as cefotaxime (100%), ceftazidime (91.67%), amoxicillin/clavulanic acid (83.33%), tetracycline (75.00%), and gentamicin (58.33%) to be very much resistant. CONCLUSION: More than 50% of the E. coli strains prevalent in the bovine milk samples were positive for ESBL production and are resistant to most of the common antimicrobials which may be alarming for human health.

Prevalence of CTX-M-Producing Klebsiella spp. in Broiler, Kuroiler, and Indigenous Poultry in West Bengal State, India.

This study was undertaken to detect the prevalence of CTX-M-producing Klebsiella spp. in healthy broiler, indigenous, and kuroiler birds reared in West Bengal (India) during November 2014-February 2015. In addition to CTX-M gene, the study was also conducted to reveal the occurrence of other ß-lactamase and class I integron genes in Klebsiella spp. isolates along with their clonal relationship. A total of 321 cloacal swabs from healthy broiler, indigenous, and kuroiler birds were collected from different places of West Bengal, India. Klebsiella spp. isolation rate varies among different types of poultry birds (43.8-72.3%). In total, 33 (10.7%) Klebsiella spp. isolates were detected phenotypically as CTX-M producers and all the isolates possessed blaCTX-M in polymerase chain reaction. Whereas 17 (51.5%) and 16 (48.5%) Klebsiella spp. isolates possessed blaSHV, and blaTEM with blaCTX-M, respectively. None of the CTX-M-producing Klebsiella spp. isolates in this study possessed class I integron gene. Randomly amplified polymorphic DNA-based phylogenetic tree revealed the presence of clonal relationship among the CTX-M-producing Klebsiella spp. isolates, recovered from broilers and indigenous birds. This study identified broilers and indigenous game birds as a potential reservoir of CTX-M-producing Klebsiella spp., which could be transmitted to the human food chain directly or indirectly.

Characterization of Salmonella Gallinarum isolates from backyard poultry by polymerase chain reaction detection of invasion (invA) and Salmonella plasmid virulence (spvC) genes.

AIM: The aim was to characterize Salmonella enterica serovar Gallinarum isolated from backyard poultry by polymerase chain reaction (PCR) detection of virulence genes invasion (invA) and Salmonella plasmid virulence C (spvC). MATERIALS AND METHODS: Two strains of Salmonella serovar Gallinarum isolates used in this study were obtained from an outbreak of fowl typhoid in backyard Vanaraja fowl. PCR technique was used for detection of invA and spvC genes using standard methodology. The invA PCR product from one representative isolate was sequenced and compared with other related Salmonella serovars in GenBank data. RESULTS: Salmonella Gallinarum produced expected amplicons of invA and spvC gene products. Nucleotide sequence of 285 bp invA gene was deposited in GenBank with accession no. KX788214. Sequence analysis of invA gene was found conserved in Salmonella serovars and demonstrated 100% homology with closely related serovars of Salmonella. CONCLUSION: Invasion gene (invA) was found to be highly conserved in Salmonella Gallinarum and highly similar with closely related serovars. The isolates also contained plasmid-mediated spvC gene indicating possession of virulence plasmid.

Molecular characterization of Myxoboluscatmrigalae (Myxosporea: Myxobolidae) infecting the gill lamellae of carp Cirrhinusmrigala (Hamilton).

The present study attempted sequencing the 18S rRNA gene of Myxoboluscatmrigalae infecting the gill lamellae of carp, Cirrhinusmrigala and compared its genetic homology and phylogenetic characteristics with 18S rRNA genes of other Myxobolus spp. The infected fish had up to 3 small, creamy white plasmodia per gill filament with 30-50 spores each. The spore size was 17.90 ± 0.70 × 7.40 ± 0.40 µm. The sporoplasm contained two large nuclei of size 0.57 ± 0.09 µm and no iodinophilous vacuole. The DNA sequence of M.catmrigalae was clustered phylogenetically with other Myxobolus spp. infecting the gills of cyprinids available in GenBank, which showed 77-87 % homogeneity. On the phylogenetic tree, M.catmrigalae (KC933944) was clustered with M.pavlovskii (HM991164) infecting the gill lamellae of silver carp, Hypophthalmichthysmolitrix. The species most closely related to M.catmrigalae in GenBank was M.pavlovskii (AF507973) infecting the gill lamellae of big head carp, Aristichthysnobilis with 87 % homogeneity. This is the first report on molecular characterization of gill lamellae infecting M. catmrigalae.

Molecular characterization of Argulus bengalensis and Argulus siamensis (Crustacea: Argulidae) infecting the cultured carps in West Bengal, India using 18S rRNA gene sequences.

The present study characterized Argulus spp. infecting the cultured carps using 18S rRNA gene sequences, estimated the genetic similarity among Argulus spp. and established their phylogenetic relationship. Of the 320 fish samples screened, 34 fish (10.6%) had Argulus infection. The parasitic frequency index (PFI) was observed to be high (20%) in Hypophthalmichthys molitrix and Labeo bata. The frequency of infection was high in September (PFI: 17%) and October (PFI: 12.9%). The 18S rRNA sequences of five A. bengalensis (KF583878, KF192316, KM016968, KM016969, and KM016970) and one A. siamensis (KF583879) of this study showed genetic heterogeneity and exhibited 77-99% homology among the 18S rRNA gene sequences of Argulus spp. of NCBI GenBank database. Among the Indian Argulus spp. the sequence homology was 87-100%. Evolutionary pair-wise distances between Indian Argulus spp. and other Argulus spp. ranged from 0 to 20.20%. In the phylogenetic tree, all the crustaceans were clustered together as a separate clade with two distinct lineages. The lineage-1 comprised exclusive of Branchiura (Argulus spp.). All Argulus bengalensis clustered together and A. siamensis (KF583879) was closely related to Argulus sp. JN558648. The results of the present study provided baseline data for future work on population structure analysis of Indian Argulus species.

