Synergistic Effect between Eugenol and 1,8-Cineole on Anesthesia in Guppy Fish (Poecilia reticulata).

This study aimed to evaluate the synergistic effect between eugenol and 1,8-cineole on anesthesia in female guppy fish (Poecilia reticulata). Experiment I evaluated the concentrations of 0, 12.5, 25, 50, and 75 mg/L of eugenol and 0, 100, 200, 300, and 400 mg/L of 1,8-cineole for times of induction and recovery from anesthesia. Experiment II divided fish into 16 study groups, combining eugenol and 1,8-cineole in pairs at varying concentrations, based on the dosage of the chemicals in experiment I. The results of the anesthesia showed that eugenol induced fish anesthesia at concentrations of 50 and 70 mg/L, with durations of 256.5 and 171.5 s, respectively. In contrast, 1,8-cineole did not induce fish anesthesia. In combination, using eugenol at 12.5 mg/L along with 1,8-cineole at 400 mg/L resulted in fish anesthesia at a time of 224.5 s. Increasing the eugenol concentration to 25 mg/L, combined with 1,8-cineole at 300 and 400 mg/L, induced fish anesthesia at times of 259.0 and 230.5 s, respectively. For treatments with eugenol at 50 mg/L combined with 1,8-cineole at 100 to 400 mg/L, fish exhibited anesthesia at times of 189.5, 181.5, 166.0, and 157.5 s. In the case of eugenol at 75 mg/L, fish showed anesthesia at times of 175.5, 156.5, 140.5, and 121.5 s, respectively. The testing results revealed that 1,8-cineole as a single treatment could not induce fish anesthesia. However, when supplementing 1,8-cineole in formulations containing eugenol, fish exhibited a significantly faster induction of anesthesia (p < 0.05). Furthermore, all fish that underwent anesthesia were able to fully recover without any mortality. However, the shorter anesthesia duration resulted in a significantly prolonged recovery time. In conclusion, eugenol and 1,8-cineole work better together as anesthetics than when used separately, and demonstrated the safety of using these anesthetic agents on guppy fish.

Self-emulsifying drug delivery systems (SEDDS) containing Cymbopogon citratus essential oil: Enhancing the stability and acaricide efficacy against Rhipicephalus (Boophilus) microplus.

The objectives of this study were to develop a self-emulsifying drug delivery system (SEDDS) to enhance the stability and efficacy of Cymbopogon citratus essential oil or lemongrass oil (LEO) against cattle tick larvae and engorged females. The system with the highest oil loading in SEDDS was composed of LEO (23.33%w/w), Tween 80: SGKH 4000 in a 2:1 ratio as surfactant (66.67%w/w), and propylene glycol as co-surfactant (10%w/w). The selected SEDDS-LEO has a particle size of 18.78 nm with a narrow size distribution (polydispersity index of 0.27). Notably, the stability of SEDDS was superior to that of the original oil, both during long-term storage and under accelerated conditions. SEDDS-LEO at oil concentrations ranging from 1.458% to 5.833% w/v showed a significantly higher percentage of egg-laying reduction against adult ticks compared with the original oil at the same concentrations (p < 0.05). Furthermore, SEDDS-LEO demonstrated greater larvicidal efficacy than the original oil, with lower LC50 and LC90 values of 0.91 mg/mL and 1.20 mg/mL, respectively, whereas the original oil's LC50 and LC90 values were 1.17 mg/mL and 1.74 mg/mL, respectively. Our findings indicate that SEDDS-LEO is a promising candidate for use as an acaricide in the control of tick populations in dairy cattle.

Effects of Encapsulation of Caesalpinia sappan L. with Cyclodextrins for Bovine Mastitis.

