Search

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.

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.

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., In veterinary medicine vaccines play a key role in control of animal health. With the advancement of science, not only whole microorganisms (inactivated or attenuated) are active substances in vaccines, but also vaccines containing parts of microorganisms (recombinant proteins or synthetic peptides) obtained by various advanced technologies are being developed. Thus, a higher degree of safety of the vaccine is ensured and better immunogenicity is achieved by the addition of an adjuvant. Adjuvants in the form of aluminum salts which have been in use for decades and are the most common in veterinary vaccines but adjuvants in the form of emulsions, including mineral oils, are also important. The negative side of use of mineral oils may be the occurrence of a local reaction at the site of application. Such changes may affect the slaughter value in fattening animals and the grade in competitions or exhibitions in show animals. Because of these local reactions, which can cause difficult applications due to viscosity, mineral oils are not often used as an adjuvant in vaccines intended for humans and pets, but are mainly used for vaccination of farm animals. The development of new technologies in production seeks to reduce the negative impact on animals, as well as possible reactions in people who use such vaccines, therefore, the package leaflet containing the mineral oil must contain a warning of the risks that may arise during administration and as well as instructions on how to act in the event of an accident.

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 phenotype of 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.

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.

Dana 1. siječnja 2020. godine u Republici Hrvatskoj na snagu je stupila Konvencija Vijeća Europe o krivotvorenju farmaceutskih proizvoda i sličnim kažnjivim djelima koja uključuju prijetnje javnom zdravlju (engl. Council of Europe Convention on counterfeiting of medicinal products and similar crimes involving threats to public health). Konvencija je potpisana 3. rujna 2015. godine, a Vlada Republike Hrvatske ju je ratificirala 20. rujna 2019. Godine. Konvencija se odnosi i na medicinske proizvode koji se koriste i u veterinarskoj medicini. Važnost borbe protiv krivotvorenih lijekova prepoznata je u Hrvatskoj već niz godina, a Hrvatska je i jedna od brojnih sudionica Interpolovih akcija protiv krivotvorenih lijekova. Prihvaćanjem ove Konvencije otvara se mogućnost učinkovitijeg nadzora nad krivotvorenim lijekovima za primjenu u humanoj i veterinarskoj medicini te bolja međunarodna suradnja

This paper presents an contemporary approach for development and validation of Loss given default (LGD) in accordance with the Basel Accords standards. The modeling data set has been based on data on recoveries of outstanding debts from corporate entities in Republic of Serbia that defaulted. The aim of the paper is to develop a LGD model capable of confirming the validity of historically observed LGD estimates on the sample of corporate entities that defaulted. The modelling approach in this research is based on average LGD without time or exposure weightening. The probability density function of realized empirical LGDs has been created by beta distribution usage. The validation process on proposed LGD model has been performed by throughout testing of: cumulative LGD accuracy ratio, mean square error calculation and regression analysis. On the basis of obtained results, the possibilities of application of the developed LGD model are proposed and discussed.

Prema definiciji Svjetske zdravstvene organizacije krivotvoreni lijek je onaj koji je u svrhu obmane lažno označen s obzirom na identitet i/ili podrijetlo. Promet i primjena takvih lijekova postali su globalni problem koji negativno utječe na zdravlje, zdravstvene sustave i ekonomiju, i zbog toga su u suzbijanje uključene brojne međunarodne ustanove. Krivotvorine mogu biti proizvodi s ispravnim, neispravnim ili čak nedozvoljenim sastojcima, bez ili sa smanjenom količinom djelatnih tvari te proizvodi u neregularnom pakiranju. Osim originalnih, i generički lijekovi mogu biti predmet krivotvorenja. U razvijenim zemljama najviše se krivotvore lijekovi za poboljšanje kvalitete života ljudi ili oni za liječenje po život opasnih bolesti, a u veterinarskoj medicini lijekovi za kućne ljubimce dok se u nerazvijenim zemljama krivotvore gotovo sve vrste lijekova. Proizvodnja, stavljanje u promet, oglašavanje, posjedovanje i primjena krivotvorenih veterinarsko-medicinskih proizvoda su zabranjeni, a svako saznanje o krivotvorinama u prometu obvezno je prijaviti nadležnim tijelima.

