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Bovine mastitis is the main cause of economic losses in dairy industry and a food safety concern due to a widespread antimicrobials use. The greatest risk lays in development of antibiotic resistant bacteria, but it also presents an obstacle for organic milk production. Propolis is a resinous mixture that honeybees collect from trees and various plants. Due to its numerous biomedical effects, it is studied as an alternative for antibiotics, with applicability in countries with high dairy production such as India.

A three-year-old male western black-tailed rattlesnake (Crotalus molossus molossus), which was refusing food and losing weight was presented with a swelling on the right side of the head below the eye. An abscess was suspected and treatment with subcutaneous enrofloxacin was started immediately. After identification of the causative agents as Pseudomonas aeruginosa and Stenotrophomonas maltophilia, and following susceptibility testing, enrofloxacin treatment was replaced with marbofloxacin, intralesional gentamicin/betamethasone treatment and gentamicin administered subcutaneously at an increased terrarium temperature of 35 °C. Seven days later, the formed pus plug was debrided and a combined marbofloxacin/gentamicin/betamethasone treatment was continued for an additional seven days. The swelling disappeared. Marbofloxacin was continued for ten more days, after which time microbiology tests were negative for S. maltophilia and P. aeruginosa. The animal began to eat and gain body weight. To our knowledge, this is the first report of an abscess treatment in a rattlesnake and the first to demonstrate the effective treatment of a S. maltophilia-induced infection with a combination of marbofloxacin and gentamicin.

Staphylococcus pseudintermedius is part of the normal flora of dogs but is also commonly found as an opportunistic pathogen. Antimicrobial resistance in S. pseudintermedius is changing over time and is generally rising steadily for those antimicrobials that are frequently used. In this work, the susceptibility of 106 canine and feline isolates of S. pseudintermedius was determined by the disk-diffusion method. Isolates were collected from various body sites of diseased dogs and cats during a six-month period in 2011. Most isolates were susceptible to amoxycillin/clavulanic acid, cephalexin, enrofloxacin and oxacillin (92.5%), followed by trimethoprimsulfamethoxazole (90.6%) and gentamicin (82.1%). Lower susceptibility was found for minocycline (67.0%), erythromycin, clindamycin and tetracycline (62.3%) and kanamycin (58.5%). Only 21.7% of the isolates were susceptible to ampicillin. Susceptibilities of isolates were compared from different isolation sites and from different temporal origins. The possible association of resistances to different antimicrobial agents is discussed.

Staphylococcus pseudintermedius is the predominant coagulase-positive species in the normal flora of dogs and cats. It can be isolated from the nares, mouth, anus, groin and forehead of healthy dogs and cats. S. pseudintermedius is an opportunistic pathogen most frequently encountered in canine and feline skin and ear infections. Methicillin-resistant S. pseudintermedius (MRSP) emerged in Brazil in the late nineties. Today, two different clones dominate in the population of dogs and cats. Dominant European clone ST71 appeared in Germany in 2005 and has rapidly spread around the world, while lineage ST68 dominates in North America. Both clones are multiresistant and present one of the biggest problems of antimicrobial resistance in the veterinary medicine. Besides all beta-lactam antimicrobials, they are typically resistant to aminoglycosides, fluoroquinolones, macrolides, lincosamides, trimethoprim-sulfamethoxazol and in many cases to tetracycline and chloramphenicol. The treatment of MRSP infections is a new challenge in veterinary medicine because of the very limited therapeutic options. The multidrug-resistance pattern results in a potential pressure for veterinarians to use antimicrobials licensed in human medicine, such as vancomycin, mupirocin and rifampicin. This opens ethical questions because of the possible emergence of resistance to these antimicrobials. Although the zoonotic potential is much lower than for MRSA, veterinarians are at a higher risk for becoming colonized and should be aware of the zoonotic risk.

Prikazano je nastavno i znanstveno djelovanje pokojnog redovitog profesora Tome Naglića

A RAPD technique was employed for molecular typing of Mycoplasma bovis strains isolated from bovine milk, nasal discharge and lungs. In addition, for the first time strains caused diverse clinical signs: pneumonia, arthritis and mastitis were compared. Also, strains isolated from Croatian cattle were compared to strains from Northern Ireland to see if there is any genetic relationship between them. 150 strains including the type strain PG45 were analyzed. The majority of Croatian strains were placed in a single group (A). Strains isolated from mastitic milk, pneumonic lungs and nasal cavities from the same farm sometimes had identical but sometimes markedly different patterns. A small portion of Croatian strains from a single herd, mainly from the years 2007-2008, clustered within the group C of Irish strains, and were more closely related to them than to other Croatian strains, possibly because of importation of animals from Western Europe. Irish strains were divided in two different groups, B and C, with isolates from the early nineties in group B, and from the years 2005 to 2010 in group C. Although no obvious relationship between geographical and/or temporal origin of strains and RAPD profiles was observed, in our study the majority of strains from different years of isolation were clearly distinguishable from each other. However, strains with identical RAPD patterns were found in different geographical locations and from distant temporal origins so for better understanding of M. bovis epidemiology further testing on greater number of strains from various locations is needed.

U predavanju su obrađeni pojmovi antimikrobni lijekovi-antibiotici, uporaba, odgovornosti veterinara, propisi, i klinički slučajevi u kojima se prikazuje rezistencija kao posljedica nerazumne uporabe antibiotika.

Tijekom šestomjesečnog razdoblja 284 izolata bakterija izdvojena su iz različitih kliničkih uzoraka podrijetlom od pasa i mačaka. Osjetljivost sojeva na cefovecin određena je disk-difuzijskom metodom. Od 284 izolata 202 bila su gram-pozitivne, a 82 gram-negativne bakterije. Najzastupljenija je bila vrsta Staphylococcuspseudintermedius (152), a na drugom mjestu streptokoki (50). Najčešće gram-negativne vrste bile su Pseudomonas aeruginosa (26), Proteus spp. (25) i E. coli (22). Najviša stopa rezistencije među gram-pozitivnim bakterijama utvrđena je u nehemolitičkih streptokoka (50%) i u vrste Staphylococcus pseudintermedius (7, 2%). Beta-hemolitički streptokoki bili su 100% osjetljivi na cefovecin. Kao što je bilo očekivano, cefovecin nije bio djelotvoran na vrste P. aeruginosa. Vrlo visok postotak rezistentnih sojeva utvrđen je i kod vrsta E. coli(40, 9%) i Proteus spp. (8%). Svi izolati P. multocida bili su osjetljivi na cefovecin. Ovi rezultati u skladu su s onima dobivenim određivanjem osjetljivosti beta-hemolitičkih streptokoka i sojeva P. multocida podrijetlom iz SAD-a i Europske Unije mikrodilucijskom metodom. To se ne može reći za izolate S. pseudintermedius, E. coli i Proteus spp., u kojih je utvrđen znatno viši postotak otpornih sojeva.

