Asian Pacific Journal of Tropical Biomedicine

889-895

Authors: Barbara Lamagna, Maria Pia Pasolini, Sandra Nizza, Karina Mallardo, Maurizio Formicola, Alessandro Costagliola, Gerardo Fatone, Filomena Fiorito, Orlando Paciello, Luisa De Martino

Objective To assess normal conjunctival cytological and bacteriological/fungal flora features in the Mediterranean buffalo (Bubalus bubalis). Methods Swabs were taken from the inferior conjunctival sac of both eyes of 57 healthy female buffaloes aged 24–36 months, with no evidence of ocular disease, farmed in Campania region (Southern Italy), for microbiological analysis. Conjunctival eye specimens of both eyes were subsequently obtained by a cyto-brush, for cytological analysis. The antimicrobial susceptibility of bacterial isolates was also determined using the disk-diffusion method on Mueller Hinton agar plates. Results Cytological examination of conjunctival swab specimens (114 eyes) revealed epithelial cells (basal, intermediate, columnar and superficial) in all samples, whereas neutrophils, lymphocytes and plasma cells were present in 70%, 10% and 2% of samples, respectively. Microorganisms, for a total of 261 aerobic bacteria and 6 fungi, were isolated from 112/114 conjunctival samples [98.25%; 95% confidence interval (CI): 93.18–99.70]. Only two conjunctival swabs did not yield bacteria and/or fungi (2/114, 1.75%; 95% CI: 0.30–6.82). Gram-positive aerobes were most commonly cultured (181/261, 69.35%; 95% CI: 63.31–74.81), with Enterococcus faecium and Staphylococcus lentus predominating. Escherichia coli was the most frequently isolated as Gram-negative bacteria (80/261, 30.65%; 95% CI: 25.19–36.69). The antimicrobial resistance patterns of the isolated bacteria showed amoxycillin/clavulanic acid and cephalothin as the least sensitive antibiotics for both Gram-positive and Gram-negative bacteria. Conclusions These results provided first information on normal conjunctival ocular microflora and cytological features in Mediterranean buffalo.

902-908

Authors: Yaser Hamadeh Tarazi, Ahmed Mahmoud Almajali, Mustafa Mohammad Kheer Ababneh, Humam Shawket Ahmed, Adnan Saleem Jaran

Objective To determine the prevalence, genetic relatedness, and pattern of antimicrobial susceptibility in methicillin-resistant Staphylococcus aureus (S. aureus) (MRSA) isolated from household dogs, farm dogs, and stray dogs, compared to isolates from their associated personnel. Methods MRSA was isolated from 250 nasal swabs (150 swabs from dogs and 100 swabs from humans). PCR assays were used to detect the presence of both the nuc and mecA genes, which confirmed the identity of S. aureus isolates and the presence of methicillin resistance, respectively. Disk diffusion was used to determine the antibiotic susceptibility against 15 antimicrobial agents along with an E-test that determined the minimum inhibitory concentration for oxacillin. Pulsed field gel electrophoresis was conducted to determine the genetic relatedness of MRSA isolates from dogs to those from associated and unassociated personnel. Results The prevalence of S. aureus in dogs and humans was 12.7% and 10.0% respectively, while the prevalence of MRSA isolates in dogs and humans was 5.3% and 5.0%, respectively. The prevalence of MRSA isolates in household dogs, farm dogs, and stray dogs was 7.8%, 4.7%, and 0.0%, respectively. MRSA isolates demonstrated a significantly higher rate of multi-resistance against three or more antimicrobial agents than methicillin-susceptible S. aureus (MSSA). Trimethoprim-sulphamethoxazole and chloramphenicol were the most effective antibiotics against all MRSA isolates. Pulsed field gel electrophoresis revealed a strong association between dog MRSA isolates and MRSA isolates from strongly associated personnel. Conclusions MRSA is prevalent in house dogs, as well as in dog rearing centers and among their strongly associated personnel. A strong association was found between the MRSA isolates from dogs and those from humans who are in close contact. In addition, MRSA isolates showed a high rate of multi-resistance compared to MSSA isolates.

915–920

Authors: I Wayan Suardana, Dyah Ayu Widiasih, I Gusti Ngurah Kade Mahardika, Komang Januartha Putra Pinatih, Budi Setiadi Daryono

Objective: To evaluate the zoonotic potency of Escherichia coli O157:H7 through arbitrarily primed-PCR (AP-PCR) methods as one of the DNA fingerprinting methods. Methods: A total of 14 isolates consisted of 11 isolates originated from human feces with renal failure symptoms, 2 isolates originated from cattle feces, and 1 control isolate were used in this study. DNA of each isolate was extracted, and their profiles were studied by using AP-PCR method with M13 F and M13 R arbitrary primers. Results: The results founded that all of 14 isolates had similarity range from 54.6% to 88.5%. Isolates KL-106(3) and KL-55(6) originated from humans showed the degree of similarity with isolates SM-25(1) and SM-7(1) originated from cattle as high as 85% and 77%, respectively. Conclusions: The high degree of similarity between isolates originated from cattle and human indicated the high potency of zoonoses. The results also concluded AP-PCR method as a briefly fingerprinting method in order to trace the epidemiological of E. coli O157:H7.

928–931

Authors: Gabriela Ulloa-Urizar, Miguel Angel Aguilar-Luis, Mar´ıa del Carmen De Lama-Odr´ıa, Jose Camarena-Lizarzaburu, Juana del Valle Mendoza

Objective: To evaluate the susceptibility of Pseudomonas aeruginosa (P. aeruginosa) in vitro to the ethanolic extracts obtained from five different Peruvian medicinal plants. Methods: The plants were chopped and soaked in absolute ethanol (1:2, w/v). The antibacterial activity of compounds against P. aeruginosa was evaluated using the cupplate agar diffusion method. Results: The extracts from Maytenus macrocarpa (“Chuchuhuasi”), Dracontium loretense Krause (“Jergon Sacha”), Tabebuia impetiginosa (“Tahuari”), Eucalyptus camaldulensis Dehn (eucalyptus), Uncaria tomentosa (“Uña de gato”) exhibited favorable antibacterial activity against P. aeruginosa. The inhibitory effect of the extracts on the strains of P. aeruginosa tested demonstrated that Tabebuia impetiginosa and Maytenus macrocarpa possess higher antibacterial activity. Conclusions: The results of the present study scientifically validate the inhibitory capacity of the five medicinal plants attributed by their common use in folk medicine and contribute towards the development of new treatment options based on natural products.

937–943

Authors: Robab Valizadeh, Ali Asghar Hemmati, Gholamreza Houshmand, Sara Bayat, Mohammad Bahadoram

Objective: To evaluate and practically demonstrate the influence of Althaea officinalis flower mucilage as a plant known in Iran's and other Middle Eastern countries' traditional medicine for its wound healing properties. Methods: Animals were divided into 6 groups of 5 cases including a non-treated group as the negative control group receiving no treatment, a group treated with eucerin as the positive control group, a phenytoin 1% group as a standard group treated topically with phenytoin 1% hand-made ointment, and treatment groups treated with hand-made Althaea officinalis flower mucilage (AFM) ointment in a eucerin base with different concentrations (5%, 10%, 15%). Results: Among the treatment groups, the AFM 15% ointment showed the best result. Wound healing duration was reduced by the surface application of these groups. Wound closure was completed on Days 14 and 15 in the AFM 15% ointment and phenytoin 1% groups, respectively. No significant difference was observed in healing period between these groups. Conclusions: In conclusion, AFM 15% ointment was found to reduce wound healing time without any significant difference with the phenytoin 1% ointment. The authors suggest increased AFM effectiveness in when combined with phenytoin or other effectual plants.