Leptospiroza je spirohetama uzrokovana zoonoza, rasprostranjena diljem svijeta, od koje obolijevaju mnoge domaće i divlje vrste životinja te čovjek. Rad prikazuje seroprevalenciju leptospiroze u uzgojima ovaca i koza na području Republike Hrvatske u razdoblju od 2008. do 2010. godine. Tijekom navedenog razdoblja u Laboratoriju za bakterijske zoonoze i molekularnu dijagnostiku bakterijskih bolesti Hrvatskog veterinarskog instituta pretraženo je ukupno 36100 krvi ovnova i jarčeva. Svi uzorci testirani su metodom mikroskopske aglutinacije (MAT) na prisutnost protutijela za jedanaest različitih seroloških varijanti (sv.) Leptospira interrogans. Pozitivne reakcije zabilježene su u 220 (0, 61%) uzoraka, a dokazana su protutijela za 7 serovara. Od ukupnog broja pozitivnih uzoraka, najveći dio dostavljen je iz 3 županije, 40 (18, 18%) iz Osječko- baranjske, 35 (15, 91%) iz Virovitičko-podravske i 34 (15, 45%) iz Bjelovarsko-bilogorske županije. Najčešće su dokazana protutijela za sv. Pomona (32, 17%), Grippotyphosa (26, 96%), Sejroe (23, 04%) i Australis (13, 04%). Najveći broj uzoraka pozitivnih na sv. Pomona zabilježen je u Virovitičko-podravskoj županiji (29, 73%), sv. Grippothyposa u Osječko-baranjskoj (35, 48%), a na sv. Sejroe u Bjelovarsko-bilogorskoj županiji (37, 74%). Serovar Australis utvrđen je u najvećem broju županija. Usprkos nižoj seroprevalenciji leptospiroze u malih preživača, ne može se zanemariti njihov značaj u održanju i širenju bolesti između divljih i domaćih životinja te ljudi na nekom području. S obzirom na to da rezultati ovog istraživanja ukazuju na postojanje prirodnih žarišta leptospiroze u kontinentalnoj regiji Republike Hrvatske, potrebno je nastaviti serološki nadzor ove zoonoze u ovaca i koza.

The aim of this study was to determine the presence of enterotoxin genes in coagulase- negative staphylococci from traditional Croatian fermented sausages. Total of 39 isolates were collected from dry sausage “kulen” and Slavonian homemade sausage as presumptive CNS, and subjected to PCR identification to the genus level. All isolates belonged to the Staphylococcus genus. Strains were tested for the presence of 13 enterotoxin genes ; seA, seB, seC, seG, seI, tsst1, seD, seE, seH, seJ, seM, seN and seO by PCR method. All strains were negative to all enterotoxin genes. Observed results indicate the absence of risk related to enterotoxogenic potential of coagulase-negative staphylococci as indigenous microbiota from autochthonous Croatian traditional sausages.

