Florfenicol (FF) is a broad-spectrum antibiotic used to treat gastrointestinal and respiratory infections in domestic animals. Considering FF's rapid elimination via urine after drug treatment, its ...use increases concerns about environmental contamination. The objective of the study was to establish a sustainable chromatographic method for simple analysis of FF in pig urine to investigate the urinary excretion of FF after a single intramuscular administration of 20 mg FF/kg body weight. The urine sample was prepared using a centrifuge and regenerated cellulose filter, and the diluted sample was analyzed. The method was validated in terms of linearity, the limit of detection (0.005 µg/mL) and quantitation (0.016 µg/mL), repeatability and matrix effect (%RSD ranged up to 2.5), accuracy (varied between 98% and 102%), and stability. The concentration-time profile of pig urine samples collected within 48 h post-drug administration showed that 63% of FF's dose was excreted. The developed method and previously published methods used to qualify FF in the urine of animal origin were evaluated by the National Environmental Method Index (NEMI), Green Analytical Procedure Index (GAPI) and Analytical GREENness Metric Approach (AGREE). The greenness profiles of published methods revealed problems with high solvents and energy consumption, while the established method was shown to be more environmentally friendly.
Studies of polyphenols in various natural products have shown their variability and often low amounts. Thus, we aimed to investigate the bioaccessibility of the main polyphenolic groups extracted ...from two poplar types of propolis collected in continental Croatia. The propolis complex, in the form of water-soluble powder of propolis extract with the carrier 2-hydroxypropyl-β-cyclodextrin (HP-β-CD), was obtained by applying ultrasonic-assisted extraction followed by lyophilization. The in vitro gastrointestinal digestion was conducted on the propolis complex extracts and a mixture of standards (pinocembrin, galangin, chrysin, caffeic acid, and caffeic acid phenylethyl ester). Fractions from the oral, gastric, and intestinal phase samples were analyzed spectrophotometrically by standard methods used to determine the content of total phenols (TPs), flavonols/flavones (TFFs), and flavanones/dihydroflavonols (TFDs). The small-intestinal phase was performed by two models: centrifugation and dialysis. The results of propolis-HP-β-CD extracts demonstrated high polyphenolic bioaccessibility during all three phases of digestion. The bioaccessibility of TPs, TFFs, and TFDs after the intestinal phase with centrifugation was from 0.02 to 8.86 times higher than in the dialysis phase, stressing that it is a better model than dialysis. Our findings show that the inclusion complex of propolis with HP-β-CD enables the good solubility of polyphenol groups.
The antimicrobial susceptibility of coagulase-negative staphylococci isolated from spontaneously fermented sausages was assessed using both traditional and molecular methods. Isolates were tested for ...sensitivity to vancomycin, ampicillin, erythromycin, tetracycline, gentamycin and oxacillin by the disk diffusion method and quantitative-qualitative epsilometer test. PCR was used for the detection of resistance genes mecA, ermB, tetK and tetM. The identified coagulase-negative staphylococci were Staphylococcus epidermidis (69 %), S. capitis (5 %) and S. warneri (2.5 %). S. epidermidis showed a high rate of phenotypical resistance to tetracycline and erythromycin (44.4 % of strains). Molecular evaluation of resistance determinants revealed tetK or tetM genes in eight S. epidermidis strains. Although S. epidermidis is not classical food poisoning bacteria, its presence in food could be of public health significance due to the possible spread of antimicrobial resistance determinants. Our findings implicate that spontaneous meat fermentation could result in products with a potential hazard to consumers.
Aujeszky’s disease (AD) is a viral infectious disease caused by Suid herpesvirus 1 (SuHV-1). Vaccination and eradication of AD in domestic pigs is possible using marker vaccines with attenuated or ...inactivated SuHV-1, or subunit vaccines. However, vaccines with attenuated SuHV-1 have shown to be more potent in inducing strong cell-mediated immune response. The studies have shown that Parapoxvirus ovis, as well as Propionibacterium granulosum with lipopolysacharides (LPS) of Escherichia coli have pronounced immunomodulatory effects and that in combination with the vaccines can induce stronger humoral and cellular immune responses than use of vaccines alone.
