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Poultry infectious and parasitic diseases cause major problems and loss during production. Because of that it is of great importance to evaluate epizootiological situation on each poultry farm and to know how to adjust the immunoprotection strategy and improve immune response of poultry accordingly. A small project was initiated in order to evaluate the epizootiological situation on poultry farms in Croatia and inform farm holders. Cloacal and pharyngeal swabs were taken on 27 farms from live poultry, poultry, rodent and wild bird faeces samples, liver, spleen, lung, intestine and skin tissue samples from death animals, samples of red mite and samples of cloacal and pharyngeal swabs from wild birds, where appropriate. After RNA/DNA isolation, a cheap, simple and fast loop-mediated isothermal amplification (LAMP) method of molecular detection was used with a set of 4 or 6 primers and Bst polymerase to detect specific microbial/parasitic RNA/DNA in 153 different samples in RealTime PCR. Final results are still pending, but preliminary results show the presence of common viral, bacterial and parasitic infections/invasions such as infectious bursal disease, Marek's disease, Newcastle disease, infectious bronchitis, coccidiosis, salmonella and campylobacter in different combinations on several farms in above mentioned samples.

In frame of the EU FP7th project "Protection of consumers by microbial risk mitigation" - PROMISE, foods of Animal origin confiscated at airports and ground ports of Austria, Croatia, Germany, Hungary and Slovenia are tested for the presence of foodborne pathogens by using standardized guidelones (ISO) for O157 verotoxinogenic E. coli and agreed protocols for non-O157 VTEC. Results of isolations and of charecteristics of the above foodborne athgens from 1800 confiscated food samples were os follows: O157 VTEC was not deteted. All together 15 strains of non-=157 VTEC were identified, mainly from food of ruminant origin. Phenotpic and genotypic (microarray) analysis of VTEC indicated an absence of antimicrobial resistance determinants but, several VTEC strains showed an abundance of E. coli fitness and virulence genes including stx and less freyuently eae. In contrast, the 28 strains of multiresistant E. coli were charecterized by a low numbers of virulence genes but by higer number of antimicrobial resistance genes and by class 1 integrons. It is concluded that the above results should not raise major food safety concerns.

Avian chlamydiosis, despite the potential risk for human health all over the world, is still not investigated enough. Especially in poultry, chlamydial infections are often misdiagnosed due to symptoms similar to those caused by other respiratory pathogens or very complex diagnosis which is also hazardous for laboratory personnel. In the last decade, molecular methods serve as most appropriate for detection of this zoonotic pathogen. In different geographic part of Croatia fecal samples were taken from 67 backyard poultry flocks. By using a Chlamydiaceae-specific real-time PCR assay targeting the 23S rRNA gene 7 samples (10.45%) were found positive. Some of these PCR positive samples belong to C. psittaci but some of them didn’t react with any of the C. psittaci specific tool targeting the ompA gene. As recently novel Chlamydophila spp. strains have been isolated from poultry some of them belong to atypical Chlamydia. Depending on the flock size and housing conditions, positive birds were treated with different antimicrobial agents (tetracycline or enrofloxacine) and bio-security measures (quarantine, regular cleaning and disinfection) were performed in all positive cases. After the treatment, the birds were examined until negative results were obtained. Further investigations are needed in attempt to clarify the role of this microorganism in poultry and prevention of potential illness in humans.

Salmonella enterica serovar Entertitidis (SE) is one of the main causes of food borne diseases, transmitted by eggs or egg products. The aim of the present study was to investigate the prevalence of SE in specimens collected from experimentally infected laying hens. In the two trials two lines of commercial laying hens at onset of the lay were orally inoculated with a single dose of 1.2x109 CFU of highly pathogenic SE. After infection the inoculated bacteria were recovered in all hens. Despite the fact that all hens became infected, we found a low number of positive eggs.

„The olm (Proteus anguinus) in Croatia – conservation research project“ is composed of the ex situ and in situ parts. The aim of the in situ part is biological and veterinarian research on flushed animals kept in specially designed „cold“ room situated at the Zagreb Zoo. The aim of this study is to learn about the normal, opportunistic and pathogenic microflora in these animals because this information is absent. In the period of November 2012 to June 2013 a total of 20 animals were tested, among them 14 flushed out at two localities and sampled immediately upon arrival to the rescue center, and 6 animals sampled at their original habitat at three localities. Oral cavity, cloacal and skin swabs were taken and analyzed by bacteriological cultures grown at 8o and 22oC for 5 days. In total, representatives of 11 bacterial genera, i.e. Acinetobacter, Aeromonas, Bacillus, Butiauxiella, Enterobacter, Escherichia, Janchtinobacterium, Micrococcus, Pseudomonas, Staphylococcus and Streptococcus were identified. Significantly more bacteria were isolated in olms flushed out than in those from their original localities. Most of them are opportunistic and grow only, or grow much faster, at higher temperatures so that they probably could not compromise the olm, unless it is flushed out. Also, most of them are soil bacteria which take part in natural elimination of organic pollution, but some of them could also compromise their health under non optimal conditions. Of particular interest could be the finding of Janchtinobacterium lividum, with known anti-fungal properties, which may help protect the olm against infection by the devastating chytrid fungus Batrachochytrium dendrobatidis.

The overall objective of the BASELINE project was to provide harmonised and validated sampling protocols structured as international standards. These procedures should support European policies in food safety and need to be suitable for food producers in order to collect comparable data improving quantitative risk analysis of selected biological and chemical agents. The public health protection in the food chain must be based on risk assessment procedures. This approach requires the standardisation and harmonisation of sampling techniques and analytical methods. New risk based criteria, named Food Safety Objectives (FSO) and Performance Objectives (PO), have complement the existing concepts of microbiological criteria (MC) and maximum residue limits (MRLs) for many chemical contaminants. The major output of the project has been to generate a proposal of improved sampling plans and related safety criteria, by using mathematical modelling for different groups of food products as seafood, eggs and egg products, fresh meats, milk and dairy products and plant products. The project results have been translated in BASELINE Recommendations for EU policymakers concerning improving food chain safety

The olm (Proteus anguinus) is the only real cave-dwelling (stygobiont) chordate found in Europe. It is adapted to a life of complete darkness in its underground habitat in caves of the Dinaric karst of Central and Southeastern Europe. The olm is extremely vulnerable to changes in its environment and is now threatened by an increasing and uncontrolled spreading of urban areas and human-made infrastructure, and the excessive pollution of water habitats. Due to its life at difficult accessible locations its biology is not well understood and its physiological microflora and the pathogens that may jeopardise its existence are completely unknown. Here we present the project of the field investigation at several localities in Croatia, aimed to increase the knowledge about the olm’s ecology and biology, including its microflora, in order to help protection and conservation of this endangered species.

Four-lined snake (Elaphe quatuorlineata) is the Europe's largest non-venomous colubrid species. Adults can grow up to 260 cm, but the average total size is 150 cm. These snakes could be found near the wood edges and hedges, in open woods, and also on rocky overgrown hillsides. They are spread over the whole coastal part of Croatia and some islands such as Olib, Cres, Silba, Hvar Vis, etc. The four-lined snake is a rather phlegmatic and relatively slow-moving, and it is considered our most docile snake that almost never bites. For better understanding of gastrointestinal microflora, some infectious diseases and parasitoses in these snakes, the oral and cloacal swabs were taken, together with the ticks from the skin for morphological identification. The snakes were caught with the safety gloves on various locations of the Cres island. Most of them were found in the fields, woods and olive groves. Two people were taking the samples together, one holding the snake and the other one taking the cloacal and oral swabs. Two parallel swabs from each site were taken, one was placed into the transport medium (Amies swabs with transport media) for the standard bacteriology, while the other one was stored for DNA isolation. Ticks from the animals were taken with the forceps and transferred into the Eppendorf tube with the moist piece of paper. The samples were stored in the refrigerator until examined. The general condition and possible health problems of each animal were assessed by the inspection and palpation of each individual. The aims of this study were (i) to get better understanding of the overall health status of four-striped snakes for the conservation purposes, and (ii) to increase the knowledge about the bacteria-related complications of potential bites and spreading of the pathogens.