In accordance with the annual orders issued by Ministry of Agriculture for years 2008, 2009 and 2010, all bucks and rams used for natural breeding and production of semen for artificial insemination have been serologically tested for leptospirosis twice each year. During that period total of 36100 blood samples were examined in the Laboratory for bacterial zoonoses and molecular diagnosis of bacterial diseases of Croatian Veterinary Institute. 16218 (44, 93%) of above mentioned samples were collected in the continental region of Croatia, and the other 19882 (55, 07%) in the coastal region. All samples were examined by microscopic agglutination test (MAT) for the presence of 11 leptospiral antigens. An initial sera dilution of 1:100 was considered as a cut-off point for a positive reaction. Positive reaction was found in 220 (0, 61%) blood samples, 212 (96, 36%) of which reacted positively with only one serovar, 6 (2, 73%) with two serovars and 2 (0, 91%) with three serovars. 207 (94, 09%) positive bloods samples came from the continental counties and the other 13 (5, 91%) from the coastal region. The majority of positive samples from continental region came from three counties (Osijek-Baranja 40 (19, 32%) ; Virovitica-Podravina 35 (16, 91%) and Bjelovar-Bilogora 34 (16, 43%)). The most frequently recorded serovars in this region were pomona (33, 18%), grippotyphosa (27, 19%), sejroe (22, 12%) and australis (13, 36%). Highest incidence of sv. pomona was found in Virovitica-Podravina county (30, 56%). Sv. grippotyphosa was the prevalent serovar in Osijek-Baranja county (37, 29%) and sv. sejroe in Bjelovar-Bilogora county (41, 67%). Sv. australis was equally distributed throughout the region. Serovars icterohaemorrhagiae, hardjo and ballum were found at much lower frequencies. 5 serovars (australis, grippotyphosa, icterohaemorrhagiae, pomona and sejroe) appeared sporadically and equally distributed in the coastal region. 7 (53, 85%) out of 13 positive blood samples from the coastal region came from Split-Dalmatia county. The endemic nature of leptospirosis, as well as prevalence of different leptospiral serovars within animal population depend primarily on combination of environmental factors such as geographical and climate conditions, exposure to different animal species, variety of present Leptospira reservoirs and farming methods. All this must be taken into consideration when assessing this surveys results, according to which the highest number of positive samples was recorded in central and eastern part of Croatia.

Leptospirosis in swine is characterised by impaired fertility, neonatal deaths, abortions and weak piglets. According to annual orders issued by Ministry of Agriculture for years 2008, 2009 and 2010, all boars used for natural breeding and production of semen for artificial insemination must be serologically tested for leptospirosis twice each year. In addition, all cases of abortion in swine must be reported and blood of each animal serologically tested for leptospirosis. During 2008, 2009 and 2010 more than 20000 swine blood samples were tested for leptospirosis. All samples were tested by microscopic agglutination test (MAT) for the presence of 11 leptospiral antigens: serovar (sv.) Icterohaemorrhagiae, sv. Ballum, sv. Australis, sv. Pomona, sv. Grippotyphosa, sv. Sejroe, sv. Saxkoebing, sv. Tarassovi, sv. Canicola, sv. Bataviae and sv. Hardjo. Initial dilution of blood samples for MAT testing was 1:100. In 2008, 10501 blood samples were tested for leptospirosis and 990 (9, 43%) were positive with at least one serovar. In 2009, out of 6387 tested blood samples, 468 (7, 33%) were positive, while in 2010, 3966 blood samples were tested and 236 (5, 95%) were positive for leptospirosis. In 2008 Međimurje county had the highest prevalence of positive blood samples (25%). In Sisak-Moslavina there were 16, 47% positive samples, in Krapina-Zagorje 13, 51% and in Varaždin county 12, 5% positive samples. In 2009 the higest prevalence of positive blood samples was in Osijek-Baranja county (12, 27%), followed by Zagreb county (9, 33%), Brod-Posavina county (8, 91%) and Međimurje county (6, 73%). Like in 2008, in 2010 the highest percentage of positive blood samples was found in Međimurje county (10, 13%). Koprivnica-Križevci county had 9, 66%, Brod-Posavina county 8, 48% and Zagreb county 8, 39% positive blood samples. During monitored period only few blood samples from coastal counties in Croatia were tested for leptospirosis and none of them was positive. In 2008 and 2009 the most frequent serovar was sv. Australis (55, 71%/2008 and 39, 66%/2009) followed by serovars Pomona (17, 26%/2008 and 21, 55%/2009), Icterohaemorrhagiae (10, 70%/2008 and 17, 24%/2009), Sejroe (7, 49%/2008 and 8, 74%/2009) or Grippotyphosa (4, 42%/2008 and 8, 99%/2009) and Hardjo (2, 28%/2008 and 3, 08%/2009). Sv. Pomona was the most frequent serovar in 2010 (28, 9%) followed by serovars Icterohaemorrhagiae (23, 02%), Sejroe (18, 16%), Australis (10, 49%), Grippotyphosa (10, 23%) and Hardjo (7, 93%). In 2008, 2009 and 2010 serovars Canicola and Saxkoebing were sporadically found, while in 2008 there were also a few samples positive with serovars Bataviae, Tarassovi and Ballum. Regarding the seroprevalence, leptospirosis in swine is common disease in continental counties in Croatia. Although leptospirosis in swine is in decrease, the risk of spreading it to other animals and humans still exists thus causing substantial economic losses in pig farming and threat to human health.