948–954

Authors: Husnul Khotimah, Mulyohadi Ali, Sutiman Bambang Sumitro, Mochamad Aris Widodo

Objective To observe the effects of Centella asiatica (C. asiatica) methanolic extract on α-synuclein aggregation and its expression in rotenone-exposed zebrafish. Methods Zebrafish (Danio rerio) were exposed to 5 μg/L rotenone for 28 days and co-incubated with 2.5, 5.0 and 10.0 μg/mL of C. asiatica methanolic extract. The medium was changed every 48 h for maintain the concentration of rotenone and extract. After 28 days zebrafish were sacrificed on the ice block and protein was isolated from zebrafish brain for ELISA of dopamine and Western blotting of α-synuclein. Immunohistochemistry was conducted to observe the α-synuclein expressions from histopathological preparation of zebrafish brain. The head were soaked in 10% formaline for less than 24 h and embedded onto paraffin block, then sliced for immunohistochemistry using anti α-synuclein antibody. We also measured zebrafish motility for 5 min in each week. Results C. asiatica has important bioactive compounds such as asiaticoside that has anti-inflammatory and antioxidant properties. It may inhibit cascade reaction due to oxidative stress induced by rotenone. Decreasing reactive oxygen species proposed probability of radical attack to α-synuclein protein that caused aggregation and increase of its expression. The motility of zebrafish was also maintained in C. asiatica groups due to the increasing dopamine level in rotenone-induced zebrafish. High level of reactive oxygen species inactivated enzyme for dopamine synthesis such as tyrosine hydroxylase, and oxidized dopamine itself. Oxidized dopamine increased α-synuclein aggregation. Thus, the dopamine level decreased in rotenone-induced zebrafish, but C. asiatica increased dopamine level. Conclusions C. asiatica has a potential to be developed as an anti-Parkinson's disease treatment due to its capability for minimized the sign of Parkinson's such as α-synuclein aggregation and expression, increasing motility and dopamine as well.

960–964

Authors: Tarek Benmeddour, Hocine Laouer, Salah Akkal, Guido Flamini

Objective To evaluate the essential oil composition and the antibacterial activity of an Algerian endemic plant, Launaea lanifera Pau (L. lanifera), grown in arid steppe regions. Methods L. lanifera essential oil was isolated from aerial parts by steam distillation and its chemical composition was evaluated by gas chromatography-flame ionization detector and gas chromatography with electron impact mass spectrometry. Furthermore, its in vitro antibacterial activity against four bacterial strains was tested following the agar disk diffusion method. Results This species had a very low essential oil yield (0.005%). Twenty-four (92.6%) individual components were identified. The main constituents were hexahydrofarnesyl acetone (31.6%), (E)-β-ionone (8.5%), (E)-β-damascenone (7.0%), 2-methyltetradecane (3.8%), n-heptadecane (3.8%), limonene (2.8%) and β-caryophyllene (2.8%). No noteworthy antimicrobial activity was observed on the tested bacteria, neither Gram negative nor Gram positive. Conclusions This is the first report on the volatile constituents and antibacterial activity of L. lanifera. The studied essential oil does not possess significant activity against the tested microorganisms.

971–973

Authors: Luis Antonio Gomez-Puerta, Armando Emiliano Gonzalez, Cesar Gavidia, Viterbo Ayvar, Hector Hugo Garcia, Maria Teresa Lopez-Urbina, for the Cysticercosis Working Group in Peru

The efficacy of oxfendazole (OFZ) on Taenia hydatigena metacestodes, also called Cysticercus tenuicollis (C. tenuicollis), was studied in 648 raising pigs. This study was performed in Tumbes Department in Peru, an endemic area for cysticercosis. Pigs were randomized in two groups; untreated group (n = 142) did not receive any treatment and treated group (n = 506) received OFZ treatment at a single dose of 30 mg/kg body weight. Six months after treatment, the pigs were necropsied. The prevalence of infection by C. tenuicollis among the pigs was 27.5% (39/142) and 2.0% (10/506) in untreated and treated groups, respectively. Untreated group was infested only with viable cysts, whereas treated group had no viable cysts. All the cysts found in treated group presented degeneration, with a thick membrane, and they contained milky fluid and fibrous tissue. A single dose of OFZ was effective against C. tenuicollis, thus providing an alternative drug for controlling this parasite in pigs.

796–800

Authors: Daniella de Sousa Mendes Moreira Alves, Rodrigo Gurgel-Gonçalves, Patr´ ıcia Albuquerque, Cesar Augusto Cuba-Cuba, Maria Imaculada Muniz-Junqueira, Selma Aparecida Souza Kuckelhaus

Objective: To evaluate whether the inoculation of contaminated cultures in the peritoneal cavity of mice could implement decontamination of Acanthamoeba cultures. Methods: Suspensions of Acanthamoeba, Acanthamoeba polyphaga ATCC 30461, or Acanthamoeba spp. isolated from soil (UnB13 strain) were inoculated in the peritoneal cavity of Swiss mice (n = 24). After 1, 6, 12, or 24 h of exposure the peritoneal cavity was washed and assessed for the presence of bacteria, fungi, and Acanthamoeba. Results: After 1 h of intraperitoneal inoculation at least 97% of the bacteria and 96% of the fungi (P < 0.05) and 99% of the bacteria (P < 0.05) were successfully eliminated from the ATCC 30461 strain and from the soil isolate UnB13 strain, respectively. This method also allowed the recovery of most trophozoites and cysts from both Acanthamoeba cultures at the end of 24 h. Conclusions: Our data demonstrated that this technique has great potential for decontamination of Acanthamoeba cultures in a short period of time.

806–811

Authors: Xian-Wen Yang, Hai-Xing Jiang, Xiao-Li Huang, Shi-Jia Ma, Shan-Yu Qin

Objective: To assess the role of Th9 and Th17 cells in malignant ascites (MA). Methods: MA from 30 hepatic carcinoma patients and benign ascites from 30 cirrhotic patients were collected. Corresponding peripheral blood samples from these hepatic carcinoma and cirrhotic patients as well as 30 healthy subjects were collected. The frequency of Th9 and Th17 cells was tested by flow cytometry. Serum levels of interleukin (IL)-9 and IL-17 were examined by ELISA. Results: The observed frequency of Th9 and Th17 cells, and the IL-9 and IL-17 serum levels were significantly higher in MA patients than those in cirrhotic patients and healthy control samples (P < 0.05). Moreover, the Th9 cells demonstrated positive correlation with Th17 cells as well as IL-9 in MA patients; however, this positive correlation was not observed in the cirrhotic patients or healthy control samples. The frequency of Th9 and Th17 cells was distinctly higher in MA patients presenting with stage III or IV malignancy and with lymph node or distant metastasis than those in patients in stage I or II and without distant metastasis (P < 0.05). Conclusions: The increased frequency of Th9 and Th17 cells in MA patients suggests that these two T cell subsets play a synergistic role in MA pathogenesis. This study also demonstrated that Th9 and Th17 cells may perform their biological functions in conjunction with IL-9 production.

