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 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.

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 trimethoprim-sulfamethoxazole (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.

During the six-month collection period, 284 bacterial strains were recovered from different clinical samples from cats and dogs. Susceptibility to cefovecin was determined by disk-diffusion method. Out of 284 bacterial isolates, 202 were gram-positive bacteria and 82 were gram-negative. The most frequently isolated bacterial species was gram-positive Staphylococcus pseudintermedius (152), followed by streptococci (50). The most common gram-negative bacteria were Pseudomonas aeruginosa (26), Proteus spp. (25) and E. coli (22). The highest resistance among gram-positive bacteria was observed in non-hemolytic streptococci (50%) and Staphylococcus pseudintermedius (7.2%). Beta-hemolytic streptococci were 100% sensitive to cefovecin. As expected, cefovecin did not show any activity against P. aeruginosa. Besides that, the highest resistance was shown by E. coli (40.9%) and Proteus spp. (8%). All P. multocida isolates were sensitive to cefovecin. The results of the conducted survey show no signifi cant differences of cefovecin activity against beta-hemolytic streptococci and P. multocida isolates from EU and USA compared to Croatian strains, when tested by the broth microdilution and disk-diffusion method. However, resistance rates in S. pseudintermedius, E. coli and Proteus spp. are much higher among the Croatian strains.

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<0.01) of ileal M cells as compared to the values obtained in the control nonprimed and challenged pigs. The proportion of these cells in this group of pigs was increased for 145%. In the levamisole-pretreated F4ac+ non-ETEC-immunized and challenged pigs only slightly increased (for 7%) proportion of M cells was recorded. However, this increase was not significantly different from the numerical values obtained for control pigs. Our finding that levamisole-adjuvanted F18ac+non-ETEC vaccine may affect de novo differentiation of antigen-sampling M cells by increasing their number in the ileum, indicated that the vaccine probably utilizes these cells as a target for entry/delivery to the nerby lymphocytes and induces protective immunity against CE. On the other hand, the failure of levamisole-adjuvanted F4ac+non-ETEC vaccine to produce a similar effect on M cells remains to be elucidated.

The bottlenose dolphin ( Tursiops truncatus ) is the only resident marine mammal species in the Croatian part of the Adriatic Sea, with an estimated number at around 220 individuals. It is an endangered and legally protected species in Croatia, and its demographic history is poorly known. This study investigates the level of genetic diversity in the bottlenose dolphin population from the Croatian part of the Adriatic Sea and a possibility of recent population size contraction, since there are indications that there has been intensive eradication operations in the mid 20th century that might have caused reductions in the effective population size and might have resulted in a loss of genetic variation. Thirty samples were genotyped at 12 dinucleotide microsatellite loci. The mean allelic richness (6.835 ± 0.705) and mean expected heterozygosity (0.692 ± 0.05) revealed high level of genetic diversity. Bottleneck analysis gave ambiguous evidence for a recent population decline in the investigated bottlenose dolphin population. The M ratio test, with two sets of parameter values, suggested a recent bottleneck; whereas the analysis by the Bottleneck program under three mutation models (TPM, SMM and IAM) showed no evidence for a genetic bottleneck. We take a more conservative approach to the interpretation of these results by accepting the evidence of a recent bottleneck. We suggest maintaining the current level of bottlenose dolphin protection in the area and careful monitoring of the population in the future.

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 the lower parts of the hind limbs. Histologically, cutaneous necrosis was found on the limbs of both animals, with blood vessel thrombosis, hemorrhages and predominantly neutrophilic infiltration, which affected the periosteum of the metatarsal bones. The surfaces of the 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 the presence of Staphylococcus aureus. The diagnosis of CHD was based on macroscopic and histopathologic findings, which were identical to those described in literature and were supported by immunohistochemical reactivity to IgM.

