Search results for: bovine mastitis

Authors: Alena Semeradtova, Marcel Stofik, Lucie Mareckova, Petr Maly, Ondrej Stanek, Jan Maly

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Recently, novel strategies based on so-called molecular evolution were shown to be effective for the production of various peptide ligand libraries with high affinities to molecular targets of interest comparable or even better than monoclonal antibodies. The major advantage of these peptide scaffolds is mainly their prevailing low molecular weight and simple structure. This study describes a new high-affinity binding molecules based immunesensor using a simple optical system for human serum albumin (HSA) detection as a model molecule. We present a comparison of two variants of recombinant binders based on albumin binding domain of the protein G (ABD) performed on micropatterned glass chip. Binding domains may be tailored to any specific target of interest by molecular evolution. Micropatterened glass chips were prepared using UV-photolithography on chromium sputtered glasses. Glass surface was modified by (3-aminopropyl)trietoxysilane and biotin-PEG-acid using EDC/NHS chemistry. Two variants of high-affinity binding molecules were used to detect target molecule. Firstly, a variant is based on ABD domain fused with TolA chain. This molecule is in vivo biotinylated and each molecule contains one molecule of biotin and one ABD domain. Secondly, the variant is ABD domain based on streptavidin molecule and contains four gaps for biotin and four ABD domains. These high-affinity molecules were immobilized to the chip surface via biotin-streptavidin chemistry. To eliminate nonspecific binding 1% bovine serum albumin (BSA) or 6% fetal bovine serum (FBS) were used in every step. For both variants range of measured concentrations of fluorescently labelled HSA was 0 – 30 µg/ml. As a control, we performed a simultaneous assay without high-affinity binding molecules. Fluorescent signal was measured using inverse fluorescent microscope Olympus IX 70 with COOL LED pE 4000 as a light source, related filters, and camera Retiga 2000R as a detector. The fluorescent signal from non-modified areas was substracted from the signal of the fluorescent areas. Results were presented in graphs showing the dependence of measured grayscale value on the log-scale of HSA concentration. For the TolA variant the limit of detection (LOD) of the optical immunosensor proposed in this study is calculated to be 0,20 µg/ml for HSA detection in 1% BSA and 0,24 µg/ml in 6% FBS. In the case of streptavidin-based molecule, it was 0,04 µg/ml and 0,07 µg/ml respectively. The dynamical range of the immunosensor was possible to estimate just in the case of TolA variant and it was calculated to be 0,49 – 3,75 µg/ml and 0,73-1,88 µg/ml respectively. In the case of the streptavidin-based the variant we didn´t reach the surface saturation even with the 480 ug/ml concentration and the upper value of dynamical range was not estimated. Lower value was calculated to be 0,14 µg/ml and 0,17 µg/ml respectively. Based on the obtained results, it´s clear that both variants are useful for creating the bio-recognizing layer on immunosensors. For this particular system, it is obvious that the variant based on streptavidin molecule is more useful for biosensing on glass planar surfaces. Immunosensors based on this variant would exhibit better limit of detection and wide dynamical range.

Keywords: high affinity binding molecules, human serum albumin, optical immunosensor, protein G, UV-photolitography

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Authors: Lizeth Fuentes-Mera, Vanessa Perez-Silos, Nidia K. Moncada-Saucedo, Alejandro Garcia-Ruiz, Alberto Camacho, Jorge Lara-Arias, Ivan Marino-Martinez, Victor Romero-Diaz, Adolfo Soto-Dominguez, Humberto Rodriguez-Rocha, Hang Lin, Victor Pena-Martinez

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Biphasic scaffolds in cartilage tissue engineering have been designed to influence not only the recapitulation of the osteochondral architecture but also to take advantage of the healing ability of bone to promote the implant integration with the surrounding tissue and then bone restoration and cartilage regeneration. This study reports the development and characterization of a biphasic scaffold based on the assembly of a cartilage phase constituted by fibroin biofunctionalized with bovine cartilage matrix; cellularized with differentiated pre-chondrocytes from adipose tissue stem cells (autologous) and well attached to a bone phase (bone bovine decellularized) to mimic the structure of the nature of native tissue and to promote the cartilage regeneration in a model of joint damage in pigs. Biphasic scaffolds were assembled by fibroin crystallization with methanol. The histological and ultrastructural architectures were evaluated by optical and scanning electron microscopy respectively. Mechanical tests were conducted to evaluate Young's modulus of the implant. For the biological evaluation, pre-chondrocytes were loaded onto the scaffolds and cellular adhesion, proliferation, and gene expression analysis of cartilage extracellular matrix components was performed. The scaffolds that were cellularized and matured for 10 days were implanted into critical 3 mm in diameter and 9-mm in depth osteochondral defects in a porcine model (n=4). Three treatments were applied per knee: Group 1: monophasic cellular scaffold (MS) (single chondral phase), group 2: biphasic scaffold, cellularized only in the chondral phase (BS1), group 3: BS cellularized in both bone and chondral phases (BS2). Simultaneously, a control without treatment was evaluated. After 4 weeks of surgery, integration and regeneration tissues were analyzed by x-rays, histology and immunohistochemistry evaluation. The mechanical assessment showed that the acellular biphasic composites exhibited Young's modulus of 805.01 kPa similar to native cartilage (400-800 kPa). In vitro biological studies revealed the chondroinductive ability of the biphasic implant, evidenced by an increase in sulfated glycosaminoglycan (GAGs) and type II collagen, both secreted by the chondrocytes cultured on the scaffold during 28 days. No evidence of adverse or inflammatory reactions was observed in the in vivo trial; however, In group 1, the defects were not reconstructed. In group 2 and 3 a good integration of the implant with the surrounding tissue was observed. Defects in group 2 were fulfilled by hyaline cartilage and normal bone. Group 3 defects showed fibrous repair tissue. In conclusion; our findings demonstrated the efficacy of biphasic and bioactive scaffold based on silk fibroin, which entwined chondroinductive features and biomechanical capability with appropriate integration with the surrounding tissue, representing a promising alternative for osteochondral tissue-engineering applications.

