Application of commercial enzyme linked immunosorbent assays (ELISA) for the detection of antibodies for foot-and-mouth disease virus in wild boar and red deer

For detecting antibodies towards foot and mouth (FMD) virus in sera collected from red deer hinds (Cervus elaphus) and wild boars (Sus scrofa), three commercially available enzyme-linked immunosorbent assays (ELISA) were used. Two ELISA kits (PrioCHECK FMDV NS and CHEKIT FMD-3ABC) were used for the detection of antibodies towards non-structural proteins of FMD virus and one assay was based on the detection of antibodies for serotype O (PrioCHECK FMDV type O). All of the sera tested in our study were negative for antibodies against FMD virus. The aim of this study was to investigate the usefulness of commercially available ELISA kits given for marketing authorization in Croatia in testing the prevalence of FMD antibodies in wild boar and red deer populations. Since the producers of ELISA kits used in our study did not declare wild animals as a target species, we hypothesised that the same kits could be used for serological diagnosis of FMD in red deer and wild boars. Our study confirmed that the kits used are acceptable for detecting antibodies in both species tested, however, the investigation highlighted the problem of validating the kits due to the absence of available positive sera originating from red deer, as well as other susceptible species, especially artiodactyls

Geometric formality of homogeneous spaces and of biquotients

We provide examples of homogeneous spaces which are neither symmetric spaces nor real cohomology spheres, yet have the property that every invariant metric is geometrically formal. We also extend the known obstructions to geometric formality to some new classes of homogeneous spaces and of biquotients, and to certain sphere bundles.Comment: 15 page

Razvoj SPE-HPLC-DAD metode za određivanje florfenikola i florfenikol amina u cerebrospinalnoj tekućini svinja

A study of florfenicol (FF) and its metabo- lite florfenicol amine (FFA) in pig cerebrospinal fluid was conducted following repeated intramuscular administration of the original (reference) and a generic veterinary medicinal product (VMP) under the same experimental conditions (20 mg FF/kg body weight, 48-hour interval). Both VMPs are solutions for injection containing FF as an active substance in the concentration of 300 mg/mL and have been authorized in Croatia for use in cattle and pigs. In this study, clinically healthy pigs were randomly divided into three groups. The first group was treated with the reference VMP, the second with the generic VMP, while the third served as the control group. Animals were sacrificed at 216, 288 and 384 hours after the first drug administration. Cerebrospinal fluid samples were analysed by the optimized and validated high-performance liquid chromatography-diode array detector method (HPLC-DAD). The solid-phase extraction (SPE) technique was chosen for sample preparation. The HPLC-DAD method provides good linearity over the concentration range of 0.05 to 5.00 μg/mL for FF and FFA. Limits of detection were 0.0023 μg/mL for FF and 0.0100 μg/mL for FFA. Extraction recoveries of FF were from 86.6% to 111.8%, and of FFA from 91.7% to 98.8%. The SPE-HPLC-DAD method has been demonstrated to be a selective, sensitive and suitable analytical method for the determination of FF and FFA in cerebrospinal fluid. The present study was based on a preliminary study that quantified FF in pig plasma at 216 hours after the first application of reference or generic VMP. However, FF and FFA were not detected in any of the cerebrospinal fluid samples during the experimental period. According to the nature of biological fluids, the SPE-HPLC-DAD method can be suitable for further pharmacokinetic studies of FF in pig plasma and serum after intramuscular administration of VMPs.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

Utjecaj levamizola na koncentraciju kortizola i promjene u perifernoj krvi svinja izloženih stresu

