Dengue is one of the major emerging viral diseases in the world, with dramatic increases in reported cases in the last few decades and annual worldwide occurrence of approximately 390 million infections. It is a highly important mosquito-vectored disease and is a problem in tropical and subtropical areas of the world. The major aim of this study was to clone and express the dengue NS3 gene, in service to its therapeutic importance for the development of stable cell lines. Blood samples from dengue fever (DF) patients were collected and subjected to PCR amplification of the NS3 gene of dengue virus serotype-2 (DENV-2). The NS3 gene was amplified using gene specific primers and cloned in the TA cloning vectors. The gene was successfully expressed in mammalian expression vector pcDNA3.1. The current finding was different from a previously reported DENV-2 strain replicon constructed in different cells, in which the whole genetic material of the virus was used instead of an active protease gene, and which gave a low yield of replicon expressing cells. Recombinant NS3 could be used to produce an antibody that is possibly helpful for developing a single step diagnostic assay to detect the dengue virus NS3 antigen in sera of dengue patients.

In recent years, there has been a great interest in biogenic amines such histamine, as they are associated with the quality and safety of some kinds of fermented foods. The aim of this study was to evaluate the effect of temperature and storage time on the content of histamine in cheeses. Samples of mould and hard cheeses were examined with RP-HPLC with an organic-aqueous mobile phase containing acidic buffer and chaotropic salt. The samples were stored either at 22 ± 2°C for 42 days (mould and hard cheeses) or at 4 ± 2°C for 112 days (mould cheeses) and 133 days (hard cheeses). The mean total histamine content in cheeses stored at 22°C was higher than the content in those stored at 4°C, with the highest concentrations found in Gorgonzola Piccante cheese (730.47 mg/kg). Histamine concentration in some types of cheeses exceeded the toxic threshold dose, indicating that after long or inadequately cool storage they may not be safe for consumers. To protect cheeses from contamination with histamine-producing bacteria and to safeguard consumers from poisoning, factors conducive to this amine’s formation should be minimised during cheese processing. Suitable temperature and time during storage of cheeses are recommended to avoid the intoxication. Monitoring of this toxin in food is necessary to ensure safety of consumers.

Inosine pranobex (Isoprinosine) stimulates cell-mediated immune responses to viral infections in humans and might have also therapeutic use in animals. The aim of this study was to compare three in vitro cytotoxicity assays on mouse embryo fibroblasts and liver cancer cells and determine their ability to detect early cytotoxic effects for inosine pranobex. BALB/3T3 clone A31and HepG2 cells were incubated with inosine pranobex at concentrations from 0.1 to 1,000 μg/mL. Cell viability was determined with the MTT reduction, the LHD release, and the NRU tests. A decrease in the cell viability was observed after incubating the BALB/3T3 clone A31and HepG2 cells with inosine pranobex. Based on the cytotoxicity endpoints measured in these investigations in BALB/3T3 clone A31cells, it can be concluded that the cell membrane may be the first part of the cell to be affected by inosine pranobex. The disintegration of lysosomes and mitochondria follows mitochondria damage. In HepG2 cells likewise, the cell membrane may be the first part of the cell to be affected by inosine pranobex. Also in liver cancer cells, the disintegration of mitochondria (assessed with the MTT reduction assay) and next of lysosomes (assessed with the NRU assay) follows mitochondria damage.

Introduction: Bisphenol A (BPA) is a substance widely used in industry for the production of polycarbonates and epoxy resins used in packaging and containers for beverages, contact lenses, compact discs (CDs), window panes, and many other elements. This compound belongs to the group of polyphenols and xenoestrogens commonly found in the human environment. What we know about BPA is still insufficient to enable us to protect our health against its adverse effects, and current knowledge of the influence of BPA on erythroblastic cell lines in bone marrow is rather fragmentary. The aim of the experiment was to assess the effect of two doses of BPA (0.05 mg/kg and 0.5 mg/kg b.w. per day) on myeloid haematopoiesis. Material and Methods: During this experiment, the number of all types of cells in the erythroblastic cell line was evaluated in porcine bone marrow before and after BPA administration. Results: The obtained results clearly indicate changes in haematopoietic activity of the bone marrow, which was demonstrated by a decrease in erythroblastic cell line production in both experimental groups. The haematological effects of the bone marrow changes were anaemia, caused by a number of erythrocytes which was depressed due to their immaturity, and a significant decrease in mean cellular volume in both groups. Conclusion: The harmful effect of high and low doses of BPA on haematopoietic processes was proved.

The efficiency of five natural antioxidants (curcumin, cranberry, pomegranate, grape seed extract (GSE), and açai berry) in reducing lipid oxidation in dog food was compared to that of the synthetic antioxidant butylated hydroxyanisole (BHA). In two different experiments content parameters were measured after 12 days of storage at 55°C. In experiment one, the natural antioxidants were added at 0.2% and BHA at 0.02% of the food (DM basis), and samples were analysed for thiobarbituric acid-reactive substances (TBARS). In experiment two, the effects of GSE and curcumin at two admixture proportions (0.1% and 0.2% of food DM) on omega-3 fatty acid (FA) content were evaluated. TBARS values were lower than the control (P < 0.01) for curcumin, cranberry, pomegranate, and GSE but not for the açai berry (P > 0.05). By day 12, although there were no significant differences (P > 0.05) between the two curcumin treatments, they preserved higher concentrations of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) (P < 0.05) than the BHA and control treatments. The addition of GSE or BHA to dog food held (P < 0.05) the concentrations of EPA higher than the control. The concentrations of EPA and DHA for the 0.2% GSE treatment were greater (P < 0.05) than the 0.1% GSE treatment. Grape seed extract at 0.2% lost less (P < 0.05) EPA concentration than BHA. The present results showed that, except for açai berry, the tested natural antioxidants could be used as a substitute for BHA in dog food.