Enzootska pneumonija proširena je u cijelom svijetu te nanosi velike gospodarske gubitke u intenzivnom svinjogojstvu. Vrsta M. hyopneumoniae patogena je isključivo za svinje, a prenosi se uglavnom putem kliconoša ili zrakom. U uzgojima gdje je enzootski prisutna, bolest je kroničnog tijeka, a stalna prisutnost grčevitog, neproduktivnog kašlja u uzgoju, slabija konverzija hrane i posljedično niži prirast često su jedini znakovi bolesti. Dodatne probleme uzrokuju vrlo česte sekundarne infekcije bakterijama i virusima. Akutni tijek bolesti javlja se u svinja koje su po prvi put došli u doticaj s vrstom M. hyopneumoniae, a vidljivi znakovi bolesti su ili vrlo blage promjene na dišnom sustavu pa sve do jake akutne upale pluća praćene visokim mortalitetom. Jasno ograničena područja konsolidiranog plućnog tkiva ljubičaste do sive boje, najčešće raspoređena na apikalnim i kranijalnim režnjevima, karakteristični su patoanatomski nalaz za enzootsku pneumoniju svinja. U dijagnosticiranju enzootske pneumonije kao najpouzdanije metode pokazale su se ELISA, lančana reakcija polimerazom (PCR) i RT-PCR (lančana reakcija polimerazom u stvarnom vremenu). Antimikrobni pripravci koji se danas najčešće koriste u suzbijanju i liječenju ove bolesti su: oksitetraciklin, klortetraciklin, florfenikol, makrolidni antibiotici, pleuromutilini iflorokinoloni. Nakon provedenog se liječenja M. hyopneumoniae još uvijek može dokazati u plućima, a recidive su vrlo česte. Mjere koje se provode u svrhu suzbijanja i iskorjenjivanja bolesti su: nadzor prometom svinjama, održavanje propisanih zoohigijenskih uvjeta, cijepljenje i preventivna medikacija.

Active immunization against porcine postweaning colidiarrhea (CD) and/or colienterotoxemia (CE) caused by F4+ and/or F18+ enterotoxigenic Escherichia coli (ETEC) is still an unsolved problem. The intestinal microfold (M) cells play role in entry/invasion of intraluminal pathogens (such as ETEC strains), in antigen sampling, and in facilitating induction of immunity to gut infections. Just as ETEC strains can exploit M cells as the portal of entry for infections such as CD and/or CE, their high transcytotic ability make them an attractive target for mucosally delivered vaccines, adjuvants and therapeutics. The objective of our study was to evaluate the effects of levamisole-adjuvanted vaccine candidate F4ac+ and F18ac+non-ETEC strains on incidence/fequency of ileal M cells and up-regulation of antigen delivery by de novo formation of these cells in weaned pigs. Conventionally reared 4-weeks-old pigs were divided into three groups of which two were parenterally and orally immunized with levamisole (at Days -2, -1 and 0) in combination with either vaccine candidate non-ETEC strain (at Day 0), respectively. The third group of pigs received saline as a placebo. Challenge was performed (at Day 7) with F4ac+ ETEC strain, and the pigs were euthanatized (at Day 13) and sampled for immunohistology. Distribution patterns of cytokeratin 18 positive M cells revealed that they are rather solitary interspersed between enterocytes than as small clusters, and most of them were found to be located at the apex of villi in the ileum of 6-weeks-old pigs. Morphometric quantification of M cells in the ileal mucosa showed that levamisole-pretreated F18ac+non-ETEC-immunized and challenged pigs had significantly increased numbers (p

Cold haemagglutinin disease (CHD) was diagnosed in two newborn French bulldog pups. Necropsy revealed cyanosis and necrosis, formation of crusts on all anatomic extremities and amputation of lower parts of hind limbs. Histologically, on the limbs of both animals cutaneous necrosis with blood vessels thrombosis, hemorrhages and predominantly neutrophilic infiltration which affected periosteum of metatarsal bones were found. Surfaces of necrotic areas were covered with thick crusts with Gram positive bacterial colonies. In the spleen and liver erythrophagocytosis was present. Lung congestion, alveolar edema and thrombosis of pulmonary artery branches, cerebral and chorioidal vessels were present. Immunohistochemicaly, lymphocytes and plasma cells in necrotic tissue were positive for IgM. Bacteriological examination of swabs from necrotic areas revealed presence of Staphylococcus aureus. The diagnosis of CHD was based on macroscopic and histopathologic findings which were identical to those described in literature and was supported by immunohistochemical reactivity to IgM.

The effect of newly synthesized derivative of 11-hydroxy-7-imino-7H-7a, 12-diazabenzo/α/anthracen-6-one, on growth and afl atoxin B1 and G1 (AFB1 and AFG1) accumulation by toxigenic mould Aspergillus fl avus ATCC 26949 was studied on a solid substrate (maize grains) to determine the possible use of this compound as a mean of controlling afl atoxin accumulation. Experiments were carried out in a stationary culture at temperature of 28oC during 21 days. Th e growth of mould was monitored by measuring the analysis of chitin as glucosamine, as a criterion, and concentration of AFB1 and AFG1 was measured by HPLC method using Hewlett-Packard instrument with fl ourescence detector. Concentration of investigated coumarine of 0.05 mmol x g-1 stimulated mould growth and aflatoxin accumulation, but concentration of 0.2 mmol x g-1 or higher produced an inhibitory effect. In the presence of 0.2 and 0.5 mmol x g-1 of this compound, mould growth was decreased by 22% and 65%, respectively. Concentration of AFB1 in these investigations was reduced by 30% and 90%, and concentration of AFG1 was reduced almost completely in respect to values obtained in control experiments. In experiments with 2.0 mmol x g-1 of this inhibitor no synthesis of both investigated aflatoxins was found in the sample although small increment of biomass was detected.