The objective of the study was to evaluate the influence of levamisole (LEV) application in pigs stressed by adrenocorticotropic hormone (ACTH) injection on the total and differential leukocyte count, blood erythrocyte count, haemoglobin concentration and cortisol level during and for 16 days after the treatment. Swedish Landrace boars aged 6 to 7 months were divided into three groups. Group 1 received levamisole 2.5 mg/kg/body mass/day intramuscularly over 3 days and ACTH 10 μg/kg/body mass/day intravenously for 3 days. Group 2 received ACTH 10 μg/kg/body mass/day intravenously for the next 3 days and group 3 received saline intramuscularly for 6 days (control group). Cortisol concentration was increased in both ACTH treated groups during all three days of administration and the day after the last ACTH treatment. A significantly increase in total leukocyte count and a decrease in lymphocyte percentages was recorded during ACTH treatment. Use of levamisole before stress induction caused an increase in total leukocyte count in the 16 day period after cessation of ACTH treatment and also a lymphocyte increase in stressed animals on the first day of ACTH injection. Pigs that received levamisol and ACTH did not show eosinophilia in contrast to pigs that received ACTH only. In both groups of stressed animals, an elevated percentage of neutrophilic granulocytes was recorded on days 2 and 3 of ACTH treatment. However, administration of levamisole led to faster normalization of neutrophils and prevented neutropenia one week after termination of stress. According to the data presented, levamisole can influence pig immunity during and after stress induction by ACTH administration.Istraživanje utjecaja levamizola na promjene u perifernoj krvi i koncentraciju kortizola provedeno je u svinja u stresu izazvanom davanjem adrenokortikotropnog hormona (ACTH). Pokus je proveden na nerastima švedskoga landrasa, u dobi 6 do 7 mjeseci, podijeljenih u tri skupine. Svinje u prvoj skupini dobivale su levamizol (2,5 mg/kg/tj. mase/dan, i.m.) tijekom tri dana i ACTH (10 μg/kg/tj. mase/dan, i.v.) tijekom 3 dana, a u drugoj skupini ACTH (10 μg/kg/tj. mase/dan, i.v.) također tijekom slijedeća 3 dana. Treća skupina bila je kontrola koja je dobivala fiziološku otopinu i.m. 6 dana. U pokusu je praćen broj leukocita, diferencijalna krvna slika, broj eritrocita, koncentracije hemoglobina i kortizola. Tijekom trodnevnog davanja ACTH kao i dan nakon zadnje primjene u obje pokusne skupine zabilježeno je povećanje kortizola u serumu. Istovremeno je primjena ACTH izazvala značajno povećanje ukupnog broja leukocita, ali i smanjenje broja limfocita. Primjenom levamizola prije izazivanja stresa izazvalo se povećanje ukupnoga broja leukocita 16 dana nakon prestanka davanja ACTH. U obje pokusne skupine svinja utvrđeno je povećanje broja neutrofila drugi i treći dan primjene ACTH. Međutim, davanje levamizola prije izazivanja stresa dovelo je do brže uspostave fiziološkoga udjela neutrofi la. U svinja koje su dobile i levamizol i ACTH nije došlo do porasta udjela eozinofila kao što je to zabilježeno u skupini koja je dobila samo ACTH. S obzirom na dobivene rezultate istraživanja može se pretpostaviti da primjena levamizola može posredno utjecati na imunosni odgovor u svinja tijekom i nakon stresa potaknutog s ACTH