Vaccination of newly hatched chicken, turkey, duck and goose already in the hatchery and achieving long lasting immunity is certainly the goal of immunoprophylaxis today. The attainment of this target is foreboded in use of micronized particles of vaccine suspension produced in an ultrasonic nebulizer. Compared to usual modes of vaccination (e.g. spray, w/v, i/m, drinking water, eye drop) small particle of vaccine suspension 2-5 μ in diameter, can reach the deepest part of respiratory system augmenting enormous the resorptive surface of the respiratory system. The specific resistance develops already after 24 hours that in cases of some disease is crucial point in immunity development. In all trials the ultrasonic nebulizer (SONOVAC® 095) was used to deliver the water suspension of different virus vaccines. The only condition to use a vaccine was its composition that avoids the foaming effect that can hinder the efficacy of nebulisation. Our best investigations example are vaccination against Marek's disease (HVT FC 126), Newcastle disease (La Sota, Ulster 2C and Queensland V4), bivalent vaccines against Newcastle disease and infectious bronchitis (La Sota or B1 + H 120) and avian pox (attenuated chicken strain). The amount of given vaccine was calculated to be one dose per bird in exposition time of 30 to 60 seconds of and was related to the type of vaccine (virus strain). Prior to antibody development the vaccinated birds should stay in hatchery for at least 24 hours before being delivered to the farms. Nebulization is an effective and safe method of specific vaccination offering welfare to poultry.

Bacterial skin infections in captive desert lizards most commonly occur secondary to traumatic injuries or burns, or may be caused by inadequate housing conditions. These infections are manifested in the form of chronic dermatitis, cheilitis or occasionally septicemia, often associated with the presence of coryneform bacteria either as causative or as complicating factors. These infections have been so far most commonly attributed to bacteria from the genus Dermabacter ; however, a new species called Devriesea agamarum was recently isolated, predominantly from skin lesions of spiny-tailed lizards (Uromastyx sp.). The most common symptoms include scaly changes around the mouth and cloaca, and over the dorsal part of the body. Sometimes septicemia can occur. Complications may include a swelling of the front limbs with the loss of toes. The disease is a highly contagious and it could easily be spread over the whole collection of animals in a short period of time. To confirm the diagnosis it is necessary to take skin scrapings from dermal changes and to incubate them on Columbia agar supplemented with 5% sheep blood under microaerophilic conditions at 37oC. After the growth of specific colonies antimicrobial susceptibility testing is suggested. Blood hematology is necessary to exclude the septicemia. Regarding the therapy ceftiofur at a dose of 5 mg/kg BW in 24-hour intervals for 18 days, or ceftazidime at a dose of 10 mg/kg BW every third day for 15 days are effective, along with the local rinsing with 0.1% chlorhexidine solution. Secondary complications like swellings should be treated surgically. It is also important to clean the animal’s environment to prevent the spreading of the disease to other animals. Since D. agamarum-associated infections were not so far reported outside Belgium, we wanted to describe the most common symptoms, diagnostics and the treatment of this relatively unknown infection, to increase the knowledge about this contagious disease.

Both wild and captive reptiles are considered reservoirs of numerous zoonoses, the most important of which is salmonellosis. Although the share of reptile-transmitted salmonellosis is low, it continuously increases with the increasing number of reptiles kept as pet animals. Many European and other countries recognized the risk of increasing number and popularity of reptiles as pet animals for public health and introduced a systemic monitoring in both veterinary and human medicine. The aim of this study was to investigate the prevalence of Salmonella serovars in captive snakes in Croatia. In a period of two years a total of 146 pharyngeal, cloacal and fecal samples were taken from 90 apparently healthy snakes belonging to private owners. For laboratory cultures Brilliant Green Agar, Xylose Lysine Deoxycholate Agar and Triple Sugar Iron Agar as selective media were used and Salmonella strains were identified by serotyping. Salmonella was detected in a total of 10% snakes. A two different serovars, S. enterica subsp. arizonae and S. enterica subsp. diarizonae were the most prevalent and these serovars have already been described as infective agents in humans. To our knowledge, this is the first report about Salmonella prevalence in pet reptiles in Croatia, indicating that salmonellosis is a frequent phenomenon in captive reptiles in Croatia so that the risk of reptile-transmitted infections increases with increasing number of reptiles in captivity.

Proteus anguinus (the olm) is one of the most mysterious amphibians in the world and the only real cave dwelling vertebrate found in Europe. Due to its specific way of life in the underground carst, the olm is extremely vulnerable to all kinds of external pollution and destruction. For the same reason it is the animal that is very difficult to investigate and protect. On the other hand, in some parts of Croatia, heavy rains or waters from melted snow wash out the olms from their underground habitat to the surface of the ground. Due to the water evaporation the animals are not able to survive on the surface and dye, sometimes in excessive numbers. Having in mind their endangered status, the Croatian Herpetological Society, Zagreb Zoo and the Faculty of Veterinary Medicine, University of Zagreb started the project of the conservation of these animals. The role of veterinarians in this project is to find out whether it is possible to bring P. anguinus back to caves without harming the existing population, and also to increase the knowledge about the animal’s normal microflora since this data is missing. Depending on the findings, one of the aims would also be to establish the treatment of the animals based on the microorganisms found and on sensitivity tests. Therefore, the standard bacteriology tests for the detection of bacteria and fungi on the skin, cloaca and the pharynx, qPCR tests for the detection of Batrachochytrium dendrobatidis, Ranavirus and Chlamydophila sp., together with parasitological tests are underway. These tests will provide information about the normal microflora of the skin and gastrointestinal system, and will allow the discrimination between normal and opportunistic microorganisms. The analyses of the above pathogens will also provide the information whether is it safe to return the animals back to the caves, if it will be necessary. This will contribute to the whole project of olm conservation, to save this population and to preserve its health.

Radiography is a very important diagnostic tool use in evaluating chelonians (tortoises and turtles), because standard clinical examination, especially palpation, is limited due to their hard exoskeleton. For the proper radographic diagnosis dorsoventral (DV), lateral (L) and cranio-caudal (CrCa) projection are used. Common radiographic findings of chelonian skeletal system are fractures of the limbs, shell or skull. Standard DV and L projection are used. For carapace and plastron fractures CrCa projection is also recommended. Decreased opacity of the appendicular skeleton due to the decalcification, primary evaluated in the shell, pectoral girdle and/or pelvis, is a typical finding in metabolic bone disease (MBD). Foreign bodies are frequent radiographic finding in chelonian gastointestinal tract. For the accurate diagnosis and treatment it is essential to perform DV and L projections, which may be supported by contrast radiography, if necessary. Frequent pathological conditions of chelonian gential tract include dystocia (egg binding). Radiography made in DV (L is non obligatory) projection is very important for diagnosis and differentiation between obstructive and non-obstructive dystocia in order to determine the appropriate treatement. To evaluate the respiratory tract of chelonians it is required to take images in CrCa and L projection. Common pathology of respiratory tract is pneumonia, characterised by focal or diffuse opacities in the lungs area. CrCa view is neccesary to differentiate unilateral and bilateral pneumonia. Because of the risk of overlooking some pathological changes, the selection of adequate projection is of great importance for proper radiographic diagnosis, due to chelonians specific anatomy and physiology.

In the autumn 2010 we investigated an outbreak of pox infection complicated with Chlamydia psittaci (C. psittaci) in a flock of free-ranged laying hens. Altogether 300 birds, kept as a backyard flock, started to show clinical signs of upper respiratory tract infection and severe purulent conjunctivitis was noticed in most of the birds. Mortality rate amounted up to 10% daily. Three laying hens were sacrificed and the samples were submitted to laboratory testings. The aerobic bacteriological examination revealed the presence of Bacillus sp. and Staphylococcus sp. in conjuntival, choanal and tracheal swabs. The conjunctival and pharyngeal swabs of two sacrificed laying hens were examined by Chlamydiaceae-specific real-time PCR, as described by Ehricht et al. (2006.) and by C. psittaci-specific real-time PCR, as described by Pantchev et al. (2008.), and reacted positive to both methods. Samples were also examined by inoculation in chicken embryos and the presence of pox virus in the chorioallantoic membrane was confirmed using specific PCR (Tadese et al., 2007.). The entire flock was treated with enrofloxacin (Enroxil 10%, Krka, Slovenija) via drinking water for 14 days and the symptoms resolved. Presence of poxvirus probably contributed to the severity of clinical signs caused by C. psittaci and enhanced the infection. Nevertheless, the pathogenicity of this C. psittaci strain remains indistinct, and we can only speculate that it could be higher then of other strains, infecting poultry. The infection route remained unclear, but due to the free-range production it could be possible that wild birds served as a source of infection or the pathogens persisted in the environment. The finding of C. psittaci in laying hens is of enormous importance from two standpoints: 1) significance for public health as a dangerous zoonosis, and 2) presence of C. psittaci in chickens which are supposed to be less prone species to this infection.