819–823

Authors: Junsei Taira, Eito Tsuchida, Masatsugu Uehara, Natsuko Ohhama, Wakana Ohmine, Takayuki Ogi

Objective: To find anti-melanogenesis materials used in whitening cosmetics. Methods: The ethanolic leaf extract of Mallotus japonicus (M. japonicus) having an anti-melanogenesis activity was separated by a sephadex LH-20 chromatography. Each fraction was measured for its tyrosinase inhibitory activity together with its polyphenol content using the Folin–Ciocalteu method. The anti-melanogenesis activity of the active fractions was determined by the melanin content in the murine B16F1 melanoma. The active fractions were put together due to similar constituents, and then separated by high performance liquid chromatography using a C-18 ODS column. The major antimelanogenesis compound was identified using 1H and 13C-NMR and liquid chromatography-mass spectrometry. Results: The ethanolic leaf extract of M. japonicus showed an anti-tyrosinase activity with a high polyphenol content, resulting in suppression of melanin production in the B16F1 melanoma. The extract was separated and the active compound was identical as rutin based on the 1H, 13C-NMR and liquid chromatography-mass spectrometry analysis data. In addition, the rutin treatment with cells reduced the melanin content in a concentration dependent manner without any cell toxicity. The leaf extract of M. japonicus containing rutin would be useful in whitening cosmetics for protection from UV-light exposure to be limiting the accumulation of melanin in skin. Conclusions: The leaf extract of M. japonicus and/or rutin isolated from the extract as a key whitening agent would be useful as a whitening cosmetic material for protecting against disorder skin due to melanin accumulation.

829–833

Authors: Samira Asgharzade, Zahra Rabiei, Mahmoud Rafieian-Kopaei

Objective: To evaluate the effect of ethanolic extract of chamomile on balance and motor learning in rats receiving scopolamine and intact rats. Methods: Fourty-two rats were divided into 6 groups (n = 7). Control group received distilled water. Rats in Group 2 were given 1 mg/kg scopolamine. Groups 3 and 4 received chamomile extract 200 mg/kg and 500 mg/kg, respectively, and scopolamine simultaneously for 20 days. Intact groups (Groups 5 and 6) only received chamomile extract 200 mg/kg and 500 mg/kg, respectively. Motor coordination of rats was assessed with rotarod apparatus. Results: According to the obtained results, compared with the control group, scopolamine significantly decreased time spent on rotarod performance (P < 0.001). Compared with scopolamine group, the strength and staying on rotarod apparatus in Group 3 significantly increased (P < 0.05). The results of this research showed that intact groups that received only chamomile extract at doses of 200 mg/kg and 500 mg/kg significantly increased time spent on rotarod, compared with scopolamine group (P < 0.001). Conclusions: The results of this study indicated the high antioxidant property and protective effect of chamomile extract on motor coordination in the groups that received scopolamine.

841–844

Authors: Zong Ning, Yu-Long Bai, Hua Lu, Kang-Lin Mo

Objective: To investigate the prognostic value of plasma C-reactive protein (CRP) level in patients with paraquat poisoning. Methods: This study included 162 patients with paraquat poisoning. The data of plasma paraquat, CRP level and arterial blood gas were analyzed. Cox regression analysis was applied to evaluate the risk factors of prognosis. Receiver operating characteristics curve analysis and area under curve were used to calculate the predictive power of significant variable. Differences in patient survival were determined using the Kaplan–Meier method and a log-rank test. Results: Plasma CRP level was significantly increased in non-survival patients compared with survival patients (P < 0.05), and positively correlated with plasma paraquat level (P < 0.05). Cox regression analysis revealed that plasma CRP level was an independent prognostic marker of mortality within 30 days. The receiver operating characteristics curve analysis indicated that area under curve of plasma CRP level was 0.867 (95% CI: 0.81–0.93), and the cut-off value was 18 mg/L, and patients with CRP level over this value had a poor survival time compared with those with less than this value. Conclusions: These results suggest that plasma CRP level is distinct increased in patients with paraquat poisoning, and the plasma CRP level may be useful for the prediction of prognosis in paraquat poisoning.

852–857

Authors: Sri Rahayu Lestari, Muhammad Sasmito Djati, Achmad Rudijanto, Fatchiyah Fatchiyah

Objective: To monitor the physiological characteristics and genes expression of obesity rat model after rambutan peel extract (RPE) treatment. Methods: Twenty-four 12-week-old male rats were divided into 4 groups: normal, obesity, obesity treated with ellagic acid (O-EA) and obesity treated with RPE30 (ORPE30). Physiological characteristics were monitored by measuring body weight, calorie intake, size of adipocyte and level of triglyceride. Peroxisome proliferator activated receptor gamma (PPARg), CCAAT/enhancer-binding proteins a and fatty acid binding protein 4 (FABP4) expression were observed using immunohistochemistry, Western blotting and quantitative RT-PCR methods. Results: Body weight gain of O-EA and O-RPE30 rats were lower than obesity group and size of adipocyte cells were smaller than obesity group (P < 0.05), but when we compared to normal group, those groups had higher body weight gain and larger adipocyte cells. The level of triglycerides, protein expression of PPARg and mRNA level of FABP4 genes were significantly downregulated on O-EA and O-RPE30 compared to obesity group (P < 0.05). Our results indicated that RPE had potential substance as inhibitor of body weight gain, declining of size of adipocyte, level of triglycerides, PPARg expression and mRNA level of FABP4 gene on obesity rat model. Conclusions: RPE have anti-obesity activity by inhibiting body weight gain, declining size of adipocyte, decreasing triglyceride, PPARg expression and mRNA level of FABP4 gene on obesity rat model.

865-869

Authors: Ferry Efendi, Retno Handajani, Nursalam Nursalam

Objective: To explore the potential of local agar of genus Gracilaria, Eucheuma, Gelidium and local brands as an alternative for imported agarose for DNA electrophoresis, and to examine their ability related to separation and migration of DNA fragments in DNA electrophoresis. Methods: Their performance at various concentrations were compared via an experimental study with a specific brand of imported commercial agarose used in molecular biology research. The measured variables were separation and migration during electrophoresis of a DNA fragment. Results: The local agar genus Gracilaria gigas, Gelidium, brand “B” and brand “S” could separate DNA fragments at a concentration between 1% and 2%, with an optimum concentration of 2% w/v, as good as a specific brand of imported commercial agarose. Conclusions: Their performance were very close to that of commercial agarose and can still be improved by further agar purification as well as by pH and sulfur control.

874–877

Authors: Mohamed Afifi, Aaser Mohamed Abdelazim

Objective: To test the ability of both zinc oxide nanoparticles (ZnONPs) and silver nanoparticles (SNPs) to ameliorate the oxidative stress resulted from diabetes in diabetic rats. Methods: Fifty male albino rats were used; ten of them were served as control group and forty, as the experiment group, were injected with streptozotocin at the single intraperitoneal dose of 100 mg/kg. Then, the experiment group was subdivided into, diabetic, diabetic + ZnONPs, diabetic + SNPs and diabetic + insulin groups. The activities and mRNA expression levels of superoxide dismutase, catalase, glutathione peroxidase and glutathione reductase were determined in brain tissues. Malondialdehyde, total antioxidant capacity, zinc and silver concentrations were estimated in the brain tissues of all rats. Results: A significant increase in the activities and mRNA expression levels of superoxide dismutase, catalase, glutathione peroxidase and glutathione reductase was shown. Malondialdehyde levels were significantly decreased while there was a significant increase in the zinc, silver concentrations and total antioxidant capacity in brain of ZnONPs and SNPs treated rats, compared with diabetic or diabetic + insulin group and their control group. Conclusions: ZnONPs and SNPs can be used to ameliorate the oxidative stress in brain resulted from diabetes mellitus.

