Calcium phosphate nanoparticles conjugated with outer membrane vesicle of Riemerella anatipestifer for vaccine development in ducklings.

Biodegradable calcium phosphate nanoparticles offer a viable substitute for traditional adjuvants such as aluminum in vaccine production. Calcium phosphate nanoparticle adjuvanted with outer membrane vesicle (OMV) of gram negative bacteria may induce efficient immune response in the host. The present study was carried out to evaluate the potential of a mucosal vaccine formulation of calcium phosphate (CAP) nanoparticle using OMV of Riemerella anatipestifer (RA) as antigen against New Duck disease in ducks. The work was initiated with isolation, identification of RA, followed by OMV production and extraction. The CAP-OMV nanoparticle was prepared and characterized. The efficacy of the vaccine formulation and toxicity were studied in ducks. The average OMV yield in terms of protein concentration was found to be 122.33 ± 3.48 mg per liter of BHI broth. In SDS-PAGE, isolated OMVs exhibited presence of 16 distinct protein bands with molecular weight ranging from 142.1 to 12.1 kDa. Seven protein bands of 74.1, 69.3, 55.5, 50.6, 45.6, 25.1 and 13.1 kDa were detected relatively more distinct. The major bands detected in our findings were 42 kDa, 37 kDa and 16 kDa that corresponds to OmpA, OmpH, P6 respectively. The mean zeta size (±SD) and potential of the nanoparticle were 246.20 ± 0.53 nm and -25.60 ± 5.97 respectively. In transmission electron microscopy (TEM), the nanoparticles exhibited an average diameter of 129.80 ± 11.10 nm and displayed spherical morphology. The median protective dose (PD50) of CAP-OMV nanoparticle was 1881.10 µg of protein. Group I ducks received 3762 µg of protein (entrapped protein in CAP-OMV nanoparticle) via intra nasal route and it showed the highest serum IgG and secretory IgA level than other immunized groups. All experimental ducks were challenged with 10 × LD50 on 35 days of post primary immunization. Group I showed 100 % survivability in the challenge study. No gross and biochemical indication of acute or chronic toxicity were recorded. In conclusion, our results suggest that CAP-OMV nanoparticle can be a safe and efficient mucosal vaccine delivery system for RA, eliciting strong immune response in the host.

Livestock, pets and humans as carriers of methicillin-resistant Staphylococcusaureus and comparative evaluation of two PCR protocols for detection.

Staphylococcus aureus are Gram positive bacteria known to acquire antibiotic resistance rapidly and pose a major challenge to clinicians worldwide. Infections by methicillin resistant Staphylococcus aureus (MRSA) are usually associated with increased mortality and prolonging of treatment. Samples (n = 706) from diverse sources (livestock, pets, animal handlers, human hospital) were collected and screened for the presence of MRSA by phenotypic and genotypic methods. The incidence of Staphylococcus aureus was greater in goats (42.00%; 28.20 - 56.80%, confidence interval [CI] 95.00%) followed by cattle (13.50%; 9.20 - 18.80%, CI 95.00%), humans (12.90%; 9.30 - 17.40%, CI 95.00%) and dogs (12.90%; 8.10 - 19.20%, CI 95.00%). Significantly higher incidence of MRSA was observed in dogs (65.00%; 40.80 - 84.60%, CI 95.00%), compared to other hosts namely cattle (48.00%; 26.50 - 64.30%, CI 95.00%), humans (35.00%; 20.20 - 52.50%, CI 95.00%) and goats (10.00%; 1.20 - 30.40%, CI 95.00%). All the S. aureus isolates were further screened for thermostable nuclease (nuc gene) by polymerase chain reaction (PCR). The incidence of nuc gene in cattle, dog, goat and human were found to be 3.30% (1.30 - 6.60%, CI 95.00%), 5.20% (2.30 - 9.90%, CI 95.00%), 28.00% (16.20 - 42.50%, CI 95.00%) and 9.10% (6.00 - 13.00%, CI 95.00%), respectively. Comparative evaluation of two PCR primers (mecA-162 and mecA-310) indicated the former one as more rational choice for detection of MRSA. Overall, the results of our study indicated possible risk of zoonotic transmission of MRSA from canines.