Salmonella enterica serovar Entertitidis (SE) is one of the main causes of food borne diseases, transmitted by eggs or egg products. Egg contamination by SE is the result of both horizontal and vertical SE transmission. In the two trials two lines of commercial laying hens at onset of the lay were orally inoculated with a single dose of 1.2x109 CFU of highly pathogenic SE. Laid eggs were collected during the 40 days post-inoculation and examined for the presence of SE on egg shells and in egg contents, by both-standard ISO method and realtime PCR. Positive eggs were laid only in narrow period of time, and in low numbers (3.0% and 0.4% respectively), showing high concordance with literature data and previous conducted studies of salmonella-contamination in salmonella-positive laying flocks.

Chlamydia psittaci is the causative agent of avian chlamydiosis, and represents a significant problem in veterinary medicine all over the world. Infection in humans is also regularly reported, especially in those closely connected to infected birds and in persons of specific occupations (veterinarians, laboratory workers, employees in poultry slaughterhouses, zoo and pet shops workers, wildlife rehabilitators). Some authors suggested that the spectrum of Chlamydiaceae spp. encountered in birds is not confined to a single species. Chlamydial infections in poultry are misdiagnosed due to symptoms similar to those caused by other pathogens, or very complex diagnosis which is also hazardous for laboratory personnel. New chlamydial agents with unknown zoonotic potential were recently detected from domestic poultry in Germany and France. Progress of diagnostic methods has also resulted in findings of some new cases of atypical chlamydial infection in hens and turkey flocks in Croatia. In different geographically part of Croatia tracheal and fecal samples were taken from two hens flocks and one turkey flock, and by using a Chlamydiaceae-specific real-time PCR assay targeting the 23S rRNA gene were found positive. Surprisingly, these PCR positive samples didn’t react with any of the C. psittaci specific tool targeting the ompA gene as well as C. abortus and C. pecorum specific tools. As recently novel Chlamydophila spp. strains have been isolated from poultry and as a specific real-time PCR has been developed targeting the conserved 16S gene and sequencing of ompA gene, the presence of novel Chlamydia sp. strain was also confirmed in turkeys and hens in Croatia. This report is our contribution to statement that the spectrum of Chlamydiaceae spp. encountered in poultry is not confined to a single species, and further investigations are needed in attempt to clarify the role of this microorganism in poultry and prevention of potential illness in humans.

Contrast radiography examination in avian patients is very important procedure for assessment of the gastrointestinal tract. A survey radiographic image in laterolateral (LL) and ventrodorsal (VD) projections should always be obtained before every gastric examination. The indications for contrast radiography of gastrointestinal tract include all of the pathological conditions characterized by morphological changes in the gastrointestinal tract or its wall. It is also used for differentiation neighboring, pathological altered organs (liver, spleen, gonads and kidney). Standard barium sulfate is often used as contrast medium, administered as 25 – 45 % suspension, in dose of 20 ml/kg body weight. Frequently diagnosed changes in the gastrointestinal tract include abnormal contents (foreign bodies, gas), displacement of all parts of the gastrointestinal tract, changes in its form or size and increased or reduced peristalsis. Timing of the radiographic exposures is based on many factors, but all studies should include LL and VD projections done immediately after the administration of the contrast medium. Transit time of contrast medium varies, depending on birds size, age, required diet, nutrition condition, contrast material, effects of systemic disease, etc. It is very difficult to propose right timing for exposures. If contrast medium has entered the intestines on the immediate postcontrast radiographs, the next images are taken in 15 minutes. Radiographs are then made every 30 minutes until contrast medium has entered the colon. In most common avian patient, budgerigar (Melopsicattus undulatus), mean time of passage the contrast are: crop 5 – 15 minutes, stomach 15 – 45 minutes, small intestines 45 – 120 minutes, colon 90 – 120 minutes and cloaca 180 – 240 minutes. Contrast radiography is needed, useful and strongly recommended as simple diagnostic tool for differentiation of many pathological conditions that affect gastrointestinal tract and other related internal organ system.

Infection caused by Salmonella, associated with egg and egg products in Europe represent the majority of the reported food-borne outbreaks (EFSA and ECDC, 2012). In EU the laying flocks should be free from S. Enteritidis and S. Typhimurium, and consequently, the product itself should be free from these serotypes. A major obstacle in control of Salmonella in eggs is an apparent low number of positive eggs (EFSA, 2007 ; Chemaly et al., 2009). The aim of this investigation was to better understand the mechanism behind the low occurrence of contaminated egg and to compare detection of Salmonella by PCR and most commonly used conventional ISO-culture methods. At 18 wk of age, 60 hens were infected in two replicate trials with oral doses of approximately 109 cells of S. Enteritidis. Shedding and colonization were measured at regular time points during 6 weeks. All laid eggs were examined for Salmonella by both, ISO 6579:2002 and PCR methods. A 5’nuclease Real-Time PCR assay targeting the ttrRSBCA locus of Salmonella spp. was applied and internal amplification control was also included in reaction, as described by Malorny et al., 2004. and Kramer et al., 2011. After infection the inoculated bacteria were recovered in all hens. Despite the fact that all hens became infected, only 8 eggs shells and one egg yolk were Salmonella positive. Surprisingly, the positive eggs were shed during a very narrow time period. Direct plating methods take several days, are expensive and labor intensive, and usually can detect only high number of bacteria. Problems related to low prevalence can be minimized if more sensitive and specific detection methods are applied for which molecular method, such as PCR, have been introduced. The method was also much quicker than conventional techniques, taking less than 24 h to obtain a result. In conclusion, both literature, and our experimental investigations of contamination of eggs from positive hens have shown that the number of contaminated eggs is low, and that the PCR was superior to ISO 6579:2002.

Protection of consumers by microbial risk mitigation through combating segregation of expertise (acronym PROMISE) is name of the EU funded project (under FP7 programs) which strategic objective is to improve and increase the integration, collaboration and knowledge transfer between the Member States including Republic of Croatia, through a collaborative workplan of exchange of expertise and dissemination actions, to tackle common food safety threats and hence to protect the European consumer. PROMISE project deal with the elucidation of neglected sources of transmission of pathogens and the respective modeling of data streams. While legal imports are well monitored for contaminations and alerts are registered through the RASFF notification systems, illegal personal food imports from all continents are not monitored at all. These uncontrolled imports present the risk that new strains of traditional pathogens will be transferred from third countries into the European Union. Analyzing, assessing and interpreting this risk of introducing new strains of pathogens are one of the main objectives of PROMISE.

Veza čovjeka s pticama, pogotovo kućnim ljubimcima, značajno se povećala posljednjih godina. S tim u vezi i bolesti koje se mogu prenjeti s ptice na čovjeka postale su značajnije. To se odnosi na broje virusne, bakterijske, gljivične i parazitarne bolesti, no skrenut ćemo pozornost samo na one od kojih je čovjek u najčešćoj opasnosti kada je u neposrednoj blizini zaražene ptice. To su: klamidioza, salmoneloza, kampilobakterioza, pseudotuberkuloza, tuberkuloza i newcastlevska bolest. Mnogo rjeđe čovjek može oboliti od kolibaciloze, vrbanca, listerioze, streptokokoze, influence, toksoplazmoze, kriptosporidioze. Samo izuzetno ptica može za čovjeka biti izvor: encefalitisa, bradavičavosti, antraksa, aspergiloze, histiplazmoze, Q groznice, sarkosporidioze ili trihofitoze. O pojavi ovih zoonoza, sustavnih epidemioloških istraživanja u Hrvatskoj za sada nema, no iz literaturnih podataka i iz podataka WHO-a za nama susjedne zemlje, vidljivo je da se moramo pripremiti i educirati veterinare i o ovoj vrsti životinja.