In 2015 in the European Union member states listeriosis caused 270 deaths. Food is the route of transmission in 99% of all human infection cases. Several studies from different countries have shown that the presence of Listeria monocytogenes in food can be as high as 58.3%. One of the most important ways to protect food from these microorganisms is to prevent the spread of the bacteria at processing plants at different stages of food production chain. The ability of L. monocytogenes to survive in extreme conditions and to form biofilms on various surfaces is a significant challenge for food safety. Removal of these bacteria from niches in processing plants is difficult and requires the use of sanitisers and precise equipment cleaning. The presence of L. monocytogenes in processing environment at slaughterhouses, deli meat factories or in retail may be a reason of cross-contamination. Proper hygienic systems applied by workers in food preparing places and knowledge about different routes of spreading of these bacteria may effectively decrease the risk of food contamination. Standardised legal regulations and control of meat product manufacture should be a fundamental way to protect food from L. monocytogenes contamination.

Introduction: Toll-like receptors (TLRs) play a key role in the rapid activation of the innate immune response to a variety of pathogens. The aim of this study was to evaluate the effect of Trichinella spiralis infection on the level of expression of the tlr4 gene in mouse intestines during the intestinal phase of experimental trichinellosis. Material and Methods: The experimental material consisted of the small and large intestines of BALB/c mice infected with Trichinella spiralis sampled at 4, 8, and 16 days post infection (dpi). Results: A statistically significant increase was demonstrated in the tlr4 mRNA level isolated from the infected mice jejunum at 4, 8, and 16 dpi over the uninfected control. Moreover, at 4, 8, and 16 dpi in the jejunum of infected mice, a strong positive reaction for the presence of TLR4 protein compared with that of uninfected mice was observed. Conclusion: Infection with T. spiralis changes the expression of the tlr4 gene in the small intestine of the mouse host.

Nerium oleander is a plant of the Apocynaceae family toxic to humans, animals, and insects. This study was performed to determine the cardiac and neurotoxicity of the plant extract by oral administration in Wistar rats and Balb/c mice and to compare the susceptibility of these animal models to oleander toxicity. Four groups of eight mice and eight rats received N. oleander extract orally while a fifth group was the control. Serum concentrations of the biochemical toxicity indicators, namely troponin and creatine kinase (CK), were determined and histopathological evaluation of the heart and brain was performed. In mice, CK and troponin concentrations were respectively 1.5 and 7 times higher than in the control group (P < 0.05), while in rats, they were 6–7 and 11 times higher. Hyperaemia, haemorrhage, and myofibrolysis, without infiltration of inflammatory cells, were observed in the heart. In the brain the authors observed hyperaemia associated with perivascular and perineuronal oedema, and in higher-dosed rats multifocal haemorrhagic and liquefactive necrotic lesions. Oleander can affect serum levels of CK and troponin due to nervous and cardiac injuries. Rats showed more severe changes in the biochemical indicators and histopathological lesions than mice. Therefore, biochemical and pathological findings indicate that Wistar rats are more susceptible to the cardiac toxicity and neurotoxicity effects of N. oleander poisoning than Balb/c mice.

Introduction: The ovarian follicular dynamics, vaginal electrical resistance (VER), progesterone (P4) and oestrogen (E2) profiles were investigated during the oestrous cycle in four indigenous ewes. Material and Methods: Daily VER values were recorded with a heat detector. The follicles were observed and measured by trans-rectal ultrasonography. Blood was collected daily for hormonal profiles. Results: A significant variation in VER values (P < 0.05) in oestrus by ewes and position in the sequence of cycles was observed. Trans-rectal ultrasonography of ovaries revealed the presence of 2–4 waves of follicular growth. Study of hormonal profiles by ELISA revealed a positive correlation between E2 concentration and development of follicles and a negative correlation between P4 concentration and their development. The concentrations of oestradiol increased in oestrus and then decreased to a basal level. Follicular growth was accompanied by a rise in the concentration of serum oestradiol. Inversely, when follicles received the stimulation for ovulation, concentration of progesterone started to fall, but after ovulation, it climbed back to its peak and remained at this state until next ovulatory follicle reached its maximum diameter. Conclusion: This study could help to set up a manipulative reproductive technique for improving genetic values in indigenous sheep.

Introduction: The aim of the study was the application and evaluation of real-time PCRs based on the fluorescence of SYBR Green I intercalating dye for the detection of three Bacillus anthracis genes in contaminated liver and blood samples. The goals for detection were rpoB gene as a chromosomal marker, pag gene located on plasmid pXO1, and capC gene located on plasmid pXO2. Material and Methods: Five B. anthracis strains were used for the experiments. Additionally, single strains of other species of the genus Bacillus, i.e. B. cereus, B. brevis, B. subtilis, and B. megaterium, and strains of six other species were used for evaluation of the specificity of the tests. Three SYBR Green I real-time PCRs were conducted allowing confirmation of B. anthracis in the biological samples. Results: The observation of amplification curves in real-time PCRs enabled the detection of the chromosomally encoded rpoB gene, pag gene, and capC gene of B. anthracis. The specificity of the tests was confirmed by estimation of the melting temperature of the PCR products. The sensitivity and linearity of the reactions were determined using regression coefficients. Strains of other microbial species did not reveal real-time PCR products. Conclusion: All real-time PCRs for the detection of B. anthracis in biological samples demonstrated a significant sensitivity and high specificity.