Bovine leptospirosis occurs throughout the world, but it is most commonly in warm climates. It is economically significant disease that results in abortions, stillbirths and loss of milk production. Therefore, the implementation of surveillance and control measures is of particular importance for the suppression of this important zoonosis. According to annual orders issued by Ministry of Agriculture for years 2008, 2009 and 2010, all bulls used for natural breeding and production of semen for artificial insemination must be serologically tested for leptospirosis twice each year. In addition, all cases of abortion in cattle must be reported and blood of each animal serologically tested for leptospirosis. Also according croatian legislation, after treatment we carry out serological blood tests twice at intervals of 21 days after completion of treatment. During the year 2008. 3024 (485 positive) cattle blood samples were tested for leptospirosis, in 2009. 3150 (538 positive) blood samples and 5373 (475 positive) in 2010. All sera were tested by microscopic agglutination test (MAT) for the presence of 12 leptospiral antigens. As antigens for testing the sera were used serovars (sv.): sv. icterohaemorrhagiae – RGA, sv. ballum – Mus 127, sv. australis – Ballico, sv. pomona – Pomona, sv. grippotyphosa – Moskow V, sv. sejroe – M84, sv. saxkoebing – M24, sv. tarassovi – Perepelicin, sv. canicola – Hond Utrecht IV, sv. bataviae – Van Tienen, sv. hardjo – Hardjoprajitno and sv. hardjo – Hardjo-bovis. An initial sera dilution of 1:100 was considered as a cut-off point for a positive reaction. In 2008. the highest prevalence had Osijek-Baranja 126 (26%), Sisak-Moslavina 108 (22.3%), Vukovar-Srijem 71 (14.6%) and Varaždin 48 (9.9%) counties. In 2009. the highest prevalence had Sisak- Moslavina 236 (43.9%), Zagreb 92 (17.1%), Bjelovar-Bilogora 46 (8.6%) and Pozega-Slavonia 38 (7.1%) counties. In 2010. counties with the the highest prevalence were Pozega-Slavonia 154 (32.4%), Osijek-Baranja 80 (16.8%), Bjelovar-Bilogora 75 (15.8%) and Sisak-Moslavina 72 (15.2%). Positive reactions for leptospirosis during the study period were not found in the Dubrovačko-neretvanska, Lika-Senj, Primorje-Gorski Kotar and Zadar County. The most frequent serovars in cattle during the three years were sv. hardjo (22.32%), sv. sejroe (22.32%), sv. saxkoebing (21.93%) and sv. hardjobovis (21.38%). Results indicate that the cattle in the continental part of the Croatia due to leptospirosis natural foci (Lonjsko Polje i Kopački rit) and the joint holding cattle, pigs and horses in the same area are exposed to leptospira infection more than animals from the croatian coast. In order to control the spread of disease within the same breeding and on the other breedings it is necessary to continue to control the disease, treat breedings with the aim of reducing the carrier state, as well as to the availability, change management to reduce the possibility of re-infection.