The main components of Caesalpinia sappan L. (CS) are brazilin and brazilein, which show high potential in pharmacologic applications. However, these have been drastically limited by the poor water solubility and stability. The present study investigates the formation of inclusion complexes F1, F2, and F3 between CS and ß-cyclodextrin (ßCD), hydroxypropyl-ß-cyclodextrin (HPßCD), and methyl-ß-cyclodextrin (MßCD), respectively. These complexes were characterized by Fourier transform infrared spectroscopy (FT-IR). The results showed that the highest encapsulation efficiency and loading capacity of CS extract were 44.24% and 9.67%, respectively. The solubility and stability of CS extract were significantly increased through complexation in phase solubility and stability studies. The complexes F1-F3 showed mainly significant antibacterial activities on gram-positive bacteria pathogens causing mastitis. Moreover, the expression levels of COX-2 and iNOS were significantly decreased in LPS-induced inflammatory cells at concentrations of 50 and 100 µg/mL. In addition, treatment of complex F3 (CS/MßCD) in bovine endothelial cells remarkably increased the chemokine gene expression of CXCL3 and CXCL8, which were responsible for immune cell recruitment (9.92 to 11.17 and 8.23 to 9.51-fold relative to that of the LPS-treated group, respectively). This study provides a complete characterization of inclusion complexes between CS extract and ßCD, HPßCD, and MßCD for the first time, highlighting the impact of complex formation on the pharmacologic activities of bovine mastitis.

Titanium Dioxide Nano-Formulation: Characterization, Antimicrobial Activity, and Wound Healing in Animals.

The use of metal oxide nanoparticles as an alternative antimicrobial agent has gained attention due to the increasing problem of antimicrobial resistance. Understanding its properties and potential benefits can contribute to the development of more effective and sustainable treatments in veterinary medicine. The aim of this study was to characterize TiO2-NP formulations and evaluate their antibacterial and wound healing abilities. The diameters and zeta potentials were determined using the Zetasizer in conjunction with dynamic light scattering. The agar-well diffusion method, time-kill kinetic assay and crystal violet assay were used to evaluate their antimicrobial activities. Wound healing assays were conducted both in vitro and in vivo. The study demonstrated that TiO2-NP formulations exhibit significant antimicrobial properties against various bacterial strains such as S. aureus and E. coli. No measurable E. coli growth was observed within a 15-min period following exposure to TiO2-NP formulations. The TiO2-NP formation can improve wound healing by enhancing cell migration and collagen formation in both in vitro and in vivo conditions. In summary, our study suggests that TiO2-NP has the potential for use as an antimicrobial agent for animal wound treatment due to its ability to suppress bacterial growth and biofilm formation, as well as to enhance wound healing.

Development of loop-mediated isothermal amplification for rapid detection of sporotrichosis caused by Sporothrix schenckii.

Background and Aim: Sporothrix schenckii is the causative agent of sporotrichosis, which most commonly causes lymphocutaneous infections in immunocompromised hosts. This pathogen infects dogs, cats, cattle, and buffaloes and can potentially infect humans. Diagnosis by fungal culture is lengthy, and although there are several clinical diagnoses and molecular methods, these are complicated and time-consuming for veterinarians. This study aimed to develop a visual diagnostic assay that is less time-consuming and can be used by veterinarians to screen for sporotrichosis. Materials and Methods: To develop a loop-mediated isothermal amplification (LAMP) assay for sporotrichosis, primers specific for fragments of the 18S rRNA gene of S. schenckii were designed. Then, the time and temperature were optimized to successfully achieve LAMP. Ten-fold serial dilutions of DNA were used to determine the detection limit using both LAMP and nested polymerase chain reaction (nPCR) assays. Results: The optimal LAMP conditions were incubation at 73°C for 30 min. Agarose gel electrophoresis revealed a ladder-like pattern of the LAMP product, and a sky-blue color indicated a positive result. A comparison of the LAMP assay with nPCR revealed that it was 10 times more sensitive than nPCR, with a detection limit of 10 pg. The use of a heat box compared with a thermocycler gave the same results. Conclusion: Loop-mediated isothermal amplification gives good results and may represent a future alternative diagnostic tool for screening fungal pathogens before the results of conventional fungal cultures are received. However, this method should be further studied to clarify its use with clinical samples.

Impact of Storage Conditions and Mold Types on Aflatoxin B1 Concentration in Corn Residue used as Dairy Feed in Small Holder Dairy Farms, Thailand.