Salmonella infections appear to be one of the most typical examples of an enteric disease that is transmitted from animals to humans. Biochemical, serological and phage typing diagnostic methods of Salmonella infections in man and animals are traditional. Unfortunately they are not enough reliable to establish differences among isolates and can not satisfactory serve in epidemiological study.Genotyping analysis of the Salmonella by plasmids and chromosome DNA restriction profiles (RFLP) has been widely used in recent years.Altogether 63 different strains of Salmonella spp. were analysed regarding plasmid content and restriction pattern of the ones containing plasmid. Bacterial strains were biochemically confirmed by using the API20E system (Bio-Merieux, France) and were serologically characterised by the slide agglutination test with Salmonella polyvalent O and group H antisera (Institute of Immunology, Zagreb, Croatia). The most strains were identified as S. enteritidis (20 human and 20 chicken isolates), while the reaming 23 strains were S. virchow, S. lichfield, S. hadar, S. typhimurium and S. gallinarum-pulorum. Plasmids harboured by Salmonella were extracted by the method of Birnboim and Doly. They were separated in horizontal 0.8% (wt/vol.) agarose gels in 1xTAE buffer at 80V for 1h and visualised by staining with 0.5 mg of ethidium bromide per ml. Total plasmid DNA was digested with EcoRI (Boehringer Mannheim Biochemicals) according to the manufacturer's recommendations. There was no differences obtained pattern for human S. enteritidis strains. So restriction enzyme digests of several strains were coprecipitated (0.3 M Na-acetate, 2V EtOH, incubated 30 min at –70 C, centrifuged at 13000g, 30 min at +4 C) and subsequently electrophoresed in a 0.8% (wt/vol.) agarose gel in 1xTAE buffer at 80V for 1h. They were visualised by staining with 0.5 mg of ethidium bromide per ml. The same was used for S. enteritidis isolated from chicken.Analysing plasmid profile of all strains investigated, 9 did not have any plasmid, 51 strains had only one of 25 kpb, and 3 had an additional plasmid of 2.5 kpb. Plasmid profile of human S. enteritidis strains compared to chicken ones did not reveal significant differences. Restriction pattern of S. enteritidis either human or chicken origin digested with EcoRI showed differences according to origin. Plasmid analysis could not be used as sole tool for epidemiological study, probably because of plasmid instability that can be loosed during manipulation in the laboratory. Plasmid restriction analysis shows differences between strains of different origin. To be able to determine relationship between the strains more precisely restriction pattern analysis of total DNA is now being used. The sensitivity of this method is increased with the number of enzymes used in the analysis.

Obligate intracellular bacterium Chlamydia psittaci (C. psittaci) is an important etiological agent in turkey respiratory diseases. It is commonly found in Europe, causing economical losses and present a possible threat for human health. Health problems occurred on the fattening turkey’s farm, particularly related to the respiratory system over several cycles of fattening. The first clinical signs have been noticed mostly at the age of 2 weeks and lasts until 17 weeks of age. Turkeys were depressed and lethargic with respiratory signs. Some of them showed unilateral swelling of the sinuses, conjunctivitis, dyspnoea, coughing and nasal discharge. At the necropsy, lesions were present in the upper and lower respiratory tract. The samples taken from dead animals were negative for most present agents of respiratory turkey diseases, both on viruses (TRT) and mycoplasma, but E. coli infections appeared on the farms. Antimicrobial therapy was conducted by using flumequine and oxytetracycline, but the clinical signs were still present. Because the symptoms were still present, two samples from trachea were taken and were found positive on Chlamydiaceae realtime PCR, but they didn’t react with C. psittaci, as well as C. abortus and C. pecorum specific diagnostic tools. As recently novel Chlamydia sp. strains has been detected from poultry and as a specific realtime PCR has been developed, the analysis of these two samples showed relatedness to newly isolated Chlamydia. Sequencing of ompA and 16S gene confirmed this data, as well as first isolation of novel Chlamydia sp. strain from turkeys in Croatia.

Probavni sustav peradi sastavljen je od vrlo složene mikroflore u kojoj pretežu bakterije. Odsustvo fiziološke mikroflore probavnog sustava preduvjet je za razvitak brojnih bolesti koje direktno utječu i na ljudsko zdravlje. Obzirom da se danas zna da preventiva antibioticima u uzgojima peradi čini više štete nego koristi, istražuju se brojni prirodni pripravci kao dostojna zamjena antibioticima. Kao biološki aktivni pripravci danas se koriste ili istražuju kompetitivna ekskluzija, probiotici, prebiotici, antibakterijski peptidi, žumanjčana protutijela, fitogeni aditivi i beta glukani.

Bacteria E. coli is important comensal in gastrointestinal tract of animals, including poultry. In parrots normal microflora represents pleomorphic Gram positive bacteria. Enterobacteriaceae and E. coli can not be frequently isolated from healthy pet birds. Imbalance of intestinal homeostasis in parrots can appears as a consequence of stress mechanism, but also of improper nutrition. Common source of E. coli for parrots represents contaminated food, water and other birds. Altogether 13 pooled fecal samples obtained from 41 pet birds of different ages and species were taken on 4 different places, from private owners and from breeders. In total, 12 bird species were examined ; 9 belonged to family Psittaciformes, and 2 to Passeriformes. The samples were taken from outdoor cages and from ones kept in owner's houses. All the samples were taken from clinically healthy birds. Bacteriological examination revealed the presence of E. coli in one sample, from two parrots of the species ring-necked parakeet (Psittacula krameri), while Bacillus sp., Staphylococcus spp. and Streptococcus spp. were isolated from remaining samples. Findings of E. coli in these two parakeets can be related to the co-habitation near thewith poultry in an outdoor cage, since the parakeets were kept in good condition, but in outdoor cage, near other poultry. The antimicrobial susceptibility of isolated strain of E. coli was determined by disk diffusion test. The strain was resistant to 6 out of 9 examined antimicrobial agents, and intermediate resistant to the enrofloxacin, doxycycline and tetracycline. From the anamnestic data there was no evidence of antimicrobial treatment of examined parrots so it could be concluded that this isolated strain originated from poultry, often treated with antibiotics. Good management practice are extremely important to obtain bird's health, and it should be stated that uncontrolled use of antibiotics lead to development of unnecessary resistance, that could be spread between species.

Bacteria and fungi isolated from pharyngeal and clocal swabs mainly represent normal microflora, but higher number of E. coli, Campylobacter sp. and Aspergillus fumigatus isolates could represent a possible danger for immunocompromised patients. High prevalence of C. psittaci positive pigeons in the City could represent possible danger for the citizens.

prvo pravilo pri hranjenju bolesnih gmazova je svakodnevno poticanje samostalnog hranjenja, stavljanjem hrane pred životinju. Ako životinja ne počne sama jesti, treba je početi hraniti na umjetan način. Gmazove je najbolje hraniti metalnom ili gumenom sondom, unoseći hranu duboko u jednjak ili izravno u želudac. Od svih gmazova najteže je umjetno hraniti i davati terapiju per os kornjačama. Kada kornjača uvuče glavu u oklop, pristup usnoj šupljini je vrlo težak. Glava se iz oklopa mora izvlačiti vrlo oprezno jer su vratni kralješci kornjače spojeni s karapaksom pa se agresivnom manipulacijom mogu ozlijediti. Da bi se olakšalo davanje lijekova i hrane kronično bolesnim kornjačama, čije liječenje traje vrlo dugo, najbolje je postaviti ezofagostomalni (ES) tubus. Odabere se meki gumeni kateter, markerom označi njegov dio do visine želuca životinje, zavinutim se hemostatom uđe kroz usnu šupljinu i jednjak do kaudolateralnog dijela vrata, vrhovi se hemaostata okrenu tako da postanu vidljivi s vanjske strane vrata i između krakova hemostata napravi incizija. Hemostatom se uhvati kraj mekanog katetera, uvuče u lumen jednjaka i usmjeri u želudac te uvuče do oznake. Oko mjesta ulaska katetera postavi se flaster u obliku leptira i pomoću njega kateter zašije za kožu, a ostatak tubusa se zalijepi s unutarnje strane plastrona na oklopu. Tubus može na svom mjestu ostati i do tri mjeseca. Pri vađenju odrežu se šavovi kojima je tubus zašiven, a životinja se nekoliko dana stavlja na antibiotik.

Most of diseases, with the exception of trauma, originate from inadequate animal housing or feeding. Such diseases develop for a long time and exist long before the first symptoms become visible to the owner. In other words, urgent cases in reptiles are usually the acute manifestations of chronic problems that require a long-term treatment and hospitalization of the patient. 1. At the presentation of diseased reptile it is of utmost importance to identify the species to which it belongs, so that the animal can be housed under optimal conditions that would facilitate its recovery. 2. Heat: This is the most important factor for the care of diseased animal. The reptiles are ectothermic animals that are completely dependent on the external sources of heat so that the assurance of optimal body temperature positively influences the immune system function and accelerates metabolic processes in the body. Prior to administration of any medications, the animal should be gradually warmed to their preferred optimal temperature. 3. Fluid therapy: It is essential in the treatment of diseased reptiles. Reptiles synthesize the water slower than mammals and the negative water balance in the body negatively affects all of the body systems. The ways of fluid administration and the choice of fluids depend on the patient's condition. 4. Feeding: Since the reptiles occasionally have been fasting for a long time before the visit to the clinic, it is not advisably to start their feeding immediately ; it is better to re-hydrate them first. After re-hydration the reptiles are encouraged to take the food independently, or they are fed by disposable or permanent tubing. 5. Further treatment: The choice of the further treatment depends on the type of pathology that is determined by a number of diagnostic procedures including microbiological swabs, x-rays, ultrasonography, blood tests etc.