Leptospiroza je zoonoza raširena po cijelom svijetu, a uzročnik je vrsta Leptospira interrogans. Svinje spadaju među najčešće domaćine leptospira tako da je leptospiroza svinja učestala bolest u svim zemljama s razvijenom svinjogojskom proizvodnjom. Svinjogojske farme idealne su za život i razmnožavanje glodavaca koji svojim izlučevinama kontaminiraju hranu i prostorije te tako podržavaju širenje leptospiroze. Leptospiroza svinja klinički se očituje pobačajima, prašenjem mrtvorođene i avitalne prasadi te neplodnošću. U Laboratorij za bakterijske zoonoze i molekularnu dijagnostiku bakterijskih bolesti je u 2011. godini radi pretrage na leptospirozu dostavljeno 2480 krvi svinja. Krvi su pretraživane metodom mikroskopske aglutinacije (MAT) na 11 antigena: serovar (sv.) icterohaemorrhagiae, sv. ballum, sv. australis, sv. pomona, sv. grippotyphosa, sv. sejroe, sv. saxkoebing, sv. tarassovi, sv. canicola, sv. bataviae i sv. hardjo. Početno razrjeđenje seruma za dijagnostiku metodom MAT bilo je 1:100. Od 2480 pretraženih krvi, 176 (7, 1%) je bilo pozitivno s najmanje jednim serovarom. Najveći udio pozitivnih krvi bio je u Međimurskoj županiji 17, 78% (od zaprimljenih 90 krvi, 16 je bilo pozitivno). Osječko-baranjska županija imala je 13, 83% pozitivnih krvi (95 od 687). Zatim slijede Virovitičko-podravska (11 od 95), Zagrebačka (23 od 205) i Krapinsko-zagorska županija (2 od 18) s oko 11% pozitivnih krvi. Iz Vukovarsko-srijemske županije zaprimljen je velik broj krvi (628) i samo 20 (3, 18%) je bilo pozitivno. U ostalim županijama postotak pozitivnih krvi je bio manji od 3%. Iz županija u obalnom dijelu Hrvatske (Istarske, Primorsko-goranske, Splitsko-dalmatinske i Šibensko-kninske) pristigao je malen broj krvi (34) i sve su bile negativne na leptospirozu. Najčešći serovar bio je sejroe (34, 02%), a nakon njega veliku učestalost imali su serovarovi saxkoebing (17, 89%), hardjo (16, 13%) i pomona (12, 61%). Učestalost serovara australis bila je 8, 50%, a učestalost serovarova icterohaemorrhagiae i grippotyphosa oko 5%. Sv. ballum, bataviae, canicola i tarassovi nisu utvrđeni ni jednom. Uzevši u obzir seroprevalenciju, leptospiroza svinja je učestala bolest u kontinentalnim dijelovima Hrvatske. Iako se broj svinja oboljelih od leptospiroze s godinama smanjuje, i dalje postoji rizik od širenja bolesti na druge životinje, ali i ljude. Tako leptospiroza u svinja ne samo da predstavlja ozbiljnu prijetnju zdravlju ljudi već uzrokuje i značajne ekonomske gubitke u uzgoju svinja.

Kontrola bruceloze svinja u Hrvatskoj propisana je godišnjom naredbom Ministarstva poljoprivrede, ribarstva i ruralnog razvoja, a prema njoj krv nerastova mora biti pretražena na brucelozu jednom godišnje, a obavezno prije početka korištenja za umjetno osjemenjivanje ili prirodni pripust. Također, svaki pobačaj mora biti prijavljen veterinaru. Od krmače koja je pobacila ovlašteni veterinar mora uzeti uzorke, te iste dostaviti u službeni laboratorij radi pretrage na brucelozu. Pretražuju se i sve novonabavljene rasplodne svinje prije uvođenja u uzgoj. Nakon utvrđenog pozitivnog serološkog nalaza svinje se isključuju iz uzgoja i prilikom klanja se uzimaju uzorci materijala radi bakteriološke pretrage na brucelozu. Tijekom 2011. godine pretraženi su organi 83 svinje iz 5 seoskih uzgoja i testesi jednog vepra. Brucela suis bv. 2 je izdvojena iz 25 (29, 8%) uzoraka dostavljenog materijala. Od 5 seoskih uzgoja infekcija Brucellom suis potvrđena je u 4 (80%) uzgoja, a uključujući i nalaz u divlje svinje infekcija je utvrđena u 5 županija. Serološka kontrola rasplodnih svinja i obavezna kontrola prilikom pobačaja osiguravaju zadovoljavajuću razinu osjetljivosti s obzirom na brzo otkrivanje infekcije u uzgoju.