Keywords: biphasic scaffold, extracellular cartilage matrix, silk fibroin, osteochondral tissue engineering

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Authors: C. I. Boshoff, S. Steenkamp-Jonker

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Cystic echinococcosis (CE), caused by Echinococcus granulosus is an important parasitic infection in livestock worldwide, with severe zoonotic potential. It is important to understand the variability of Echinococcus granulosus, as genotype variations may influence lifecycle patterns, development rate, and transmission. Cystic Echinococcus samples were collected from domestic animals in Eastern Cape Province, South Africa. A molecular study was performed on 14 hydatid cysts obtained from caprine, ovine and bovine livers in order to determine the Echinococcus granulosus strain present in these hosts. The sequencing of the mitochondrial cytochrome C oxidase subunit I (coxI) gene of the hydatid cysts produced sequences of 400 bp for each sample analysed. These sequences were aligned with those present in GenBank and a phylogenetic tree was constructed. Based on coxI genotype the isolates could be grouped into E. granulosus sensu stricto. The findings of the study represent a pilot molecular study on Echinococcus from domestic animals undertaken in South Africa.

Keywords: Echinococcus granulosus, genotypes, livestock, South Africa

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Authors: Binnur Sagbas

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Artificial joint replacements such as total knee and total hip prosthesis have been applied to the patients who affected by osteoarthritis. Although different material combinations are used for these joints, biopolymers are most commonly preferred materials especially for acetabular cup and tibial component of hip and knee joints respectively. The main limitation that shortens the service life of these prostheses is wear. Wear is complicated phenomena and it must be considered with friction and lubrication. In this study, micro wave (MW) induced argon+oxygen plasma surface modification were applied on ultra-high molecular weight polyethylene (UHMWPE) and vitamin E blended UHMWPE (VE-UHMWPE) biopolymer surfaces to improve surface wettability and wear resistance of the surfaces. Contact angel measurement method was used for determination of wettability. Ball-on-disc wear test was applied under 25% bovine serum lubrication conditions. The results show that surface wettability and wear resistance of both material samples were increased by plasma surface modification.

Keywords: artificial joints, plasma surface modification, UHMWPE, vitamin E, wear

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Authors: Mosaab A. Omar, Mohammad Saleh Al-Aboody, Mohmed A. Rizk, Shimaa M. Elsayed, Ahmed ElSify, Naoaki Yokoyama, Ikuo Igarashi

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Enoxacin is a broad-spectrum 6-fluoronaphthyridinone antibacterial agent (fluoroquinolones) structurally related to nalidixic acid used mainly in the treatment of urinary tract infections and gonorrhea. Also, it has been shown recently that it may have cancer inhibiting effect. The primary antibabesial effect of Enoxacin is due to inhibition of DNA gyrase subunit A, and DNA topoisomerase. In the present study, enoxacin was tested as a potent inhibitor against the in vitro growth of bovine and equine Piroplasms. The in vitro growth of five Babesia species that were tested was significantly inhibited (P<0.05) by micromolar concentrations of enoxacin (IC50 values= 13.5, 7.2, 7.5, and 24.2 µM for Babesia bovis, Babesia bigemina, Babesia caballi, and Theileria equi, respectively). Enoxacin IC50 values for Babesia and Theileria parasites were satisfactory as the drug is a potent antibacterial drug with minimum side effects. Therefore, enoxacin might be used for the treatment of Babesiosis and Theileriosis especially in case of mixed infections with bacterial diseases or in the case of animal sensitivity against diminazin toxicity.

Keywords: enoxacin, Babesia, Theileria, IC50 and dimenazin

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Authors: Rima Zakzouk, Noriko Hiraishi, Mohamed Mahdi Alshahni, Koichi Makimura, Junji Tagami

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Background and Purpose: Silver diamine fluoride (SDF) is used to prevent and arrest dental caries. The aim of this study is to evaluate the effect of SDF on reducing Candida albicans adhesion on dentin. Materials and Methods: Bovine dentin disks (6×6 mm) were cut by Isomet and polished using grit silicon carbide papers down to 2000 in order to obtain flat dentin surfaces. Samples were divided into two groups. The first group (SDF group) was treated with 38% SDF for 3 min, while the other group (control group) did not undergo SDF treatment. All samples were exposed to C. albicans suspension, washed after 6 hours incubation at 30 °C before to be tested using XTT (2,3-Bis-(2-Methoxy-4-Nitro-5-Sulfophenyl)-2H-Tetrazolium-5-Carboxanilide) and real time PCR approaches. Statistical analyses of the results were performed at the significance level α = 0.05. Results: SDF inhibited C. albicans adhesion onto dentin. A significant difference was found between the SDF and control groups in both XTT and real time PCR tests. Conclusion: Using SDF to arrest the caries, could inhibit the Candida growth on dentin.

Keywords: silver diamine fluoride, dentin, real time PCR, XTT

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Authors: Mohammad Asadpour, Gholamreza Razmi, Gholamreza Mohammadi, Abolghasem Naghibi

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Cryptosporidium Spp., protozoan parasites of the phylum Apicomplexa, have a wide spectrum of hosts including humans, domestic animals and wild mammals, birds, reptiles, amphibians and fish. Dairy cattle have been identified in numerous reports as a major source of environmental contamination with this pathogen. In this study, a Polymerase Chain Reaction (PCR), Restriction Fragment Length Polymorphism (RFLP) analysis of the Small-Subunit (SSU) rRNA gene was used to detect and identify Cryptosporidium Spp. in 300 fecal specimens from 1 to 30 days pre-wean calves in 10 farms in Mashhad, Iran. Eighty five (28.3%) and forty five (15%) of the specimens were positive for Cryptosporidium by microscopic and PCR examination respectively. Restriction digestion of the PCR products by VSPI and Ssp1 restriction enzymes and analysis of sequence data revealed the presence of C. parvum, bovine genotype in all isolates. Our findings suggest that cattle can be a source of Cryptosporidial infections for humans and animals in Mashhad area. This is the first published description of Cryptosporidium sub genotyping in Mashhad.