The aims of this study were to determine the impact of storage practice and mold types on mold growth and aflatoxin B1 (AFB1) concentration in corn residue from local seed corn plants, the main roughage source of dairy farms in the northern region in Thailand. A total of 223 samples from 2 types of corn residue - dried and wet - were collected. Mold contamination was determined by spread plate technique, and aflatoxin B1 (AFB1) quantification was performed by a commercial enzyme-linked immunosorbent assay. Multivariate linear models were created to determine factors associated with fungal quantity and AFB1 concentration. Results showed that the presence of Cladosporium spp. in the samples was associated with a lower risk of AFB1 contamination (P<0.05). In addition, appropriate storage practices, e.g. keeping feeds under a roof and using floor canvas under feed piles, gave lower risk of mold contamination and decreasing AFB1 contamination.

Performance of Loop-Mediated Isothermal Amplification Technique in Milk Samples for the Diagnosis of Bovine Tuberculosis in Dairy Cattle Using a Bayesian Approach.

This study aimed to estimate the sensitivity (Se) and specificity (Sp) of loop-mediated isothermal amplification (LAMP) and single intradermal tuberculin (SIT) tests for the diagnosis of bovine tuberculosis (bTB) in dairy cattle in Thailand using a Bayesian approach. The SIT test was performed in 203 lactating dairy cattle from nine dairy farms located in Chiang Mai province, Thailand. Milk samples were collected for the LAMP test. Kappa analysis was performed to determine the agreement between the two tests. A one-population conditional independence Bayesian model was applied to estimate the Se and Sp of the two tests. Of 203 dairy cattle, 2 were positive for the SIT test using standard interpretation, whereas 38 were positive for the LAMP test. A poor agreement (kappa = 0) was observed between the two tests. The median Se and Sp of the SIT test using standard interpretation were 63.5% and 99.1%, respectively. The median Se and Sp of the LAMP test were 67.2% and 82.0%, respectively. The estimated true prevalence of bTB was 3.7%. The LAMP test with milk samples can potentially be used as a non-invasive screening test for the diagnosis of bTB in dairy cattle.

Elucidating Pathway and Anesthetic Mechanism of Action of Clove Oil Nanoformulations in Fish.

Clove oil (CO), an essential oil of Syzygium aromaticum, has been reported as an anesthetic for many fish species. However, its insoluble properties require a suitable delivery system for its application. In the present study, nanoformulations of CO as a nanoemulsion (CO-NE), a self-microemulsifying drug-delivery system (CO-SMEDDS), and a self-nanoemulsifying drug-delivery system (CO-SNEDDS) were prepared for delivering CO. Zebrafish were used as a fish model to investigate oil pathways. The result shows fluorescence spots of fluorescence-labeled CO accumulate on the gills, skin, and brain. All CO nanoformulations significantly increased penetration flux compared to CO ethanolic solution. Investigation of the anesthetic mechanism of action using a rat brain γ-aminobutyric acid subtype A (GABAA) receptor-binding test demonstrates that CO and its major compound, eugenol, modulate [3H]muscimol binding. CO-NE exhibited a concentration-dependent binding activity with an EC50 value of 175 µg/mL, significantly higher than CO solution in dimethyl sulfoxide. In conclusion, CO enters the fish through the skin and gills. The anesthetic mechanism of action of CO is based on modulation of [3H] muscimol binding to GABAA receptors. Among three nanoformulations tested, CO-NE is the most effective at increasing permeability and enhancing the receptor-binding activity of the oil.

Prevalence of Microsporum canis from Pet Cats in Small Animal Hospitals, Chiang Mai, Thailand.

Dermatophytosis is a disease caused by dermatophytes, a group of fungi that can cause disease both in humans and animals. The important genera that are pathogenic in animals include Trichophyton and Microsporum. Microsporum canis is an important species because it can cause zoonosis and is commonly found in domestic animals. Cats, which live very close to humans, may expose humans to this pathogen. This research focused on the epidemiology of M. canis found in cats. Hair samples were collected via the Mackenzie technique from cats with and without skin lesions, preliminarily examined with 10% KOH preparation, and cultured for fungal identification. Samples were confirmed with molecular techniques including polymerase chain reaction, gel electrophoresis, and sequencing. Samples were collected from 138 cats located in 93 households, 43 from cats with skin lesions (31.16%) and 95 from cats without skin lesions (68.84%). Eighteen cats with lesions (13.04%) and ten cats without lesions (7.2%) were found to carry M. canis. In eleven of the eighteen cats both with skin lesions and positive for M. canis (61.11%), the pathogen was found both at the site of the lesion and at other sites in the body. Because the pathogen can be found in the hair of cats with and without skin lesions, owners, keepers, veterinarians, and others who come into contact with these animals are at risk of infection if they are not aware or do not take precautions after contact with them.