Tij ekom prve godine programa certifi kacij e stada goveda slobodnih od tuberkuloze bakteriološki je u Laboratorij u za bakterij ske zoonoze i molekularnu dij agnostiku bakterij skih bolesti bio pretražen materij al 314 goveda iz 100 uzgoja koja su reagirala pozitivno na tuberkulin bilo monotestom ili komparativnom metodom. Mikobakterij e su izdvojene iz 129 (41, 1%) uzoraka. Vrste iz M. tuberculosis kompleksa (M. bovis, M. caprae i M. tuberculosis) su tij ekom 2010. godine izdvojene iz materij ala 117 goveda (90, 7%) iz 23 uzgoja (23%) u 7 županij a. M. caprae je utvrđen u 83 goveda (70, 9%) iz 9 uzgoja iz 4 županij e, M. bovis u 33 goveda (28, 2%) podrij etlom iz 14 uzgoja u 5 županij a, a M. tuberculosis u jednog goveda (0, 85%). Saprofi tske mikobakterij e izdvojene su iz 12 uzoraka goveda (10, 3%) dostavljenih iz 12 uzgoja (12%). Identifi cirane su sljedeće vrste mikobakterij a: M. terrae (4), M. gordonae (1), M. peregrinum (1), M. chitae (1), M. nonchromogenicum (1), M. parafortuitum (1), M. vaccae (1), M. thermoresistibile (1) i jedna neidentifi cirana vrsta (M. sp.) Mikobakterij e nisu izdvojene iz 185 (58, 9%) uzoraka materij ala goveda. Veliki broj uzgoja u kojima su izdvojene saprofi tske vrste mikobakterij a (12), visok postotak bakterioloških pretraga koje nisu završile s izdvajanjem Mycobacterium sp. (58, 9%) i 65% uzgoja s negativnom bakteriološkom pretragom ukazuje na visoku osjetljivost i nisku specifi čnost metode monotesta. Ovu činjenicu trebalo bi u budućnosti uzeti u obzir prilikom suzbij anja tuberkuloze u stadima u kojima nismo ustvrdili patognomonične promjenena liniji klanja i/ili laboratorijskim pretragama iz materijala izdvojili uzročnika tuberkuloze goveda.

This study evaluates the effects of total splenectomy and autologous spleen transplantation in a porcine model on complete red blood cell count, platelet count, percentage of reticulocytes and red blood cell morphology. Nineteen pigs observed in the research were randomly divided into three groups. The first group underwent sham surgery, the second group was splenectomized and the third group, after being splenectomized, had 20 % of autologous spleen tissue transplanted into the greater omentum. Blood was sampled prior to surgery and on the 1st, 5th, 12th and 26th days after surgery. Erythrocyte count, indices and morphology, hemoglobin, hematocrit and platelet count were assayed. Erythrocyte count was significantly decreased postoperatively in sham-operated pigs (day 5) and in autotransplanted pigs (days 5 and 12), while in splenectomized pigs a significant decrease of erythrocyte count was present on all postoperative days of the experiment. Hemoglobin values and hematocrit changed according to changes in red blood cell count during the whole period of the experiment. Erythrocyte indices of splenectomized pigs were significantly increased postoperatively (MCV on the 12th and 26th days, MCH and MCHC on day 5). Autotransplanted pigs had significantly lower MCV (days 1, 5 and 12) and MCH (days 1 and 12), but significantly higher MCHC (5th day). The platelet count was significantly increased (12th day) in splenectomized pigs. The percentage of reticulocytes in the blood, including polychromasia, was significantly increased in all three groups of pigs on all postoperative days of the experiment. Morphological abnormalities of erythrocytes were found on the blood smears of all investigated groups of pigs, with prevalence on the smears of splenectomized pigs. The anemia and retikulocytosis found in blood samples of all investigated groups of pigs may have been physiological results showing the classical postoperative organism reaction to blood loss and surgical trauma. The more frequent appearance of variations in red blood cell morphology, such as the appearance of leptocytes, codocytes and Howell-Jolly bodies in splenectomized pigs when compared with the other two experimental groups, suggests that this was a change specific for splenectomy.