Keywords: cryptosporidium, genotype, dairy calves, 18S rRNA, Mashhad

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Authors: Farhan Ahmad Atif, Abd Ur Rehman, Muhammad Babir

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Dietary cation anion difference (DCAD) is an important aspect of dairy nutrition, especially in the transition period. Sahiwal cattle is the highest milk producing breed among Zebu cattle. We planned first study on transition Sahiwal cattle to determine the effects of feeding varying levels of negative DCAD. For this purpose, twenty pregnant cows (at the 250th day of gestation) were selected and randomly divided into 5 groups comprising four animals each. Five iso-caloric (2100 Kcal) and iso-nitrogenous (12%) diets were formulated and each diet was allotted to each group. The animals received positive DCAD diet served as control. Diets were supplemented with NutriCAB® to attain 0, -15, -30 and -45 DCAD levels. Experimental diets were fed at ad-libitum upto parturition and data regarding feed intake were recorded on daily. Post-partum incidence of milk fever, dystocia, retention of placenta (RP), mastitis as well as milk production, milk fat percentage and serum Ca levels were recorded. Urine and blood pH were determined weekly during the last month of pregnancy. Results showed that prepartum feed intake and blood pH were not affected (P > 0.05), while urine pH was significantly reduced (P < 0.05) by lowering DCAD levels. Post parturient blood calcium level linearly increased (P < 0.05) with decreasing DCAD. Pre-partum negative DCAD feeding had no effect (P > 0.05) on post-parturient milk production and fat percentage. However, parturient related problems decreased with decreasing DCAD feeding. It was concluded that negative DCAD feeding raised serum calcium level and reduced the incidence of post-parturient problems in Sahiwal cattle.

Keywords: dairy cattle, transition, metabolic diseases, reproductive disorders, incidence

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Authors: S. Md. Shaarani, J. Md. Jahim, R. A. Rahman, R. Md. Illias

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Modern developments in biotechnology have paved the way for extensive use of biocatalysis in industries. Although it offers immense potential, industrial application is usually hampered by lack of operational stability, difficulty in recovery as well as limited re-use of the enzyme. These drawbacks, however, can be overcome by immobilization. Cross-linked enzyme aggregates (CLEAs), a versatile carrier-free immobilization technique is one that is currently capturing global interest. This approach involves precipitating soluble enzyme with an appropriate precipitant and subsequent crosslinking by a crosslinking reagent. Without ineffective carriers, CLEAs offer high enzymatic activity, stability and reduced production cost. This study demonstrated successful CLEA synthesis of recombinant xylanase from Trichoderma reesei using ethanol as aggregating agent and glutaraldehyde (2% (v/v); 100 mM) as crosslinker. Effects of additives including proteic feeder such as bovine serum albumin (BSA) and poly-L-Lysine were investigated to reveal its significance in enhancing the performance of enzyme. Addition of 0.1 mg BSA/U xylanase showed considerable increment in CLEA development with approximately 50% retained activity.

Keywords: cross-linked, immobilization, recombinant, xylanase

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Authors: Jessica P. Fiorotti, Maria C. Freitas, Caio J. B. Coutinho-Rodrigues, Mariana G. Camargo, Emily S. Mesquita, Amanda R. C. Corval, Ricardo O. B. Bitencourt, Allan F. Marciano, Diva D. Spadacci-Morena, Patricia S. Golo, Isabele C. Angelo, Vania R. E. P. Bittencourt

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The bovine tick, Rhipicephalus microplus, is an arthropod of great importance in veterinary medicine leading to anemia, weight loss, animals' leather depreciation and also acting as a vector of many pathogens. In this way, the parasitism causes a loss of 3.24 billion dollars per year in Brazil. Knowingly, entomopathogenic fungi act as natural controller of some arthropods, acting mainly by active penetration through the cuticle. However, it can also act on the hemolymph and through the production of mycotoxins. Hemocytes are responsible for the cellular immune response and participate in the processes of phagocytosis, nodulation and encapsulation and may undergo changes when challenged by pathogens. The aim of the present study was to evaluate changes in R. microplus hemocytes after inoculation of Metarhizium robertsii using transmission electron microscopy. The isolate ARSEF 2575 and 200 engorged R. microplus females were used. The groups were divided into control, in which the females were inoculated with 5 μL of sterile distilled water solution and 0.1% Tween 80, and a group inoculated with 5 μL of fungal suspension at the concentration of 10⁷ conidia mL⁻¹. The experiment was performed in duplicate and each group contained 50 females. Twenty-four hours after fungal inoculation, hemolymph was collected through the cuticle dorsal surface perforation of the tick females. After collection, the hemolymph samples were centrifuged at 500 x g for 3 minutes at 4 °C, the plasma was discarded and the hemocyte pellet was resuspended in 50 μl PBS. The suspension material was fixed in 2% glutaraldehyde in Millonig buffer for three hours. After fixation, the material was centrifuged at 500 x g for 3 minutes, the supernatant was discarded and the cells were resuspended in a wash solution. Subsequently, the cells were post-fixed with 1% osmium tetroxide in phosphate buffer for one hour at room temperature and dehydrated in increasing concentrations of ethanol, and then embedded in Epon resin. The ultrathin sections were examined under the LEO EM 906E transmission electron microscopy at 80kV. The ultrastructural results revealed that.in control group, the cells were considered intact, in which the granulocytes were observed with granules of different electrodensities, intact mitochondria and cytoplasm without vacuolization. In addition, granulocytes showed plasma membrane projections similar to pseudopodia. Plasmatocytes presented as irregularly shaped cells, with the eccentric nucleus, agranular cytoplasm and some cells presented pseudopodia. Nevertheless, in the group exposed to the fungus, most of the cells presented in degeneration. The granulocytes found had fewer granules in the cytoplasm and more vacuoles. Plasmatocytes, after treatment, presented many vacuoles also in the cytoplasm and the lysosomes presented great amount of electrodense material in their interior. Thus, the results suggest that the fungus has a depressant action in the immune system of the tick, not only by the cell degranulation, but also suggesting that this leads to morphological changes in the hemocytes and may even trigger processes such as phagocytosis.

