Improved procedure for the detection of C. difficile in samples with low number of bacteria was described. Preenrichment step was added prior to LightCycler rtPCR (LC rtPCR) with the purpose to increase the number of C. difficile in samples. One day enrichment culture for C. difficile followed by LC rtPCR assay targeting all three toxin genes can be applied as accurate and rapid screening test especially in samples with low number of C. difficile, as no culture positive/LC rtPCR negative samples were observed. It can be applied to different samples, which could broaden the knowledge about prevalence of C. difficile in food and environmental samples. Furthermore, there is no standard approach for the detection of C. difficile in food; therefore, application of improved procedure could enable comparison of results obtained in different studies.
COBISS.SI-ID: 3637882
Mycoplasma synoviae and Newcastle disease virus (NDV) are 2 avian pathogens that cause modulation in expression of a variety of cytokine and chemokine genes in chickens. However, there is limited data about gene modulation after coinfection with these 2 pathogens and even less data about gene modulation after infection of chicken embryos. In this study, the effect of M. synoviae type strain WVU 1853 and lentogenic LaSota vaccine strain of NDV infection on cytokine and chemokine gene expression in chicken embryos was analyzed in the liver, spleen, bursa of Fabricius, and thymus by using quantitative real-time PCR. Mycoplasma synoviae infection of embryos resulted in intensive upregulation of cytokine and chemokine genes, including interferon (IFN)-γ, IL-1β, IL-6, IL-12p40, IL-16, IL-18, MIP-1β (CCL4), inducible nitric oxide synthase (iNOS), XCL1, and lipopolysaccharide-induced tumor necrosis factor-α factor (LITAF), with different expression profiles in the 4 organs. Inoculation of lentogenic NDV significantly upregulated IFN-γ, IL-6, and IL-16 genes in spleen and IFN-γ, IL-1β, IL-2, IL-16, IL-21, XCL1, and MIP-1β (CCL4) genes in the thymus, but to a lesser extent than M. synoviae. Overall effect of NDV inoculation, regarding the number of modulated cytokine and chemokine genes and the extent of expression, was lower than M. synoviae. When NDV was introduced after on-going M. synoviae infection, most M. synoviae-induced cytokine and chemokine genes were significantly downregulated.
COBISS.SI-ID: 3303304
In 2009, a surprisingly high number of animals seropositive for equine infectious anaemia virus (EIAV; 26 horses from 13 farms) were detected in Slovenia. The aim of this study was to develop a polymerase chain reaction (PCR) assay for the detection of the proviral nucleic acid and characterisation of the Slovenian EIAV strains. In total, 26 horses (including 2 foals and 4 pregnant mares) and 4 fetuses were examined in this study. A PCR assay using the EIAV F1 and EIAV R1 primers was designed and tested using genomic DNA extracted from 28 spleen samples, 18 whole blood samples and 17 peripheral blood leucocyte samples. Amplicons of 22 PCRs obtained from the spleen samples were subjected to direct DNA sequencing and phylogenetic analysis. All spleen samples from 22 adult animals were positive for EIAV by PCR, whereas whole blood and the peripheral blood leucocyte samples were positive from only 4 animals. Spleen samples from foals and fetuses were negative by PCR. The Slovenian EIAV sequences could be mapped into 9 different branches of the phylogenetic tree. The PCR was able to detect different EIAV strains from spleen samples of seropositive animals detected in Slovenia. Phylogenetic analysis revealed high genetic diversity of the EIAV strains detected in Slovenia, with their closest relatives being European strains. In utero transmission in pregnant mares did not occur.
COBISS.SI-ID: 3711354
Lasalocid is a veterinary ionophore antibiotic used for prevention and treatment of coccidiosis in poultry. It enters the environment with the use of contaminated manure on agricultural land. Despite its extensive use, the effects of lasalocid on non-target soil organisms are poorly explored. We used classical subleathal ecotoxicity tests to assess the effects of lasalocid on earthworms (Eisenia andrei) and isopods (Porcellio scaber) and compared the results with tests using avoidance behaviour as the endpoint. The results showed that avoidance is a much more sensitive endpoint. For earthworms, EC50 for avoidance (12.3 mg kg-1 dry soil) was more than five times lower than EC50 for reproduction (69.6 mg kg-1 dry soil). In isopods the sensitivity of the behavioural response test was even higher. While the highest lasalocid concentration 202 mg kg-1 had no significant effects on isopod growth or survival, already the lowest used concentration in the behavioural assay (4.51 mg kg-1) caused significant impact on isopod behaviour. However, the EC50 value for isopod avoidance could not be calculated. Using the avoidance test results for calculating the predicted no-effect concentration (PNEC) of lasalocid to soil invertebrates, the value is close to the predicted environmental concentration (PEC). This indicates that the use of lasalocid-contaminated manure could potentially impair the habitat function of agricultural soils.
COBISS.SI-ID: 3667834
The validation of analytical procedure for the determination of aflatoxin B1 in eggs of laying hens was presented. The procedure consisted of the extraction of the analyte from the sample, immunoaffinity column clean-up and liquid chromatography with postcolumn bromination and fluorescence detection. The parameters obtained in the first (LOD 2 ng/kg, LOQ 6 ng/kg, RSDR 11%, izkoristek 70%) and in the second laboratory (LOD 2 ng/kg, LOQ 5 ng/kg, RSDr 20%, izkoristek 67%) indicate that both versions of the procedure are suitable for the determination of aflatoxin B1 in eggs of laying hens.