In 2010, Croatia initiated the proceedure of verification of officially tuberculosis- free herds. Cattle older than 6 weeks were tested with tuberculin skin test (monotest) twice in period of at least 6 months between testings. Cattle with suspicious reaction on monotest were re-tested using comparative method (bovine and avian tuberculin). All skin tests were performed according to the European Council Directive 64/432/EEC and Croatian ordinance on animal health requirements applicable to trade in bovine animals and swine from year 2008. Cattle with positive tuberculin skin test reactions (monotest or comparative method) were removed from breeding. Tissue and organ samples were taken during slaughter for bacteriological isolation of causative agents of bovine tuberculosis– Mycobacterium (M.) sp. Isolates were identified by microscopic examination of suspicious colonies, as well as molecular examintion for the presence of 16SrRNA. GenoType MTBC Kit (Hain, Germany) was used for identification of species belonging to the M. tuberculosis complex. Additional identification of nontuberculous mycobacteria (NTM) isolates was performed by classical bacteriological methods. Total of 314 cattle samples from from 100 breedings were examined. M. tuberculosis complex species (M. bovis, M. caprae and M. tuberculosis) were isolated from 117 cattle samples (37.3%) coming from 23 breedings (23%) in 7 counties. NTM were isolated from samples collected from 12 cattle (3.8%) belonging to 12 flocks (12%) and were: M. terrae (4), M. gordonae (1), M. peregrinum (1), M. chitae (1), M. nonchromogenicum (1), M. parafortuitum (1), M. vaccae (1), M. thermoresistibile (1) and one unindentified species (M. sp.). Mycobacteria were not isolated in 185 (58.9%) bovine samples taken from animals with positive tuberculin reaction. Bacterial infection with any kind of mycobacteria was not identified in 64 breedings (64%). Breedings in which identified causative agent of bovine tuberculosis belonged to the M. tuberculosis complex were free of NTM. Infection of cattle with numerous NTM could partially be the reason for lowered tuberculin skin test specificity and should be concidered in planing of programms like verification of cattle herds officially free from tuberculosis.

Many hematological changes have been noted in the peripheral blood following removal of the spleen, but there are diverse reports of certain hematological parameters in various species. When total splenectomy is unavoidable, spleen autotransplantation can be performed in order to restore the function of the intact spleen, however, there is a still controversy about effectiveness of transplanted splenic tissue. The study was conducted on nineteen Landras piglets aged three months. Piglets were randomly divided in to three groups: sham-operation (n=6), total splenectomy (n=6), and splenic autotransplantation (n=7) with spleen chips autotransplanted into the greater omentum. The blood samples were taken just before the surgery, and on 1st, 5th, 12th, 26th and 40th postoperative day. EDTA blood samples were used to determine complete blood count using hematological counter SERONO – 9120 Baker System. White blood cell differentiation and reticulocyte count was performed microscopically on blood smears stained with May-Grünwald-Giemsa method, and methylene blue vital dye. Postoperative erythrocyte, hemoglobin and hematocrit depressions were found in all groups up to the 12th day of experiment. After that, erythrocyte parameters recovery was noted only in the control group. Reticulocyte number was increased during whole experiment in the splenectomized and autotransplanted group ranged from 1.5 to 10 times the preprocedure level, subsequent with sporadic finding of circulating normoblasts, and appearance of Howell-Yolly bodies. In control piglets, marked reticulocytosis was diminished at 40th postoperative day. Platelet number ranged between reference values in all investigated piglets. Following the operation, all groups showed leukocytosis with an increase of the absolute neutrophil number and initial decrease of the lymphocyte number which was followed by the later recovery. Noted leukocytosis was accompanied with increased band neutrophil number up to 12th postoperative day. At the 12th postoperative day reactive reactive lymphocytes were frequently found in the autotransplantated group. These results are widening the knowledge about hematological effects of splenectomy and splenic implants.

