β-lactamases are a diverse class of enzymes produced by bacteria that present a major cause for resistance to β-lactams. In this study we analysed 159 fecal samples from dairy cows, for the presence ...of presumptive ESBL, AmpC, and carbapenemase-producing
. Phylotyping was done using Clermont phylo-typing method, targeting arpA, ChuA, and YjaA genes, along with the DNA fragment TspE4.C2. Convetional PCR method was used to confirm the presence of
genes among 39 phenotypically confirmed ESBL producing
The results showed presence of CTX-M, SHV, TEM and OXA1
genes in 28 (71.79%), 1 (2.56%), 29 (74.35%), 2 (5.12%) of isolates, respectively Twenty (51.28%) isolates showed presence of both
CTX-M and TEM genes. The strain that carried the
SHV gene was found to carry
TEM gene as well, while one of the strains that carried
OXA1 gene was also carrying
CTX-M and TEM gene. The ration between isolates and phylo-groups was as follows: 9 (23.07%) strains were assigned to phyllo-group D; 14 (35.89%) to phyllo-group B; 16 (41.02%) to phyllo-group A. Out of the 39 strains where
genes were identified, 29 (74.35%) were categorized as multi drug resistant.
The primary objective of our study was to detect the occurrence of enterotoxigenic
in diverse types of cheese (cow’s milk cheese and mixed milk cheese) samples from R.N. Macedonia. Cheese samples ...were analyzed for enumeration and isolation of the
strains according to ISO 6888-1. We detected the toxigenic potential of the strains by the use of the Enzyme Link Fluorescent Assay VIDAS system, and we confirmed the presence of the SEs (
) genes by multiplex PCR. The results showed that out of 270 samples of cheese, coagulase-positive staphylococci (CPS) were detected in 27 (10%), and coagulase-negative staphylococci in five samples (1.8%). Biochemically, all 27 CPS samples were confirmed to be
With VIDAS SET2 test we confirmed that 11 isolates are producers of one of the toxins limited by the test. With the conventional PCR we confirmed genes in only 7 isolates. Most common detected gene was
n=3 (42.8%), followed by
n=2 (28.6%), and
n=2 (28.6%). Additionally,
and
genes were not detected in any of the
isolates. Discrepancies between the two test methods for detection of enterotoxigenic potential are not uncommon. The presence of viable
cells that have enterotoxin potency demonstrates the importance of appropriate hygiene practices in the diary process and also the maintenance of the products in order to obtain a safe final product for the consumers.
As the number of genetically modified crops increases rapidly, their accurate detection is significant for labelling and safety assessment. Currently, real-time PCR is the “golden standard” method ...for GMO detection. Hence, extraction of high quality DNA represents a crucial step for accurate and efficient DNA amplification. For GMO presence evaluation in the extracted DNA from raw corn kernels and roasted soybean, we used real-time PCR method, in consistent with the ISO17025 accreditation standards. As for DNA extraction, modified basic SDS protocol by adding
enzyme in different steps of the protocol, with different time and temperature of incubation was used. The results showed as most suitable, the protocol where 10 µl of
enzyme was added together with the lysis buffer at 65 °C for 30 minutes. Data for DNA yield and purity for roasted soybean was 469.6±3.3 µg/ml with A260/280 absorbance ratio 1.78±0.01. Suitability of DNA extracts for GMO analysis was assessed by screening for the presence of 35S promotor and Tnos terminator. Diluted extracts in concentrations 10, 1, 0.1, 0.01 and 0.0027 ng/µl, were tested in six replicates. Positive signal of amplification (LOD) was detected in all concentrations for both genetic elements in both matrices. The LOQ for 35S and Tnos for both matrices was 0.1 ng, while for Tnos in raw corn kernels was 0.01 ng. This in-house developed DNA extraction method is simple and obtains high-quality DNA suitable for GMO screening of 35S promotor and Tnos terminator in both raw and processed matrices.