Analiza florfenikola u plazmi svinja primjenom validirane PPT-HPLC-DAD metode

High performance liquid chromatography (HPLC) has proven to be an effective tool for examining the disposition kinetics of florfenicol (FF), a synthetic broad-spectrum antibiotic used to treat infectious diseases in veterinary medicine. Modification and optimisation of the protein precipitation (PPT) sample preparation procedure and HPLC with diode array detector (DAD) instrumental method were carried out to ensure conditions suitable for FF analyses in pig plasma samples. Stable supernatants with good plasma mean recoveries of FF (99.8% ± 0.7%RSD) were achieved using 1% v/v phosphoric acid solution in methanol and 10% w/v sodium chloride in aqueous solution. The PPT-HPLCDAD method’s detection limit of 0.004 μg/mL and quantification limit of 0.013 μg/mL provides high sensitivity for analyses of FF in plasma samples. In addition, the optimisation of method conditions resulted in shorter extraction and analysis time and less solvent consumption, which stresses the sustainability of this method in analytical chemistry. The optimised and validated PPT-HPLC-DAD method was applied in a comparative study of FF in pig plasma after administration of veterinary medicinal products. The study was conducted on fattening pigs following repeated intramuscular administration of two similar solutions for injection at a dose of 20 mg FF/kg bodyweight (test groups 1 and 2). The solutions for injection contained the same FF concentration, i.e., 300 mg/mL, but differed in excipients. The aim was to examine the influence of administrated solutions for injection on the extent of exposure to FF in pig plasma. The dynamics of kinetic profiles of FF in pig plasma from both treatments correspond to the FF kinetic profiles published in similar studies. However, differences were observed in the concentrations of FF, which were constant throughout the study, and statistical differences between the test groups were confirmed (P<0.05). Though these findings suggest the possible influence of excipients, a full comprehensive conclusion on the influence of administrated solutions for injection on FF exposure in pig plasma requires additional research.Tekućinska se kromatografija visoke djelotvornosti (HPLC, engl. High-Performance Liquid Chromatography) pokazala učinkovitim alatom za ispitivanje kinetike florfenikola (FF), sintetitčkog antibiotika širokog spektra koji se u veterinarskoj medicini koristi za liječenje zaraznih bolesti. Modifikacija i optimizacija postupka pročišćavanja uzoraka krvi tehnikom precipitacije proteina (PPT) i instrumentalne metode sustava HPLC-a uz detekciju UV-detektorom s nizom fotodioda (DAD, engl. Diode Array Detector) provedene su sa svrhom postizanja pouzdane analitičke metode za određivanje FF-a u plazmi svinja. PPT postupkom priprave uzoraka dodavanjem 1% otopine fosforne kiseline u metanolu (V/V) i 10% vodene otopine natrijevog klorida (m/V) dobiveni su stabilni nadtalozi s dobrim srednjim analitičkim povratima FF-a iz plazme (99,8%±0,7%RSD). Postignuta granica detekcije PPT-HPLC-DAD metode od 0,004 μg/mL i granica određivanja od 0,013 μg/mL omogućuju dobru osjetljivost određivanja FF-a u plazmi; optimizacijom uvjeta metode skraćeno je vrijeme ekstrakcije i analize te je smanjena količina potrošnje otapala čime je postignuta održivost metode u analitičkoj kemiji. Primjenom optimirane i validirane PPT-HPLC-DAD metode provedena Tekućinska se kromatografija visoke djelotvornosti (HPLC, engl. High-Performance Liquid Chromatography) pokazala učinkovitim alatom za ispitivanje kinetike florfenikola (FF), sintetitčkog antibiotika širokog spektra koji se u veterinarskoj medicini koristi za liječenje zaraznih bolesti. Modifikacija i optimizacija postupka pročišćavanja uzoraka krvi tehnikom precipitacije proteina(PPT) i instrumentalne metode sustava HPLC-auz detekciju UV-detektorom s nizom fotodioda (DAD, engl. Diode Array Detector) provedene su sa svrhom postizanja pouzdane analitičke metode za određivanje FF-a u plazmi svinja. PPT postupkom priprave uzoraka dodavanjem 1% otopine fosforne kiseline u metanolu (V/V) i 10% vodene otopine natrijevog klorida (m/V) dobiveni su stabilni nadtalozi s dobrim srednjim analitičkim povratima FF-a iz plazme (99,8%±0,7%RSD). Postignuta granica detekcije PPT-HPLC-DAD metode od 0,004 μg/mL i granica određivanja od 0,013 μg/mL omogućuju dobru osjetljivost određivanja FF-a u plazmi; optimizacijom uvjeta metode skraćeno je vrijeme ekstrakcije i analize te je smanjena količina potrošnje otapala čime je postignuta održivost metode u analitičkoj kemiji. Primjenom optimirane i validirane PPT-HPLC-DAD metode provedena FF-a u plazmi svinja