U predavanju su obrađeni najčešći uzročnici mastitisa u mliječnih krava:mastitis, Staphylococcus aureus, Streptococcus agalactiae, S. dysgalactiae, Mycoplasma bovis s mikrobiološkog gledišta. Navode se čimbenici virulencije i patogeneza bolesti s podacima o liječenju i profilaksi

Staphylococcus pseudintermedius (S. pseudintermedius) je gram-pozitivna kugličasta bakterija koju je pod nazivom Staphylococcus intermedius prvi put opisao Hajek 1976. godine. Sasaki i suradnici su 2007. na osnovi podataka dobivenih DNA-DNA hibridizacijom utvrdili da vrsta S. intermedius nije jedinstvena i predložili novu klasifikaciju. Predložili su da se naziv S. intermedius ubuduće koristi samo za sojeve izdvojene iz divljih golubova, a da sojevi podrijetlom iz pasa i mačaka budu izdvojeni u novu vrstu, Staphylococcus pseudintermedius. Sojevi koji se mogu izdvojiti iz domaćih golubova, konja i minka svrstani su u vrstu Staphylococcus delphini, s dvije grupe, A i B. U literature se često za ove tri vrste koristi skupni naziv „Staphylococcus intermedius group“ (SIG). Bakterija S. pseudintermedius izrazito je vrsno specifična i dio je fiziološke mikroflore kože i sluznica zdravih pasa i mačaka. Uvjetno je patogena bakterija, uzročnik površinskih i dubokih piodermija, upala vanjskog zvukovoda, ždrijela, nosnica i očnih spojnica. Rjeđe se može izdvojiti kod upala mokraćnog i spolnog sustava. Posljednjih godina otkriveni su sojevi bakterije S. pseudintermedius rezistentni na meticilin (meticilin rezistentni Staphylococcus pseudintermedius – MRSP), a učestalost izdvajanja iz zdravih i bolesnih pasa je u porastu. Rezistencija na meticilin u stafilokoka nije posredovana enzimom, nego promjenom ciljnog mjesta u staničnoj stijenci bakterije na koje se veže antibiotik. Sojevi otporni na meticilin imaju promijenjen protein koji veže penicilin (PBP), a označavamo ga kao PBP2a koji, u usporedbi s PBP, ima vrlo slabi afinitet za beta-laktamske antibiotike te se oni na njega ne mogu vezati i spriječiti sintezu stanične stijenke. U Hrvatskoj su prvi put meticilin-rezistentni sojevi S. pseudintermedius izdvojeni iz bolesnih pasa i mačaka 2008. godine (MATANOVIĆ i sur., 2009.). Razlog sve učestalijeg izdvajanja MRSP zasigurno je prečesta uporaba antibiotika širokog spektra, nedovoljno dugo liječenje i hipodoziranje. Osobit problem kod liječenja kućnih ljubimaca predstavlja korištenje antibiotika širokog spektra koji se nalaze na listi iznimno važnih antimikrobnih lijekova u humanoj medicini, primjerice cefalosporina i fluorokinolona. Meticilin-rezistentni sojevi S. pseudintermedius redovito su otporni i na mnoge antimikrobne lijekove iz drugih skupina. Zbog iznimne važnosti za zdravlje ljudi i životinja potrebno je pratiti širenje meticilin-rezistentnih stafilokoka u populaciji. Za genetsku tipizaciju izolata S. pseudintermedius koriste se metode poput gel-elektroforeze u pulsirajućem polju (PFGE, engl. Pulsed field gel electrophoresis), tipizacija na osnovi sekvencioniranja više genskih lokusa (MLST, engl. Multi locus sequence typing) i spa-tipizacija. Tipizacija sojeva osobito je važna za praćenje epizootiološke situacije kod infekcija meticilin-rezistentnim sojevima S. pseudintermedius. Dosadašnja istraživanja na osnovi tipizacije spa gena i MLST pokazala su da su europski i sjevernoamerički sojevi genetski različiti i da je u Europi dominantan klon t02-ST71, dok u Sjevernoj Americi prevladava tip t06-ST68. Liječenje životinja inficiranih meticilin-rezistentnim sojevima S. pseudintermedius iznimno je zahtjevno. Svi dosad izdvojeni sojevi u Hrvatskoj bili su multirezistentni i u većini slučajeva ni jedan antimikrobni lijek registriran za uporabu u veterinarskoj medicini nije bio djelotvoran.

A three-year-old male western black-tailed rattlesnake refusing food and losing weight presented with a swelling on the right side of the head below the eye. An abscess was suspected and treatment with subcutaneous enrofloxacin was started immediately. After identification of the causative agents as Pseudomonas aeruginosa and Stenotrophomonas maltophilia, and following susceptibility testing enrofloxacin was replaced by marbofloxacin and intralesional treatment with gentamicin/betamethasone was introduced, along with gentamicin administered subcutaneously at an increased terrarium temperature of 35 C.

Bakterije svrstane u red Chlamydiales mogu uzrokovati bolest (klamidiozu) u ljudi, brojnih vrsta domaćih i divljih životinja, kao i u ptica. Jedna od značajnijih vrsta, patogenih za životinje, ali i za čovjeka, je bakterija Chlamydophila (Cp.) psittaci. Dijagnoza klamidioze u ptica oslanja se, između ostalog i na imunološke dijagnostičke postupke. U ovom radu prikazat ćemo rezultate postupka izravne imunofluorecencije (DIF) za dokaz uzročnika, kao i rezultate serološkog postupka posredne imunofluorescencije (IIF) za dokaz protutijela na bakteriju Cp. psittaci u različitih vrsta ptica.

Background: Infections with antimicrobial-resistant bacteria are a great challenge in both human and veterinary medicine. Pseudomonas aeruginosa, an opportunistic, ubiquitous bacterial pathogen is becoming increasingly important due to its antibiotic resistance and ability to cause serious infections in animals and people. Objectives: Comparison of in vitro activities of enrofloxacin, ciprofloxacin, ticarcillin, ceftazidime, gentamicin and cefepime against 96 strains of P. aeruginosa isolated from dogs with otitis externa during the years from 1999 to 2008. Methods: Antimicrobial susceptibility was evaluated by E test1 in accordance with the guidelines of the National Committee for Clinical Laboratory Standards. Results: The most active compound in vitro was ceftazidime with 100% efficiency. The majority of tested strains were susceptible to ticarcillin (89.58%), followed by ciprofloxacin (87.50%), cefepime (58.33%) and gentamicin (56.25%). The highest resistance was observed towards enrofloxacin (53.13%), cefepime (33.33%), gentamicin (18.75%), ticarcillin (10.42%) and ciprofloxacin (9.38%). Only one strain (1.04%) showed sensitivity to enrofloxacin. Conclusions: This study showed that among all tested antimicrobial agents, ceftazidime was the most effective against P. aeruginosa strains. At the same time resistance to enrofloxacin was unexpectedly high.