A Comprehensive Review on Pharmacologically Active Phyto-Constituents from Hedychium species.

In this review, we describe and discuss the phytoconstituents present in Hedychium species and emphasize their potential as drug candidates. Though they are widely validated in vitro and in vivo models, to date, no efforts have been made to compile in a single review all the pharmacologically active phytoconstituents from Hedychium species, and their pharmacological and toxicity profile. In this study, we present a reinvestigation of the chemical constituents present in Hedychium species obtained from the essential oil and solvent extraction of the flowers, leaves and rhizomes under consideration. Key databases such as PubMed, Science Direct, Scopus, and Google Scholar amongst others were probed for a systematic search using keywords to retrieve relevant publications on this plant. An exhaustive electronic survey of the related literature on Hedychium species resulted in around 200 articles. Articles published between the years 1975-2021 were included. The studies conducted on either crude extracts, solvent fractions or isolated pure compounds from Hedychium species reported with a varied range of biological effects such as anti-inflammatory, analgesic, antidiabetic, potentially anti-asthmatic, and cytotoxic, among other related activities of the chemical constituents present in its essential oil and solvent extract deployed in this review. Traditional and herbal medication around the world that uses different parts of Hedychium species were considered for anti-inflammatory, skincare, analgesic, anti-asthmatic, anti-diabetic, antidotal uses, among others. These uses support the idea that chemical constituents obtained from solvent extraction may also exert the same action individually or in a synergistic manner. The review concluded that there is scope for computation and biological study to find out possible new targets for strengthening the potency and selectivity of the relevant compounds, and to find a commercial method for extraction of active pharmaceutical ingredients.

Immunopotential of Pasteurella multocida bivalent outer membrane protein-based vaccine entrapped in aluminum hydroxide nanoparticles.

Swine pasteurellosis is one of the most economically important diseases of pig caused by Pasteurella multocida (P. multocida) capsular types A and D. These organisms are commensals and opportunistic pathogens in the upper respiratory tract in pig. In the present study, we extracted whole outer membrane proteins (OMP) from P. multocida capsular types A and D and were mixed together in the ratio of 1:1 forming bivalent outer-membrane proteins. The bivalent OMP was adsorbed onto aluminum hydroxide nanoparticles. The size of aluminum hydroxide nanoparticles adsorbed outer membrane protein was found to be in the range of 125 to 130 nm. We observed that aluminum hydroxide nanoparticles adjuvanted bivalent OMP-based vaccine elicited quicker immune kinetics in terms of IgG response as compared to aluminum hydroxide microparticles adjuvanted bivalent bacterin vaccine against P. multocida capsular type A and D.

Densitometric analysis of rep-PCR data: Insight into genetic variability and transmission of Clostridium perfringens typed with an improved multiplex PCR.

An epidemiological study was conducted in North-East India (part of Indo-Burma biodiversity hotspot) to better understand the distribution, diversity, and transmission of Clostridium perfringens among livestock, pets, wild animals (captive), and humans. A total of 160 C. perfringens isolates were recovered from 642 diarrhoeic faecal samples with an isolation rate of 24.92%. Isolation rate was the highest among captive wild animals (37.5%) followed by dog (34.6%), human (33.8%), pig (32.7%), cattle (20.8%), goat (18.3%) and poultry (9.3%). Isolates were toxin typed using a seven gene multiplex PCR designed for simultaneous detection of cpa, cpb, cpb2, etx, iap, cpe and netB. The majority of isolates, 128 (80%) were of type A, followed by 17 (10.62%), 5 (3.12%), 4 (2.5%), 3 (1.87%), 2 (1.25%) and 1 (0.63%) isolates of type C, D, E, G, F and B, respectively. Beta 2 toxin gene was present in 65 (50%) of type A isolates, followed by 7 (41.2%), 4 (80%), 1(25%), and 1 (100%) of type C, D, G and B isolates, respectively. Beta 2 toxin has a high prevalence among dogs (28.6%), cattle (27.3%), and pig (20.8%) compared to humans, goat, wild animals, and poultry (1.2-14.3%). The prevalence of CPE and NetB toxin-positive strains was low, with only 3 (1.8%) and 5 (3.1%) isolates, respectively. Association of C. perfringens with diarrhoea in Civet Cat, Golden Langur, and Gray Langur has been reported for the first time. The genetic diversity and transmission of isolates were investigated using automated rep-PCR (Diversilab®, bioMérieux) using two densitometry-based matrices: modified Kullback-Leibler (KL) and Pearson's correlation (PC). The PC and modified KL matrices formed three distinct clusters with 59% and 27.2% similarity, respectively. C. perfringens diversity and transmission were best studied using modified KL matrix that placed more emphasis on the presence of bands rather than intensity. However, the PC method was found to be more suitable for differentiating strains within a toxin type, with slightly higher D-values.