Silver nanoparticles (AgNPs) fabricating potential of aqueous shoot extract of Aristolochia bracteolata and assessed their antioxidant efficiency.

This research was performed to evaluate the silver nanoparticles (AgNPs) fabricating potential of aqueous shoot extract of Aristolochia bracteolata and also assess the free radicals scavenging potential of synthesized AgNPs. The results obtained from this study showed that the aqueous shoot extract of A. bracteolata has the potential to synthesize the AgNPs and it was initially confirmed by color change in the reaction blend as yellow to dark brownish. Subsequently, a clear absorbance peak was found at 425 nm in UV-visible spectrum analysis. The functional groups involved in the capping and stabilization of AgNPs were confirmed by Fourier Transform-Infrared spectroscopy (FTIR) analysis and recorded about 10 sharp peaks 3688, 3401, 2980, 2370, 1948, 1642, 1480, 1280, 782, and 628 cm-1. The Scanning Electron Microscope (SEM) and Transmission Electron Microscope (TEM) observations revealed that the predominant shape of the AgNPs was spherical and size ranged from 41.43 to 60.51 nm. Interestingly, the green fabricated AgNPs showed significant free radicals scavenging activity and were confirmed with ferric reducing assay, 1, 1-diphenyl-2-picryl-hydrazyl (DPPH), H2O2 radicals, and 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radicals scavenging activity. Thus, after a few in-vivo antioxidant studies, Aristolochia bracteolata-mediated AgNPs can be considered as an antioxidant agent.

Fabrication, characterization, anti-inflammatory, and anti-diabetic activity of silver nanoparticles synthesized from Azadirachta indica kernel aqueous extract.

The Azadirachta indica is an excellent and pharmaceutically valuable phytochemicals enriched traditional medicinal plant. The purpose of the research was to assess the ability of A. indica aqueous kernel extract to synthesize silver nanoparticles as well as their anti-inflammatory and anti-diabetic activity in vitro. The obtained results state that the aqueous kernel extract of A. indica can fabricate the silver nanoparticles and be confirmed by standard analytical techniques. Under UV-visible spectrophotometer analysis, the absorbance peak was found at 430 nm was related to the surface plasmon resonance of silver nanoparticles. The FTIR (Fourier-transform infrared spectroscopy) analysis revealed that numbers of functional groups belong to the pharmaceutically valuable phytochemicals, which act as reducing, capping, and stabilizing agent on silver nanoparticles synthesis. The size and shape of the silver nanoparticles were examined as 19.27-22.15 nm and spherical in shape. Interestingly, this kernel fabricated silver nanoparticles possess a reasonable anti-inflammatory (69.77%) and anti-diabetic (73.5%) activity at 100 µg mL-1 and these were partially comparable with standards (anti-inflammatory: 81.15%; anti-diabetic: 87.9%). Thus, the aqueous kernel extract fabricated silver nanoparticles can be considered for further in-vivo study to assess the practical possibility to promote as a pharmaceutical agent.

Green synthesis of Zirconium nanoparticles using Punica granatum (pomegranate) peel extract and their antimicrobial and antioxidant potency.

The biosynthesis of metal oxide nanoparticles provides an excellent alternative to the chemical synthesis approach. The aim of the current study was a green and eco-friendly synthesis of zirconium nanoparticles (ZrNPs) from fruit peels of Punica granatum (Pomegranate). The synthesis of ZrNPs was confirmed using a UV-visible spectrophotometer. The functional groups present on surface of ZrNPs were analyzed using FTIR. The average size of obtained ZrNPs was analyzed using SEM and DLS and it was around 20-60 nm. The antimicrobial activity of obtained ZrNPs was tested against Gram-positive strains (Bacillus subtilis and Staphylococcus aureus), Gram-negative strains (Escherichia coli and Klebsiella pneumoniae) and Fungi (Aspergillus niger) by agar well diffusion method. ZrNPs showed maximum zone of inhibition against S. aureus (19 mm) and A. niger (18 mm) at the maximum concentration of 200 µg/mL. The antioxidant scavenging activity of obtained ZrNPs was analyzed using the following methods: DPPH radical scavenging activity, Hydroxyl radical scavenging activity, Ferric reducing antioxidant power and hydrogen peroxide radical scavenging activity. This the first and foremost study on ZrNPs synthesized using P. granatum fruit peel extract reporting their efficacy as antimicrobial agents against Bacteria and Fungi. Considering the tolerance of zirconium towards human body, it can also be used as antimicrobial coating material on human implants.