Mačke su odnosu na pse relativno rijetki pacijenti, pogotovo kada je riječ o dermatitisima i otitisima uzrokovanih bakterijskom vrstom Staphylococcus. Stoga smo odlučili istražiti prisutnost bakterija vrste Staphylococcus, odnosno ustanoviti koje sve vrste naseljavaju kožu i zvukovode zdravih mačaka. U našem istraživanju uzeli smo obriske kože i zvukovoda 39 zdravih mačaka i iz 15 mačaka izdvojili 20 sojeva vrste Staphylococcus sp. Najviše izolata (13) pripadalo je koagulaza-pozitivnim vrstama S. intermedius (9) i S. aureus (4). Koagulaza- negativnim stafilokokima pripadalo je sedam izolata od kojih je najzastupljeniji bio S. hominis (5 sojeva), a po jedan izolat vrsti S. epidermidis i S. hylosus. Ovi su rezultati u suglasju s rezultatima sličnih istraživanja kod kojih su S. intermedius i S. aureus bile najzastupljenije koagulaza-pozitivne vrste, a S. hominis, S. epidermidis i S. hylosus koagulaza-negativne vrste. Mačke su relativno rijetko nosioci koagulaza-pozitivnih i koagulaza- negativnih stafilokoka. Vrsta Staphylococcus sp. nije izdvojena s kože i zvukovoda 24 zdrave mačke.

U radu su prikazani rezultati istraživanja in vitro osjetljivosti bakterije Pseudomonas aeruginosa na antipseudomonasne lijekove pomoću E testa, disk difuzijskog testa i dilucijskom metodom Najučinkovitiji antimikrobni lijekovi prema rezultatima većine objavljenih istraživanja bili su imipenem te fluorokinolonski antibiotici marbofloksacin i ciprofloksacin. Rezultati najnovijih istraživanja su pokazali da se rezistencija bakterije P. aeruginosa na antimikrobne lijekove drastično povećava pa tako ti isti antibiotici uskoro neće biti učinkoviti u liječenju pseudomonasnih infekcija. Usporedbom rezultata određivanja osjetljivosti različitim postupcima pokazalo se da je E test precizna i točna metoda za određivanje minimalne inhibicijske koncentracije, a zbog jednostavnijeg izvođenja i praktičnija od ostalih.

U radu je opisana epizootija antraksa koja se javila na području Bobovca (Sunja) tijekom 2006. i 2007. godine. Pretraženo je 14 uški s okolnim mišičjem, od čega 11 tijekom 2006. godiene i 3 tijekom 2007. godine. Određena je osjetljivost 6 izdvojenih sojeva prema 13 antimikrobnih lijekova. Prvim pozitivnim nalazima u kolovozu 2006. potvrđena je bedrenica, nakon čega se donose mjere radi suzbijanja iste. Mjere se nisu pokazale učinkovite kao prilikom suzbijanja epizootije bedrenice u Jasenovcu 2002. godine. Kada je nakon provedene imunoprofilakse jedna životinja uginula od bedrenice u travnju 2007. godine, prekontrolirana je i vakcina korištena za imunoprofilaksiu životinja na području Bobovca. Utvrđeno je da vakcina nije imala dostatan broj živih spora te da je to najvjerojatniji uzrok zbog kojeg životinje nisu bile potpuno imune prema ovoj bolesti. Nakon kontrole vakcine povećane su doze vakcine i ponovljena je vakcinacija kako bi životinje primile zadovoljavajući broj živih spora za kvalitetno stjecanje imunosti. Nakon toga je epizootija prestala. Određivanjem osjetljivosti šest izolata bakterije Bacillus anthracis utvrđeno je da su svi sojevi bili osjetljivi prema svim testiranim antimikrobnim lijekovima osim sulfametoksazola s trimetoprimom, prema kojem su svi testirani izlati bili rezistentni.

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Mycoplasma bovis was first isolated from milk of cows with mastitis in USA. Throughout the years, its significance has also been proved in arthritis, pneumonia, otitis and conjunctivitis in calves. In recent years, M. bovis has been frequently isolated from nasal cavities and/or pneumonic lungs of calves in many countries in North America, Europe, and Asia. The first isolation of M. bovis in Croatia was in year 2002 during the first outbreak of mycoplasma bovine mastitis recorded in the country. Since then milk from cows with history of mastitis from selected large milk farms has been monitored for mycoplasma and M. bovis has been recovered from 209 out of 1764 milk samples examined. Unfortunately the significance of M. bovis in calf pneumonia in Croatia is poorly appreciated, as demonstrated by the low numbers of clinical samples submitted to the Institute for diagnostic investigation. In a preliminary investigation to confirm the contribution of M. bovis to calf pneumonia in Croatia, four farms on which calves from various sources were raised and which had experienced continuous problems with chronic pneumonia were selected for study. Sixty six nasal swabs from 4 farms were collected and examined for the presence of M. bovis using enrichment and capture sandwich ELISA, culture in Hayflick broth and culture on mycoplasma agar. After isolation on solid agar and identification by sELISA, 20 isolates were randomly selected and tested by PCR. M. bovis was detected in 39 out of 74 (52, 7 %) nasal swabs from all 4 farms and 5 out of 11 (45, 5 %) lung samples from 4 farms by enrichment and capture sELISA. All samples positive in sELISA yielded growth of mycoplasma colonies on mycoplasma agar plates. All 20 selected isolates that were previously identified by ELISA were confirmed as M. bovis by PCR.

Mycoplasma bovis was first isolated from milk of cows with mastitis in USA. Throughout the years, its significance has also been proved in arthritis, pneumonia, otitis and conjunctivitis in calves. In recent years, M. bovis was frequently isolated from nasal cavities and/or pneumonic lungs of calves in many countries in North America, Europe, and Asia. In our previous work we confirmed that M. bovis is a significant and widespread cause of bovine mastitis in Croatia, however there are no data about its role in respiratory diseases of calves. The aim of this study was to investigate the prevalence of nasal carriage of M. bovis and to confirm its role in pneumonia of calves in Croatia. Four farms on which calves from various sources were raised and had continuous problems with chronic pneumonia were selected for the study. M. bovis was suspected as a cause of pneumonia because there were no improvements after prolonged antibiotic therapy. Sixty six nasal swabs and 9 lung samples of calves from 4 farms were obtained and investigated for the presence of M. bovis using enrichment and capture ELISA, culture in Hayflick broth and culture on mycoplasma agar. 10 nasal swabs and all lung samples were also plated on Columbia agar with 5% sheep blood (Biorad, France) for routine bacteriological analysis. After isolation on solid agar and identification by ELISA, 20 isolates were randomly selected and tested by polymerase chain reaction specific. M. bovis was detected in 36 out of 66 (54.5%) nasal swabs and 3 out of 9 (30%) lung samples by enrichment and capture ELISA. All samples positive in ELISA yielded growth of mycoplasma colonies on mycoplasma agar plates. All 20 selected isolates that were previously identified by ELISA were confirmed as M. bovis by PCR. Out of 10 tested nasal swabs, 90% were positive for both M. bovis and Pasteurella multocida. P. multocida and Mannheimia haemolytica only were cultured from one lung sample each, while other 7 samples were negative for bacteria. This work confirmed our assumption that M. bovis is a frequent colonizer of nasal cavities of calves on Croatian farms. M. bovis was isolated for the first time from pneumonic lungs of calves in Croatia thus confirming its role as a cause of pneumonia in calves. However, as there is no routine testing of pneumonic lungs for the presence of mycoplasmas, this problem is often overlooked as more common pathogens such as P. multocida, M. haemolytica and Histophilus somni are more easily isolated and attributed to pathologic conditions. There is an urgent need to stop the spreading of M. bovis by identifying heavily infected herds and establishing prevention and control measures in transport and herd market.

