Wasana Chaisri

Preparation and Characterization of Cephalexin Loaded PLGA Microspheres

The aim of this study was to evaluate the effects of emulsion type and process parameters on the properties of CPX-loaded PLGA microspheres in order to obtain delivery systems suitable for the treatment of dairy mastitis. The microsphere size was analyzed by photon correlation spectrophotometry. Determination of the drug loading was achieved by HPLC. It was found that CPX-loaded PLGA microspheres prepared using a w/o/w double emulsion technology were slightly larger (approximately 3-5 microm) but much higher in drug content (approximately 18% w/w) than those obtained using o/w single emulsion preparation technology (average size was 2 microm, encapsulation efficiency was less than 2 %). It was also demonstrated that stirring during emulsification and a change in both the internal and external phase of the emulsion, affected the size and the drug entrapment efficiency of the microspheres obtained. A 60/40 v/v mixture of chloroform and acetone was found to be the best organic solvent system for creating the primary emulsion. To obtain a high yield (>90%) of microspheres with a desirable size and high drug entrapment efficacy, a stirring rate of 8,000-10,000 rpm gave the best results. It is concluded CPX-loaded PLGA microspheres with suitable characteristics for the treatment of cows with dairy mastitis can be prepared by a w/o/w double emulsion preparation method.

Enhanced gentamicin loading and release of PLGA and PLHMGA microspheres by varying the formulation parameters

The purpose of this study was to develop a suitable formulation for gentamicin sulfate (GS) that gives a sustained release of the drug. Therefore this drug was loaded into poly(D,L-lactide-co-glycolide) (PLGA) and poly(lactic-co-hydroxymethyl glycolic acid) (PLHMGA) microspheres. The effects of various formulation parameters (ethanol, surfactant, osmotic value of the external phase, polymer type and concentration) on particle characteristics (size, loading and release) were investigated. The GS loaded microspheres were prepared using a double emulsion evaporation technique. The results demonstrate that neither ethanol nor surfactants had beneficial effects on the drug loading efficiency (around 4-10%). However, an increase in buffer concentration (and thus osmotic pressure) of the external phase resulted in a substantial increase of GS-loading (from 10 to 28%). Further, an increase of concentration of PLGA in DCM from 10% to 15/20% caused a 4-time increase of the drug loading. The best formulation identified in this study had a loading efficiency of around 70% resulting in PLGA microspheres with a 6% (w/w) loading. The particles showed a burst release of the drug depending on their porosity, followed by a phase of 35 days where hardly any release occurred. The drug was then slowly released for around 25 days likely due to degradation of the microspheres. The drug loading efficiency of GS in PLHMGA was not significantly different from PLGA microspheres (64%). The release of GS from PLHMGA microspheres was faster than that of PLGA because the degradation rate of PLHMGA is more rapid than PLGA. This study shows that prolonged release of gentamicin can be obtained by loading this drug into microspheres made of biodegradable aliphatic polyesters.

Effects of Alpinia galanga oil on anesthesia and stress reduction in Oreochromis niloticus

Oreochromis niloticus (Nile tilapia) is one widely cultured fish in Thailand. Handling processes and transportation causes high stress in Nile tilapia. This study explores anesthetic effect and stress reduction of Alpinia galanga oil (AGO) on Nile tilapia. The anesthetic activity was evaluated by the time for fish induction to anesthesia and full recovery. It was found that the suitable dose of AGO that caused desirable anesthesia of Nile tilapia was 700 mg/L. This dose gave induction and recovery times of approximately 257 and 438 sec, respectively. Blood glucose and plasma cortisol of the fish anesthetized with AGO showed nearly normal levels indicating that the fish stress during handling was not increased. Study on loading densities of fish mimicked general fish transportation and showed that loading density of fish was a crucial factor on fish stress. The highest water quality was found in the lowest loading density of fish. Water containing AGO at a concentration of 150 mg/L showed significantly higher potential for reducing fish activity and water improvement than without AGO. Therefore, AGO is a promising natural edible plant oil for anesthesia in Nile tilapia.

Antimicrobial Resistance of Biofilm-Forming Streptococcus agalactiae Isolated from Bovine Mastitis

Streptococcus agalactiae is a contagious mastitis pathogen commonly found in dairies in northern Thailand. During infection, S. agalactiae may form biofilms which is known to be associated with increased antimicrobial resistance of bacteria. We aimed to investigate changes in antimicrobial resistance of biofilm producing S. agalactiae associated with bovine mastitis. We measured biofilm formation, Minimum Inhibitory Concentrations (MIC) and Minimal Bactericidal Concentrations (MBC), and Minimum Biofilm Eradication Concentration (MBEC) of 56 archived isolates from bovine milk in Chiang Mai, Thailand. Quantitative biofilm evaluation found no (0%) strong, 21 (37%) moderate, and 30 (54%) weak biofilm producers, as well as 5 (9%) non-biofilm producers. Qualitative biofilm assay found only 11 isolates (20%) to be biofilm producers; these were further investigated for resistance to ampicillin, cloxacillin, cephalexin, gentamicin and tetracycline. All 11 isolates showed higher MBECs compared to MICs and MBCs. Some S. agalactiae strains from cows with clinical or subclinical mastitis can produce biofilms in vitro, and these appear more resistant to common antibiotics. Such resistance can be an obstacle in the eradication of S. agalactiae from infected herds. Determination of biofilm formation by S. agalactiae cultured from milk may be useful for creating an effective treatment plan and prognosis of bovine mastitis.

