The aim of this study was to construct two plasmid-specific shRNA transcripts of the bcl-2 gene in order to prepare for reverse of cell apoptosis. The plasmid was designed according to a previously published sequence of interfering RNA following an appropriate reference, using appropriate software. By annulling complementary oligonucleotides, double-stranded inserts were formed. Recombinant shRNA-encoding plasmids were constructed by digestion of psiRNA-x7SKGFPzeo plasmid (psiRNA-x7SKGFPzeo, with restrictive endonuclease BbsI electrophoresis in ultra-pure agarose with low melting point (LMP-Agarose). For each of the constructs, a suitable double-stranded insert downstream of x7SK (strong RNA III promoter) with T4 DNA ligase was cloned. The control plasmid psiRNAScr was used directly for transformation. The PC-3 cell lines were transfected with 2 plasmids, psiRNA-Bcl-2 and psiRNAScr to suppress the bcl-2 gene construct. The results have shown that the lowest level of bcl-2 genes was 48 h, and even lower 72 h after the transfer, and the mRNA levels returned to normal in 120 h. An increase in the percentage of cells with spontaneous apoptosis has been observed with successful inhibition of the bcl-2 gene. The induction of apoptosis in transfected cells increased the percentage of necrotic cells proportionally. The percentage of apoptotic cells transfected with psiRNA-bcl-2 plasmid increased proportionally to the increase of hydrogen peroxide concentration. The transfection of the PC-3 cell line from prostate cancer with constructed shRNA plasmid has induced suppression of bcl-2 gene expression versus control Scr plasmid. Suppression of bcl-2 gene expression significantly increased cell sensitivity to apoptosis induction.

The aim of this study was to investigate the effect of oregano and chitosan applied as edible coatings on the chemical and microbiological properties of different type of meat products. The main focus from the microbiological aspect was on Listeria monocytogenes as a pathogen that can survive for a long period on this type of product and, hence, present a risk for consumers. In vitro testing showed chemical quality after 7 and 14 days’ storage of the three types of products was stable. Promising results were obtained for the microbial analysis, whereby large reductions in L. monocytogenes numbers after 7 and 14 days’ storage at 4°C was measured in the coated products. The results indicated the chitosan and oregano combination can be an effective inhibitor of L. monocytogenes growth in chilled meat products.

Staphylococcus aureus is an important foodborne pathogen due to toxin-related virulence, invasiveness and antibiotic resistance. The ability of S. aureus strains to produce one or more staphylococcal enterotoxins (SEs) in food has been associated with the occurrence of staphylococcal food poisoning (SFP), which is the most common foodborne intoxication worldwide. The study aimed to determine the count of S. aureus strains in samples of raw cow’s milk and various cheeses produced in R. North Macedonia and to detect their ability to produce enterotoxins by passive agglutination SET RPLA (OXOID, UK) and by enzyme-linked fluorescence assay (ELFA) VIDAS SET 2 (Biomerieux, France). A total of 130 S. aureus strains were analyzed. The ability to produce SEs was determined in 17 (13.1%) strains using the SET RPLA detection kit and in 20 (15.4%) strains using the VIDAS SET 2. The study detected enterotoxigenic strains in cheese samples, despite the low count of S. aureus which was below the detection limit according to the Book of rules for microbiological criteria (Off. G. of R.M no 100/2013). Based on these and similar findings, S. aureus must be considered as a possible cause of intoxication, despite the undetected and underreported cases of SFP in the scientific literature.

The aim of the study was to identify the isolation rate of thermotolerant campylobacters in a small-scale broiler-meat production farm over a one-year period. The second deliverable of the study was to determine the potential virulence markers. The laboratory investigation was performed on 283 samples (cloacal swabs, caeca, carcass swabs) collected on three sampling points (farm, slaughter line, and cold storage). The isolates obtained with the conventional microbiological method were confirmed with multiplex PCR for identification of campylobacters. The presence of 10 virulence genes was analyzed in the C. jejuni isolates ( flaA, racR, virB11, dnaJ, wlaN, cadF, ciaB, cdtA, cdtB, cdtC). Out of 283 samples, 169 (59.7%) were confirmed as Campylobacter spp., 111 (39.2%) C. jejuni, and 43 (15.2%) C. coli. C. jejuni was the most prevalent in all sampling points. Campylobacter spp. showed a characteristically seasonal prevalence with the highest isolation rate during the warmer period of the year. We detected the cadF and ciaB genes in all C. jejuni isolates. The flaA gene was present in 50% of the examined strains. The cdt genes (cdtA, cdtB, and cdtC) were confirmed in 52.8%, 52.8%, and 47.2% of the C. jejuni strains, respectively. C. jejuni showed 15 profiles of virulence patterns with four predominant profiles.

Subclinical mastitis is an asymptomatic udder infection distributed worldwide with enormous losses in the dairy industry. The study’s objective was to determine the presence of this pathological condition in small dairy farms in the R. of N. Macedonia and to identify the most common associated bacteria. Milk samples were obtained from 96 dairy cows (378 udder quarters) in seven dairy farms, in 3 consecutive samplings 24–72 hours apart. The samples were cultured on routine bacteriological growth media and incubated for 24–48 hours. The isolates were identified by AximaiD Plus MALDITOF MS Platform. Subclinical mastitis was found in 49 animals (51%) and 104 infected quarters (27%). The most frequent isolated bacteria on cow level were Streptococcus uberis (19.4%), Staphylococcus haemolyticus (13.4%), Staphylococcus aureus (7.4%) and Staphylococcu ssimulans (7.4%). On quarter level, the most isolated pathogen was Streptococcus uberis (35.6%) followed by Staphylococcu shaemolyticus and Staphylococcus aureus (10.3% and 9.2% respectively). Subclinical mastitis was found to be highly present in the selected small dairy farms. The most prevalent bacteria identified in the dairy farms (Streptococcus uberis, Staphylococcus aureus and coagulase–negative staphylococci) indicate that poor management and udder health practices, inadequate milking procedures and lack of mastitis control strategies greatly contribute to occurrence and persistence of subclinical mastitis.