Keywords: bovine tick, cellular defense, entomopathogenic fungi, immune response

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Authors: J. Suwanprateeb, F. Thammarakcharoen

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Calcium phosphate coating (CaP) has been employed for protein delivery, but the typical direct protein adsorption on the coating led to low incorporation content and fast release of the protein from the coating. By using bovine serum albumin (BSA) as a model protein, rapid biomimetic co-precipitation between calcium phosphate and BSA was employed to control the distribution of BSA within calcium phosphate coating during biomimetic formation on titanium surface for only 6 h at 50 oC in an accelerated calcium phosphate solution. As a result, the amount of BSA incorporation and release duration could be increased by using a rapid biomimetic co-precipitation technique. Up to 43 fold increases in the BSA incorporation content and the increase from 6 h to more than 360 h in release duration compared to typical direct adsorption technique were observed depending on the initial BSA concentration used during co-precipitation (1, 10, and 100 microgram/ml). From X-ray diffraction and Fourier transform infrared spectroscopy studies, the coating composition was not altered with the incorporation of BSA by this rapid biomimetic co-precipitation and mainly comprised octacalcium phosphate and hydroxyapatite. However, the microstructure of calcium phosphate crystals changed from straight, plate-like units to curved, plate-like units with increasing BSA content.

Keywords: biomimetic, Calcium Phosphate Coating, protein, titanium

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Authors: Jong Kook Lee, Sangcheol Eum, Jaehong Kim

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Wollastonite/hydroxyapatite composite coatings on zirconia were obtained by room temperature spray process. Wollastonite powder was synthesized by solid-state reaction between calcite and silica powder. Hydroxyapatite powder was prepared from bovine bone by the calcination at 1200oC 1h. From two starting raw powders, three kinds of powder mixture were obtained by the ball milling for 24h. By using these powders, wollastonite/hydroxyapatite coatings were fabricated on zirconia substrates by a room temperature spray process, and their microstructure and biological behavior were investigated and compared with pure wollastonite and hydroxyapatite coatings. Wollastonite/hydroxyapatite coatings on zirconia substrates were homogeneously formed in microstructure and had a nanoscaled grain size. The phase composition of the resultant wollastonite/hydroxyapatite coatings was similar to that of the starting powders, however, the grain size of the wollastonite or hydroxyapatite particles was reduced to about 100 nm due to their formation by particle impaction and fracture. The wollastonite/hydroxyapatite coating layer exhibited bioactivity in a stimulated body fluid and forming ability of new hydroxyapatite precipitates of 25 nm during in vitro test in SBF solution, which was enhanced by the increasing wollastonite content.

Keywords: wollastonite, hydroxyapatite composite coatings, room temperature spay process, zirconia

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Authors: Tety Hartatik, Dian Kurniawati, Adiarto

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The aim of the research was to determine the associations between polymorphism of the bovine growth hormone (GH) gene (Leu/Val, L/V) and milk production of Friesian Holstein Cattle. A total of 62 cows which consist of two Friesian Holstein groups (cattle from New Zealand are 19 heads and cattle from Australia are 43 heads). We perform the PCR and RFLP method for analyzing the genotype of the target gene GH 211 bp in the part of intron 4 and exon 5 of GH gene. The frequencies of genotypes LL were higher than genotype LV. The number of genotype LL in New Zealand and Australia groups are 84% and 79%, respectively. The number of genotype LV in New Zealand and Australia groups are 16% and 21%, respectively. The association between Leu/Val polymorphism on milk production, fat and protein content in both groups does not show the significant effect. However base on the groups (cows from New Zealand compare with those from Australia) show the significant effect on fat and protein content.

Keywords: Friesian Holstein, fat content, growth hormone gene, milk production, PCR-RLFP, protein content

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Authors: Jesus Hernandez, Zead Elzoeiry, Md. S. Islam, Abel E. Navarro

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The adsorption of bovine serum albumin (BSA) and human hemoglobin (Hb) on naturally-occurring adsorbents was studied to evaluate the potential recovery of proteins from meat industry residues. Spent peppermint tea (PM), powdered purple corn cob (PC), natural clay (NC) and chemically-modified clay (MC) were investigated to elucidate the effects of pH, adsorbent dose, initial protein concentration, presence of salts and heavy metals. Equilibrium data were fitted according to isotherm models, reporting a maximum adsorption capacity at pH 8 of 318 and 344 mg BSA/g of PM and NC, respectively. Moreover, Hb displayed maximum adsorption capacity at pH 5 of 125 and 143 mg/g of PM and PC, respectively. Hofmeister salt effect was only observed for PM/Hb system. Salts tend to decrease protein adsorption, and the presence of Cu(II) ions had negligible impacts on the adsorption onto NC and PC. Desorption experiments confirmed that more than 85% of both proteins can be recovered with diluted acids and bases. SEM, EDX, and TGA analyses demonstrated that the adsorbents have favorable morphological and mechanical properties. The long-term goal of this study aims to recover soluble proteins from industrial wastewaters to produce animal food or any protein-based product.

Keywords: adsorption, albumin, clay, hemoglobin, spent peppermint leaf

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Authors: Nasiruddin Khan

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The action of (-)-epicatechin, a cocoa (Theobroma cacao) flavanol that modulates redox/oxidative stress are contributed mainly to their antioxidant properties. The present study investigates the concentration and time dependent effect of (-)-epicatechin metabolites 3MeEc, 4MeEc, and 4SulEc on the production of ROS on BAEC using L-012, Lucigenin as chemiluminescence dye and XO/HX system. Our result demonstrates that 3MeEc shows significant (P <0.05) lowering effect of ROS production in BAEC with increasing concentration of metabolite while L-012 was used as chemiluminescence dye but not in the case of Lucigenin. In XO/HX system, using L-012 as chemiluminescence dye, 3MeEc and 4MeEc showed significant lowering effect on ROS production with increasing concentration from 100-500nM as compared to the positive control (SOD). When Lucigenin was used as chemiluminescence dye, 3MeEc exerted significant lowering effect with increasing concentration when compared to the positive control (SOD) whereas 4MeEc showed significant lowering effect in ROS production from 250 nM on as compared to positive control. For 4SulEc, a significant lowering effect of ROS production was only observed at 100 and 250 nM. Overall, although each metabolite shows considerable effect, 3MeEc exhibited more pronounced effect on decreasing the production of ROS as compared to other two metabolites.