Toxinotyping and molecular characterization of antimicrobial resistance in Clostridium perfringens isolated from different sources of livestock and poultry.

The present study was designed to understand the presence of antimicrobial resistance among the prevalent toxinotypes of Clostridium perfringens recovered from different animals of Tamil Nadu, India. A total of 75 (10.76%) C. perfringens were isolated from 697 multi-species fecal and intestinal content samples. C. perfringens type A (90.67%), type C (2.67%), type D (4%) and type F (2.67%) were recovered. Maximum number of isolates were recovered from dog (n = 20, 24.10%) followed by chicken (n = 19, 5.88%). Recovered isolates were resistant to gentamicin (44.00%), erythromycin (40.00%), bacitracin (40.00%), and tetracycline (26.67%), phenotypically and most of the isolates were found to be resistant to multiple antimicrobials. Genotypic characterization revealed that tetracycline (41.33%), erythromycin (34.66%) and bacitracin (17.33%) resistant genes were present individually or in combination among the isolates. Combined results of phenotypic and genotypic characterization showed the highest percentage of erythromycin resistance (26.66%) among the isolates. None of the isolates showed amplification for lincomycin resistance genes. The correlation matrix analysis of genotypic resistance showed a weak positive relationship between the tetracycline and bacitracin resistance while a weak negative relationship between the tetracycline and erythromycin resistance. The present study thus reports the presence of multiple-resistance genes among C. perfringens isolates that may be involved in the dissemination of resistance to other bacteria present across species. Further insights into the genome can help to understand the mechanism involved in gene transfer so that measures can be taken to prevent the AMR spread.

Phenolic rich Cocos nucifera inflorescence extract ameliorates inflammatory responses in LPS-stimulated RAW264.7 macrophages and toxin-induced murine models.

Anti-inflammatory and antinociceptive effects of the acetone extract of Cocos nucifera (CnAE), an important ingredient in several traditional drugs, have been studied using different in vitro and in vivo models. CnAE did not show any observable toxicity in RAW264.7 macrophages by MTT assay. The calorimetric analysis (total COX, 5-LOX, MPO, iNOS and NO), ELISA (IL-1ß, IL-6, TNF-α and PGE2) and qRT-PCR (IL-1ß, IL-6, TNF-α and NF-κB) were performed in LPS-induced RAW264.7 macrophages. Phosphorylation of NF-κBp65 and IκB was determined by western blotting. CnAE (100 µg/mL) remarkably inhibited total COX (68.67%) and 5-LOX (63.67%) activities, and subsequent release of iNOS, NO and PGE2 (p ≤ 0.05) in RAW264.7 cells treated with LPS. ELISA showed CnAE markedly decreased the level of pro-inflammatory cytokines IL-1ß (p ≤ 0.001), IL-6 (p ≤ 0.001) and TNF-α (p ≤ 0.001) in LPS treated RAW264.7 cells. CnAE (100 µg/mL) also significantly down-regulated the mRNA expressions of pro-inflammatory cytokines (IL-1ß, p ≤ 0.05; IL-6, p ≤ 0.01 and TNF-α, p ≤ 0.001) and NF-κB (p ≤ 0.001) against LPS-induction. Moreover, LPS-induced phosphorylation of IκB-α and NF-κB p65 was significantly inhibited by CnAE (100 µg/mL). In vivo anti-inflammatory studies showed that CnAE (400 mg/kg) significantly inhibited carrageenan-induced acute paw oedema (59.81%, p ≤ 0.001) and formalin-induced chronic paw oedema (52.90%, p ≤ 0.001) in mice. CnAE at a dose of 400 mg/kg also showed a significant anti-nociceptive effect on acetic acid-induced writhing (48.21%, p ≤ 0.001) and Eddy's hot plate methods. These findings suggest that CnAE has significant anti-inflammatory and anti-nociceptive properties, mainly attributed to the inhibition of NF-κB/IκB signalling cascade.