Subcutaneous tumor in a 17-year-old female cockatiel is described. The yellowish, elliptic tumor measuring 4×3 cm was noticed by the owner of this bird, on the right side of the cloaca. The skin was damaged and the wound was bleeding. After the clinical examination, surgical removal of the tumor was suggested. Histopathological examination revealed the presence of a xanthoma. In a period of one year, neither the reoccurrence of the skin xanthoma nor any health problems were observed.

Chlamydiosis is a zoonosis caused by intracellular bacteria Chlamydophila psittaci which represents a significant problem in veterinary and humane medicine all over the world. More the 467 different bird species can be carriers of the pathogen. This zoonosis is very important in parrots and pigeons, but it still is an important disease in the intensive and especial free range breeding of turkey, geese, ducks and chickens. The unique biphasic lifestyle includes parasitic intracellular and infective extracellular phase, renders the respective diseases difficult to control. In the Republic of Croatia, prevention and the illness control is carried out according to the law provisions (mandatory quarantine, intrastate movement of infected birds, e.i.). The national laboratory for the diagnosis of Chlamydia (CHLAMLab) was established in 2006 and in 2011 received accreditation. During the years samples have been collected from various species of wild, pet birds and domesticated fowl. A variety of assays and techniques that directly detect antigen in tissue and swab samples from birds are available but we suggest using a real-time PCR method as a standard. I our laboratory the TaqMan 5' nuclease PCR method was used described by Ehricht et al. (2006), based on detection of 23 rRNA gen and reaction was carried out in Mx3005P cycler (Stratagene, USA). Positive samples have been detected from a wide range of avian species and the estimate of infection varies from 2.2% to 28.0 %. Depending on the flock size and housing conditions, positive birds were treated therapeutically with different antimicrobial agents (tetracycline or enrofloxacine) and bio-security measures (quarantine, regular cleaning and disinfection) were performed in all positive cases. After the treatment, the birds were examined until negative results were obtained. Our results clearly point the need for urgent and certain diagnostics, as a postulate for protection of human health, especially the ones working with the birds. If the birds are showing any respiratory or gastrointestinal symptoms, chlamydiosis should be tested. Not only the number of the birds positive for chlamydiosis but also the data on the people fallen ill with psittacosis showed that this infection is not only a problem of veterinary service, but also a public health service problem.

Bolest u ljudi prouzročena bakterijama roda Campylobacter spp. predstavlja značajan problem javnog zdravstva diljem svijeta, s iznimnim sociološko-ekonomskim učinkom. Meso peradi smatra se glavnim izvorom termofilnih kampilobaktera, posebno ono svježe, a ljudi se najčešće zaraze prilikom pripreme ili jedući ga nedovoljno termički obrađenog. Smanjujući broj zaraženih jata peradi ili smanjujući broj bakterija na trupovima peradi u klaonici, smanjuje se i rizik za potrošače, a sve se to može učiniti primjenom strogih biosigurnosnih mjera u primarnoj peradarskoj proizvodnji. Bakterije roda Campylobacter često su neosjetljive na brojne antimikrobne tvari, što je posljedica nekontrolirane uporabe antibiotika, posebice u tovu pilića. Stoga, uporaba lijekova u peradarstvu mora biti ciljana i pod strogim nadzorom stručnjaka, a istraživanje osjetljivost na najčešće primjenjivane pripravke sustavno i temeljito.

The infection caused by Chlamydophila psittaci (C. psittaci) has to be mandatory reported in Croatia, and previous investigations revealed frequent presence of C. psittaci in birds, especially in city pigeons, but also in turkeys and laying hens. Because of the zoonotic potential of C. psittaci, it is important to make a rapid, definitive diagnosis in birds. Most commercially available tests are based on monoclonal antibodies against genus-specific epitope of chlamydial LPS, like ELISA test, but from the previous investigations it is well known that sometimes non-specific positive results can appears. Altogether 411 samples (feces, cloacal or so called triple swabs-conjunctival, pharyngeal and cloacal swab together) were taken from pigeons, parrots, canaries and finches. The samples were taken in pet shops, at the breeders, in the Clinic for birds of the Faculty of Veterinary Medicine University of Zagreb, and in the ZOO. The birds were examined for their health status or as sick ones in the Clinic for Birds. From that, 169 samples were from racing pigeons, 177 from parrots, 58 from canaries and 7 from finches. For the detection of specific C. psittaci antigen commercial ELISA kit was used- IDEIATM PCE Chlamydia kit (DAKO Cytomation Ltd., UK). The samples that were by ELISA test described as suspicious positive were also examined by PCR as described by Hewinson et al. (1997). Total DNA was extracted by commercial DNeasy® Blood & Tissue Kit (Qiagen, USA). The expected PCR amplicon were at 264 bp corresponding to the ompA gene. All of the 411 samples were examined by ELISA test to reveal the presence of specific antigen of C. psittaci. Out of them, 70 were positive (17.03%). Regarding the sampling place, majority of the positive samples were found at the breeders (18.91%), in the pet stores (17.39%), while there was only few positive samples from the Clinic and ZOO (both 8.33%). According to the bird species, the most positive were pigeons (23.08%), followed by canaries (15.52%) and parrots (12.43%). All the samples from finches were negative. Extremely high titre of positive samples obtained by ELISA test were considered as suspicious due to the fact that LPS antigens of some other bacteria can cross-react with monoclonal antibodies specific for chlamydial LPS. For that reasons, all suspicious samples (26) were also examined by PCR. Out of 18 suspicious samples from the pigeons only one was described as PCR positive. On the other hand, 8 suspiciously high positive samples from the parrots and canaries were PCR positive in 75% and 100%, respectively. Finding of 17.03% positive samples can represent a potential threat for human health, but has also confirmed the need for monitoring of the prevalence of Chlamydia in different bird species, both at the breeders and in pet stores. The PCR has been shown as the method of choice when is necessary to clarify nonspecific or suspicious results, obtain by other methods.

Successful improvement of the Croatian poultry production, particularly of the family farms, demands availability of the so-called knowledge capital. Based on this assumption, the research is focused on the analysis of the existing poultry formal and non formal educational system and need for general and specialized education of the poultry producers. The analysis shows that current practice in formal education system and transfer knowledge does not demonstrate widespread integration of management, marketing, food quality and environmental topics into poultry educational programs. There is a comparatively small number of additional educational forms and intellectual services for farmer professional training and permanent education. This demands the development of a flexible and multy-functional education subsystem based on setting up of functional relations between knowledge and demand of modern poultry production, aimed at strengthening of the basic know-how and capacities, encouraging of initiatives, realistic synergy compatible with the dynamical nature of the scientific and technological achievements. It is necessary to incorporate inter-disciplinary approach into vocational educational programs and to expand public and private self-education sources for poultry farmers.

Bacteria Campylobacter spp. are the leading causes of acute bacterial gastroenteritis all over the world. Most of the human outbreaks are related to preparation and/or consummation of poorly cooked poultry meat. Overuse of different antibiotics in poultry production caused increasing number of resistant strains that could be also transmissioned to human. The antimicrobial susceptibility to azithromycin, ciprofloxacin, enrofloxacin, erythromycin, gentamicin and tetracycline of altogether 56 C. jejuni and C. coli strains isolated from 616 samples of poultry of different species, age and production type were detected by using three methods –disc diffusion, agar dilution and E test. The highest in vitro resistance by using disk diffusion method was observed to azithromycin (from 90.9% up to 100%), erythromycin (from 63.3% up to 65.4%), but also to tetracycline (from 65.4% to 76.6%) and gentamycine (from 11.1% up to 62.5%). The study determined also the high frequency of resistant Campylobacter strains (from 40% to 43.3%) to enrofloxacin and ciprofloxacin. Altogether 20 strains were multiresistant, to two or more antibiotics. Out of them, four C. jejuni strains isolated from toms, two from parent turkey flocks and 12 strains isolated form broilers of 10 weeks of age were resistant to all five antibiotics. The highest sensitivity was observed to gentamycin and eritromycin. Correlation between disk diffusion and agar dilution method revealed statistically significant results, and disk diffusion method can be used as relevant for the antimicrobial susceptibility testing. High percentage of resistant strains to all antibiotics tested, revealed the uncontrolled use of medicaments in poultry production. Antimicrobial agents will continuously be used in human and veterinary medicine, but it should be judiciously and under control, together with routinely susceptibility testing.