Istraživanje je provedeno na dvadeset svinja pasmine križani landras u dobi oko tri mjeseca. Svinje su podijeljene u tri skupine. Svinjama iz prve (kontrolne) skupine učinjena je lažna operacija, drugoj skupini svinja kirurški je odstranjena slezena, a trećoj skupini svinja nakon izvršene splenektomije u trbušnu šupljinu vraćeno je 20% tkiva odstranjene slezene. Krv je svinjama izvađena neposredno prije operativnih zahvata te prvog, petog, dvanaestog i dvadeset i šestog dana postoperativno. U uzorcima krvi je određen ukupan broj eritrocita, koncentracija hemoglobina, hematokrit, eritrocitne konstante, te broj retikulocita i trombocita. Također su promatrane i zabilježene promjene morfologije stanica krvi te prisutnost razvojnih oblika. Anemiju i retikulocitozu kojima su na operativni zahvat reagirale sve tri skupine svinja smatramo posljedicama obrambene reakcije organizma na operativne zahvate i gubitak krvi. Zabilježene su brojne morfološke promjene crvenih krvnih stanica u sve tri skupine svinja, ali njihov najučestaliji nalaz u splenektomiranih svinja smatramo obilježjem specifičnim za splenektomiju. S obzirom na učestaliju pojavu patoloških inkluzija te abnormalnih morfoloških oblika eritrocita na razmascima periferne krvi svinja s autotransplantiranim tkivom slezene, nego na razmascima kontrolne skupine, smatramo da je autotransplantat bio nefunkcionalan, odnosno da je količina transplantiranog tkiva od 20% ukupne težine slezene, bila premala da bi se očuvala njena filtracijska sposobnost.

Vector borne diseases are classical emerging infectious diseases in human and animal populations. Ticks represent perfect vectors for number of bacteria, parasites and viruses. The genera Anaplasma, Ehrlichia and recently specified cluster ‘Candidatus Neoehrlichia’ comprise all bacteria from family Anaplasmataceae, transmitted by ixodid ticks to mammalian hosts causing infection in humans and animals. ‘Candidatus Neoehrlichia mikurensis’ is one of emrgening vector borne zoonosis recently recognized as human pathogen. First case of human infection was reported from Sweden in 2010 and up to now six human infections were described in Czech Republic, Germany and Switzerland. In 2011 first case of septicaemia in two dogs was reported indicating ability of Neoehrlichia to infect various mammalian species, while different rodent species may act as reservoir host. The aim of this study was to identify species from family Anaplasmataceae from different species of wild animals (851). The presence of selected pathogens from wild cervids, wild boars, small rodents, muflons, chamois, martens, bears, badgers, wolves, jackals and foxes were determined by performing PCR on spleen samples and subsequent sequencing of fragment of 16S rRNA gene. Sequence analysis revealed presence of N. mikurensis, two strains of Anaplasma phagocytophilum, Anaplasma centrale, A. bovis and Ehrlichia sp. Small rodents were infected mainly with N. mikurensis together with A. phagocytophilum. Neoehrlichia mikurensis was single species detected in wild boars, bears and badgers, which represent first finding in these mammalian species. In chamois all species have been found except A. bovis. Wild cervids were harbouring both strains of A. phagocytophilum, while muflons were infected with A. centrale, A. phagocytophilum, A. bovis and N. mikurensis. Martens and jackals were free from pathogens while sequencing failed in wolf samples. Foxes were infected with both strains of A. phagocytophilum and interestingly in single animal A. bovis was confirmed. Two isolates Ehrlichia sp. from roe deer and chamois were identical to Ehrlichia sp. from Sika deer from Japan. Data obtained from this study clearly present presence of two zoonotic pathogens: A. phagocytophilum, causative agent of human granulocytic anaplasmosis (HGA) and newly recognized pathogen N. mikurensis in various species. These results also imply importance of wildlife as potential reservoir for ticks and potential transmission to humans.