Enhanced Tribological and Bacterial Resistance of Carbon Nanotube with Ceria- and Silver-Incorporated Hydroxyapatite Biocoating.

Pertaining to real-life applications (by scaling up) of hydroxyapatite (HA)-based materials, herein is a study illustrating the role of carbon nanotube (CNT) reinforcement with ceria (CeO2) and silver (Ag) in HA on titanium alloy (TiAl6V4) substrate, utilizing the plasma-spraying processing technique, is presented. When compared with pure HA coating enhanced hardness (from 2.5 to 5.8 GPa), elastic modulus (from 110 to 171 GPa), and fracture toughness (from 0.7 to 2.2 MPa·m1/2) elicited a reduced wear rate from 55.3 × 10-5 mm³·N-1·m-1 to 2.1 × 10-5 mm³·N-1·m-1 in HA-CNT-CeO2-Ag. Besides, an order of magnitude lower Archard's wear constant and a 41% decreased shear stress by for HA-CNT-CeO2-Ag coating depicted the effect of higher hardness and modulus of a material to control its wear phenomenon. Antibacterial property of 46% (bactericidal) is ascribed to Ag in addition to CNT-CeO2 in HA. Nonetheless, the composite coating also portrayed exaggerated L929 fibroblast cell growth (4.8 times more than HA), which was visualized as flat and elongated cells with multiple filopodial protrusions. Hence, synthesis of a material with enhanced mechanical integrity resulting in tribological resistance and cytocompatible efficacy was achieved, thereupon making HA-CNT-CeO2-Ag a scalable potent material for real-life load-bearing implantable bio-coating.

Antioxidant and antibacterial hydroxyapatite-based biocomposite for orthopedic applications.

Post-implantation, vicinity acquired oxidative stress and bacterial infections lead to apoptosis with eventual bone-resorption and implant failure, respectively. Thus, in order to combat aforementioned complications, present research aims in utilizing antioxidant ceria (CeO2) and antibacterial silver (Ag) reinforced hydroxyapatite (HA) composite with enhanced mechanical and cytocompatible properties. Highly dense (>90%) spark plasma sintered HA-based composites elicits enhanced elastic modulus (121-133 GPa) in comparison to that of HA. The antioxidant activity is quantified using ceria alone, wherein HA-ceria and HA-ceria-Ag pellets exhibits ~36 and 30% antioxidant activity, respectively, accrediting ceria as a scavenger of reactive oxygen species, which was corroborated with the % Ce3+ change quantified by X-ray photoelectron spectroscopy. The HA-Ag pellet shows antibacterial efficacy of ~61% for E. coli and ~53% for S. aureus, while a reduction of ~59% for E. coli and ~50% for S. aureus is observed for HA-ceria-2.5Ag pellet, affirming Ag reinforcement as an established bactericidal agent. The enhanced hydrophobicity on all the HA-based composites affords a high protein adsorption (24 h incubation). Further, elevated hFOB cell count (~6.7 times for HA-ceria-Ag on day 7) with filopodial extensions (60-150 µm) and matrix-like deposition reflect cell-substrate intimacy. Thus, synergistic antioxidant ceria and antibacterial Ag reinforcement with enhanced mechanical integrity can potentially serve as cytocompatible porous bone scaffolds or bioactive coatings on femoral stems.

Comparative evaluation of hypoglycemic and hypolipidemic activity of various extract of Anogeissus latifolia bark in streptozotocin-induced diabetic rats.