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.

Iskustvo stečeno radom u Ambulanti za ptice pri Zavodu za bolesti peradi Veterinarskog fakulteta u Zagrebu autorica je pretočila u ovu knjigu. Štivo je potkrijepila originalnim fotografijama iz bogate vlastite zbirke. Opisala je osnovna načela držanja i hranidbe ptica, rukovanja prilikom kliničkog pregleda i terapije. Opisala je najčešće bolesti i učestale ozljede, a posebnu pažnju posvetila je zoonozama. Čitatelj također doznaje podatke o osnovnim anatomskim i fiziološkim osobitostima te o specifičnostima liječenja i anestezije ptica kućnih ljubimaca.

Bakterije roda Campylobacter uobičajeni su komenzali u crijevima zdravih kokoši, no vrlo često ova bakterija uzrokuje bolest u čovjeka. Broj prijavljenih slučajeva kampilobakterioze u ljudi raste zadnjih godina u čitavom svijetu, pa je tako i u Hrvatskoj druga po redu najučestalija zoonoza - toksoinfekcija koja se prenosi hranom. Epidemiološke studije pokazuju da je meso peradi najčešći izvor infekcije ljudi ovom termofilnom bakterijom. Nekontrolirana uporaba antibiotika, posebice u tovu pilića sve češće dovodi do razvoja rezistencije uzročnika. Obrisci kloake, porijeklom od kokoši različitih dobnih i proizvodnih kategorija, pogodni za izdvajanje kampilobaktera, uzeti su na farmama i drugim objektima u Republici Hrvatskoj, te su dostavljeni na pretragu u bakteriološki laboratorij Zavoda za bolesti peradi s klinikom. Izdvojeni sojevi kampilobaktera pretraženi su difuzijskim postupkom na osjetljivost prema šest antimikrobnih tvari: azitromicin, ciprofloksacin, enrofloksacin, eritromicin, gentamicin i tetraciklin. Istraživanje osjetljivosti bakterija roda Campylobacter spp. izdvojenih iz kokoši dokazalo je pretpostavljenu njihovu vrlo visoku rezistenciju na antibiotike (od 21, 05 % pa sve do 100, 00%). Uporaba fluorokinolona u liječenju peradi u Hrvatskoj vrlo je učestala, pa rezistencija istraživanih sojeva kampilobaktera (od 21, 05 % pa do 61, 11 %) na enrofloksacin i ciprofloksacin nikako nije zanemariva. U našem istraživanju visoka rezistencija kampilobaktera očitovana je i na eritromicin, azitromicin i gentamicin (od 52, 63 % za C. jejuni pa do 100, 00 % za C. coli) koji su vrlo važna sredstva izbora u liječenju ljudi od kampilobakterioze. Svjesni smo da ćemo još dugo morati koristiti lijekove u liječenju bakterijskih bolesti peradi, no njihova uporaba mora biti ciljana i pod strogim nadzorom stručnjaka, a sustavno istraživanje njihove osjetljivost na najčešće primjenjivane lijekove je dužnost znanstvenika koji se bave ovom problematikom.

Wild birds can carry a wide range of viral and other zoonotic agents, which may be transmitted to humans. From October 2005 to March 2006 HPAI H5N1 virus was isolated from wild birds (mute swans, black-headed gulls and a mallard duck) in Croatia at five locations. After isolation of H5N1 virus at 2006 from mallard duck near City of Zagreb (capital of Croatia) Department of Poultry Diseases with Clinic at the Faculty of Veterinary Medicine, has conducted monitoring of avian viruses that could endanger human health. Samples (999 pharyngeal and cloacal swabs) from 23 wild bird species were taken. After year 2006 Croatia has regular monitoring for avian influenza in wild birds and poultry (especially in the backyard flocks). During 2007 (6, 928 wild birds and 18, 000 blood samples from poultry) and 2008 (2, 486 wild birds ; 20, 000 blood samples and 1, 500 cloacal swabs from poultry) were taken. Isolation was performed with classical virus detection method by inoculation of 10 day old chicken embryos, and molecular methods by conventional PCR and Real Time PCR (M gene, H5, H7 and N1 genes), and serological methods by antibody detection from blood samples (inhibition hemagglutination and ELISA). All samples were HPAI virus negative but investigators from the Poultry Centre of the Croatian Veterinary Institute isolated from wild birds LPAI viruses: H2N3, H3N8, H5N3 and H10N7. The results obtained by these investigations and monitoring revealed the need for permanent monitoring of wild bird’ s health status, especially the water birds species. Vaccination against AI is never practiced in Croatia. Quick and accurate detection of wild migratory birds infected with the H5N1 virus prevented the spread of the virus to the domestic poultry in Croatia which would have had enormous consequences.

The aim of the study was to evaluate the presence of clinically relevant Campylobacter spp. in poultry in Croatia. The zoonosis campylobacteriosis, caused by thermophilic Campylobacter spp., is an important public health problem in most areas of the world. Poultry meat and products appears to be a major source of campylobacteriosis through cross-contamination of ready-to-eat foods, direct hand-to-mouth transfer during food preparation, and to a lesser extent, through the consumption of undercooked poultry meat. Campylobacteriosis is the second reported food-borne infection in Croatia but the national action plan for surveillance of campylobacter doesn’t exist, and epidemiological studies weren’t conducted so far to reveal the real prevalence of these bacteria in poultry. Cloacal swabs were taken on the farms from altogether 91 broiler chickens and 28 laying hens at the end of the laying period. All swabs were subjected first to bacteriological examination using modified Charcoal Cefoperazone Desoxycholate Agar (mCCDA) and incubated in microaerobic atmosphere. The identification of species was obtained by the multiplex PCR (mPCR). Altogether 30 isolated strains (13 C. jejuni and 17 C. coli) were subjected to antimicrobial sensitivity testing using the disk diffusion method. Antimicrobial agents on disk and concentrations were as follows: azithromycin (15 μg, Bio-Rad, USA), ciprofloxacin (5 μg, Bio-Rad, USA), enrofloxacin (5 μg, Bayer, Germany), erythromycin (15 IU, Becton Dickinson, UK), gentamicin (10 μg, Bio-Rad, USA), and tetracycline (30 μg, Bio-Rad, USA). From 91 chicken swabs 35 (38.46%) isolates were found mPCR positive, whereas 18 were detected as C. coli and 17 as C. jejuni. Out of 28 laying hen swabs, 14 were Campylobacter spp. positive, and by mPCR 8 (57.14%) were identified as C. coli, and 6 (42.86%) as C. jejuni. The study determined the high frequency of resistant Campylobacter strains (from 46.15 % to 100 %) to all five tested antibiotics. Fluoroquinolones and other antimicrobial agents will continuously be used in human and veterinary medicine, but it should be judiciously and under control, together with routinely susceptibility testing. The relatively high number of campylobacter positive samples points that the approach to the control of campylobacter in Croatia should be changed and that the Action plan, similar to others in European Countries is needed, to assure healthy and safe poultry meat for consumers.

Chlamydiosis is a zoonosis caused by intracellular bacteria Chlamydophila psittaci (C. psittaci), which represents a significant problem in veterinary and humane medicine all over the world. Numerous wild and domesticated bird species fall ill, and the diagnostics, compared to many other bacterial illnesses, is very complex and dangerous for laboratory personnel. In the Republic of Croatia, prevention and the illness control is carried out according to the law provisions. The national laboratory for the diagnosis of Chlamydia was established in 2006. During the 2007 and 2008 samples have been collected from various species of wild and domesticated fowl. In order to confirm the infection of birds by Chlamydia, samples (droppings, the swap of conjunctiva, pharynx and cloaca, the organs of perished birds) were examined by means of different diagnostic procedures ; the methods applied were ELISA, Clearview test, conventional PCR and Real Time PCR. Of the totally 1789 examined samples, 232 (12.9 %) were positive. Positive samples has been detected from a wide range of avian species, but especially positive turkey and laying hens flocks suggested that further investigations are needed in attempt to detect the role of Chlamydia in clinically respiratory syndrome. Depending on the flock size and housing conditions, positive birds were treated therapeutically with different antimicrobial agents (tetracycline or enrofloxacine) and bio-security measures (quarantine, regular cleaning and disinfection) were performed in all positive cases. After the treatment, the birds were examined until negative results were obtained. Not only the number of the birds positive for chlamydiosis but also the data on the people fallen ill with psittacosis during the period from 1997 through 2008 showed that this infection is not only a problem of veterinary service, but also a public health service problem.