Seroprevalence of bluetongue in ruminants of Jharkhand.

AIM: This study was carried out to assess the presence of anti-bluetongue (BT) antibodies in sheep, goat and cattle of different agro-climatic zones of Jharkhand. MATERIALS AND METHODS: Serum samples were collected from apparently healthy as well as suspected sheep, goat and cattle from different districts of Jharkhand covering different agro-climatic zones. Serum samples were screened by indirect enzyme linked immunosorbent assay (iELISA) for detecting anti-BT antibodies. RESULTS: Out of a total of 480 animal serum samples (sheep-190, goats-210 and cattle-80) screened, 83 (43.68%) of sheep, 91 (43.33%) of goat and 46 (57.50%) of cattle sera were found positive. The % positivity ranged between 41% and 51% in different agro-climatic zones. The results showed slight higher seroprevalence, although not significantly, in cattle than sheep and goats in different agro-climatic zones of Jharkhand. CONCLUSIONS: The above data indicate widespread prevalence of BT virus antibodies in studied areas. The incidence of BT is not detected officially, so far. The present seroprevalence status of BT in Jharkhand indicates presence of BT infection in the state for the first time.

Isolation and purification of beta-lactoglobulin from cow milk.

AIM: The present study was undertaken to standardize a convenient method for isolation and purification of ß-lactoglobulin (ß-lg) from cow milk keeping its antigenicity intact, so that the purified ß-lg can be used for detection of cow milk protein intolerance (CMPI). MATERIALS AND METHODS: Raw milk was collected from Gir breed of cattle reared in Haringhata Farm, West Bengal. Milk was then converted to skimmed milk by removing fat globules and casein protein was removed by acidification to pH 4.6 by adding 3 M HCl. ß-lg was isolated by gel filtration chromatography using Sephacryl S-200 from the supernatant whey protein fraction. Further, ß-lg was purified by anion-exchange chromatography in diethylaminoethyl-sepharose. Molecular weight of the purified cattle ß-lg was determined by 15 percent one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and was analyzed by gel documentation system using standard molecular weight marker. RESULTS: The molecular weight of the purified cattle ß-lg was detected as 17.44 kDa. The isolated ß-lg was almost in pure form as the molecular weight of purified ß-lg monomer is 18kDa. CONCLUSION: The study revealed a simple and suitable method for isolation of ß-lg from whey protein in pure form which may be used for detection of CMPI.

Molecular and phylogenetic characterization of Thelohanellus qadrii (Myxozoa, Myxosporea, Bivalvulida) infecting the secondary gill epithelium of Indian major carp, Catla catla (Hamilton, 1822).

Myxosporean taxonomy which is traditionally based on the morphology of the myxospore stage, is in a state of flux given new insights provided by the expanding dataset of DNA sequences. To date, more than 40 species of Thelohanellus from India have been described according to morphometric characteristics. Nevertheless, molecular data on these histozoic myxosporean parasites of freshwater fish are scarce. In the present study, molecular characterizations of Thelohanellus qadrii infecting the secondary gill epithelium of Indian major carp Catla catla (Hamilton, 1822) and its phylogenetic relationship is reported. The sub-adult cultured catla were observed to have low to moderate gill myxosporean infections. The morphometry of mature spores was in compliance with original descriptions of T. qadrii. Based on the analysis of 18S rRNA gene, phylogenetic clusters which were established according to a consensus sequence, illustrated the taxonomic placement of a series of myxobolids. The DNA sequence homogeneity of T. qadrii (KF170928) with other Thelohanllus spp. ranged from 78% to 95% and formed a dichotomy with cyprinid gill lamellae infecting T. toyamai (HQ338729). Distance matrix results indicated a high genetic diversity among myxosporeans. The present report is the first on the molecular and phylogenetic characterizations of T. qadrii.

Evaluation of autologous bone marrow in wound healing in animal model: a possible application of autologous stem cells.

A study was conducted to evaluate the potential of autologous bone marrow-derived cells in comparison with buffy coat of autologous blood for rapid cutaneous wound healing in rabbit model. Three square full-thickness skin excisional wounds were created in 15 selected experimental animals (rabbit) divided randomly into three groups. The wound was treated with autologous bone marrow cells in plasma (group 1), buffy coat of blood in plasma (group 2) and autologous plasma as control (group 3). Wounds were observed for 30 days for granulation tissue formation, biochemical, histomorphological and histochemical evaluation. In this study, granulation tissue appeared significantly lesser in wounds of group 3 animals followed by group 2 and 1 animals. Neovascularisation, granulation tissue formation, denser, thicker and better arranged collagen fibres, reticulin fibres and elastin fibres formation was more in group 1 as compared with other groups. It was concluded that the application of bone marrow-derived nucleated cells into the wound margins resulted in early and significantly faster rate of complete healing as compared with buffy coat of autologous blood and autologous plasma (control). This approach may be beneficial in various surface wounds that heal at a slower rate and recommended for healing of various complicated wound in future.