Background Anogeissus latifolia Wall. (A. latifolia) bark has been traditionally used in the treatment of various diseases which includes diabetes and general debility. The present study was aimed to investigate the comparative hypoglycemic and hypolipidemic activity of various extracts of A. latifolia bark in streptozotocin-induced type 1 diabetic rats. Methods Acute toxicity was carried out at 2 g/kg dose of petroleum ether extract of A. latifolia bark (PEALB), chloroform extract of A. latifolia bark (CEALB) and methanol extract of A. latifolia bark (MEALB) in rats. Diabetes was induced by streptozotocin (STZ, 60 mg/kg, i.p.) and it was confirmed at 72 h. Diabetic rats received above extracts at 100 and 200 mg/kg doses for 28 days. Body weight and blood glucose level were determined at every week after the treatment schedule. Serum biochemical parameters and lipid profile levels were estimated at the end of the study. Results PEALB, CEALB and MEALB were non-toxic and no death was observed at 2 g/kg dose. Administration of MEALB at 100 and 200 mg/kg showed significant (p< 0.01, p< 0.05) improvement in body weight and reduction in blood glucose at third and fourth week of treatment. Altered serum biochemical parameters and lipid profiles level were brought to near normal level significantly (p<0.001) compared to diabetic control rats after the administration of both doses of MEALB. However, PEALB and CEALB did not exhibit significant hypoglycemic and hypolipidemic activity. Conclusions Our findings revealed that long-term (28 days) treatment of MEALB possesses significant hypoglycemic and hypolipidemic activity compared to PEALB and CEALB in type 1 diabetic rats and given evidence to the traditional use of A. latifolia bark in diabetes.

Curcumin Ameliorates Cisplatin-Induced Nephrotoxicity and Potentiates Its Anticancer Activity in SD Rats: Potential Role of Curcumin in Breast Cancer Chemotherapy.

Breast malignant neoplastic disease is one of the most complex diseases, as it is a multifactorial disease in which virtually all the targets are instantly or indirectly inter-reliant on each other. Cisplatin (CIS), an inorganic antineoplastic agent is widely utilized in the treatment of various solid tumors including breast cancer. Despite everything, its clinical use is limited, due to ototoxicity, peripheral neuropathy, and nephrotoxicity. The present work was directed to assess the combined result of curcumin (CUR) and CIS in 7, 12-dimethyl benz[a]anthracene (DMBA) induced breast cancer in rats and the prevention of nephrotoxicity induced by the latter. CIS-induced nephrotoxicity was assessed by change in body weight, kidney weight, altered levels of BUN, creatinine, TNF-α, IL-6, IL-8, IL-10, and histopathology of the kidney. Anticancer activity was assessed by measurement of tumor weight, tumor volume, % tumor inhibition, levels of PPAR-γ, and BDNF in mammary tumors and histopathology of mammary tumors. CUR pre-treatment mitigated nephrotoxicity by reducing the inflammatory markers (TNF-α, IL-6, and IL-8; p < 0.001). Further, it reduced mammary cancer via increasing the expression of PPAR-γ (p < 0.001) and decreasing the expression of BDNF (p < 0.001) in mammary tumors. It also reduced tumor volume, further postulating that CUR might adjunct the anticancer activity of the CIS. To the best of our knowledge, this is the first report, which showed that CUR ameliorated CIS-induced nephrotoxicity and improved its anticancer activity in DMBA induced breast cancer in female Sprague-Dawley rats.

Effect of ZnO morphology on affecting bactericidal property of ultra high molecular weight polyethylene biocomposite.

Bacterial infection of implants can be controlled by selective trapping of bacteria, followed with consequent killing by targeted antibacterial agents. Herein, the role of various ZnO morphologies, viz. micro-rods (R), nanoparticles (NP), and micro-disks (D) on antibacterial efficacy of ZnO via release of Zn(2+) and H2O2 is assessed, both as isolated powders and via incorporating them in cytocompatible ultra high molecular weight polyethylene (UHMWPE). Though ZnO is antibacterial, interestingly, all ZnO morphologies elicited a supportive growth of gram-negative bacteria (Escherichia coli) in culture medium (until 28-35 µg/ml). But, all ZnO morphologies did elicit bactericidal effect on gram positive bacteria (Staphylococcus aureus or Staphylococcus epidermidis) both in culture medium (for 0-2.5 µg/ml) or when incorporated (5-20 wt.%) into UHMWPE. The bactericidal mechanisms were quantified for various ZnO morphologies via: (i) H2O2 production, (ii) Zn(2+) release, and (iii) the presence of surface oxygen vacancies. On one hand, where only ZnO(NP) elicited release of H2O2 in the absence of light, maximum Zn(2+) release was elicited by ZnO(D). Interestingly, when ZnO is incorporated as reinforcement (5-20 wt.%), its antibacterial action against E. coli was vividly observed due to selective proliferation of bacteria only on friendly UHMWPE matrix. Hence, luring bacteria on affable UHMWPE surface can be complemented with their targeted killing by ZnO present in composite.