Ptice burno reagiraju na svaki stres, a nerijetko izazvana bol u postupku liječenja dovodi do šoka te posljedično uginuća ptice. Stoga se u ptica anestezija primjenjuje vrlo često ne samo radi kirurških zahvata, već ponekad i kod kliničkih pregleda te primjene lijekova. Mogući postupci brojni su, no svode se na davanje anestetika parenteralno, ili što je u ptica mnogo sigurnije, inhalacijom. U radu su opisana vlastita iskustva i iskustva drugih autora u primjeni različitih anestetičkih sredstava i postupaka u ptica. Posebno je obrađeno područje potpore, stabilizacije i premedikacije pri primjeni anestetika, a opisane su i specifične životne funkcije ptica. Navedeni su mogući postupci i učinci onih anestetika koji se najčešće koriste u praksi i koji se primjenjuju u Ambulanti za bolesti ptica pri Zavodu za bolesti peradi, Veterinarskog fakulteta u Zagrebu. Obzirom da je anestezija ptica po mnogočemu specifična, opisane su i specifičnosti pri oporavku pacijenata, navedena je procjena rizika, te specifične potrebe nekih dobnih kategorija i vrsta ptica. Posebno se ističu povoljni učinci inhalacijskog anestetika izoflurana, ekstrapulmonalnog disocijacijskog pripravka ketamina, te lokalnog anestetika lindokaina.

Klamidioza je zoonoza uzrokovana intracelularnom bakterijom Chlamydophila psittaci (Cp. psittaci), koja predstavlja značajan problem u veterinarskoj i humanoj medicini diljem svijeta. Obolijevaju brojne vrste divljih ptica i peradi, a dijagnostika je za razliku od mnogih drugih bakterijskih bolesti vrlo složena i opasna po laboratorijsko osoblje. U Republici Hrvatskoj se preventiva i suzbijanje bolesti obavlja prema zakonskoj odredbi. Prikupljeno je 1068 uzoraka od različitih vrsta ptica i peradi iz različitih županija Republike Hrvatske. Da bi dokazali infekciju ptica klamidijama, uzorci (izmet, obrisak konjunktive, ždrijela i kloake, organi uginulih ptica) pretraženi su različitim dijagnostičkim postupcima. Korištene su metode ELISA, Clearview test i PCR. Od ukupno 1068 pretraženih, 178 uzoraka (16, 67 %) je bilo pozitivno na bakteriju Cp. psittaci. Prema županijama najveći broj zabilježenih pozitivnih uzoraka potječe iz Požeško-slavonske županije (50%). Prema mjestu uzorkovanja najveći broj pozitivnih nalaza (17, 21%) je potjecao od uzgajivača. Dijagnostička metoda PCR za dokazivanje prisutnosti Cp. psittaci pokazala su se kao specifična, osjetljiva i primjenjiva u dijagnostici klamidioze u ptica. Ne samo broj pozitivnih ptica na klamidiozu već i podaci o oboljelim ljudima od psitakoze u razdoblju od 1997. do 2007. godine pokazuju da ova infekcija nije samo problem veterinarske službe, već je i javno zdravstveni problem te je nužna uspostava boljeg praćenja epidemiološke situacije u Republici Hrvatskoj na državnom nivou u zajedništvu veterinarskog i javnog zdravstva.

Megabakterioza je bolest papiga tigrica, kanarinaca, nimfi, agapornida, a postoje izvješća i o infekcijama velikih papiga i drugih vrsta ptica. Ova gljivična infekcija može biti akutna do kronična, ali i usputni nalaz u klinički zdravih različitih vrsta ptica. Uzročnik bolesti je Macrorhabdus (M.) ornithogaster, izrazito veliki gram-pozitivan ili gram-labilan štapićasti mikroorganizam za kojeg se u početku mislilo da je izrazito velika bakterija, pa je po tome i dobio naziv, a tek nedavno prepoznat je kao anamorfna askomicetna gljivica. Točan način prijenosa ove gljivice na ptice još uvijek nepoznat, a klinička je slika najčešće nespecifična. Najčešće M. ornithogaster uzrokuje sindrom polaganog slabljenja (engl. going light syndrome), tešku bolest koja završava uginućem. Dijagnoza se potvrđuje pregledom mikroskopskog preparata izmeta ili ispirka voljke, no bolesne ptice tek povremeno izlučuju megabakterije. Radiografskom pretragom nalazi se proširenje žljezdanog želuca. Nema uspješnog liječenja, terapija antimikoticima je proturječna, a uspješna primjena amfotericina B opisana je jedino kod ptica u ranim stadijima bolesti. Od 2003. godine u Ambulanti za ptice Veterinarskog fakulteta dijagnosticirana je megabakterioza u 122 ptice različitih vrsta (najčešće tigrice, kanarinci i agapornisi). Bez obzira na način liječenja, većina ptica je uginula, pretpostavljamo zato što su se vlasnici prekasno obratili za pomoć u ambulantu. Obzirom na učestalost ove bolesti u ptica kućnih ljubimaca, prepoznavanje kliničkih znakova od strane vlasnika u ranom stadiju bolesti i pravovremeni početak terapije smatramo neophodnim.

Posljednjih godina diljem svijeta Campylobacter spp. se spominje kao jedan od najznačajnijih uzročnika akutnih bakterijskih enteritisa u ljudi. Većina istraživanja pokazala je da je konzumacija nedovoljno termički obrađenog i/ili priprema sirovog mesa peradi najznačajniji izvor zaraze. U jatima peradi koja se drže slobodno (« ; ; ; free-range» ; ; ; ) čini se da je zbog veće izloženosti okolišu i veća pojavnost Campylobacter pozitivnih jedinki, nego li je to u onih držanih u intenzivnom tovu. Da bi se odredila učestalost nalaza bakterije vrste Campylobacter u jatu tovnih pilića, držanih na malom obiteljskom domaćinstvu u „ semi-free range“ uvjetima, neposredno pred kraj tova, uzeti su obrisci kloake 31 tovnog pileta dobi 10 tjedana. Bakteriološkom pretragom iz svih uzetih obrisaka (ukupno 31) izdvojen je Campylobacter spp. Sojevi su podvrgnuti i daljnjoj identifikaciji pomoću mPCR, te je 16 sojeva identificirano kao C. coli, 9 kao C. jejuni, dok preostalih šest sojeva nismo bili u mogućnosti do kraja identificirati. Ovim istraživanjem potvrđena je sumnja da je u tzv. « ; ; ; free-range» ; ; ; uzgojima peradi infekcija bakterijama češća, bilo da se radi o ubikvitarnim bakterijama ili onim vrlo zaraznim i već zaboravljenih u industrijskoj proizvodnji peradi. Molekularne metode identifikacije kampilobaktera pokazale su se jednostavnima i sigurnima, te će se i u buduće koristiti u istraživanjima.

The paper investigated Croatian foreign trade of poultry products with Mediterranean countries over the period 2002-2006. Analysed were the value of export and import of poultry products, its share in Croatian total foreign trade exchange, principal importers of Croatian poultry products as well as exporters. Statistical data from the Croatian Bureau for Statistics were used in analysing. Data were processed by standard mathematic-statistical methods for economic analyses. In that period, the total Croatian export value of poultry products was 105.2 million US$, while the value of import was 87.1 million US$. Foreign trade exchange of poultry products was carried out with 14 Mediterranean countries. The export value of poultry products in those countries was 52.3 million US$, what was 49.8% of total export value of Croatian poultry products. The greatest importers were Bosnia and Herzegovina, Slovenia and Serbia and Montenegro. The main export products were poultry meat and poultry meat processing. The import value of poultry products was 27.8 million US$ or 31.9% of total Croatian import of poultry products. Slovenia, Italy and France were the main exporters. The most significant imported products were poultry eggs and poultry meat processing. The foreign trade balance of poultry products with Mediterranean region was positive over the entire research period.