A novel synthesis, analysis and evaluation of Musa coccinea based zero valent iron nanoparticles for antimicrobial and antioxidant.

Zerovalent Iron Nanoparticles (MC-ZVI NPs) were synthesized from Musa coocinea peel extract as reducing and stabilizing agent using a novel synthesis technique. The synthesis of MC-ZVI NPs was confirmed using UV-vis spectroscopy showing a sharp absorption peak at 341 nm. Further the chemical and structural characterization of MC-ZVI NPs were performed using Fourier Transform Infrared spectroscopy (FTIR), Scanning Electron Microscopy (SEM), and Dynamic Light Scattering technique (DLS). FTIR analysis revealed the presence of phytochemical molecules associated with the MC-ZVI NPs. SEM analysis revealed the synthesized MC-ZVI NPs were in spherical shaped, while DLS analysis confirmed the synthesis of poly dispersed and non-homogenous MC-ZVI NPs. The antimicrobial efficacy of MC-ZVI NPs synthesized using Musa coccinea peel extract was tested against bacterial (Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, and Bacillus subtilis) and fungal (Aspergillus niger) pathogens. But MC-ZVI NPs exhibited maximum of 19 mm zone of inhibition against B. subtilis and A. niger. Further the free radical scavenging activity MC-ZVI NPs was confirmed using DPPH, hydroxyl radical, hydrogen peroxide, FRAP assay showing displayed effective antioxidant activity. Thus, the present idea will give a fast and cost effective approach to synthesize MC-ZVI NPs with antimicrobial property for application in biomedical purposes.

Phytochemical and Safety Evaluations of Zingiber ottensii Valeton Essential Oil in Zebrafish Embryos and Rats.

Zingiber ottensii Valeton (ZO) exhibits pharmacological activity and has long been used in traditional medicine. However, reports about its safety profiles are limited. The present study aimed to evaluate the phytochemical profile and the toxic effects of ZO essential oil on the development of zebrafish and acute oral toxicity in rats. The essential oil was isolated from ZO rhizomes, and phytochemicals were analyzed using a gas chromatography-mass spectrometer (GC-MS). The embryotoxic and teratogenic effects of ZO essential oil were evaluated in zebrafish embryos and larvae and the acute oral toxicity was determined in rats. GC-MS results showed the essential oil contained zerumbone as a major phytoconstituent (24.73%). The zebrafish embryotoxicity of ZO essential oil appeared to be concentration- and time-dependent manner, with a moderate LC50 (1.003 µg/mL). Teratogenicity in zebrafish embryos also included morphological defects, decreased hatchability, and reduced heart rate. In rats, ZO essential oil (2000 mg/kg, p.o.) resulted in no mortality or significant toxicities. These findings suggest that ZO has embryotoxic and teratogenic effects in zebrafish embryos but does not result in death or acute oral toxicity in rats. Further long-term toxicity studies are needed to confirm the safety of products developed from ZO essential oil.

First Evidence of Carp Edema Virus Infection of Koi Cyprinus carpio in Chiang Mai Province, Thailand.

The presence of carp edema virus (CEV) was confirmed in imported ornamental koi in Chiang Mai province, Thailand. The koi showed lethargy, loss of swimming activity, were lying at the bottom of the pond, and gasping at the water's surface. Some clinical signs such as skin hemorrhages and ulcers, swelling of the primary gill lamella, and necrosis of gill tissue, presented. Clinical examination showed co-infection by opportunistic pathogens including Dactylogyrus sp., Gyrodactylus sp. and Saprolegnia sp. on the skin and gills. Histopathologically, the gill of infected fish showed severe necrosis of epithelial cells and infiltrating of eosinophilic granular cells. Electron microscope examination detected few numbers of virions were present in the cytoplasm of gill tissue which showed an electron dense core with surface membranes worn by surface globular units. Molecular detection of CEV DNA from gill samples of fish was performed by polymerase chain reaction (PCR) and confirmed by nested-PCR. Phylogenetic analyses revealed that CEV isolate had 99.8% homology with the CEV isolated from South Korea (KY946715) and Germany (KY550420), and was assigned to genogroup IIa. In conclusion, this report confirmed the presence of CEV infection of koi Cyprinus carpio in Chiang Mai province, Thailand using pathological and molecular approaches.

