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, the use of bone scaffolds as bone tissue substitutes, especially the use of such as hydroxyapatite and tricalcium phosphate, has been very popular. Today, the use of modern engineering techniques and advances in nanotechnology have expanded the use of nanomaterials as bone scaffolds for bone tissue applications. This study was performed on 60 adult male New Zealand rabbits divided into four experimental groups: the control group without any treatment, the second group receiving hydroxyapatite, the third group treated with β-tricalcium phosphate, and the fourth group receiving nanocomposite polycaprolactone (PCL) scaffold. In a surgical procedure, a defect 6 mm in diameter was made in a hind limb femur. Four indexes were used to assess histopathology, which were union index, spongiosa index, cortex index, and bone marrow. The results showed that nanocomposite PCL and control groups always had the respective highest and lowest values among all the groups at all time intervals. The histopathological assessment demonstrated that the quantity of newly formed lamellar bone in the nanocomposite PCL group was higher than in other groups. All these data suggest that PCL had positive effects on the bone healing process, which could have great potential in tissue engineering and clinical applications.

Introduction: Highly pathogenic Asian H5-subtype avian influenza viruses have been found in poultry and wild birds worldwide since they were first detected in southern China in 1996. Extensive control efforts have not eradicated them. Vaccination prevents such viruses infecting poultry and reduces the number lost to compulsory slaughter. The study showed the efficacy of inactivated H5 vaccine from the H5N8 virus against highly pathogenic H5N8 and H5N6 avian influenza viruses in chickens. Material and Methods: Reverse genetics constructed an H5 vaccine virus using the HA gene of the 2014 H5N8 avian influenza virus and the rest of the genes from A/PR/8/34 (H1N1). The vaccine viruses were grown in fertilised eggs, partially purified through a sucrose gradient, and inactivated with formalin. Chickens were immunised i.m. with 1 µg of oil-adjuvanted inactivated H5 antigens. Results: Single dose H5 vaccine recipients were completely protected from lethal infections by homologous H5N8 avian influenza virus and shed no virus from the respiratory or intestinal tracts but were not protected from lethal infections by heterologous H5N6. When chickens were immunised with two doses and challenged with homologous H5N8 or heterologous H5N6, all survived and shed no virus. Conclusion: Our results indicate that two-dose immunisations of chickens with H5 antigens with oil adjuvant are needed to provide broad protection against different highly pathogenic H5 avian influenza viruses.

Introduction: Traditionally, evolutionary analysis of equine influenza virus (EIV) is based on the HA gene. However, the specificity of the influenza virus enables the classification of viral strains into different phylogenetic groups, depending on the gene being analysed. The aim of the study was to analyse phylogenetic paths of EIV based on M gene with reference to the HA gene. Material and Methods: M gene of Polish isolates has been sequenced and analysed along with all M sequences of EIV available in GenBank database. Phylogenetic analysis was performed using BioEdit, ClustalW, and MEGA7 softwares. Results: The clustering of the strains isolated not only from Asia but also from Europe into one common Asian-like group of EIV was observed. Twelve nucleotide substitutions in the M gene of strains from the Asian-like group were crucial for the evolutionary analysis. We also observed homology in the M gene of the Asian-like and H7N7 strains. Conclusions: M gene specific for the Asian-like group is present in strains recently isolated in Europe and Asia, which were classified previously in the Florida 2 clade based on HA. Therefore, Asian-like group does not seem to be assigned to a specific geographical region. Traces of H7N7 strains in more conservative genes like M of some contemporary EIV strains may indicate the link between the old phylogenetic group and recent H3N8 strains. Analysis of conservative genes may be more useful in tracking the direction of virus evolution than in the genes where the high variability rate may blur the original relationships.

In recent years, the high sensitivity and specificity of novel miRNA biomarkers have been utilised for early diagnosis and treatment monitoring of various diseases. Previous reports showed that abnormal expression of miR-208 in mice resulted in the development of an aberrant cardiac conduction system and consecutive arrhythmias. On the other hand, a study on infarcted human heart tissue showed upregulation of miR-208a in subjects with ventricular tachyarrhythmias compared to healthy controls. We prospectively investigated the expression of miR-208a and -208b in the serum of dogs presenting different cardiac arrhythmias. A total of 28 dogs with atrial fibrillation (n = 8), ventricular premature contractions (n=6), conduction system disturbances (n = 7), and free of heart conditions (as controls) (n = 7) were enrolled in the study. Total RNA was extracted from serum samples and miR-208a and -b, miR-16 as well as a cel-miR-39-5p spike-in were analysed with qPCR and ddPCR. miR-208a and miR-208b were not expressed in any of the samples. The calculated ddPCR miR-16 relative expression (normalised with cel-miR-39 spike-in) showed a good correlation (r = 0.82; P < 0.001) with the qPCR results. This outcome warrants further investigation, possibly focusing on tissue expression of miR-208 in the canine heart.

Electrical cardioversion is a therapeutic procedure used to convert various types of arrhythmias back to sinus rhythm. It is used to restore the sinus rhythm in dogs with atrial fibrillation. The effect of the electrical energy used during cardioversion on red blood cells (RBC) is not fully understood. Studies on humans reported lysis of RBC following electrical cardioversion. Similar studies have not been carried out on dogs. The aim of the study was to assess the effect of electrical cardioversion on chosen RBC parameters. The study was carried out on 14 large and giant breed dogs weighing from 30 to 84 kg with lone atrial fibrillation (lone AF). Electrical cardioversion was carried out under general anaesthesia by biphasic shock with 70–360 J of energy. Blood was collected at T0 – during atrial fibrillation, prior to cardioversion, and at T1 – 30 min after electrical cardioversion. Complete blood counts as well as total and direct bilirubin concentrations were evaluated. A maximum output of 360 J was used. In all cases, electrical cardioversion was effective, and no significant changes in the number of RBC and RBC indices were noted. Similarly, there were no statistically significant differences in the levels of total and direct bilirubin. Electrical cardioversion in dogs led neither to statistically nor clinically significant RBC lysis.

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.

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.

Xylazine, a type of α₂-adrenoceptors, is a commonly used drug in veterinary medicine. Xylazine-induced changes in the content of amino acid neurotransmitters – glycine (Gly) and aspartic acid (Asp), in different brain regions and neurons were studied. Wistar rats were administered 50 mg/kg or 70 mg/kg of xylazine by intraperitoneal injection. In addition, in vitro experiments were conducted, in which neurons were treated with 15 μg/mL, 25 μg/mL, 35μg/mL, and 45 μg/mL of xylazine. Test methods were based on the enzyme-linked immunosorbent assays (ELISA). During anaesthesia, Asp levels in each brain area were significantly lower compared to the control group. Except for the cerebrum, levels of Gly in other brain areas were significantly increased during the anaesthesia period. In vitro, xylazine-related neuron secretion of Gly increased significantly compared to the control group at 60 min and 90 min. Moreover, xylazine caused a significant decrease in the levels of Asp secreted by neurons at 20 min, but gradually returned to the level of the control group. The data showed that during anaesthesia the overall levels of Asp decreased and overall levels of Gly increased. In addition, the inhibitory effect of xylazine on Asp and the promotion of Gly were dose-dependent. Our data showed that different effects of xylazine on excitatory and inhibitory neurotransmitters provided a theoretical basis for the mechanism of xylazine activity in clinical anaesthesia.

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.