880–884

Authors: Sylvester Okhuelegbe Eraga, Mathew Ikhuoria Arhewoh, Rosemary Ngozi Chibuogwu, Magnus Amara Iwuagwu

Objective: To investigate the pharmaceutical equivalence of ten brands of ibuprofen tablets (400 mg) purchased from pharmacies in Benin City, Nigeria. Methods: The drug samples were subjected to uniformity of weight, crushing strength, friability, melting point, disintegration and dissolution tests following acceptable and official protocols. The ibuprofen content was determined using UV and high performance liquid chromatography method. Results: Crushing strength values of the drug samples ranged between 6 and 16 kp while the disintegration times were between 7.43 and 10.40 min (for uncoated tablets) and 3.25–37.32 min (for coated tablets). Friability values were less than 1% and the melting points of recrystallized ibuprofen from the samples ranged from 73.5 to 76.0
C. The amount of ibuprofen released within 1 h ranged between 18% and 102% and two brands failed the content of active ingredient in the UV method of assay while all the brands passed the test using HPLC. Conclusions: Ibuprofen (400 mg) tablets marketed in Benin City, Nigeria vary in pharmaceutical quality.

702–706

Authors: Rosario Galarza-Seeber, Juan David Latorre, Xochitl Hernandez-Velasco, Amanda Drake Wolfenden, Lisa Renee Bielke, Anita Menconi, Billy Marshall Hargis, Guillermo Tellez

Objective: To evaluate the ability of Bacillus spp. as direct-fed microbials (DFM) to biodegrade aflatoxin B1 (AFB1) by using an in vitro digestive model simulating in vivo conditions. Methods: Sixty-nine Bacillus isolates were obtained from intestines, and soil samples were screened by using a selective media method against 0.25 and 1.00 mg/mL of AFB1 in modified Czapek-Dox medium. Plates were incubated at 37
Cand observed every two days for two weeks. Physiological properties of the three Bacillus spp. candidates were characterized biochemically and by 16S rRNA sequence analyzes for identification. Tolerance to acidic pH, osmotic concentrations of NaCl, bile salts were tested, and antimicrobial sensitivity profiles were also determined. Bacillus candidates were individually sporulated by using a solid fermentation method and combined. Spores were incorporated into 1 of 3 experimental feed groups: 1) Negative control group, with unmedicated starter broiler feed without AFB1; 2) Positive control group, with negative control feed contaminated with 0.01% AFB1; 3) DFM treated group, with positive control feed supplemented with 109 spores/g. After digestion time (3:15 h), supernatants and digesta were collected for highperformance liquid chromatography fluorescence detection analysis by triplicate. Results: Three out of those sixty-nine DFM candidates showed ability to biodegrade AFB1 in vitro based on growth as well as reduction of fluorescence and area of clearance around each colony in modified Czapek-Dox medium which was clearly visible under day light after 48 h of evaluation. Analysis of 16S-DNA identified the strains as Bacillus amyloliquefaciens, Bacillus megaterium and Bacillus subtilis. The three Bacillus strains were tolerant to acidic conditions (pH 2.0), tolerant to a high osmotic pressure (NaCl at 6.5%), and were able to tolerate 0.037% bile salts after 24 h of incubation. No significant differences (P > 0.05) were observed in the concentrations of AFB1 in neither the supernatants nor digesta samples evaluated by high-performance liquid chromatography with fluorescence detection between positive control or DFM treated groups. Conclusions: In vitro digestion time was not enough to confirm biodegradation of AFB1. Further studies to evaluate the possible biodegradation effects of the Bacillus-DFM when continuously administered in experimentally contaminated feed with AFB1, are in progress.

714–719

Authors: Haytham Ahmed Zakai, Wajihullah Khan

Objective: To analyse the efficacy of diethylcarbamazine (DEC), tetramisole and chlorpromazine on the longevity and activity of glucose-6-phosphatase and succinate dehydrogenase in the microfilariae recovered from the peripheral circulation of the rats before and after the treatment. Methods: Setaria cervi worms were implanted in white rats via laparotomy and microfilaraemic rats were divided into 4 groups. Groups 1, 2 and 3 were treated with DEC, tetramisole and chlorpromazine respectively, while Group 4 served as infected control. Longevity of microfilariae and differential leucocyte counts were recorded till the disappearance of microfilariae from peripheral blood. Glucose-6-phosphatase and succinate dehydrogenase enzymes were localized in the microfilariae recovered from normal and treated rats. Results: The microfilariae survived for 48 days in untreated rats while survival was reduced to 15, 21 and 27 days after treatment with DEC, tetramisole and chlorpromazine, respectively. Eosinophils and neutrophils increased during 2nd and 3rd weeks, whereas the lymphocytes increased during 4–7 weeks. DEC treatment resulted in slight decrease in the localization of succinate dehydrogenase but not in glucose-6-phosphatase. Tetramisole and chlorpromazine treatment did not show any appreciable change in the localization of both the above enzymes. Conclusions: DEC proved the most effective drug which cleared the microfilaraemia within 15 days and reduced the activity of succinate dehydrogenase to some extent followed by tetramisole and chlorpromazine which took more time for the clearance of microfilariae and had no effect on the localization of both glucose-6-phosphatase and succinate dehydrogenase.

728–732

Authors: Farida Hussan, Rina Haryani Osman Basah, Mohd Rafizul Mohd Yusof, Nur Aqilah Kamaruddin, Faizah Othman

Objective: To determine the effect of Plantago major (P. major) extract on the liver injury following acetaminophen (APAP) toxicity. Methods: The male Sprague Dawley rats (n = 38) were randomly divided into normal control (n = 6) and experiment (n = 32) groups. The latter was subdivided into four groups and induced with APAP (1000 mg/kg) per oral, followed by P. major extract and N-acetylcysteine orally to the respective groups for six days. Results: On the seventh day, the serum bilirubin, liver enzymes and tissue malondialdehyde were increased in APAP groups whereas the total protein in serum, tissue superoxide dismutase and glutathione levels were reduced. The plant extract treatment reduced the histological deteriorations such as aggregation of hepatocellular cords, formation of binucleated cells and vacuolisation of the cells with scanty cytoplasm. It also revealed significant reduction of malondialdehyde and increased level of superoxide dismutase and glutathione. The findings in the extract treated groups were comparable to the group treated with N-acetylcysteine. Conclusions: In conclusion, P. major can enhance innate antioxidant activity and ameliorate the APAP-induced liver injury.

738–743

Authors: Hataipan Phienwej, Ih-si Swasdichira, Surattana Amnuoypol, Prasit Pavasant, Piyamas Sumrejkanchanakij

Objective: To investigate the effect of Tinospora crispa (T. crispa) extract on matrix metalloproteinase-13 (MMP-13) expression and cell migration. Methods: The cytotoxicity of T. crispa extract was examined by 3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyltetrazolium bromide assay on head and neck squamous cell carcinoma (HNSCC) cell lines. The effect on expression of MMP-13 was analysed by RT-PCR and ELISA. The migration was assessed by wound healing assay. Results: MMP-13 mRNA was highly expressed in the metastatic human HNSCC cell lines, HN22 and HSC-3. T. crispa extract at a concentration of 100.0 mg/mL caused about 50% reduction of cell survival. T. crispa extract at a non-toxic concentration of 12.5, 25.0 and 50.0 mg/mL significantly suppressed MMP-13 mRNA expression and secreted MMP- 13 in both HN22 and HSC-3. The expression of tissue inhibitors of metalloproteinase-2 (TIMP-2) by HSC-3 cells was attenuated by 25.0 and 50.0 mg/mL of T. crispa extract. Addition of the extract to cells in a wound healing assay showed inhibition of cell migration by HN22 cells. Conclusions: These data suggest that T. crispa could be considered as a potential therapeutic drug to prevent metastasis of HNSCC.