Emergence of oriental theileriosis in cattle and its transmission through Rhipicephalus (Boophilus) microplus in Assam, India.

AIM: The aim of the present study was to investigate the presence of Theileria in blood samples of crossbred and indigenous adult cows raised under unorganized small scale farming system in a Babesia and Anaplasma endemic geographical area from Assam, India and to see its transmission through Rhipicephalus (Boophilus) microplus ticks. MATERIALS AND METHODS: For the present study, 57 clinical cases of cattle suspected to be of hemoparasitic infections were taken into consideration. The parasites were identified based on morphology in giemsa stained blood smear followed by polymerase chain reaction (PCR). Sera samples were tested for T. annulata antibodies in plate and Dot-ELISA. PCR was also conducted in eggs of Rhipicephalus (Boophilus) microplus tick collected from a Theileria orientalis positive animal. RESULTS: PCR amplified 1124, 776, and 160 bp DNA fragments of B. bigemina (64.91%), T. orientalis (21.05%) and A. marginale (14.03%), respectively. This assay further conducted in 12 T. orientalis positive blood samples with primers of Buffeli, Chitose, and Ikeda variants of T. orientalis showed 3 samples positive to Ikeda type and none for Buffeli and Chitose. Babesia bovis and Theileria annulata specific primers also did not amplify any fragment during the PCR assay of the blood samples. Further, all sera samples tested negative to T. annulata antibodies in Plate and Dot-ELISA. PCR conducted in eggs of R (B).microplus tick collected from a T. orientalis positive animal revealed presence of the parasite DNA. Gradual improvement in physical condition leading to complete recovery in 10 out of 12 T. orientalis infected clinical cases treated with buparvaquone(at 2.5mg/kg.b.wt I/M) was the feedback obtained from field veterinarians and the cattle owners. CONCLUSION: The present investigation represents the first report of occurrence of T. orientalis in cattle of Assam with involvement of pathogenic Ikeda strain in clinical outbreaks and its possible natural transmission by R (B). microplus through the transovarian mode.

Topical amiloride delays healing of deep thermal wounds in albino rabbits.

The present investigation was undertaken to explore the ulcer healing properties of three dosage schedules of different strengths of topically administered amiloride solution in thermally produced skin ulcers on the side of the midline on the back of the rabbits. Four circular thermal burns about 4.0 cm in diameter (two on either side of the midline) were made 4.0 cm apart on the pre shaved back of each anaesthetized (with thiopentone sod., 45 mg/kg, ip) rabbit with a heated template, applied for 30 sec. Three days later under anaesthesia, the burns were excised removing epidermis, dermis and subcutaneous tissue. The animals were randomly divided into various groups. Ulcers on one side of the midline were treated with normal saline and served as control, whereas those on the other side were treated with amiloride solutions. Each ulcer was observed for its size, slough formation and any sign of irritation on alternate days until healing was complete. Healing of ulcers were significantly delay in comparison to control group with all the strength of amiloride (0.02%, 0.5% and 1%) in all the dosage schedule (o.d., b.i.d. and q.i.d.) in terms of days required for complete healing (except with b.i.d. dosage schedule of 0.5% & q.i.d. dosage schedule of 1% amiloride solution), ulcer size and area under the size time curve (except with b.i.d. &q.i.d. dosage schedule of 1% amiloride solution). Thus, it is concluded that unlike mechanical ulcers topical amiloride delays wound repair in deep burn lesions.