Keywords: epicatechin metabolites, HO-1, Nrf2, ROS

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Authors: Al-Braa Kashegari, Ali M. El-Halawany, Akram A. Shalabi, Sabrin R. M. Ibrahim, Hossam M. Abdallah

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Chemical investigation of Chrozophora oblongifolia resulted in the isolation of five major compounds that were identified as apeginin-7-O-glucoside (1), quercetin-3-O-glucuronic acid (2), quercetin-3-O-glacturonic acid (3), rutin (4), and 1,3,6-trigalloyl glucose (5). The identity of isolated compounds was assessed by different spectroscopic methods, including one- and two-dimensional NMR. The isolated compounds were tested for their antioxidant activity using different assays viz., DPPH, FRAP, ABTS, ORAC, and metal chelation effects. In addition, the inhibition of target enzymes involved in the metabolic syndrome, such as alpha-glucosidase and pancreatic lipase, were carried out. Moreover, the effect of the compounds on the advanced glycation end-products (AGEs) as one of the major complications of oxidative stress and hyperglycemia in metabolic syndromes were carried out using BSA‐fructose (bovine serum albumin), BSA-methylglyoxal, and arginine methylglyoxal models. The pure isolates showed a protective effect in metabolic syndromes as well as promising antioxidant activity. The results showed potent activity of compound 5 in all measured parameters meanwhile, none of the tested compounds showed activity against pancreatic lipase.

Keywords: Chrozophora oblongifolia, antioxidant, pancreatic lipase, metabolic syndromes

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Authors: Natesan Balasubramanian, Palpandi Pounpandi, Venkatraman Thamil Priya, Vellasamy Shanmugaiah, Karubbiah Balakrishnan, Mandayam Anandam Thirunarayan

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Streptococcus agalactiae is commonly known as Group B Streptococcus (GBS) and it is the most common cause of life-threatening bacterial infection. GBS first considered as a veterinary pathogen causing mastitis in cattle later becomes a human pathogen for severe neonatal infections. In this present study, a total of 20 new clinical isolates of S. agalactiae were collected from male (6) and female patient (14) with different age group. The isolates were from Urinary tract infection (UTI), blood, pus and eye ulcer. All the 20 S. agalactiae isolates has clear hemolysis properties on blood agar medium and were identified by serogrouping and MALTI-TOF-MS analysis. Antibiotic susceptibility/resistance test was performed for 20 S. agalactiae isolates, further phenotypic resistance pattern was observed for tetracycline, vancomycin, ampicillin and penicillin. Genotypically we found two antibiotic resistance genes such as Betalactem antibiotic resistance gene (Tem) (70%) and tetracycline resistance gene Tet(O) 15% in our isolates. Six virulence factors encoding genes were performed by PCR in twenty GBS isolates, cfb gene (100%), followed by, cylE(90.47%), lmp(85.7%), bca(71.42%), rib (38%) and low frequency in bac gene (4.76%) were determined. Most of the S. agalactiae isolates produced strong biofilm in the polystyrene surface (hydrophobic), and low-level biofilm formation was found in glass tube (hydrophilic) surface. lytR is secreted protein and localized in bacterial cell wall, extra cellular membrane, and cytoplasm. In silico docking studies were performed for lytR protein with four antibiofilm compounds, including a peptide (PR39) with the docking study showed peptide has strong interaction followed by ellagic acid and interaction length is 2.95, 2.97 and 2.95 A°. In ligand EGCGO10 and O11 two atoms intract with lytR (Leu271), with binding bond affinity length is 3.24 and 3.14. The aminoacid Leu 271 is act as an impartant aminoacid, since ellagic acid and EGCG interact with same aminoacid.

Keywords: antibiotics, biofilms, clinical isolates, S. agalactiae, virulence

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Authors: Nikoo Soleimani

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Background: Reducing torque during bone drilling is one of the effective factors in reaching to an optimal drilling process. Methods: 15 bovine femurs were drilled in vitro with a drill bit with a diameter of 4 mm using two methods of torsional ultrasonic assisted drilling (T-UAD) and convent conventional drilling (CD) and the effects of changing the feed rate and rotational speed on the torque were compared in both methods. Results: There was no significant difference in the thrust force measured in both methods due to the direction of vibrations. Results showed that using T-UAD method for bone drilling at feed rates of 0.16, 0.24 and 0.32 mm/rev led for all rotational speeds to a decrease of at least 16.3% in torque compared to the CD method. Further, using T-UAD at rotational speeds of 355~1000 rpm with various feed rates resulted in a torque reduction of 16.3~50.5% compared to CD method. Conclusions: Reducing the feed rate and increasing the rotational speed, except for the rotational speed of 500 rpm and a feed rate of 0.32 mm/rev, resulted generally in torque reduction in both methods. However, T-UAD is a more effective and desirable option for bone drilling considering its significant torque reduction.