In our study distribution of peripheral blood T cell subpopulations was analysed after administration of vaccine alone (attenuated SuHV-1), immunostimulators (inactivated Parapoxvirus ovis or combination of an inactivated P. granulosum and detoxified LPS of E. coli) and combinations of vaccine with each immunostimulator to the 12-week old piglets. Throughout the study no significant changes were found in the proportions of γδ and most αβ T cell subpopulations analysed. However, on the seventh day of the study combination of an inactivated P. granulosum and LPS of E. coli with vaccine induced transient but significant increase of the proportions of CD4+CD8α+ and CD4−CD8α+ αβ T cells, that have been strongly associated with early protection of SuHV-1 infected pigs.
Our findings indicate that combination of inactivated P. granulosum and detoxified E. coli LPS could be used for enhancement of a cellular immune response induced by vaccines against AD.
Spectrophotometric procedures for the rapid characterization of propolis have been performed on propolis samples from different regions of Croatia. In order to determine the major groups of bioactive ...compounds in propolis, the following optimised and validated spectrophotometric methods were carried out: the Folin-Ciocalteu method for the content of total phenolics (TPs) and two distinct methods for the content of total flavonoids: aluminium chloride (AlCl3 ) complexation method for total flavones/flavonols (TFFs) and the 2.4-dinitrophenylhydrazine (2.4-DNPH) method for total flavanones/dihydroflavonols (TFDs). Validation parameters, including linearity, sensitivity, range, accuracy, limit of detection, limit of quantification, precision and robustness were implemented. The following polyphenol standards were used for the validation procedure: gallic acid (GA), pinocembrin (PC), galangin (GN), quercetin (QC) and a mixture of PC and GN. Validated methods were applied to analyse six samples of raw propolis from Croatian continental and Adriatic regions. The high qualitative/quantitative variability of the TP, TFF and TFD content was observed. Although the method of extraction (ultrasonic-assisted extraction or microwave assisted extraction) showed a non-significant effect on extraction yield (P>0.05) and the polyphenolic concentrations obtained of each sample in general, ultrasonic extraction was found to be more selective. Furthermore, the calibration compound used for constructing the calibration curve highly influenced the final concentrations of TPs and TFFs. The study showed good linearity, accuracy, repeatability, intermediate precision, LOD and LOQ for all three spectrophotometric methods, considering that these analyses are the basis for further research into the individual polyphenolic compounds in the propolis samples covered by this research.
•We have genotyped Coxiella burnetii strains for the first time in Croatia.•For the genotyping we used MLVA.•We have identified 13 novel C. burnetii genotypes that appear to be unique to Croatia.
...Although Q fever affects humans and animals in Croatia, we are unaware of genotyping studies of Croatian strains of the causative pathogen Coxiella burnetii, which would greatly assist monitoring and control efforts. Here 3261 human and animal samples were screened for C. burnetii DNA by conventional PCR, and 335 (10.3%) were positive. Of these positive samples, 82 were genotyped at 17 loci using the relatively new method of multi-locus variable number tandem repeat analysis (MLVA). We identified 13 C. burnetii genotypes not previously reported anywhere in the world. Two of these 13 genotypes are typical of the continental part of Croatia and share more similarity with genotypes outside Croatia than with genotypes within the country. The remaining 11 novel genotypes are typical of the coastal part of Croatia and show more similarity to one another than to genotypes outside the country. Our findings shed new light on the phylogeny of C. burnetii strains and may help establish MLVA as a standard technique for Coxiella genotyping.