748–755

Authors: Anastasia Wheni Indrianingsih, Sanro Tachibana, Rizna Triana Dewi, Kazutaka Itoh

Objective: To isolate and investigate antioxidant and a-glucosidase inhibitor compounds in the leaves of Quercus gilva Blume (Q. gilva). Methods: Dry leaves of Q. gilva were extracted with methanol and the methanolic extract was further separated by silica gel column chromatography using several solvents with increasing polarity. The antioxidant activities of the isolated compounds were evaluated using various in vitro assays: 1,1-diphenyl-2-picrylhydrazyl radical scavenging activity, hydrogen peroxide radical scavenging activity, b-carotene bleaching assay, and reducing power assay. The a-glucosidase inhibitory assay was conducted against aglucosidase from Saccharomyces cerevisiae. Results: Three compounds were isolated and their structures were identified as catechin (1), epicatechin (2), and tiliroside (3) using an instrumental analysis. Compound 2 had higher antioxidant activity with inhibitory concentrations (IC50) of (22.55 ± 2.23) mmol/L than that of quercetin, which was used as the standard, with an IC50 of (28.08 ± 2.39) mmol/L, followed by compound 1 with IC50 of (40.86 ± 3.45) mmol/L. On the other hand, compound 3 had the lowest antioxidant activity with an IC50 of (160.24 ± 8.15) mmol/L. However, compound 3 had the highest a-glucosidase inhibitory activity with an IC50 of (28.36 ± 0.11) mmol/L, followed by compounds 1 and 2 with (168.60 ± 5.15) and (920.60 ± 10.10) mmol/L, respectively. Conclusions: The results obtained for the antioxidant activities and a-glucosidase inhibitory activities in a methanolic extract from the leaves of Q. gilva confirmed the potential of this plant as a source of natural antioxidants and antidiabetic medicine.

761–767

Authors: Hai Nguyen Thanh, Hue Pham Thi Minh, Tuan Anh Le, Huong Duong Thi Ly, Tung Nguyen Huu, Loi Vu Duc, Thu Dang Kim, Tung Bui Thanh

Objective: To investigated the protective potential of ethanol extracts of Scutellaria baicalensis (S. baicalensis) against lipopolysaccharide (LPS)-induced liver injury. Methods: Dried roots of S. baicalensis were extracted with ethanol and concentrated to yield a dry residue. Mice were administered 200 mg/kg of the ethanol extracts orally once daily for one week. Animals were subsequently administered a single dose of LPS (5 mg/ kg of body weight, intraperitoneal injection). Both protein and mRNA levels of cytokines, such as tumor necrosis factor alpha, interleukin-1b, and interleukin-6 in liver tissues were evaluated by ELISA assay and quantitative PCR. Cyclooxygenase-2, inducible nitric oxide synthase, and nuclear factor-kB protein levels in liver tissues were analyzed by western blotting. Results: Liver injury induced by LPS significantly increased necrosis factor alpha, interleukin-1b, interleukin-6, cyclooxygenase-2, inducible nitric oxide synthase, and nuclear factor-kB in liver tissues. Treatment with ethanol extracts of S. baicalensis prevented all of these observed changes associated with LPS-induced injury in liver mice. Conclusions: Our study showed that S. baicalensis is potentially protective against LPSinduced liver injury in mice.

773–777

Authors: Mustafa Kemal Çelen, Murat Sayan, Tuba Dal, Celal Ayaz, Alicem Tekin, Tuncer O¨ zekinci, Suda Tekin Koruk, Tunga Barcin, Recep Tekin, Mehmet Sinan Dal, Sevgi Kalkanlı

Objective: To detect the subtype characterization and drug-resistant mutations in HIV-1 strains after the refugee movement from Syria to Turkey between 2011 and 2014 in south east border lines. Methods: A total of 65 patients were included in this study, of which 57 (88%) patients were antiretroviral therapy-naive patients. HIV-1 RNA was detected and quantified by real-time PCR assay. HIV-1 subtypes and circulating recombinant forms (CRFs) were identified by phylogenetic analysis (neighbor-joining method), and drug-resistant mutations were analyzed. Results: Three major HIV groups were indicated. Two of these groups were located in subtype B. The other group showed heterogeneity. Subtype B (48/65, 73.8%), followed by CRFs (12/65, 18.5%) was the most common strain. Subtype of CRFs consisted of CRF01_AE (9/65, 13.8%) and CRF02_AG (3/65, 4.6%). Subtype C (1/65, 1.5%), sub-subtypes A1 (2/65, 3.1%) and F1 (2/65, 3.1%) were also detected with low prevalence. The rate of overall primary antiretroviral resistance was 4.9% (3/61). Drug-resistant rate for non-nucleoside reverse transcriptase inhibitors was 4.9%. The thymidine analogue mutation rate was 13.1% (8/61). Conclusions: HIV molecular epidemiology studies are necessary to determine transmission patterns and spread. Subtype B and CRF01_AE, CRF02_AG are the most prevalent strains in the south-east of Turkey. However, subtype C, sub-subtypes A1 and F1 are of low prevalence but persist in the south-east of Turkey. In the near future, changing of HIV epidemiology will be possible in Turkey due to migration movement in border lines and resistance testing will play an important role in HIV management.

781–784

Authors: D´ebora Oro, Andreia Heissler, Eliandra Mirlei Rossi, Diane Scapin, Patrıcia da Silva Malheiros, Everton Boff

Objective: To evaluate the antifungal effect of Cinnamomum zeylanicum (C. zeylanicum) and Melaleuca alternifolia essential oils and honey against strains of Candida sp. from HIV-positive patients in order to subsidize new therapeutic strategies for candidiasis. Methods: The study evaluated the antifungal effect of natural antimicrobials against 30 strains of Candida sp. isolated from oral cavities in HIV-infected patients. Then, they were compared to the action of fluconazole and amphotericin B. Antifungal susceptibility was evaluated by the broth macrodilution technique and the minimum inhibitory concentration and the minimum fungicidal concentration were determined. Results: Among all antifungals evaluated in this study, amphotericin B was the one showing the best results; however, all compounds studied here showed inhibitory activities against isolates of Candida sp. Honey (0.0313–64 mg/mL) demonstrated fungistatic activity inhibiting 70% of the isolates. C. zeylanicum essential oil (0.0313–64 mg/ mL) inhibited 93.3% of the Candida strains and Melaleuca alternifolia essential oil (0.0313–64 mg/mL) was able to inhibit 73.3% of them. Conclusions: Therefore, all natural compounds evaluated in this study, especially C. zeylanicum essential oil, may become promising agents for oral candidiasis therapy including in HIV-positive patients.