Keywords: torsional ultrasonic assisted drilling, torque, bone drilling, rotational speed, feed rate

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Authors: Muhammad Waqar Ashraf, Atiq A. Mian

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In the present study, accumulation of eight heavy metals, lead (Pb), cadmium (Cd), manganese (Mn), copper (Cu), zinc (Zn), iron (Fe), nickel (Ni) and chromium (Cr)was determined in kidney, heart, liver and muscle tissues of Lethrinus miniatus fish caught from Arabian Gulf. Metal concentrations in all the samples were measured using Atomic Absorption Spectroscopy. Analytical validation of data was carried out by applying the same digestion procedure to standard reference material (NIST-SRM 1577b bovine liver). Levels of lead (Pb) in the liver tissue (0.60µg/g) exceeded the limit set by European Commission (2005) at 0.30 µg/g. Zinc concentration in all tissue samples were below the maximum permissible limit (50 µg/g) as set by FAO. Maximum mean cadmium concentration was found 0.15 µg/g in the kidney tissues. Highest content of Mn in the studied tissues was seen in the kidney tissue (2.13 µg/g), whereas minimum was found in muscle tissue (0.87 µg/g). The present study led to the conclusion that muscle tissue is the least contaminated tissue in Lethrinus miniatus and consumption of organs should be avoided as much as possible.

Keywords: lethrinus miniatus, arabian gulf, heavy metals, atomic absorption spectroscopy

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Authors: John O. Akindoyo, M. D. H. Beg, Suriati Binti Ghazali, Nitthiyah Jeyaratnam

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Regeneration of bone due to the many health challenges arising from traumatic effects of bone loss, bone tumours and other bone infections is fast becoming indispensable. Over the period of time, some approaches have been undertaken to mitigate this challenge. This includes but not limited to xenografts, allografts, autografts as well as artificial substitutions like bioceramics, synthetic cements and metals. However, most of these techniques often come along with peculiar limitation and problems such as morbidity, availability, disease transmission, collateral site damage or absolute rejection by the body as the case may be. Hydroxyapatite (HA) is very compatible and suitable for this application. However, most of the common methods for HA synthesis are expensive and environmentally unfriendly. Extraction of HA from bio-wastes have been perceived not only to be cost effective, but also environment-friendly. In this research, HA was produced from bio-waste: namely bovine bones through a combination of hydrothermal chemical processes and ordinary calcination techniques. Structure and property of the HA was carried out through different characterization techniques (such as TGA, FTIR, DSC, XRD and BET). The synthesized HA was found to possess similar properties to stoichiometric HA with highly desirable thermal, degradation, structural and porous properties. This material is unique for its potential minimal cost, environmental friendliness and property controllability. It is also perceived to be suitable for tissue and bone engineering applications.

Keywords: biomaterial, biopolymer, bone, hydroxyapatite

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Authors: Muhammad Waqar Ashraf

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In the present study, accumulation of eight heavy metals, lead (Pb), cadmium (Cd), manganese (Mn), copper (Cu), zinc (Zn), iron (Fe), nickel (Ni) and chromium (Cr)was determined in kidney, heart, liver and muscle tissues of Lethrinus Miniatus fish caught from Arabian Gulf. Metal concentrations in all the samples were measured using Graphite Furnace Atomic Absorption Spectroscopy (GF-AAS). Analytical validation of data was carried out by applying the same digestion procedure to standard reference material (NIST-SRM 1577b bovine liver). Levels of lead (Pb) in the liver tissue (0.60µg/g) exceeded the limit set by European Commission (2005) at 0.30 µg/g. Zinc concentration in all tissue samples were below the maximum permissible limit (50 µg/g) as set by FAO. Maximum mean cadmium concentration was found to be 0.15 µg/g in the kidney tissues. Highest content of Mn in the studied tissues was seen in the kidney tissue (2.13 µg/g), whereas minimum was found in muscle tissue (0.87 µg/g). The present study led to the conclusion that muscle tissue is the least contaminated tissue in Lethrinus Miniatus and consumption of organs should be avoided as much as possible.

Keywords: Arabian gulf, Lethrinus miniatus, heavy metals, atomic absorption spectroscopy

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Authors: O. O. Leigh, L. C. Agbugba, A. O. Oyewunmi, A. E. Ibiam, A. Hassan

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Accuracy of insemination timing is a key determinant of high pregnancy rates in livestock breeding stations. The estrous detector probes are a recent introduction into the Nigerian livestock farming sector. Many of these probes are species-labeled and they measure changes in the vaginal mucus resistivity (VMR) during the stages of the estrous cycle. With respect to size and shaft conformation, the Draminski® swine estrous detector probe (sEDP) is quite similar to the bovine estrous detector probe. We investigated the reliability of the sEDP at insemination time on two farms designated as FM A and FM B. Cows (Bunaji, n=20 per farm) were evaluated for VMR at 16th h post standard OvSynch protocol, with concurrent insemination on FM B only. The difference in the mean VMR between FM A (221 ± 24.36) Ohms and FM B (254 ± 35.59) Ohms was not significant (p > 0.05). Sixteen cows (80%) at FM B were later (day 70) confirmed pregnant via rectal palpation and calved at term. These findings suggest consistency in VMR evaluated with sEDP at insemination as well as a high predictability for VMR associated with good pregnancy rates in dairy cattle. We conclude that Draminski® swine estrous detector probe is reliable in determining time of insemination in cattle breeding stations.

Keywords: dairy cattle, insemination, swine estrous probe, vaginal mucus resistivity

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Authors: Zarmina Gillani, Nuzhat Huma, Aysha Sameen, Mulazim Hussain Bukhari

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Whey is an excellent food ingredient owing to its high nutritive value and its functional properties. However, composition of whey varies depending on composition of milk, processing conditions, processing method, and its whey protein content. The aim of this study was to prepare a whey powder from raw whey and to determine the influence of different processing temperatures (160 and 180 °C) on the physicochemical, functional properties during storage of 180 days and on whey protein denaturation. Results have shown that temperature significantly (P < 0.05) affects the pH, acidity, non-protein nitrogen (NPN), protein total soluble solids, fat and lactose contents. Significantly (p < 0.05) higher foaming capacity (FC), foam stability (FS), whey protein nitrogen index (WPNI), and a lower turbidity and solubility index (SI) were observed in whey powder processed at 160 °C compared to whey powder processed at 180 °C. During storage of 180 days, slow but progressive changes were noticed on the physicochemical and functional properties of whey powder. Reverse phase-HPLC analysis revealed a significant (P < 0.05) effect of temperature on whey protein contents. Denaturation of β-Lactoglobulin is followed by α-lacalbumin, casein glycomacropeptide (CMP/GMP), and bovine serum albumin (BSA).