Analiza fenotipa CD3+CD16+ limfocita periferne krvi svinja

The phenotype of porcine peripheral blood T cells and natural killer (NK) cells has been well-studied over the past three decades, though porcine peripheral blood lymphocytes with mixed T/NK-cell phenotype within perforin- and NKp46- positive CD3+ populations have also been identified. Despite the mixed phenotype, both populations showed in vitro NK cell-like major histocompatibility complex-unrestricted cytolysis. In this study, the peripheral blood lymphocytes of 15 crossbreed, 12-week-old pigs of both sexes, were analysed by flow cytometry for the expression of leukocyte surface antigens (cluster of differentiation, CD) that can be found on porcine T cells (CD3, TCR-γδ and CD4), NK cells (CD16) or on both cell populations (CD8α and SLA-DR). We found the presence of a minor population of CD3+CD16+ cells within peripheral blood lymphocytes (2.84%). Peripheral blood CD3+CD16+ lymphocytes consisted of all four subpopulations with respect to the expression of surface antigens CD4 and CD8α; most were CD4-CD8α+ (60.64%) and CD4-CD8α-(36.77%). While the proportion of SLA- DR+ cells within both subpopulations was similar (8.01% of CD3+CD16+CD4-CD8α+ lymphocytes and 7.41% of CD3+CD16+CD4- CD8α- lymphocytes), the proportion of TCR-γδ+ cells was noticeably higher within CD3+CD16+CD4-CD8α+ (43.48%) than CD3+CD16+CD4-CD8α- (16.55%) lymphocytes. When the expression of individual surface antigens was analysed on peripheral blood CD3+CD16+ lymphocytes, most were CD8α+ (62.44%), though some were also TCR- γδ+ (32.56%), SLA-DR+ (7.55%) or CD4+ (2.59%). Expression of CD8α on CD3+CD16+ lymphocytes was not related to co-expression of other surface antigens, though most CD3+CD16+TCR-γδ+ lymphocytes (81.04%) and most CD3+CD16+CD4+ lymphocytes (69.50%) expressed CD8α. Expression of SLA-DR was not related to the co-expression of TCR-γδ or CD8α, or to the co-expression of both antigens (TCR-γδ and CD8α) on CD3+CD16+CD4- lymphocytes. The results also showed the presence of peripheral blood lymphocytes with the combined phenotypeof T cells and NK cells in three-month old pigs. Though a functional analysis of the investigated cells was not performed in this study, future investigations should provide more insight about the functional properties of porcine peripheral blood CD3+CD16+ lymphocytes with distinct phenotypic characteristics, especially concerning antigen- specific responses and whether the results presented here are solely age-related.Fenotip T limfocita i NK stanica (engl. Natural Killer Cells) periferne krvi svinja dobro je istražen tijekom zadnja tri desetljeća. Međutim, unutar subpopulacija limfocita periferne krvi svinja koje iskazuju površinski antigen CD3 i perforin ili receptor NKp46 ustvrđeni su i limfociti s mješovitim fenotipom T limfocita i NK stanica. Unatoč mješovitom fenotipu, obje subpopulacije limfocita su in vitro pokazale citolitički učinak koji nije bio usmjeren na molekule glavnog sustava tkivne podudarnosti, odnosno citolitički učinak sličan NK stanicama. U našem istraživanju protočnom citometrijom analizirani su limfociti periferne krvi od petnaest svinja oba spola, križane pasmine i u dobi dvanaest tjedana, s obzirom na iskazivanje površinskih antigena koji mogu biti prisutni na T limfocitima (CD3, TCR-γδ i CD4), na NK stanicama (CD16) ili na obje navedene populacije stanica svinja (CD8α i SLA-DR). Naše je istraživanje pokazalo da limfociti periferne krvi sadržavaju manji udio subpopulacije koja iskazuje površinske antigene CD3 i CD16 (2,84%). Limfociti periferne krvi s fenotipom CD3+CD16+ sastojali su se od sve četiri subpopulacije s obzirom na iskaziva- nje površinskih antigena CD4 and CD8α, od kojih je većina bila CD4-CD8α+ (60,64%) i CD4- CD8α- (36,77%). Iako je udio stanica koje iskazuju SLA-DR u obje navedene subpopulacije bio sličan (8,01 % od CD3+CD16+CD4-CD8α+ limfocita i 7,41 % od CD3+CD16+CD4-CD8α- limfocita), udio stanica koje iskazuju TCR-γδ je bio primjetno veći u CD3+CD16+CD4-CD8α+ limfocitima (43,48 %), nego u CD3+CD16+CD4- CD8α- limfocitima (16,55 %). Analiza iskazivanja pojedinih površinskih antigena na CD3+CD16+ limfocitima periferne krvi poka- zala je da ih je većina bila CD8α+ (62,44 %), ali također i TCR-γδ+ (32,56 %), SLA-DR+ (7,55 %) i CD4+ (2,59 %). Iskazivanje površinskog antigena CD8α na CD3+CD16+ lim- focitima nije bilo povezano s iskazivanjem drugih površinskih antigena. Međutim, ve- ćina CD3+CD16+TCR-γδ+ limfocita (81,04 %) i većina CD3+CD16+CD4+ limfocita (69,50 %) iskazivali su i površinski antigen CD8α. Iska- zivanje molekule SLA-DR nije bilo povezano s iskazivanjem površinskog antigena TCR-γδ ili CD8α, niti s oba površinska antigena (TCR- γδ i CD8α) na CD3+CD16+CD4- limfocitima. Naše je istraživanje pokazalo i prisutnost limfocita s kombiniranim fenotipom T limfocita i NK stanica u perifernoj krvi svinja, u dobi od tri mjeseca, međutim funkcija navedenih stanica nije istraživana. Stoga se nadamo se da ćemo budućim istraživanjima moći pojasniti jesu li dobiveni rezultati vezani uz dob svinja. Nadalje, očekujemo da će dodatna istraživanjima omogućiti bolje spoznaje o funkcijama pojedinih subpopulacija CD3+CD16+ limfocita periferne krvi svinja različitog fenotipa, posebice o odgovoru navedenih stanica specifičnom za određeni antigen