612–618

Authors: Wan Fatma Zuharah, Chan Jia Ling, Nurfazlina Zulkifly, Nik Fadzly

Objective: To evaluate the lethal concentration, oviposition deterrence and ovicidal activity of acetone extracts of Melanochyla fasciculiflora (M. fasciculiflora) leaf and Gluta renghas (G. renghas) leaf against Aedes albopictus (Ae. albopictus). Methods: To determine the lethal concentration of Anacardiaceae, ten test concentrations of the extracts ranging from 200 to 650 mg/L were selected for larvicidal bioassays and 25 early fourth instar larvae were exposed to the extracts for 24 h. The sub-lethal concentrations used for oviposition deterrence was the value of LC25, LC50 and LC75 from above study which is 235 mg/L, 470 mg/L and 705 mg/L for M. fasciculiflora extract and 187.5 mg/L, 375 mg/L and 562.5 mg/L for G. renghas extract, respectively. Twenty gravid Ae. albopictus were allowed to oviposit in different treated concentrations. For oviciding procedure, a total of 300 eggs of Ae. albopictus were soaked in solution with each treated concentration as mentioned above for 24 h. After 24 h, eggs were sieved and soaked in seasoned water, and hatching rates were calculated. For comparison, only seasoned water was used in control experiment. Results: G. renghas demonstrated lower LC50 value of 372.80 mg/L compared to M. fasciculiflora (467.90 mg/L). The activity index of negative oviposition revealed the deterrent effect and thus, caused a remarkable negative response resulting in oviposition of fewer eggs compared with control (without plant extract). The acetone extract of M. fasciculiflora was more effective than G. renghas extract in displaying oviposition deterrence potential since the latter did not possess the deterring effect within the concentration range tested. However, both plant extracts exhibited excellent oviciding effect as 92.33% of eggs failed to be hatched when treated with 705.0 mg/L of M. fasciculiflora and 86.67% with 562.5 mg/L of G. renghas. The oviposition deterrence and percentage of egg mortality were directly proportional to the concentrations of extracts in both plants tested. Conclusions: These results clearly indicate that the acetone extract of G. renghas could be served as potential larvicide, whereas M. fasciculiflora has better sub-lethal effect for oviposition deterrence and against Ae. albopictus as an oviciding agent.

624–628

Authors: Mohamed Habib Oueslati, Faraj A. Al-Ghamdi, Adel Noubigh

Objective: To isolate and characterize the bioactive secondary metabolites from aerial parts of widespread Chenopodiaceae taxa growing in Saudi Arabia: Salsola villosa Delile. ex Schul. Methods: Antibacterial activities of chloroformic extract, fractions and isolate compounds was evaluated against five bacterial strains (Escherichia coli, Pseudomonas aeruginosa, Salmonella typhimurium, Staphylococcus aureus and Staphylococcus epidermidis), using a paper disc diffusion method. The purification of compound(s) of chloroform extract was done by chromatographic column of silica gel. The structure elucidation was determined by extensive spectroscopic analysis (1H and 13C nuclear magnetic resonance, correlation spectroscopy, heteronuclear multiple bond correlation, heteronuclear multiple quantum coherence and nuclear overhauser enhancement spectroscopy) and high resolution electrospray ionization mass spectroscopy analysis. Results: Bioactivity guided fractionation of the chloroformic extract led to the isolation of two bioactive compounds: 4-(40-hydroxy-20-methylcyclopent-20-enyloxy)- 4-methylcyclopent-2-enol (1) named salsolanol and 40-[3-(hydroxymethyl)oxiran-2-yl]-3- [(E)-3-hydroxyprop-1-en-1-yl]-6, 20-dimethoxy [1, 10-biphenyl]-2-ol (2) named biphenylsalsinol. The antibacterial effects of the chloroform extracts, fractions and isolated compounds 1 and 2 were also evaluated in this work. Results showed that the compounds 1 and 2 exhibited antibacterial activities against four strains: Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli and Pseudomonas aeruginosa with diameter of zone of inhibition ranging between (9.33 ± 0.94) to (26.33 ± 0.94) mm. Conclusions: Based on data presented here, two new natural compounds secondary cyclic alcohol 1 and biphenylpropanoid 2 isolated from bioactive chloroformic extract from aerial parts of Salsola villosa can be responsible for its antibacterial activities.

636–644

Authors: Tarik Khouya, Mhamed Ramchoun, Abdelbassat Hmidani, Souliman Amrani, Hicham Harnafi, Mohamed Benlyas, Younes Filali Zegzouti, Chakib Alem

Objective: To evaluate the anti-inflammatory, anticoagulant and antioxidant effects of aqueous extracts of thyme varieties from Moroccan. Methods: The aqueous extracts of tree medicinal plants [Thymus atlanticus (T. atlanticus), Thymus satureioides and Thymus zygis (T. zygis)] were screened for their antioxidant activity using 1,1-diphenyl-2-picrylhydrazyl radical-scavenging, ferric reducing antioxidant power assay, radical scavenging activity method, the inhibition of 2,2'-azobis (2-amidinopropane) dihydrochloride that induces oxidative erythrocyte hemolysis and thiobarbituric acid reactive substances assay. The anti-inflammatory activity of aqueous extracts was evaluated in vivo using croton oil-induced ear edema and carrageenan-induced paw edema in mice and rats, respectively. This extracts were evaluated in vitro for their anticoagulant activity at the different concentrations by partial thromboplastin time and prothrombin time activated. Results: All thyme varieties were found to possess considerable antioxidant activity and potent anti-inflammatory activity in the croton oil-induced edema. Administration of aqueous extracts of two varieties (50 mg/kg) (T. zygis and T. atlanticus) reduced significantly the carrageenan-induced paw edema similar to non-steroidal anti-inflammatory drug (indomethacin, 10 mg/kg). In partial thromboplastin time and prothrombin time tests, T. atlanticus and T. zygis extracts showed the strongest anticoagulant activity. In contrast, Thymus satureioides did not show the anticoagulant activity in these tests. Conclusions: All aqueous extracts possess considerable antioxidant activity and are rich in total polyphenol and flavonoid but they act differently in the process of inflammatory and coagulation studied. This study shows great variability of biological activities in thyme varieties.

650–657

Authors: Atamgba Agbor Asuk, Margaret Akpana Agiang, Kayode Dasofunjo, Amonor James Willie

Objective: To analyse the phytochemical contents of leaf, stem bark and root of Jatropha curcas (J. curcas) in four solvent extracts and their proximate and mineral compositions. Methods: Standard analytical procedures were used for the determination of phytochemicals, proximate and mineral compositions of the leaf, stem bark and root extracts of J. curcas. Results: Results of the analysis showed the presence of polyphenols, flavonoids, alkaloids, cardiac glycosides, coumarins, saponins, terpenoids, steroids, triterpenoid saponins, carotenoids, phlobatannins and tannins in the leaf, stem bark and root of all the solvent extracts. Flavonoids were present in the highest amount in the ethyl acetate extracts of the leaf (7.35% ± 0.02%), stem bark (4.12% ± 0.01%) and root (3.35% ± 0.02%) followed by polyphenols in the methanol extracts of leaf (4.62% ± 0.02%), stem bark (2.77% ± 0.05%) and root (2.49% ± 0.02%). Poly-acetylated compounds were absent in all the solvent extracts of the leaf, stem bark and root. However, some anti-nutritional agents such as oxalates, phytates and cyanates were present in all the solvent extracts of the leaf, stem bark and root except the ethyl acetate. Phytates were high in the aqueous solvent of the leaf (6.12% ± 0.00%) but low in the stem bark (1.00% ± 0.05%) and root (0.89% ± 0.03%). Proximate composition showed appreciable amounts of total carbohydrate (36.33% ± 0.72%), crude protein (26.00% ± 0.47%) and reducing sugars (5.87% ± 0.14%) in the leaf, while crude fat was more in the stem bark (16.70% ± 0.30%). There was corresponding substantial energy in the leaf [(1514.77 ± 20.87) kJ/100 g] and stem bark [(907.00 ± 8.52) kJ/100 g]. Moisture and ash contents of the leaf, stem bark and root were within acceptable limits for the use in drugs formulation. The mineral composition showed substantial amounts of important elements such as Fe, Ca, Na, Mg and Zn. Others were P, K and Se. Conclusions: The outcome of this study suggests that the leaf, stem bark and root of J. curcas have very good medicinal potentials, meet the standard requirements for drug formulation and serve as good sources of energy and nutrients except for the presence of some anti-nutritional elements predominant in the leaf.