Keywords: whey powder, temperature, denaturation, reverse phase, HPLC

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Authors: Sri Rahayu, M. Dwi Susan, Aris Soewondo, W. M. Agung Pramana

Abstract:

Semen is an organic fluid (seminal plasma) that contain spermatozoa. Proteins are one of the major seminal plasma components that modulate sperm functionality, influence sperm capacitation and maintaining the stability of the membrane. Semen freezing is a procedure to preserve sperm cells. The process causes decrease in sperm viability due to temperature shock and oxidation stress. Oxidation stress is a disturbance on phosphorylation that increases ROS concentration, and it produces lipid peroxide in spermatozoa membrane resulted in high MDA (malondialdehyde) concentration. The objective of this study was to examine the effect of freezing on protein and MDA profile of bovine sperm cell and seminal plasma after freezing. Protein and MDA of sperm cell and seminal plasma were isolated from 10 sample. Protein profiles was analyzed by SDS PAGE with separating gel 12,5 %. The concentration of MDA was measured by spectrophotometer. The results of the research indicated that freezing of semen cause lost of the seminal plasma proteins with molecular with 20, 10, and 9 kDa. In addition, the result research showed that protein of the sperm (26, 10, 9, 7, and 6 kDa) had been lost. There were difference MDA concentration of seminal plasma and sperm cell were increase after freezing. MDA concentration of seminal plasma before and after freezing were 2.2 and 2.4 nmol, respectively. MDA concentration of sperm cell before and after freezing were 1,5 and 1.8 nmol, respectively. In conclusion, there were differences protein profiles of spermatozoa before and after semen freezing and freezing cause increasing of the MDA concentration.

Keywords: MDA, semen freezing, SDS PAGE, protein profile

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Authors: Peter James C. Icalia, Agapita J. Salces, Loida Valenzuela, Kangseok Seo, Geronima Ludan

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This study evaluated polymorphism of 11 microsatellite markers in four local genetic groups of cattle. Batanes cattle which has never been studied using microsatellites is evaluated for its genetic distance from the Ilocos cattle while Brahman and Holstein-Sahiwal are also included as there were insemination programs by the government using these two breeds. PCR products that were genotyped for each marker were analyzed using POPGENEv32. Results showed that 55% (Fst=0.5501) of the genetic variation is due to the differences between populations while the remaining 45% is due to individual variation. The Fst value also indicates that there were very great differences from population to population using the range proposed by Sewall and Wright. The constructed phylogenetic tree based on Nei’s genetic distance using the modified neighboor joining procedure of PHYLIPv3.5 showed the admixture of Brahman and Holstein-Sahiwal having them grouped in the same clade. Batanes and Ilocos cattle were grouped in a different cluster showing that they have descended from a single parental population. This would presumably address the claim that Batanes and Ilocos cattle are genetically distant from other groups and still exist despite the artificial insemination program of the government using Brahman and other imported breeds. The knowledge about the genetic structure of this population supports the development of conservation programs for the smallholder farmers.

Keywords: microsatellites, cattle, Philippines, populations, genetic structure

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Authors: Ramalingam Boobalan, Chinpiao Chen, Jui-I. Chiao

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A series of erlotinib analogues that have structural modification at 6,7-alkoxyl positions is efficiently synthesized. The key reactions that involved in synthesis are one-pot oxime formation-dehydration for the formation of nitrile, quinazoline ring formation reaction between aniline and o-cyanoaniline via formamidine intermediate, Fe/NH4Cl catalyzed reduction-hetereocyclization-reductive ring opening reaction for the formation of o-aminobenzamide, high yielding seal tube reactions for O-demethylation, sodium iodide substitution, ammonia substitution. The in vitro anti-tumor activity of synthesized compounds is studied in two non-small cell lung cancer (NSCLC) cell lines (A549 and H1975). Among the synthesized compounds, the iodo compound 6 (ETN-6) exhibits higher anti-cancer activity compared to erlotinib. An efficient method is developed for the conjugation of erlotinib analogue-4, alcohol compound, with protein, bovine serum albumin (BSA), via succinic acid linker. The in vitro anti-tumor activity of the protein attached erlotinib analogue, 8 (ETN-4-Suc-BSA), showed stronger inhibitory activity in both A549 and H1975 NSCLC cell lines.

Keywords: anti-cancer, BSA, EGFR, Erlotinib

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Authors: Oustani Mabrouka, Halilat Md Tahar, Hannachi Slimane

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This study was conducted on representative and contrasting soils of arid regions. It focuses on the compared influence of two organic fertilizers: poultry manure (PM) and bovine manure (BM) on improving the microbial functioning of non-saline (SS) and saline (SSS) soils, in particularly, the process of mineralization of nitrogen and carbon. The microbiological activity was estimated by respirometric test (CO2–C emissions) and the extraction of two forms of mineral nitrogen (NH4+-N and NO3--N). Thus, after 56 days of incubation under controlled conditions (28 degrees and 80 per cent of the field capacity), the two types of manures showed that the mineralization activity varies according to type of soil and the organic substrate itself. However, the highest cumulative quantities of CO2–C, NH4+–N and NO3-–N obtained at the end of incubation were recorded in non-saline (SS) soil treated with poultry manure with 1173.4, 4.26 and 8.40 mg/100 g of dry soil, respectively. The reductions in rates of release of CO2–C and of nitrification under saline conditions were 21 and 36, 78 %, respectively. The influence of organic substratum on the microbial density shows a stimulating effect on all microbial groups studied. The whole results show the usefulness of two types of manures for the improvement of the microbiological functioning of arid soils.