Colistin, a last defence polypeptide antibiotic against invasive gram-negative bacteria

Kolistin je polimiksin, polipeptidni baktericidni antibiotik širokog spektra djelovanja protiv Gram-negativnih bakterija, uključujući vrste Acinetobacter spp., Pseudomonas aeruginosa, Klebsiella spp. i Enterobacter spp. Njegova antibakterijska aktivnost je prepoznata u četrdesetim godinama prošlog stoljeća. Međutim, zbog znatnih nuspojava kao što su nefrotoksičnost i neurotoksičnost, polimiksini su se rijetko koristili od 1970-ih kada su se na tržištu pojavili manje toksični aminoglikozidi i drugi antibiotici. Intravenski se kolistin primjenjivao jedino u liječenju pacijenata s infekcijom pluća Gram-negativnim bakterijama, odnosno cističnom fibrozom. Mehanizam antimikrobne aktivnosti kolistina je vezanje na lipopolisaharide i fosfolipide u vanjskoj staničnoj membrani Gram-negativnih bakterija. Oni kompetitivno istiskuju dvovalentne katione iz fosfatnih skupina membranskih lipida, što dovodi do poremećaja vanjske stanične membrane, propuštanja unutarstaničnih sadržaja i smrti bakterija. Komercijalno su dostupna dva oblika kolistina: kolistin sulfat i natrijev kolistimetat. Vezano za doziranje kolistina i danas postoji potreba za studijama optimizacije režima doziranja i učinkovitosti. Danas širom svijeta veliki problem predstavlja pojava teških infekcija Gram-negativnim bakterijama Pseudomonas aeruginosa i Acinetobacter baumannii, otpornih na većinu klasa komercijalno dostupnih antibiotika, kao i nedostatak novih antibiotika s aktivnošću protiv tih bakterija. Stoga su ponovno razmatrane terapeutske mogućnosti polimiksina i došlo je do ponovne uporabe ovih polipeptidnih antibiotika, odnosno polimiksina B i kolistina. U veterinarskoj medicini, kolistin je korišten za liječenje infekcija prouzročenih Enterobacteriaceae na farmama životinja. Danas je jedan od pet najčešće korištenih antibiotika u životinja namijenjenih za hranu unutar EU. Potrošnja kolistina u životinja daleko je veća od one u ljudi budući da se primjenjuje uglavnom za liječenje stada ili jata. Njegova uporaba se povećala posljednjih godina djelomično i zbog razvoja otpornosti na druge klase antibiotika. Europska agencija za lijekove (EMA) je izradila izvješće o prodaji antimikrobnih veterinarskih lijekova za 30 zemalja EU za razdoblje 2010.- 2015. U R. Hrvatskoj kolistin se koristi u formi kolistin sulfata u liječenju crijevnih infekcija prouzročenih neinvazivnom E. coli osjetljivom na kolistin u teladi, svinja, kokoši i purana, dok se više ne koristi u liječenju crijevnih infekcija ždrjebadi niti za liječenje infekcija prouzročenim salmonelama. U 2015. u R. Hrvatskoj je zabilježena prodaja od 2,5% polimiksina u ukupnoj prodaji veterinarskih lijekova, dok je na primjer u Španjolskoj bila 8,7% ukupne prodaje. EMA je preporučila da se lijekovi koji sadrže kolistin trebaju koristiti samo za tretman drugog reda u životinja te da bi se njihova prodaja trebala svesti na minimum u svim državama članicama EU kako bi se smanjio rizik od antimikrobne rezistencije. Također, u veterinarskoj medicini nije dopuštena primjena kombinacije kolistina s drugim farmakološki djelatnim tvarima. S obzirom na postojeće podatke vjeruje se da je kolistin antibakterijski terapeutik “posljednje obrane” u 21. stoljeću protiv Gram-negativnih patogena rezistentnih na više lijekova.Colistin is a polymyxin, a bactericidal antibiotic polypeptide with wide spectrum action against Gram-negative bacteria including Acinetobacter spp., Pseudomonas aeruginosa, Klebsiella spp. and Enterobacter spp. Its antibacterial activity was already recognized in the 1940s. However, due to significant side effects such as nephrotoxicity and neurotoxicity, polymyxins have seldom been used since the 1970s, when less toxic aminoglycosides and other antibiotics were made available on the market. Colistin was only used intravenously in the treatment of patients with lung infections with Gram- negative bacteria or cystic fibrosis. The mechanism of antimicrobial activity of colistin is that it binds to the lipopolysaccharides and phospholipids in the outer cell membrane of Gram-negative bacteria. They competitively expel bivalent cations from the phosphate groups of membrane lipids, leading to external cell membrane disorders, leakage of intracellular content, and bacterial death. Two forms of colistin, colistin sulphate and colistimethate sodium are commercially available. With regard to colistin dosage, dosing and efficacy dosing optimization studies are still required The occurrence of severe infections with Gram-negative bacteria, such as Pseudomonas aeruginosa and Acinetobacter baumannii, is a major problem worldwide, as they are resistant to most classes of commercially available antibiotics, and new antibiotics with antibacterial activity against these bacteria are lacking. Therefore, the therapeutic possibilities of polymyxins were re-examined and the reuse of these polypeptide antibiotics, i.e. polymyxin B and colistin, considered. In veterinary medicine, colistin has been used for the treatment of infections caused by Enterobacteriaceae in farm animals. Today, it is one of the five most commonly used antibiotics in food-producing animals within the EU. Consumption of colistin in animals is far greater than in humans. Its use has increased in recent years partly due to the development of resistance to other classes of antibiotics. The European Medicines Agency (EMA) presented a report on the sale of antimicrobial veterinary drugs using data from 30 EU countries for the period 2010–2015. In Croatia, colistin is used in the form of colistin sulphate in the treatment of intestinal infections caused by non-invasive, colistin-sensitive E. coli in cattle, pigs, hens and sheep. In 2015, sales of polymyxins accounted for 2.5% of the total sales of veterinary drugs in Croatia, while in Spain, sales accounted for 8.7% of the total sales. The EMA has recommended that colistin-containing medicines should only be used for second-line treatment in animals and that their sales should be minimized in all EU Member States in order to reduce the risk of antimicrobial resistance. Given the existing data, it is believed that colistin is a “last defence” antibacterial therapeutic against Gram-negative pathogens resistant to multiple drugs in the 21st century