Staphylococcus pseudintermedius is an opportunistic pathogen most frequently isolated from canine and feline skin and ear infections. Methicillin resistance in staphylococci is mediated by mecA gene, which encodes for production of an altered penicillin binding protein that has a low affinity for all beta-lactam antibiotics. Methicillin-resistant Staphylococcus aureus (MRSA) is a tremendous problem both in human and veterinary medicine ; however methicillin-resistant S. pseudintermedius (MRSP) is emerging as a significant threat in household pets and is reported more frequently during last years. From November 2008 to June 2009 seven multiresistant S. pseudintermedius strains were isolated from external ear canal (2), pharynx, nasal cavity, skin (2) and conjunctival sac of diseased dogs and one strain from a pharynx of diseased cat. They were identified on the basis of biochemical properties. All isolates were DNase and coagulase positive, clumping factor negative or weakly positive, hemolytic on blood agar and produced beta-galactosidase, while none of them produced acetoin. Antibiotic susceptibility testing was performed by disc diffusion on Mueller-Hinton agar according to CLSI guidelines. All strains were resistant to methicillin, oxacillin, penicillin, amoxicillin-clavulanic acid, cefalexin, cefovecin, gentamicin, lincomycin, clindamycin, erythromycin, enrofloxacin, ciprofloxacin, marbofloxacin and trimethoprim-sulfamethoxazole and, with the exception of one, to chloramphenicol. All strains were susceptible to mupirocin, bacitracin, rifampicin, fucidic acid and amikacin, and all except one to tetracycline. Multidrug-resistant MRSP represent a challenge for antimicrobial therapy in veterinary medicine due to the limited treatment options and absence of effective antimicrobials labeled for use in animals.

This paper presents our observations during the second outbreak of bovine mastitis in Croatia. On a farm with approximately 700 dairy cows in different stages of lactation a significant increase in severe clinical mastitis cases resistant to antibiotic therapy was observed. The animals were of Friesian Holstein breed and some had been purchased from Western Europe. The acute form of mastitis was characterised by a rapid decrease in milk production, and a rapid spread of infection from affected to adjacent quarters. The animals were unsuccessfully treated with different antibiotics for several months. Combinations of tetracycline, neomycin and bacitracin ; penicillin G, dihydrostreptomycin, novobiocin and neomycin ; clavulanic acid-amoxicillin, cephalosporines ; and lincomycin were used. To investigate a possible role of mycoplasma as a causative agent of a large number of mastitis cases, 421 udder secretions were microbiologically examined. Mycoplasma colonies of typical appearance were cultured from 77 milk samples. Mycoplasmas were identified directly from milk samples by antigen capture ELISA test and polymerase chain reaction for routine identification of M. bovis. All isolated mycoplasmas were identified as Mycoplasma bovis. Mastitic bovine milk originated from a large number of farms in Croatia has been routinely cultured for mycoplasma on appropriate media at Department of Microbiology and Infectious diseases for the last 30 years. On this basis, it is likely that mycoplasma mastitis in Croatia was brought into into the country with the imported animals. As bovine mycoplasma mastitis might be a major udder health problem in dairy cattle there is an urgent necessity of coordinated action of veterinary diagnostic laboratories and veterinary authorities in identifying the distribution and control of this infection in Croatia.

The level of environmental pollution with airborne emissions from the barns can be assessed by comparing the fungi count in the barn air with the fungi pattern in the immediate environmental atmosphere. Therefore, the aim of the study was to determine fungi count in a dairy barn and its immediate environment, to help establish the borderline value in line with EU recommendations on airborne emissions from animal housings.

Mastitis uzrokovan bakterijom Mycoplasma bovis vrlo je kontagiozan i u zaraženom uzgoju oboli preko 20% krava. Nagli pad mliječnosti uz karakteristične promjene konzistencije mlijeka i neosjetljivost na klasičnu terapiju antibioticima karakteristični su simptomi mastitisa uzrokovanog vrstom Mycoplasma bovis. U radu je opisana epizootija mikoplazmalnog mastitisa uzrokovanog bakterijom Mycoplasma bovis na farmi mliječnih krava u Hrvatskoj. Na farmi se nalazi veći broj krava Holštajnskofrizijske pasmine. Posljednjih mjeseci zabilježen je porast broja mastitisa kod kojih klasična terapija antibioticima nije dovela do izlječenja. Zbog sumnje na mastitis uzrokovan mikoplazmama, 51 uzorak mlijeka dostavljen je u bakteriološki laboratorij Zavoda za mikrobiologiju i zarazne bolesti Veterinarskoga fakulteta Sveučilišta u Zagrebu radi pretrage na vrstu Mycoplasma bovis. Uzorci mlijeka nasađeni su na čvrstu hranjivu podlogu za uzgoj mikoplazmi (PPLO agar) te na tekuću hranjivu podlogu (PPLO bujon) u deseterostrukim razrjeđenjima i inkubirani na 37&ordm ; ; C u mikroaerofilnim uvjetima do pojave tipičnih kolonija mikoplazmi, odnosno do isteka 10. dana u slučaju negativnih uzoraka. Usporedno, uzorci mlijeka nasađeni su u jažice mikrotitracijske ploče obložene specifičnim protutijelima za M. bovis i inkubirani u mikroaerofilnim uvjetima pri 37&ordm ; ; C tijekom tri dana (enrichment and capture ELISA). Nakon inkubacije, postupak dokazivanja izveden je uz pomoć monoklonskih protutijela za M. bovis koristeći streptavidin peroksidazu i tetrametilbenzidin kao supstrat. U 22 od 51 (43%) pretraženih uzoraka mlijeka dokazali smo mikoplazme vrste M. bovis. Nakon četiri dana inkubacije kolonije tipične za mikoplazme izrasle su iz 16 uzoraka nasađenih na PPLO agar, a iz preostalih šest nakon sljedeća tri dana i prethodnog obogaćivanja u tekućoj hranjivoj podlozi. Enrichment and capture ELISA pokazala se kao brz, osjetljiv i specifičan test za dokazivanje M. bovis izravno u uzorcima mlijeka. Postotak od 43 posto pozitivnih uzoraka veći je od prosjeka koji je utvrđen na farmama mliječnih krava u zapadnoj Europi što se može dovesti u vezu s unosom M. bovis u nezaraženu populaciju krava. Predlažemo obvezno pretraživanje junica i krava na M. bovis prije uvođenja novih životinja na farmu te nužnost monitoringa mikoplazmalnog mastitisa u krava na području cijele Hrvatske kako bi se spriječilo širenje u nezaražene uzgoje.