The probability of contamination of non-transgenic varieties with genetically modified (GM) products increase as a result of global expansion of areas sown with transgenic crops. DNA-based methods as ...accurate, efficient and reliable methods are preferable for detection of GM material in raw or highly processed foods. Isolation of high quality DNA with a suitable and efficient DNA extraction protocol is crucial for getting precise results in DNA amplification. In this study, we performed modifications of previously known Sodium dodecyl sulfate (SDS)-based DNA extraction method regarding the incubation period, DNA pellet washing and addition of organic solvent extraction, to improve DNA quality and to reduce costs. Raw corn kernels and roasted soybean seed were used as samples. DNA was extracted following three protocols, modifications of Edwards protocol. The type of detergent used in raw corn sample did not cause significant effects on extracted DNA yield and purity, while in roasted soybean samples the 2% (w/v) SDS lysis buffer gave the highest DNA yield. The additional incubation step raised the DNA yield from raw corn for 121%, while the purest DNA from soybean sample was obtained using organic solvent extraction. Electrophoretic determination of DNA integrity showed varying degree of DNA smearing from roasted soybean. Contrary, all extraction protocols used on raw corn kernels produced a high molecular weight DNA. Thus, our in-house DNA extraction protocol is as efficient but more cost effective compared to commercial kits and can be used for raw corn, while the protocol for roasted soybean needs further improvement.
The sector of small, medium enterprises and entrepreneurs has an important role in economic development of all countries. In recent years, they have become carriers of economic growth, regardless ...these are countries with developed economies, developing countries or countries in transition. These businesses entities contribute to increasing dynamics of the economy and improving its competitiveness. Their basic role in the economic system is to solve the problem related to low economic activity, low innovation, low level of competitiveness, unequal distribution of wealth and income, as well as high unemployment rate through the creation of new jobs. In Serbia, they also represent an important economic factor of development. Given that, they have the potential for further growth and development, this paper is about analysis of the development of the entrepreneurial sector in Serbia and neighboring countries, with special reference to the period from 2015 to 2020 years. The sector of small and medium enterprises is an extremely important economic segment, as the analysis of the development of this sector will show.
Due to the actuality of spongiform encephalopathies and their proven spreading by means of animal feed containing meat and bone meal, the description and measurement of osteocytic lacunae contributes ...to more easily distinguish bone fragments in meat and bone meal. Transmissible spongiform encephalopathies (TSEs) have attracted a lot of attention, especially after 1986, when the first case of BSE (bovine spongiform encephalopathy) was detected. Since the outbreak of spongiform encephalopathy (BSE), the use of animal protein including bone meal as an ingredient in animal feed has been controlled by several regulations including Regulation (EC) 999/2001, Regulation (EC) 1774/2002, and Regulation (EC) 1234/2003. The classical microscopic method is the only official method for detecting animal protein in animal feed in the European Union (Commission Regulation (EC) 152/2009). By applying the microscopic method to the animal feed samples, we performed detection in order to determine the presence of animal proteins that originate from mammals and fish. The microscopic analysis of all 421 samples, of which 115 were raw materials for the production of animal feed, 230 were concentrates for ruminant nutrition and 76 were concentrates for non-ruminant nutrition (32 concentrates for laying hens and 44 concentrates for pigs), did not provide positive results, that is, no remains of animal tissues of mammalian origin were found in any specimen. Whereas in 10 out of 32 (31.25%) concentrates intended for non-ruminant nutrition (laying hens), pieces of fish tissue were found. In these samples, we usually detected the presence of fish bones, gills and scales.
Yersinia enterocolitica is one of the priority biological hazards in pork inspection. Persistence of the pathogen, including strains resistant to antimicrobials, should be evaluated in pigs from ...different housing systems for risk ranking of farms. In this 2019 study, tonsils were collected from 234 pigs, of which 69 (29.5%) were fattened on 3 big integrated farms, 130 (55.5%) on 10 medium-sized farms, and 35 (15%) on 13 small family farms. In addition, 92 pork cuts and minced meat samples from the same farms were tested for the presence of Y. enterocolitica using the culture method. Phenotypic and genetic characteristics of the isolates were compared with previously collected isolates from 2014. The overall prevalence of Y. enterocolitica in pig tonsils was 43% 95% CI 36.7−49.7. In pigs from big integrated, medium-sized, and small family farms, the prevalence was 29%, 52%, and 40%, respectively. All retail samples of portioned and minced pork tested negative for pathogenic Y. enterocolitica, likely due to high hygienic standards in slaughterhouses/cutting meat or low sensitivity of culture methods in these matrices. The highest recovery rate of the pathogen from tonsils was found when alkali-treated PSB and CIN agar were combined. The biosecurity category of integrated and medium farms did not affect the differences in prevalence of Y. enterocolitica (p > 0.05), in contrast to family farms. Pathogenic ail-positive Y. enterocolitica biotype 4 serotype O:3 persisted in the tonsils of pigs regardless of the type of farm, slaughterhouse, and year of isolation 2014 and 2019. PFGE typing revealed the high genetic concordance (80.6 to 100%) of all the Y. enterocolitica 4/O:3 isolates. A statistically significant higher prevalence of multidrug-resistant Y. enterocolitica 4/O:3 isolates was detected in the tonsils of pigs from big integrated farms compared to the other farm types (p < 0.05), with predominant and increasing resistance to nalidixic acid, chloramphenicol, and streptomycin. This study demonstrated multidrug resistance of the pathogen in pigs likely due to more antimicrobial pressure on big farms, with intriguing resistance to some clinically relevant antimicrobials used in the treatment of yersiniosis in humans.