663–670

Authors: King Shimumbo Nalubamba, Eugene Chisela Bwalya, Ntombi Basimbi Mudenda, Hetron Mweemba Munangandu, Musso Munyeme, David Squarre

Objective: To determine the gastrointestinal tract helminthic fauna in domestic and wild guineafowl in Zambia. Methods: Post-mortem and laboratory parasitological examinations for helminth identification and enumeration were conducted on 198 guineafowls (148 domestic and 50 wild) from November 2010 to October 2011. Results: All guineafowls were infested with one or more helminths. Eleven helminth species, namely, Raillietina echinobothrida, Raillietina tetragona, Raillietina cesticillus, Ascaridia galli, Allodapa suctoria, Gongylonema ingluvicola, Tetrameres spp., Heterakis spp., Acuaria spiralis, Syngamus trachea, and Streptocara pectinifera were identified with no trematodes recorded. Mean nematode burden between domestic and wild fowl showed no differences having 113.7 [confidence interval (CI) 98.9–128.6] and 108 (CI 76.6–139.5) nematodes respectively. In contrast, female guineafowls had a mean of 151.9 (CI 128.4–177.8) nematodes per host which was significantly more than the males that had a mean of 79.6 (CI 66.8–94.4). However, there were differences in helminth species richness between domestic and wild guineafowls with domestic guineafowls having more species present at a mean of 4.2 (CI 3.91–4.44) than the wild ones at a mean of 3.4 (CI 2.92–3.88) but there were no sex differences. Eight of the eleven helminth species cooccurred in domestic and wild fowl and five of the helminth species had higher prevalence in domestic guineafowls. Conclusions: Syngamus trachea, Streptocara pectinifera and Acuaria spiralis are reported for the first time in domestic poultry in Zambia. This study represents the first comparative study of helminths in domestic and wild guineafowls at an interface area and adds to the knowledge base in a discipline where a dearth currently exists.

676–683

Authors: Essa Ajmi Alodeani, Mohammad Arshad, Mohammad Asrar Izhari

Objective: To deal with the anti-uropathogenic and in silico screening of (E-)-N0- (substituted-benzylidene)-2-(quinolin-8-yloxy)acetohydrazide analogues in order to search the potential anti-uropathogenic agents. Methods: Three (E-)-N0-(substituted-benzylidene)-2-(quinolin-8-yloxy)acetohydrazide analogues were synthesized. Structure elucidation was done using various spectroscopic techniques including infrared radiation, 1hydrogen-nuclear magnetic resonance, carbon- 13 nuclear magnetic resonance, etc. Physicochemical score, bioactivity score and molecular docking studies were carried out using Lipinski's rule of five, Molinspiration (web based software), Autodock 4.2 tools. In vitro anti-uropathogenic activity was carried out against four pathogens named as Staphylococcus aureus (S. aureus), Staphylococcus epidermidis, Proteus mirabilis and Escherichia coli by disc diffusion method and macrodilution test following their morphological and biochemical characterization. Results: The formation of (E-)-N0-(substituted-benzylidene)-2-(quinolin-8-yloxy)acetohydrazide is confirmed from the spectroscopic results. All the compounds were found in compliance with Lipinski's rule of five and exhibited bioactivity score from −0.50 to 0.00. Docking results revealed that compound-1 is forming one hydrogen bond with TYR 576 and two hydrogen bond with GLU 569, while compound-2 is forming one hydrogen bond with ARG 599, and compound-3 forming 0 hydrogen bond. The anti-uropathogenic evaluation exhibited that compound one exhibited better activity against S. aureus, while it was found to possess moderate to good activity against both Gram-positive bacteria and Gram-negative bacteria excluding S. aureus. Conclusions: Our study revealed that compound one exhibited better activity than the standard in case of S. aureus and moderate to good activity against rest of the pathogens. Molecular docking, physicochemical and bioactivity studies strongly supported the experimental results. From the well obtained results it was concluded that compound-1 can lead as potential anti-uropathogenic agents.

689–693

Authors: Pascalina Chanda-Kapata, William Ngosa, Albertina Ngomah Moraes, Nicole Maddox, Nathan Kapata

Objective: To assess the prevalence of HIV infection, to highlight HIV-testing refusal rates among participants in a population-based tuberculosis survey and to assess the implication for programme implementation. Methods: This cross-sectional study on the characteristics of participants who refused HIV testing was conducted in a national survey in Zambia. All eligible participants were aged above 15 years and included in the analysis. Results: Out of the 44 791 tuberculosis survey participants, 14 164 (31.6%) refused to participate in HIV testing. The unemployed, rural dwellers, married, and those aged 15– 24 years were associated with higher refusal rates. Conclusions: Strategies to improve HIV testing acceptance are necessary. Qualitative research is recommended to understand the reasons for testing refusals so that remedial interventions can be implemented.

510-514

Authors: Sameer Rushdi Organji, Hussein Hasan Abulreesh, Khaled Elbanna, Gamal Ebrahim Haridy Osman, Manal Khider

Objective: To investigate the true incidence of Bacillus cereus (B. cereus) in food and children diarrhea cases. Methods: A total of 110 samples of various dairy products such as raw milk, long life pasteurized milk, yoghurt and infant powdered milk formulas, raw rice, and feces were examined for the presence of B. cereus by selective plating on mannitol-egg-yolkpolymyxin agar. Confirmation of B. cereus was carried out by biochemical tests and PCR. Identification of non-B. cereus isolates was carried out by 16S rDNA sequencing. Antimicrobial susceptibility was done by disk diffusion method. Results: Overall 35 samples (31.8%, n = 110) yielded Bacillus-like growth. Of which 19 samples (54.28%) were positive for B. cereus. All isolates were positive for enterotoxin production. No psychrotolerant B. cereus strains were detected in all samples. All B. cereus isolates were resistant to penicillin G, but susceptible to vancomycin, erythromycin and clindamycin. Conclusions: The results of this study confirm the importance of including B. cereus in disease control and prevention programs, as well as in routine clinical and food quality control laboratories in both Saudi Arabia and Egypt.

525-532

Authors: Demetrio L. Valle Jr., Jeannie I. Andrade, Juliana Janet M. Puzon, Esperanza C. Cabrera, Windell L. Rivera

Objective: To investigate the antibacterial activities of crude ethanol extracts of 12 Philippine medicinal plants. Methods: Crude ethanol extracts from 12 Philippine medicinal plants were evaluated for their antibacterial activity against methicillin-resistant Staphylococcus aureus, vancomycinresistant Enterococcus, extended spectrum β-lactamase-producing, carbapenem-resistant Enterobacteriaceae and metallo-β-lactamase-producing Pseudomonas aeruginosa and Acinetobacter baumannii. Results: The leaf extracts of Psidium guajava, Phyllanthus niruri, Ehretia microphylla and Piper betle (P. betle) showed antibacterial activity against the Gram-positive methicillinresistant Staphylococcus aureus and vancomycin-resistant Enterococcus. P. betle showed the highest antibacterial activity for these bacteria in the disk diffusion (16-33 mm inhibition diameter), minimum inhibitory concentration (19-156 μg/mL) and minimum bactericidal concentration (312 μg/mL) assays. P. betle leaf extracts only showed remarkable antibacterial activity for all the Gram-negative multidrug-resistant bacteria (extended spectrum β-lactamaseproducing, carbapenem-resistant Enterobacteriaceae and metallo-β-lactamase-producing) in the disk diffusion (17-21 mm inhibition diameter), minimum inhibitory concentration (312-625 μg/mL) and minimum bactericidal concentration (312-625 μg/mL) assays. Conclusions: P. betle had the greatest potential value against both Gram-negative and Grampositive multidrug-resistant bacteria. Favorable antagonistic activities were also exhibited by the ethanol extracts of Psidium guajava, Phyllanthus niruri and Ehretia microphylla.