Keywords: Salinity, Organic matter, Microorganisms, Mineralization, Nitrogen, Carbon, Arid regions

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Authors: Shokoufeh Roudashti, Shahin Bahari, Fakhri Haghi, Habib Zeighami, Ghazal Naderi, Paniz Shirmast

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Background: Mycobacterium tuberculosis complex (MTBC) causes tuberculosis (TB) in humans and animals. Mycobacterium MTBC is one of the most important species of zoonotic pathogens that can be transmitted from cattle to humans. The disease can transmit to human by direct contact with the infected animals, drinking unpasteurized milk and consumption of uncooked meat. The presence of these opportunistic, pathogenic bacteria in bovine milk has emerged as a public-health concern, especially among individuals who consume raw milk. Tuberculosis MTBC is the predominant infectious cause of morbidity and morality worldwide, It is estimated that one third of the world population (approx. 1.8 billion persons) is infected with M. tuberculosis and each year there are 8 million new cases worldwide. The aim of this study, to detect Mycobacterium MTBC in raw milk samples using polymerase chain reaction (PCR). Materials and Methods: In the present study, 60 raw milk samples were collected from rural areas in Zanjan, Iran. After extraction of DNAs and using special primers for Is6110 gene as a marker, PCR was applied to detect the presence or non-presence of the related gene. Results: According to the findings of this study, 8 (13.5 %) out of 60 milk samples were positive for Mycobacterium spp (P < 0.1). Conclusions: The Outbreak of genus Mycobacteria spp in milk samples were determined to be relatively high in Zanjan, Iran.

Keywords: Mycobacteria spp, raw milk, PCR, Zanjan

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Authors: A. Ameur Ameur, F. Chougrani, M. Halbouche

Abstract:

In order to characterize the sheep's milk, we analyzed and compared, in a first stage of our work, the physical and chemical characteristics in two Algerian sheep breeds: Hamra race and race Ouled Djellal breeding at the station the experimental ITELV Ain Hadjar (Saïda Province). Analyses are performed by Ekomilk Ultra-analyzer (EON TRADING LLC, USA), they focused on the pH, density, freezing, fat, total protein, solids-the total dry extract. The results obtained for these parameters showed no significant differences between the two breeds studied. The second stage of this work was the isolation and characterization of milk proteins. For this, we used the precipitation of caseins phi [pH 4.6]. For this, we used the precipitation of caseins Phi (pH 4.6). After extraction, purification and assay, both casein and serum protein fractions were then assayed by the Bradford method and controlled by polyacrylamide gel electrophoresis (PAGE) in the different conditions (native, in the presence of urea and in the presence of SDS). The electrophoretic pattern of milk samples showed the presence similarities of four major caseins variants (αs1-, αs2-β-and k-casein) and two whey proteins (β-lactoglobulin, α-lactalbumin) of two races Hamra and Ouled Djellal. But compared to bovine milk, they have helped to highlight some peculiarities as related to serum proteins (α La β Lg) as caseins, including αs1-Cn.

Keywords: Hamra, Ouled Djellal, protein polymorphism, sheep breeds

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Authors: Adela Diepoltova, Klara Konecna, Ondrej Jandourek, Petr Nachtigal

Abstract:

A loss of control over antibiotic-resistant pathogens has become a global issue due to severe and often untreatable infections. This state is reflected in complicated treatment, health costs, and higher mortality. All these factors emphasize the urgent need for the discovery and development of new anti-infectives. One of the most common pathogens mentioned in the phenomenon of antibiotic resistance are bacteria of the genus Staphylococcus. These bacterial agents have developed several mechanisms against the effect of antibiotics. One of them is biofilm formation. In staphylococci, biofilms are associated with infections such as endocarditis, osteomyelitis, catheter-related bloodstream infections, etc. To author's best knowledge, no validated and standardized methodology evaluating candidate compound activity against staphylococcal biofilms exists. However, a variety of protocols for in vitro drug activity testing has been suggested, yet there are often fundamental differences. Based on our experience, a key methodological step that leads to credible results is to form a robust biofilm with appropriate attributes such as firm adherence to the substrate, a complex arrangement in layers, and the presence of extracellular polysaccharide matrix. At first, for the purpose of drug antibiofilm activity evaluation, the focus was put on various conditions (supplementation of cultivation media by human plasma/fetal bovine serum, shaking mode, the density of initial inoculum) that should lead to reproducible and robust in vitro staphylococcal biofilm formation in microtiter plate model. Three model staphylococcal reference strains were included in the study: Staphylococcus aureus (ATCC 29213), methicillin-resistant Staphylococcus aureus (ATCC 43300), and Staphylococcus epidermidis (ATCC 35983). The total biofilm biomass was quantified using the Christensen method with crystal violet, and results obtained from at least three independent experiments were statistically processed. Attention was also paid to the viability of the biofilm-forming staphylococcal cells and the presence of extracellular polysaccharide matrix. The conditions that led to robust biofilm biomass formation with attributes for biofilms mentioned above were then applied by introducing an alternative method analogous to the commercially available test system, the Calgary Biofilm Device. In this test system, biofilms are formed on pegs that are incorporated into the lid of the microtiter plate. This system provides several advantages (in situ detection and quantification of biofilm microbial cells that have retained their viability after drug exposure). Based on our preliminary studies, it was found that the attention to the peg surface and substrate on which the bacterial biofilms are formed should also be paid to. Therefore, further steps leading to the optimization were introduced. The surface of pegs was coated by human plasma, fetal bovine serum, and L-polylysine. Subsequently, the willingness of bacteria to adhere and form biofilm was monitored. In conclusion, suitable conditions were revealed, leading to the formation of reproducible, robust staphylococcal biofilms in vitro for the microtiter model and the system analogous to the Calgary biofilm device, as well. The robustness and typical slime texture could be detected visually. Likewise, an analysis by confocal laser scanning microscopy revealed a complex three-dimensional arrangement of biofilm forming organisms surrounded by an extracellular polysaccharide matrix.

Keywords: anti-biofilm drug activity screening, in vitro biofilm formation, microtiter plate model, the Calgary biofilm device, staphylococcal infections, substrate modification, surface coating

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