Bacteriological examination of an abscess found between blubber and musculature of a striped dolphin (Stenella coeruleoalba) found dead on 30th March 2002 in the Adriatic Sea, Croatia was performed and an aerotolerant, nontoxogenic Clostridium tertium was isolated in pure culture. National Collections of Industrial Food and Marine Bacteria (NCIMB Ltd.), Aberdeen, Scotland, UK, confirmed the results. The sequencing results showed it to be C. tertium with 100% similarity. The strain was named Clostridium tertium strain Zagreb, acceded to the culture collection and assigned the accession number NCIMB 13970. This is the first record of C. tertium in marine mammals.

Es wirde ein Fall der perakuten Clostridiuminfektion bei einem etwa / Jahre alte Trampeltier (Camelus bactrianus) aus dem Zagreber Zoo beschireben. Das Tier verendete plotzlich, phne vorherige klinische Symptome. Es lebte zusammen mit zwei weiteren Kamelen, bei denen keine Krankheitszeichen beobachtet werden konnten. Die Obduktion ergab folgende makroskopische Veranderungen: Degeneration des Harzmuskels mit disseminierten Petechien subepikardial im linken Herzventrikel, Blutungen im Laryinx und Pharinx, starke Nephrose beider Nieren und hamorrhagische Enteritis des Abomasus. Samtliche Organe wurden auch histologisch untersucht. Mithilfe bakteriologisher Untersuchungen der Darm- und Lungenproben wurde Clostridium perfringens diagostiziert.

Ciprofloxacin, enrofloxacin, gentamicin, cefipime ceftazidime and ticarcillin-clavulanic acid were tested by the E-test against 84 Pseudomonas aeruginosa strains. Ceftazidim was the most active agent with 100% of isolates having a MIC ≤ 8 mg/L (range 0.38 – 6). Ciprofloxacin and ticarcillin-clavulanic had similar activity 76% and 73% were inhibited by a MIC ≤ 1 mg/L (range 0.064 -1) and a MIC ≤ 64 mg/L (range 4 -64) respectively. Cefipime inhibited 50% of tested strains with a MIC ≤ 8 mg/L of (range 0.75 – 8). Gentamicin was the least active with 42% of P. aeruginosa isolates having a MIC ≤ 4 (range 0.125 – 16). Interpretative criteria for enrofloxacin are provide by the CLSI veterinary guidelines (M31-A2, 2002) and refer to the specific animal being treated. The CLSI guidelines state that for canine and feline Gram negative enteritic bacilli and other susceptible microorganisms, the breakpoints are S≤ 0.5, F= 1-2 and R ≥ 4, where F indicates the availability of FDA-approved labeling and those microorganisms should be considered susceptible if dosing modifications found in the package insert are applied. According to CLSI none of 84 tested strains were susceptible to enrofloxacin, 57% (48 strains) were resistant with a MIC range 3 – 32 mg/L and 43% (36 strains) having a MIC range 0.75 – 3mg/L, were ranged as category F.

Clinical characteristics of mastitis supported by the results of microbiological examination, definitely confirm our supposition that a great number of animals housed on farm resisting antibiotic therapy are infected with M. bovis. This is the first finding of mycoplasmal mastitis caused by this M. bovis in Croatia. During last 30 years (Naglić, 2005) the great number of bovine milk originated from large and small farms was on appropriate media unsuccessfully examined for mycoplasmal. Infection in Bacteriological laboratory of our Department. It is obviously that infection with M. bovis was imported in recent years with imported cows from abroad. As bovine mycoplasmal mastitis might be a major udder health problem in dairy cattle there is an urgent necessity of coordinated action of veterinary diagnostic laboratories and veterinary authorities in identifying the distribution and control of this infection in Croatia.

During six months 50 strains of Staphylococcus intermedius were isolated from different pathological specimens originated from dogs. Antimicrobial susceptibility patterns against three fluoroquinolones were obtained on 50 Staphylococcus intermedius strains. In vitro antimicrobial susceptibility testing was performed employing the disk diffusion method (Kirby-Bauer). The in vitro sensitivity against enrofloxacin and marbofloxacin showed that 98.0% of S. intermedius strains were sensitive and 96% were sensitive to ciprofloxacin. One of tested strains shoved multiple resistance to all tested fluoroquinolone.

Izvješće se odnosi na opis prvog nalaza bakterije Mycobacterium africanum iz pećinara-Procavia capensis u svijetu.

U radu su iznijeti podaci stručne i znanstvene literature o etiologiji i proširenosti mikoplazmoznog mastitisa u krava koji uzrokuje vrsta Mycoplasma bovis. Opisani su klinički znakovi, način dijagnostike i iskorjenjivanja te bolesti koja se ističe nemogućnošću liječenja. Bolest je u nas etiološki ustanovljena u velikog broja životinja na jednoj farmi krava muzara.

The paper describes and outbreak of Salmonellosis in chinchilla caused by Salmonella enteritidis and S. sofia.

Fifteen strains were identified as members of Mollicutes (primers MW 28/29). No strains were found to belong to MmmSC (SC3NEST1-L/R)The examined were identified as MmmLC/Mmcapri. No strains were found to belong to MmmSC (SC3NEST1-L/R.

Komercijalnim pripravkom imunoenzimnog testa ustanovljene su salmonele u 1.55 posto pretraženih uzoraka. Kulturelnom pretragom salmonele su dokazane ustanovljene u 0.99 posto tih uzoraka. Zbog visoke podudarnosti rezultata, smatra se opravdanim uporaba imunoenzimnog testa u dokazivanju salmonela u hrani za pse.