Raspodjela florfenikola (FF) i njegovog metabolita florfenikol amina (FFA) u cerebrospinalnoj tekućini svinja istražena je nakon dvokratne primjene u mišić s 48-satnim razmakom originalnog i ...generičkog veterinarsko-medicinskog proizvoda (VMP) iste terapijske doze (20 mg FF/kg tjelesne mase) u istim uvjetima pokusa. Oba VMP-a imaju odobrenje za stavljanje u promet u Hrvatskoj te su po farmaceutskom obliku otopine za injekcije i sadržavaju 300 mg FF/mL. Pokus je proveden na klinički zdravim svinjama raspoređenim u dvije pokusne i jednu kontrolnu skupinu. Prvoj pokusnoj skupini životinja primijenjen je originalni, drugoj skupini generički lijek, dok je treća skupina bila kontrolna. Žrtvovanje životinja uslijedilo je nakon 216, 288 i 384 sata od prvog davanja VMP-a. Uzorci cerebrospinalne tekućine prikupljeni su u trenutku žrtvovanja i analizirani su optimiranom i validiranom tekućinskokromatografskom metodom uz detekciju UV-detektorom s nizom fotodioda (HPLC-DAD). Obrada uzoraka cerebrospinalne tekućine provedena je ekstrakcijom na čvrstoj fazi C18 (SPE). Primjenom SPE-HPLC- DAD metode ustvrđeno je da matrica ne utječe na linearnost FF i FFA u radnom području od 0,05 do 5,00 μg/mL te je postignuta granica detekcije od 0,0023 μg/mL za FF i od 0,0100 μg/mL za FFA. Vrijednosti analitičkih povrata kreću se od 86,6 % do 111,8 % za FF, odnosno od 91,7% do 98,8% za FFA. U ovom radu je dokazano da je SPE-HPLC-DAD metoda selektivna, osjetljiva i pouzdana analitička metoda za određivanje FF i FFA u cerebrospi- nalnoj tekućini. S obzirom na rezultate našeg preliminarnog istraživanja FF u plazmi svinja tretiranih originalnim, odnosno generičkim VMP-om, cilj ovog istraživanja bila je i usporedba distribucije FF u uzorcima plazme i cerebrospinalne tekućine svinja nakon 216. sata od primjene VMP-a. Međutim, u uzorcima cerebrospinalne tekućine svinja žrtvovanih u navedenom pokusnom razdoblju nisu detektirani ni FF ni FFA. Zbog sličnosti matrica, SPE-HPLC-DAD metoda mogla bi poslužiti u budućim farmakokinetičkim studijama FF u uzorcima plazme i seruma dobivenim od svi- nja nakon primjene u mišić.
U veterinarskoj medicini cjepiva imaju presudnu ulogu u očuvanju zdravlja životinja. Napretkom znanosti djelatne tvari u cjepivima nisu više samo cijeli mikroorganizmi (inaktivirani ili atenuirani), ...već se razvijaju cjepiva koja sadržavaju dijelove mikroorganizama (rekombinirani proteini ili sintetski peptidi) dobivena različitim naprednim tehnologijama. Na taj je način osiguran daleko veća neškodljivosti cjepiva, a njegova imunogenost nastoji postići dodatkom adjuvansa. Uz adjuvanse u obliku aluminijevih soli, a koji su u uporabi već desetljećima i prvi po zastupljenosti u veterinarskim cjepivima, značajni su i adjuvansi u obliku emulzija, a između ostalih i mineralna ulja. Negativna strana primjene mineralnih ulja može biti pojava lokalne reakcije na mjestu primjene. Ovakve promjene mogu u tovnih životinja utjecati na klaoničku vrijednost, a u izložbenih životinja prilikom natjecanja ili izložbi na ocjenu. Zbog navedenih lokalnih reakcija koje mogu izazvati i otežane primjene zbog viskoznosti, mineralna ulja nisu često korišteni adjuvans u cjepivima namijenjenim ljudima i kućnim ljubimcima, već se uglavnom koriste za cijepljenje farmskih životinja. Razvojem novih tehnologija u proizvodnji nastoji se umanjiti negativan utjecaj na životinje, kao i moguće reakcije u osoba koje primjenjuju takva cjepiva, stoga uputa za cjepivo koje sadržava mineralno ulje mora sadržavati upozorenje tijekom primjene o rizicima koji mogu nastati i upute postupanja u slučaju reakcije.
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