538-543

Authors: Innocent Onyesom, Ejovi Osioma, Precious Chiamaka Okereke

Objective: To assess the effects of hot water leaf extract of Nauclea latifolia (N. latifolia) on antioxidant status, lipid peroxidation values and parasite levels in hepatic and brain tissue of experimental mice (BALB/c) infected with Plasmodium berghei (P. berghei) malaria. Methods: Forty nine mice were divided into seven groups (n = 7) and used for the study. Group A (control) were given 0.2 mL/kg phosphate buffer saline; Group B mice were infected with P. berghei and treated with phosphate buffer saline. Groups C and D mice were also infected but treated with 200 and 300 mg/kg body weight of leaf extract respectively. Groups E and F mice were not infected, but received 200 and 300 mg/kg of leaf extract respectively. Group G mice were infected and treated with chloroquine (5 mg/kg). Liver and brain tissues of mice were prepared for both biochemical assay and microscopic examination. Results: Results showed that P. berghei malaria infection induced oxidative stress in both liver and brain tissues as evidenced by the significant (P < 0.05) decrease in antioxidants: superoxide dismutase, reduced glutathione and catalase. These reductions perhaps caused compromise in membrane integrity as indicated by the significant increase in lipid peroxidation product malondialdhyde. Malaria parasites were also identified in these tissues. However, N. latifolia treatment eliminated the parasites in tissues and protected them from oxidative damage even better than chloroquine treatment did, whose anti-malarial potency also cleared tissue parasites. The measurement of protection by N. latifolia against damage was strengthened by the insignificant micro structural alterations. Conclusions: The bioactive phytochemical(s) in N. latifolia should be structured and the mechanism(s) of its antimalarial tendency should be further investigated.

547-552

Authors: Jicheng Yu, Liu Zhang, Qiu Liu, Xiaohui Qi, Ying Ji, Beom Seok Kim

Objective: To evaluate various types of samples from the different marine environments as sources of actinomycetes from the Yalujiang coastal wetland, North China, and to screen their antimicrobial properties. Further, the identified actinomycetes were characterized based on morphological, biochemical, and physiological characteristics. Methods: Eight different production media were used to isolate actinomycets from different stations of marine soil sediments in Yalujiang coastal wetland and the genotypic positions were established by 16S rDNA. Results: A total of 172 actinomycetal isolates were obtained from 13 samples using five media. The most effective culture media in the isolation of actinobacteria were Gause’s Synthetic agar and Starch-casein agar. Among 172 isolates, 46 isolates (26.74%) showed antibacterial activity, 70.93% belonged to the genus Streptomyces, others were Micromonospora spp. and Rhodococcus spp. Out of the 46 isolates, two cultures were further supported by morphological characterization analysis. Conclusions: This is the first report about actinomycetes isolated from Yalujiang coastal wetland and it seems that the promising isolates from the unusual/unexplored wetland may prove to be an important step in the development of microbial natural product research.

560-565

Authors: Ali Rafati, Mahboobeh Erfanizadeh, Ali Noorafshan, Saied Karbalay-Doust

Objective: To investigate the effects of benzene on rat’s cerebellum structure and behavioral characteristics, including anxiety and motor impairment. Methods: Twenty rats were randomly allocated into two groups orally receiving distilled water and benzene (200 mg/kg/day). A total of 10 rats were used at the beginning of benzene exposure. Two rats died during benzene treatment and 8 rats remained for evaluation of the behavioral test and finally 6 rats underwent histological assessment. At the end of the 4th week, motor function and anxiety were evaluated in rotarod test and elevated plus maze, respectively. Besides, the cerebellum was dissected for structural assessment using stereological methods. Results: Performance of the benzene-treated rats in fixed and accelerating speed rotarod was impaired and their riding time (endurance) was lower compared to the control group (P = 0.02). The benzene-treated rats also spent less time in the open arms and had fewer entrances to the open arms in comparison to the control group, indicating anxiety (P = 0.01). The total volume of the cerebellar hemisphere, its cortex, intracerebellar nuclei, total number of the Purkinje, Bergmann, Golgi, granule, neurons and glial cells of the molecular layer, and neurons and glial cells of the intracerebellar nuclei were reduced by 34%-76% in the benzene-treated rats in comparison to the distilled water group (P = 0.003). The most cell loss was seen in Bergmann glia. Conclusions: The structure of cerebellum altered after benzene treatment. In addition, motor impairment and anxiety could be seen in benzene-treated rats.

571-575

Authors: Ivan Viktorovich Borozda, Mikhail Anatolievich Danilov, Kirill Sergeevich Golokhvast

Objective: To propose an original method of surgical treatment for the acromial extremity of the clavicle rupture (Rockwood II-VI) with modeled Kirschner wire and cortical screw. Methods: Anatomical study and a test method were applied to 43 cadavers of both sexes. During the period between 2000 and 2013, 34 patients of both sexes were operated upon using the new method. In the comparison group (n = 120), the fixation of the acromial extremity of the clavicle rupture was performed with hamate plate, Lee hook and Kirschner wires. Results: Its application allows, according to the evaluation scale of Constant and Murley (1987), 10% more preservation of the function of the shoulder compared with traditional methods of surgical treatment, and shortens the required hospital treatment and temporary disability periods. Conclusions: It is shown that the proposed author’s method combines low invasiveness, minimum dimensions of the construction and low-cost treatment.

581-586

Authors: Rina Girard Kaminsky, Renato Valenzuela Castillo, Coralia Abrego Flores

Objective: To document epidemiologic data, clinical manifestations and treatment in hospitalized children with Trichuris dysenteric syndrome from Honduras during 2010-2012. Methods: Severe trichuriasis cases were identified by routine stool examinations from hospitalized patients (12 years old or less). Relevant epidemiologic, clinical and management data were obtained from review of clinical histories in the ward. Results: Of 122 Trichuris infections diagnosed in 11 528 (1.0%) stool samples for all ages, 81 (66.4%) were identified in the age group 2-12 years old, 21 (25.9%) of which were severe (≥ 100 eggs in 2 mg of feces). Thirteen of those 21 patients collaborated in this study. Patients (9 males and 4 females) were of rural precedence, from large poor or very poor families, chronically parasitized, and between 2 and 12 years old. Dysentery of months duration, severe anemia and stunting were common complaints; clinical characteristics associated with heavy Trichuris infections included egg counts from 232-3 520 eggs per direct smear, hemoglobin from 3.4-10.8 g/dL, eosinophilia up to 43%, severe malnutrition and growth stunting. Orally administered drugs mebendazole, albendazole, metronidazole, nitaxozanide, and piperazine were prescribed at different dosages and duration other than recommended; no cure or egg excretion control was exercised before patient release. A range of 340 to about 10 000 worms were recovered after treatment from 8 patients. Conclusions: This report underlines the need for detailed community studies in trichuriasis morbidity, effective treatment assessment and clinical response in severely malnourished parasitized children.