To determine in vitro sensitivity to selected antimicrobial agents, during seven years, strains of Pseudomonas (P.) aeruginosa were collected from clinical specimens. From 1993 through 2000 from 5344 examined clinical specimens total of 155 strains belonging to this species were isolated. The majority of strains (123 strains) originated from ear discharge of dogs with otitis externa, and the other strains were isolated from pathologically changed organs of dead animals, milk samples, urine, faeces and wounds. The strains were tested for susceptibility by disk-diffusion method using Mueller-Hinton agar against seven agents of known antipseudomonas activity, i.e. imipenem, pipemidic acid, carbenicillin, ceftazidime, cefoperazone, amikacin, and gentamicin. Imipenem was the most active of tested compounds, with 151 strains ( 97.4%) sensitive, one strain (0.6%) partial sensitive and three strains (1.9%) were resistant. Against cephalosporin cefoperazon (third generation) 140 strains (90.3%) showed sensitivity, eight were partial sensitive (5.2%) and seven (4.5%) were resistant. Aminoglycoside amikacin was active against 134 isolates (86.5%), nine of them were partial sensitive (5.8%), and 12 (7.7%) were resistant. Ceftazidime was active against 132 strains (85.2%), eight strains (5.2%) were partial sensitive and 15 were resistant (9.7%). To aminoglycoside gentamicin 129 strains (83.2%) were sensitive, nine (5.8%) were partial sensitive and 17 (10.6%) were resistant. Against carbenicillin 118 strains (76.1%) were sensitive, nine (5.8%) were partial sensitive and 28 strains (18.1%) were resistant. Significant difference in antipseudomonas activity to all antimicrobial agents was observed in pipemidic acid. Out of 155 tested strains, 62 were resistant (40.0%), 11 were partial sensitive (7.1%) and 82 strains (52.9%) were sensitive. Susceptibility tests showed that the majority of strains were sensitive to specific antipseudomonas agents, except the less active pipemidic acid.

U ovom strčnom uvodniku ukratko su iznijeti podatci o rodu Listeria i najznačajnijoj vesti L. monocitogenes te kninička očitovanja u životinja inficiranih ovom intracelularnom, uvjetno patogenom bakterijom.

Listeriosis is an infectious disease of domestic and wild animals coused by Listeria monocytogenes, and is transferable to humans. There are two clinical forms of listeriosis: septicemic and encephalitic. This paper deals with two cases of listeriosis in chinchillas (Chinchilla laniger), originating form a farm in the County of Međimurje in Croatia, and were pathomorphologically and bacteriologically examinated. This particular breed consisted of 64 chinchillas, 34 of which have died due tue same symptoms. History reveals that animals had suffered loss of appetite, and abortion occurred about 5 days before dead. Gross lesions included general anaemia and cachexia. Whithis-grays nodules the size of millet grain or little larger were noticed along the capsule of liver, serosa of colon and in mesenterial lymph nodes. Numerous vaiably-sized granulomas in liver, intestines, lymph nodes and kidneys, with no special pattern, were observed histopathologically. They consisted of necritic cells with somewhat more granulocytes in the center and scattered individual giagantocytes. Bacteriological examination of liver specimens revealed Listeria monocytogenes in pure culture in both cases. This is the first description of listeriosis in chinchillas in Croatia.

The most of 9 tested antimicrobial agents showed considerable in vitro effectiveness against tested M. conjunctivae isolates. The most effective were enrofloxacin, tiamulin, chlortetracycline and oxytetracycline, followed by tylosin and gentamicin. The highest resistance was recorded with chloramphenicol, neomycin, and streptomycin Until now only a limited number of reports on sensitivity testing of M. conjunctivae isolates are available. Remarkable in vitro sensitivity of our M. conjunctivae isolates to tetracyclines was verified by good results obtained by topic and systemic administration of these drugs during the first outbreak of ovine infectious keratoconjunctivitis in Croatia. It can be concluded from our study that enrofloxacin, tiamulin, tetracyclines and tylosin are promising for the local and/or systemic treatment of IKC in small ruminants. Taking into account the broader antibacterial activity and possibilities of incorporation in ophthalmic preparations, tetracyclines may be preferred.

Conected with the previously report on an outbreak of OIKC in Croatia, further investigation in epidemiology and microbiology was carried out. M. conjunctivae was not isolated from the animals derived from previously infected flocks. However many of the examined animals with a mild conjunctivitis were infected with B. ovis.

Connected with the import of several thousand sheep from Australia and N Zealand in Croatia during 1995 a great number of native sheep that have been in contact with the imported animals acquired a severe ocular disease closely resembling OIKC. M. conjunctivae was isolated from 44 per cent and B. ovis from 56 per cent of microbiologically examined conjunctival swabs. For many resons most of the farmers ekiminated imported animals from their flocks and only sporadic cases of OIKC were recognised in 1996. By the end of 1997 all inspected flocks were clinacally free from OIKC. At this time conjunctival swabs were taken for microbiological examination from 63 animals originated from 6 previously infected flocks, and from 28 animals derivad from the flocks with no history of OIKC. The age of animals varied from several months to five years. All examined animlas were free from M. conjunctivae infection. Two Acheloplasma strains and two glucose- and arginin-negative mycoplasma isolates were ascertained in one flocks. B. ovis was cultured from 42, 9 per cent of examined animals from the flocks with previous history of OIKC. Heavy growth of B. ovis was generally associated with mild conjunctivitis. Independently of clinical signs of conjunctivitis this bacteria was isolated in a small number of colonies from the conjunctivas of only one animal derived from the flocks with no history of OIKC.

Vegetative valvular endocarditis involving the aortic valves was diagnosed clinically, radiographically and echocardiographically in a 3-years-old male bull-terrier. ECG findings showed monomorphic ventricular extrasystoly while denivelation of the ST segment pointed to the possibility of the subendocardial heart infarction. The dog had been treated with ampicilline, gentamicine and enalapril during 20 days, and then the therapy was continuated with ampicilline during the next 15 days. Bacteriological examination of the blood was negative. Despite an initial therapeutic responst, the general condition of the dog after one month of therapy deteriorated and the owner asked for the animal to be euthanized. Post-mortem (PM) examination confirmed aortic valvular endocarditis, and the bacteriological examination of the affected valves showed bacterial infection with Enterococcus faecalis. Due to left coronary artery embolisation myocardial recernt infarction of the left ventricle and septum was noted. Other PM findings included renal recent infarctions, lung oedema, heart hypertrophy and villous fibrotic aortic endoarteritis near the valves.