Spektrofotometrijsko određivanje glavnih polifenolnih skupina u uzorcima propolisa iz različitih područja Republike Hrvatske

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.U ovom su radu provedene spektrofotometrijske metode za brzu analizu uzoraka propolisa iz različitih područja Republike Hrvatske. S ciljem određivanja glavnih skupina bioaktivnih spojeva u propolisu, optimizirane i validirane su tri spektrofotometrijske metode: Folin-Ciocalteuova metoda za određivanje ukupnih fenola i dvije različite metode za sadržaj ukupnih flavonoida - metoda kompleksiranja s aluminijevim kloridom za ukupne flavone/ flavonole te 2,4-DNPH metoda za ukupne flavanone/dihidroflavonole. Validacijom su određeni sljedeći parametri: linearnost, osjetljivost, raspon, točnost, granica detekcije, granica kvantifikacije, preciznost i robusnost. Za provedbu validacijskog protokola korišteni su standardi polifenola, uključujući galnu kiselinu (GA), pinocembrin (PC), galangin (GN), kvercetin (QC) te smjesu pinocembrina i galangina (PC-GN). Validiranim metodama analizirano je šest uzoraka sirovog propolisa iz kontinentalne i jadranske regije Hrvatske. Primijećena je velika raznolikost u sadržaju ukupnih fenola, ukupnih flavona/flavonola te ukupnih flavanona/dihidroflavonola među uzorcima iz dviju hrvatskih regija. Iako metode ekstrakcije (ultrazvučna ekstrakcija ili ekstrakcija potpomognuta mikrovalovima) nisu pokazale statistički znakovitu razliku u obliku prinosa ekstrakcije (P > 0,05), te gledajući koncentracije polifenola cjelokupno, ultrazvučna je ekstrakcija bila selektivnija. Nadalje, velik utjecaj na konačne koncentracije ukupnih fenola i ukupnih flavona/flavonola imao je standard korišten za izradu kalibracijske krivulje. Dobivena je dobra linearnost, točnost, ponovljivost, srednja preciznost, granica detekcije i granica određivanja u svim trima spektrofotometrijskim metodama te su ovi rezultati temelj za daljnja istraživanja pojedinačnih polifenolnih spojeva u uzorcima propolisa