Factors in Dry Period Associated with Intramammary Infection and Subsequent Clinical Mastitis in Early Postpartum Cows

The objective of this study was to determine cow characteristics and farm management factors during the dry period associated with early postparturient intramammary infection (IMI) and subsequent clinical mastitis (CM). Data were collected three times: before drying off (P1), during the dry period (P2), and 5 to 14 days after calving (P3), using questionnaires and farm investigation. Milk samples were aseptically collected for bacterial identification at P1 and P3. Factors associated with IMI and CM were analyzed using multiple logistic regression models. The final model showed that IMI in early postpartum was significantly associated with full insertion of dry cow antibiotic, dry cows in barns with a combination of tie and free stalls, body condition score (BCS) in dry period and after calving, and milk yield before drying off. For IMI cows, factors significantly associated with clinical expression of mastitis were having daily barn cleaning, teat disinfected with alcohol before administration of dry cow therapy, BCS before drying off, milk yield before drying off, and days in milk at drying off. In conclusion, both cow and farm management factors are associated with the IMI rate and subsequent expression of clinical signs of mastitis in early postpartum cows.

Aflatoxin B1 Contamination of Dairy Feeds after Storage in Farm Practice in Tropical Environmen

　The objective of this study was to determine the contamination of aflatoxin B1 (AF-B1) when keeping various dairy feeds in a farm environment. The study was carried out from March to May 2011 and involved 63 small holder dairy farms belonging to a single dairy cooperative in Chiang Mai province, Thailand. All feed samples used for milking cows including 4 commercial concentrates (CC1 to CC4), by-products from local corn processing factories fermented in plastic bags (SIL), and corn and cob meal or corn dust (CCD). Feed samples were collected 2 times at before and after storage. Farmers were requested to store CC1 to CC4 and CCD for a month and SIL for a week using their routine on-farm storage arrangements. All samples were measured for their AF-B1 concentrations by ELISA. Results showed that AF-B1 concentrations of CC1 to CC4, SIL and CCD before storage were 5.1, 4.1, 4.0, 4.2, 5.5 and 5.5 μg/kg, respectively, and after storage the concentration of AF-B1 were 9.7, 6.5, 9.8, 12.3, 11.4 and 20.0 μg/kg, respectively. CCD at after storage was the only feed that had mean level more than 20 μg/kg. Concentrations of AF- B1 at before storage in all feeds were significantly lower than after storage (P<0.01), and the increased ratio of AF-B1 levels was approximately 2 to 3 times. The study concluded that increased AF-B1 levels are related to feed types and farm conditions.

Investigation of antiaromatase activity using hepatic microsomes of Nile tilapia (Oreochromis niloticus)

Microsomal aromatase enzymes of humans and rats have been used in antiaromatase assays, but enzyme activity is species-specific. The current study extracted hepatic microsomes of Nile tilapia (Oreochromis niloticus) to investigate and compare the antiaromatase activity of chrysin, quercetin, and quercitrin. This activity was evaluated using a dibenzylfluorescein (DBF) assay. Results revealed that the age and body weight of Nile tilapia affected the yield of extracted microsomes. Extraction of hepatic microsomes of Nile tilapia was most effective when using a reaction medium with a pH of 8.0. A DBF assay using Nile tilapia microsomes revealed significant differences in levels of antiaromatase activity for chrysin, quercetin, and quercitrin. Chrysin was the most potent aromatase inhibitor, with an IC50 of 0.25 mg/mL. In addition, chrysin is an aromatase inhibitor that also inhibits the proliferation of cancer cells. Hepatic microsomes of Nile tilapia can be used to investigate and compare the antiaromatase activity of different compounds.

Nanoemulsion: A suitable nanodelivery system of clove oil for anesthetizing Nile tilapia

Clove oil ethanolic solution (CL-EtOH) have always been used for fish anesthesia. However, ethanol causes major side effect of fish hypersensivity. In this study, clove oil loaded nanoemulsion (CLN) was developed in order to enhance water miscibility of clove oil without using ethanol in the preparations. The obtained CLN was characterized in terms of droplet size, size distribution expressed as polydispersity index (PDI), and zeta potential. The anesthetic effect of CLN in comparison with CL-EtOH on Oreochromis niloticus (Nile tilapia) was investigated. The results showed that the best CLN was composed of 20% w/w clove oil and 15% w/w polysorbate 20. This CLN has internal droplet size of 63.2 ± 1.0 nm, PDI of 0.31 ± 0.04, and zeta potential of - 30.3 ± 8.1 mV. GC-MS analysis indicated that eugenol was the main compound in clove oil. It was found that the induction time to anesthesia for Nile tilapia that received this CLN was shorter than that received CL-EtOH at the same eugenol concentration. The results of this study showed the potential of nanoemulsion on water miscible and efficacy enhancing of clove oil without using ethanol. The obtained CLN from this study is a promising formulation for fish aquaculture where fish sedation is required.