Streptoccocus suis is responsible for a wide variety of infection in domestic pigs.It has been associated with meningitis, bronchopneumonia, endocarditis, arthritis, abortion and septicaemia.(Clifton-Hadley and Alexander 1988)In the most of the countries. including Croatia, where epidemiological studies have been reported, S. suis capsular type 2 appears to be the most frequently isolated type(Clifton-Hadley and Alexander 1988, Touil and others 1988) S., suis capsular type 3 has been isolated mainly from the lungs (bronchopneumonia) and less often from meninges/brain, heart and other tissue of diseased domestic pigs(ouil and others 1988)This study presents a case of streptococcal pneumonia in a young wild boar (Sus scrofa) . In a hunting -ground area of 10,000 hectares near by Zagred about 20 herds of wild boar and domestic pigs co-exist.A three month old boar died in a small herd which consisted of eight animals.The carcasae was very emaciated and dirty. The eyes were stuck together with a purulent dischrage and the conjuctivae were extremely pale.At necropsy lobar fibrinous , partialy hemorrhagic pneumonia was found together with serofibrinous pleuritis and pericarditis. The lungs, spleen and liver were submitted for bacterioligical examination.All the organs were inoculated onto horse blood agar.After 24 hours incubation at 37 C in aerobic conditions, large numbers of alpha-haemolytic streptococci were recovered from the lungs along with a coimparable number of colonies of E. coli.Altough E. coli is a common bacteria and has been formerly isolated together with S., suis.(Higgins and others 1997). is not easy to verify its role in this particular case.There were bacteria no recovered from the spleen.The alpha -haemolytic bacteria was identified biochemically according to the method described by Vecht and others (1985) as Streoptococcus suis and was sent for serotyping.The isolated strain was confirmed as a S suis capsular 3. This is believed to the first report of of S. suis from an animal other than domestic animals in Croatia and also the first report of the isolation of capsular type 3 in wild boar. The authors would like to thank Dr J Henrichsen, Statens Serum institute, Copenhagen, Denmark, for serotyping

Susceptibility of epidemiologically unrelated clinical canine and feline isolates of S. intermedius were routinelly tested by agar diffusion method over a period two years. The strains were tested against 13 known antistaphylococcal agents irrespectivelly of their clinical applicability for the treatment of infections caused by S. intermedius.

U biologiji, veterini, ili agronomiji znanstvena istraživanja, u kojima se rabe pokusne životinje, obično su zamišljena tako da se s pomoću njih prikupe svrsishodni i značajni podaci za životinju ili životinjsku vrstu na kojoj se pokus obavlja. Danas se većina pokusnih životinja rabi u istraživanjima lijekova, provjerama valjanosti cjepiva, ispitivanjima toksičnosti različitih pripravaka i istraživanjima karcinoma u kojima životinje služe kao model koji zamjenjuje istraživanja na ljudima. Zbog velikog broja životinja koje se rabe u znanstvenim istraživanjima nametnula su se i brojna pitanja o dobrobiti pokusnih životinja. Riječ je o zakonskim propisima koji određuju pojedinosti o ponašanju prema pokusnim životinjama tijekom i nakon pokusa, njihovom odabiru s obzirom na vrstu, broju životinja koje će biti uporabljene u pokusu, njihovu smještaju, hranidbi, njezi, anesteziji, analgeziji i eutanaziji. Uporaba životinja u različitim istraživanjima ima dugu povijest, a temelji Zapadne medicine kao i uporaba životinja imaju korjene u dalekoj prošlosti. Tijekom 18. stoljeća na ovamo, postupno je usvajano mišljenje da rezultati pokusa na životinjama doprinose dobrobiti ljudi i poboljšanju životnih uvjeta, a također je postalo razvidno da razvoj medicine, odnosno njezina budućnost, uvelike ovisi o rezultatima pokusa provedenim na životinjama. S porastom broja pokusa na životinjama pojavile su se i prve udruge za zaštitu životinja i njihovih prava. Sve brži razvoj brojnih grana biomedicinskih znanosti, primjerice farmakologije, toksikologije, virologije, imunologije kao i dijela farmaceutske industrije uzrokovao je povećanje broja laboratorijskih životinja u različitim pokusima što je imalo za posljedicu brojne rasprave o moralnoj opravdanosti njihove uporabe u pokusima, a pitanje pokusnih životinja i njihova uporaba u različitim eksperimentima postalo je i političko pitanje. U ovom su radu prikazani rezultati nekih znanstvenih istraživanja usmjerenih na pronalaženje alternativnih postupaka koji bi uspješno zamijenili istraživanja in vivo. Iako se posljednjih destetljeća ulažu veliki napori u pronalaženju spomenutih postupaka oni imaju brojne nedostatke u odnosu na istraživanja provedena na životinjama i još uvijek ne mogu u mnogim istraživanjima zamjeniti in vivo modele.

Epizootical and clinical observations during an outbreak of ocular disease in sheep were indicative in ovine infectious keratoconjunctivitis. Clinical diagnosis was cinfirmed by numerous isolation of mycoplasmas and Branhamella ovis. All investigations clearly indicated that the epizootic of OIKC in native sheep was related to the import of infected animals. Introduction of imported sheep and rams into the floks and direct contact with them were mostly responsible for the appearance of the disease in native sheep. From the glucose-fermenting arginin-negative strains, 10 out of 12 isolates were identified by PCR as M. conjunctivae. It therefore can be assumed that the majority of the other isolates with the same characteristics morphological and physiological properties also belonged to this species.The finding of M. conjunctivae in a large number of animals from the affected native floks, especially in those untreated with antibiotics, and the absence of infection in the healthy native floks supported the hypothesis that mycoplasmas were the primary cause of ovine keratoconjunctivitis. Although Branhamella ovis could not be assumed as primary etiological agens of OIKC as shown by others, its incidence rate their close relation with the disease.

At the begining of 1995. approximately 6 000 sheep from Australia and New Zealand were imported in Croatia as donation and distributed to private farmers all over the country. During the spring and summer of the same year a great number of native seep thate were in conctat with imported animals developed the signs of an ocular disease closely resembling to ovine keratoconjunctivitis. This disease has not been yet etiologically diagnosed in Croatia. Microbiological examination of conjunctical smears revealed a great number of animals infected with glucose-fementing mycoplasmas and Branchamella ovis. The infection with Chlamydia psittaci was not establishesd in any examined case. At the time of sampling the imported animals did not reveal clear-cut signs of disease, but were frequently found infected with mycoplasma and branhamella. Besides the results of microbiological investigation of epizootia, an outline of literature data on infectious ovine keratokonjunctivitis was given in the paper.