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
isolates (
). Out of 283 samples, 169 (59.7%) were confirmed as
spp., 111 (39.2%)
, and 43 (15.2%)
.
was the most prevalent in all sampling points.
spp. showed a characteristically seasonal prevalence with the highest isolation rate during the warmer period of the year. We detected the
and
genes in all
isolates. The
gene was present in 50% of the examined strains. The
genes (
,
, and
) were confirmed in 52.8%, 52.8%, and 47.2% of the
strains, respectively.
showed 15 profiles of virulence patterns with four predominant profiles.
Analytical methods based on immunoaffinity column clean-up and quantitative determination with liquid chromatography-fluorescence detection were used to determine aflatoxins and ochratoxin A in liver ...samples. The validation of the procedures was performed. The linearity of the methods was checked, and a good coefficient of correlation was found for all aflatoxins and OTA as well. The LOD and LOQ were acceptable: 0.003 µg/kg and 0.009 µg/kg for AFB
; 0.001 µg/kg and 0.005 µg/kg for AFB
; 0.006 µg/kg and 0.020 µg/kg for AFG
; 0.007 µg/kg and 0.022 µg/kg for AFG
; 0.08 µg/kg and 0.27 µg/kg for OTA. The results for the repeatability estimated by the relative standard deviation (RSD
) were satisfactory and the obtained values were in the acceptable range (1.97–14.41% for all aflatoxins and 3.76-8.31% for OTA) at three proposed concentration levels. RSD
values showed acceptable correlation between two analysts for all four aflatoxins and OTA. The RSD
values were as followed: 2.37% and 5.60% for AFB1, 6.71% and 8.78% for AFB
, 4.40% and 7.00% for AFG
and 10.30% and 13.91% for AFG
(for the first and second analyst, respectively). The RSDR values for OTA were 4.91% and 3.15% (1 µg/kg); 3.76% and 4.12% (5 µg/kg) and 8.31% and 8.21% (10 µg/kg). The mean recovery for total aflatoxins and OTA were 78.10% and 93.34%, respectively. All validation parameters were in accordance to European legislation. They indicate that the proposed analytical procedures are suitable and they could be methods of choice for the determination of aflatoxins and OTA in liver samples.
In recent years many researchers have described the reduced mycotoxin toxicity caused by probiotic bacteria. Since reduction under gastrointestinal conditions of the bioavailability of mycotoxins by ...probiotics is not fully investigated in birds, the aim of the study was to determine the effect of probiotic on T-2 mycotoxicosis induced apoptosis in broiler’s liver. For the study, twelve 1-days old broilers were divided equally into T-2 toxin (T2) and probiotic with T-2 (P+T2) groups. From the first experimental day, probiotic Enterococcus faecium DSM 7134 was administered in drinking water to P+T2 group. From the fourth day, T-2 toxin was given for three consecutive days to T2 toxin group. At 8th experimental day chicken were sacrificed, liver was fixed in buffered 10% formalin, embedded into paraffin, slices 5 μm in thickness were cut followed by immunohistochemical staining with polyclonal primary antibodies p21 and p53 (Abcam, UK) according to the manufacturers’ guidelines (IHC kit, Abcam, UK). Blood samples were taken by cardiac puncture to measure liver enzymes. Immunohistochemical staining revealed strong expression of p53 and p21 antibodies in hepatocytes nuclei as well as around blood vessels in T-2 toxin group’s chicken liver tissue. Staining by both antibodies was less intensive in P+T2 group. Enzyme analysis showed significantly increased (p<0.05) blood aspartate transaminase and alanine transaminase concentrations by 33.87% and 68.03% respectfully in T2 toxin group, while enzyme concentrations were decreased in P+T2 group. The obtained results showed reduced features of liver apoptosis in treatment with probiotic bacteria.
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