Staphylococcus pseudintermedius (S. pseudintermedius) je gram-pozitivna kuglicasta 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 ubuduce koristi samo za sojeve izdvojene iz divljih golubova, a da sojevi podrijetlom iz pasa i macaka budu izdvojeni u novu vrstu, Staphylococcus pseudintermedius. Sojevi koji se mogu izdvojiti iz domacih golubova, konja i minka svrstani su u vrstu Staphylococcus delphini, s dvije grupe, A i B. U literature se cesto za ove tri vrste koristi skupni naziv „Staphylococcus intermedius group“ (SIG). Bakterija S. pseudintermedius izrazito je vrsno specificna i dio je fizioloske mikroflore kože i sluznica zdravih pasa i macaka. Uvjetno je patogena bakterija, uzrocnik povrsinskih i dubokih piodermija, upala vanjskog zvukovoda, ždrijela, nosnica i ocnih spojnica. Rjeđe se može izdvojiti kod upala mokracnog i spolnog sustava. Posljednjih godina otkriveni su sojevi bakterije S. pseudintermedius rezistentni na meticilin (meticilin rezistentni Staphylococcus pseudintermedius – MRSP), a ucestalost izdvajanja iz zdravih i bolesnih pasa je u porastu. Rezistencija na meticilin u stafilokoka nije posredovana enzimom, nego promjenom ciljnog mjesta u stanicnoj stijenci bakterije na koje se veže antibiotik. Sojevi otporni na meticilin imaju promijenjen protein koji veže penicilin (PBP), a oznacavamo 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 sprijeciti sintezu stanicne stijenke. U Hrvatskoj su prvi put meticilin-rezistentni sojevi S. pseudintermedius izdvojeni iz bolesnih pasa i macaka 2008. godine (MATANOVIC i sur., 2009.). Razlog sve ucestalijeg izdvajanja MRSP zasigurno je precesta uporaba antibiotika sirokog spektra, nedovoljno dugo lijecenje i hipodoziranje. Osobit problem kod lijecenja kucnih ljubimaca predstavlja koristenje antibiotika sirokog 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 sirenje meticilin-rezistentnih stafilokoka u populaciji. Za genetsku tipizaciju izolata S. pseudintermedius koriste se metode poput gel-elektroforeze u pulsirajucem polju (PFGE, engl. Pulsed field gel electrophoresis), tipizacija na osnovi sekvencioniranja vise genskih lokusa (MLST, engl. Multi locus sequence typing) i spa-tipizacija. Tipizacija sojeva osobito je važna za pracenje epizootioloske situacije kod infekcija meticilin-rezistentnim sojevima S. pseudintermedius. Dosadasnja istraživanja na osnovi tipizacije spa gena i MLST pokazala su da su europski i sjevernoamericki sojevi genetski razliciti i da je u Europi dominantan klon t02-ST71, dok u Sjevernoj Americi prevladava tip t06-ST68. Lijecenje životinja inficiranih meticilin-rezistentnim sojevima S. pseudintermedius iznimno je zahtjevno. Svi dosad izdvojeni sojevi u Hrvatskoj bili su multirezistentni i u vecini slucajeva ni jedan antimikrobni lijek registriran za uporabu u veterinarskoj medicini nije bio djelotvoran.

The effect of newly synthesized derivative of 11-hydroxy-7-imino-7H-7a, 12-diazabenzo/α/anthracen-6-one, on growth and aflatoxin 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 aflatoxin accumulation. Experiments were carried out in a stationary culture at temperature of 28oC during 21 days. The 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 fluorescence 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.

Similarly as in other organ structures stem cells are present in cornea residing basal epithelial layer termed palisade of Voight. A growing interest in allografts and xenografts implies a thorough study of regenerative potentials of these cells, as well as a clear description of their patterns in in vitro tissue cultures to be grafted. Recently we have developed a simple method for cultivation of porcine corneal epithelial stem cells obtained by biopsy from the limbal region. Eight enucleated eyes were obtained from four slaughtered pigs. 5 mm2 samples of limbal epithelium were taken by keratotomy method. The primary cultures of these cells showed phenotypic and morphometric characteristics of porcine corneal epithelial cells following May-Grunwald-Giemsa staining. After 5d of sowing they reached 80% of confluence. With the «Night & Day» lenses a total confluence was achieved 5d earlier in comparison to the cells that were grown in the secondary cultures. Accordingly, the use of porcine limbal stem cells has potentials in veterinary medicine (as novel approach in reparative/regenerative medicine of pets, horses and selected breeds), but also are in accordance with the extensive studies on the potential use of xenografts, mainly swine tissues/organs, in humans.