Occurrence and Antimicrobial Susceptibility Profiles of Multidrug-Resistant Aeromonads Isolated from Freshwater Ornamental Fish in Chiang Mai Province.

Antimicrobials are commonly used to prevent and treat disease in the ornamental fish industry. However, the indiscriminate and comprehensive overuse of unregulated antimicrobials without appropriate diagnostic examination could contribute to the development of antimicrobial-resistant strains of bacterial pathogens. Moreover, human infections caused by pathogens transmitted from fish or the aquatic environment are quite common. The frequent detection of antimicrobial resistance in ornamental fish and their environments are inevitable so as to decrease the transfer of antimicrobial-resistant bacteria from aquatic sources to other environments. This study evaluated the prevalence of common bacteria species and the antimicrobial susceptibility profile in ornamental fish that were sold in an ornamental fish shop in Chiang Mai, Thailand. Aeromonas spp. were the most dominant of the isolated species from the ornamental fish samples and accounted for 68.09% of the total. Other species detected included Vibrio spp., Pseudomonas spp., and Citrobacter spp. A high percentage of resistance to amoxicillin (93.75%), oxytetracycline (79.69%), and erythromycin (75.00%) was observed among the Aeromonas spp. The antimicrobial resistance information for ornamental fish is very limited, and the results from this study indicate that the Aeromonas spp. are highly resistant to several important antibiotics. The results suggest that additional steps should be taken to educate store owners to reduce the indiscriminate use of these antibiotics to decrease the antimicrobial resistance in ornamental fish to potentially improve public health.

Antibacterial Activities of Acetic Acid against Major and Minor Pathogens Isolated from Mastitis in Dairy Cows.

The present study evaluated the antimicrobial activities of acetic acid against bovine mastitis pathogens compared to lactic acid and lauric and caprylic saturated fatty acids. Eleven mastitis pathogens were isolated from sub-clinical and clinical bovine mastitis cases for the study. An initial screening of their antibacterial activities by agar well diffusion method was performed. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of each acid were obtained using a microdilution method; each acid was diluted from stock solution and then were diluted with culture broth to reach concentrations ranging from 4 to 0.004% w/v. The results showed acetic acid had the highest zone of inhibition against all pathogens except Escherichia coli compared with lauric and caprylic acids. The MIC and MBC were lowest for acetic acid against both Gram-positive (except Staphylococcus chromogenes from the coagulase negative staphylococci (CNS) group) and Gram-negative pathogens, intermediate for lactic and caprylic acids and greatest for lauric acid. In conclusion, acetic acid had antimicrobial activities against most mastitis pathogens compared with other acids. Further studies are needed to optimize the formulation and concentration of acetic acid for teat-dipping agent in the future.

Phytochemical and Safety Evaluations of Volatile Terpenoids from Zingiber Cassumunar Roxb. On Mature Carp Peripheral Blood Mononuclear Cells and Embryonic Zebrafish.