Wild birds can carry a wide range of viral, bacterial, fungal and protozoan zoonotic agents, which may be transmitted to humans. Many of them cover long distances during annual migrations, and can potentially play a role in the epidemiology of zoonoses. In the locations around City of Zagreb during 2004 and 2006, 73 and 273 wild birds of different species were captured, respectively, and pharyngeal and cloacal swabs were taken. Birds were belonging to the species: Blackcap (Sylvia atricapilla), Robin (Erithacus rubecula), Chiffchaff (Phylloscopus collybita), Blackbird (Turdus merula), Garden Warbler (Sylvia borin), Sedge Warbler (Acrocephalus schoenobaenus), Great Tit (Parus major), Nightingale (Erithacus megarhynchos), Great Reed Warbler (Acrocephalus arundinaceus), Pied Flycatcher (Ficedula hypoleuca), Wren (Troglodytes troglodytes), Song Thrush (Turdus philomelos), Greenfinch (Carduelis chloris), and Goldcrest (Regulus regulus). All swabs were streaked directly on nutrient agar (Becton Dickinson, Cockeysville, USA) BPLS-Agar (Merck, Darmstadt, Germany), and Sabouraud Dextrose Agar (Becton Dickinson, Cockeysville, USA). Also, cloacal swabs were examined for the presence of Campylobacter spp. The taxonomic classification of isolated bacteria was based on colony morphology, microscopically and biochemical characteristic, mostly using API 20 E and API 20 NE strips (BioMerieux, Lyon, France). Fungi were identified macroscopically, based on colony morphology and microscopically. From pharyngeal swabs bacteria E. coli, Staphylococcus spp., Streptococcus spp., and Bacillus sp., and fungi Candida spp., Cladosporium sp., Mucor sp., Alternaria sp., Aspergillus (A.) flavus, and Penicillium sp. were isolated. From cloacal swabs bacteria Bacillus sp., E. coli, Staphylococcus spp., Streptococcus spp., and Pseudomonas sp., and fungi A. fumigatus, A. niger, Candida sp., C. albicans, Cladosporium sp., Mucor sp., Penicillium sp., Alternaria sp., Scapulariopsis sp. and Rodotorula rubra were isolated. All examined cloacal swabs were Campylobacter negative. Comparing the results from two different years, similarities of bacterial and fungal species and their number is evident, even on a higher number of the samples. All isolated bacterial and fungal species are opportunistic, but they can also be obligatory pathogens, especially E. coli and Aspergillus species, and may cause diseases in poultry and humans, especially in elderly or immunocompromised ones. The results obtained by these two investigations revealed the need for permanent monitoring of wild bird’ s health status, especially the species that are more infected and are in the closer contact with the humans.

Određivanje spola u ptica vrlo često je zahtjevan i nesiguran zadatak, a nužan je ne samo radi ptica u uzgoju već i onih u divljini. Iako postoje različite metode, one se međusobno bitno razlikuju po jednostavnosti i brzini izvođenja, pouzdanosti, te konačno i veoma značajno, razini stresa kojem se ptica podvrgava prilikom pregleda ili uzimanja uzoraka za analizu. Pregled kloake, morfometrijska analiza, laparoskopija, analiza steroida, kariotipizacija, protočna citometrija i DNA fingerprinting, neke su od metoda identifikacije spola u ptica. U laboratoriju Zavoda za bolesti peradi s klinikom Veterinarskog fakulteta u Zagrebu koristi se postupak lančane reakcije polimerazom (PCR). Spol u ptica određuju Z i W spolni kromosomi. Regija koja je sačuvana na W i Z kromosomu, a koja je izrazito konzervirana, je regija CHD gena. Dio gena koji varira u broju pb između W i Z kromosoma je intronska regija CHD gena. Ona je dulja na W kromosomu pa je stoga pogodna za PCR pretragu primjenom univerzalnih početnica. Specifične početnice (klice) umnažaju jedan produkt kromosoma Z u mužjaka, dok u ženki umnažaju dva produkta, jedan kao i u mužjaka, koji je produkt Z kromosoma, a drugi produkt W kromosoma. Produkti PCR reakcije primjenom specifičnih početnica pokazuje razlike u veličini i intenzitetu, što je odraz razlika u genomu analiziranih vrsta, a što nam ujedno omogućava razlikovanje pojedinog spola. Iako je za izolaciju DNK za PCR pretragu moguće koristiti različite materijale, u našem laboratoriju se najčešće koriste badrljice rastućih pera uzetih sa prsne regije. Naša iskustva određivanja spola u ptice PCR-om dokazuju da je ovaj postupak vrlo pouzdan, jednostavan, brz i relativno jeftin.

Avian Clinic, as part of Department of Poultry Diseases, exists at the Faculty of Veterinary Medicine, University of Zagreb until 2003. Before that the companion birds were treated occasionally on the Department or other clinics at the Faculty. Since 2002 one of the elective courses in X. semester is &laquo ; ; ; ; Diseases of cage birds&raquo ; ; ; ; where students gain theoretical knowledge about avian medicine, and opening of the Avian Clinic enables applying of gained skills, but also to hold seminars and master classes for veterinarians. Treatment of companion birds is primary task, but in addition, care for wild birds, which need special restraint and treatment is also provided. Together with clinical examination, complete laboratory diagnostic (bacteriological, mycological and viral- molecular laboratory), X- ray, surgery and other special services are available in our Clinic and at the Faculty. Hospitalization can also be provided on site. Our experts are member of the AAV, WPSA, WVPA, Croatian Veterinary Chamber and Croatian Microbiological Society. The number of patients, belonging to different species, increases every year, what suggests that more and more bird owners in Croatia seek adequate care and advice for their companion birds.

Tijekom devet mjeseci, od ožujka do prosinca 2006. godine, ulovljeno je 860 divljih ptica pripadnica 14 vrsta u širem i užem području grada Zagreba. Skupni obrisak ždrijela i kloake svake ptica korišten je u pokušaju izdvajanja virusa newcastleske bolesti (VNB), paramiksovirusa serotipa 1 (PMV 1) na kokošjim zamecima. Istodobno je izvađena i krv radi određivanja specifičnih protutijela ovog virusa probom inhibicije hemaglutinacije (HI). Virusološkom pretragom niti u jednom uzorku nije dokazan paramiksovirus. Protutijela virusa NB dokazana su u šest od ukupno 11 pretraženih vrsta ptica, a titrevi HI protutijela kretali se u rasponu od 1:2(1) do 1:2(9). U najvećem broju slučajeva (35%) ovim virusom bili su zaraženi golubovi. Postignuti rezultati neosporno dokazuje postojeću zarazu u pretraživane populacije opisujući je tako kao stalni rezervoar ove paramiksovirusne infekcije.

Introduction Campylobacteriosis is the second reported human food- borne infection in Croatia (www.publichealth-zagreb.hr), and represent an important public health problem in most areas of the world. According to the EFSA, in 2005, campylobacteriosis overtook salmonellosis as the most reported zoonotic disease in humans in the EU (EFSA, 2006). Numerous investigations confirmed that consumption of turkey meat can be associated with a disease outbreak in human (Shandera et al., 1992). Comprehensive epidemiological investigation that will reveal the prevalence of Campylobacter in turkey flocks weren’ t conducted in Croatia recently. The principal purpose of this study was to evaluate the prevalence of Campylobacter spp. in fattening turkeys, on-farm prior to transport at the slaughterhouse. Materials and methods Altogether 60 cloacal swabs were taken from 19 and 20 weeks old toms. Samples were taken in 5 separated facilities located at two different farms. Pre-enrichment and isolation of Campylobacter spp. was conducted by using Campylobacter Selective Enrichment Broth and modified Charcoal Cefoperazone Desoxycholate Agar plates (Oxoid, Unipath, UK). All presumptive colonies were identified as Campylobacter spp. by standard microbiological and biochemical procedures (Bolton et al., 1992). From isolated strains the DNA was extracted as described by Jackson et al. (1993) and multiplex PCR was conducted as described by Manfreda et al. (2003). Results and discussion From all 5 facilities, altogether 36 (60%) cloacal swabs were Campylobacter spp positive. All of these strains were examined by multiplex PCR and 3 of them were identified as C. jejuni. The identification of other isolated strains is still in progress. Program for the surveillance of Campylobacter in poultry flocks doesn’ t exist in Croatia, but the positive results clearly stated that control of the infection at the farm level, prior to slaughter, is also preferable method for the effective monitoring of epidemiological situation. Large-scale investigation will be conducted to reveal the real prevalence of Campylobacter in turkey production.