Utjecaj imunomodulatora virusnog ili bakterijskog podrijetla i cjepiva protiv bolesti Aujeszkoga na udio B limfocita periferne krvi na tovljenu prasad

The consequences of infection by Suid herpesvirus type 1 (SuHV-1) that causes Aujeszky’s disease (AD) are well studied, however, the effects of immunomodulators (IMs) of microbial origin (viral and bacterial) when administered solely or in combination with the attenuated SuHV-1 vaccine are less known. The effects of parenteral administration of IMs, inactivated Parapoxvirus ovis (P. ovis) or a combination of inactivated Propionibacterium granulosum (P. granulosum) and detoxified Escherichia coli lipopolysaccharide (LPS) and attenuated SuHV-1, strain Bartha, on the proportion of peripheral blood CD3- CD21+ B cells were analysed in 30 crossbred, 3-month old pigs using flow cytometry (FCM). Specific antibodies for gE and gB of SuHV- 1 were detected using the enzyme-linked immunosorbent assay (ELISA). Data were compared among six experimental groups: (1) pigs that separately received the vaccine, (2) IM of bacterial origin, (3) IM of viral origin, (4) simultaneous administration of the vaccine and bacterial IM, (5) simultaneous administration of the vaccine and viral IM, and (6) the control group of untreated pigs. Comparison of B cell proportions and the detection of specific antibodies in blood samples of vaccinated pigs on Day 11 of the experiment showed a transient decrease in B cell contents, though this could not be assumed to be related since the control group showed a decrease in B cell proportion on the same day. The results showed that the use of IMs alone or in combination with the attenuated SuHV-1 vaccine did not have a significant impact on the proportion of peripheral blood B cells in growing pigs.Posljedice infekcije herpes virusom-1 svinja (SuHV-1) uzročnikom bolesti Aujeszkoga (BA) dobro su istražene, ali, manje je poznat učinak imunomodulatora (IM) mikrobnog podrijetla (virusnog ili bakterijskog) primijenjenih u kombinaciji s atenuiranim cjepivom protiv BA. Učinak parenteralne primjene IM-a, inaktiviranog virusa Parapoxvirus ovis ili kombinacije inaktivirane bakterije Propionibacterium granulosum i lipopolisaharida bakterije Escherichia coli i cjepiva koje sadržava atenuirani virus BA (SuHV-1, soj Bartha) na udio CD3-CD21+ B limfocita periferne krvi analiziran je protočnom citometri¬jom u krvi 30 komercijalnih križanaca svinja u dobi od tri mjeseca bez protutijela za virus BA. Prisustvo je protutijela za glikoproteine B (gB) i E (gE) virusa BA provjereno imunoenzimnim testom (engl. Enzyme Linked Immunosorbant Assay, ELISA). Dobiveni su rezultati uspoređeni između 6 pokusnih skupina tj. između svinja koje su primile: cjepivo (1. skupina), IM bakterijskog podrijetla (2. skupina), IM virusnog podrijetla (3. skupina), svinja koje su istovremeno primile cjepivo i IM bakterijskog podrijetla (4. skupina) ili cjepivo i IM viruisnog podrijetla (5. skupina) te netretirane, kontrolne skupine svinja (6. skupina). Usporedba udjela B limfocita u uzorcima krvi cijepljenih svinja 11. dan pokusa pokazali su prolazno smanjenje sred¬nje vrijednosti udjela B limfocita za koje se nije moglo pretpostaviti da je povezano s pojavom specifičnih protutijela, jer je smanjenje srednje vrijednosti udjela B limfocita bilo i u kontrolnoj skupini istoga dana. Naši su rezultati pokazali da primjena samo IM-a, kao i njihovih kombinacija s atenuiranim cjepivom protiv BA, nisu imali značajan utjecaj na udio B limfocita periferne krvi u tovljene prasadi