Pharmaceutical products of essential oil from Zingiber cassumunar Roxb. are extensively being developed, while the research on their safety is seldom documented. The aim of the present study was to evaluate the phytochemical profile and the effect of cassumunar ginger oil on cell-based assay and the zebrafish model. The essential oil was isolated from fresh rhizomes of Z. cassumunar using simultaneous steam-distillation. Chemical composition was analyzed using gas chromatograph coupled to a mass spectrometer (GC-MS). Effect of cassumunar ginger oil on adult carp fish peripheral blood mononuclear cells (PBMCs) was investigated using MTT assay. The embryotoxic and teratogenic effects of cassumunar ginger oil were studied in zebrafish embryos. GC-MS results showed that the essential oil was composed of sabinene (43.54%) and terpinen-4-ol (29.52%) as the major phytoconstituents. No fish PBMC cytotoxic effect was observed with the concentration less than 50 µg/mL of cassumunar ginger oil. Our results showed for the first time the embryotoxic and teratogenic effects of cassumunar ginger oil in zebrafish embryos. The result indicated that the cassumunar ginger oil induced zebrafish embryotoxicity in a concentration-dependent manner. At 500 µg/mL of cassumunar ginger oil demonstrated significantly moderated embryotoxicity within 24 h (p < 0.05). The survival rate of 100 µg/mL of cassumunar ginger group was markedly declined to zero at 96-h post-fertilization (log-rank test, p = 0.001). However, survival rates of zebrafish embryo in the 1 and 10 µg/mL cassumunar ginger groups were more than 90% throughout the trial period. Moreover, very low teratogenicity to the zebrafish embryo was also observed in 1 and 10 µg/mL of cassumunar ginger groups. Our findings suggest that there is hardly any cytotoxicity, embryotoxicity and teratogenicity at concentrations less than 10 µg/mL of cassumunar ginger oil. However, the toxicity assessment of its pharmaceutical product should prove for further consumer protection.

Antiseptic effect of natural teat dip containing lactic acid against mastitis-causing Escherichia coli.

AIM: This study aimed to estimate the enumeration of total bacteria and coliform on teat skin from dairy cows and evaluate the efficacy of the natural rice gel containing 5% v/v lactic acid (NGL) against Escherichia coli standard and field strains isolated from bovine teat skin. MATERIALS AND METHODS: A total of 100 bacterial teat skin samples (25 cows) were collected from dairy cows in smallholder farm. The cows were housed in freestall barns. The colonization of total bacteria and E. coli on teat skin was measured by 3M Petrifilm method. The minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) of lactic acid were evaluated for reference strain of E. coli ATCC 25922 and two field strains of E. coli. The natural teat sanitizer was formulated using 5% NGL with modified rice gel. In vitro antiseptic efficacy of 5% NGL was determined by time-kill kinetic assay. E. coli morphology after exposure with 5% NGL was examined under a scanning electron microscope (SEM). RESULTS: The total bacteria and coliform counts from bovine teat skin were 2.11×104 and 1.54×101 colony-forming units/ml, respectively. The MIC and MBC of lactic acid on the tested bacteria were 0.5% v/v. The natural teat dip was successfully prepared with minimum change in consistency after 1 year of storage at 4°C. The reduction rate of 5% NGL on E. coli ATCC 25922 and field strain showed 32.77% and 27.58%, respectively. An appearance under SEM of non-viable E. coli after being incubated with 5% NGL clearly showed atypical form and rough surface cell membrane. CONCLUSION: The rice gel containing 5% v/v lactic acid is a promising preparation as a natural teat antiseptic for reducing bacteria on teat skin. It was shown to be effective against E. coli causing bovine mastitis in dairy cows.

Inhibitory Effects and Killing Kinetics of Lactic Acid Rice Gel Against Pathogenic Bacteria Causing Bovine Mastitis.

Staphylococcus aureus and S. epidermidis are the major teat skin bacteria and lead to severe bovine mastitis. Teat antiseptic is an important tool for controlling intramammary infection. The antibacterial activity of lactic acid (LA) against one reference strain of S. aureus ATCC 25923 and two field strains including S. aureus and S. epidermidis was investigated using the broth microdilution method. Its minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were 0.5% for strains belonging to both species. An antiseptic preparation containing 5% LA with modified rice gel (LA-RG) was successfully prepared. Rheological behavior of LA-RG was found to be a pseudoplastic flow with thixotropy with viscosity of approximately 0.007 Pas. LA-RG exhibited a sufficient adhesive property in the rolling ball test with a length of 9.67 ± 0.04 cm. Killing kinetic studies of LA-RG showed that the killing rate of LA-RG was significantly faster than that of LA. After 32 min of exposure to LA-RG, approximately 86% and 60% of S. aureus and S. epidermidis were reduced, respectively. Abnormal bacterial cell surface after exposure to LA-RG was observed by scanning electron microscopy. It is concluded that LA-RG is a promising preparation as an alternative product for preventing mastitis in dairy cattle.