ENTERIC BACTERIA IN FECAL SAMPLES OF EURASIAN GRIFFON VULTURES

Fecal samples originating from 15 Eurasian griffon vultures were collected during June 2012 in the territory of special nature reservation Uvac anđ examineđ for presence of enteric bacteria Escherichia coli anđ Salmonella spp. Salmonellas were isolateđ from five samples (33.3%) anđ serologically typed as Salmonella enterica subsp. enterica ser. Veneziana. E. coli was isolated from four samples (26.6%). Antimicrobial susceptibility testing revealed resistance to one anđ more antibiotics only in E. coli isolates.


Introduction
Wild birds play an important role in the ecology and circulation of human and animal pathogens including viruses, bacteria, fungi and protozoa (Hubalek, 2004;Abulreesh et al., 2007;Benskin et al., 2009). In some European countries, wild birds (particularly gulls) are identified as reservoirs of enteric pathogens such as various serotypes of Salmonella (Kapperud et al., 1998;Hubalek, 2004;Čižek et al., 2007) and toxin-producing Escherichia coli strain (Abulreesh et al., 2007), which could be also multiple resistant (Palmgren et al., 2006;Čižek et al., 2007;Dolejska et al., 2007). Prevalence of salmonellosis among the wild-bird population has shown increasing tendency over the past 40 years as a result of artificial feeding by humans (Tizard, 2004). Wide range of Salmonella serotypes was isolated in wild birds, including multidrug-resistant strains of S. Typhimurium DT104 (Reche et al., 2003;Čižek et ак, 2007). Enteric pathogens mainly reside in the intestinal tract of raptors or opportunistic carrion eating birds (such as vultures, crows and gulls), who feed on the ground, at sites where human waste is released or live by the fecally contaminated waters (Gerlach 1994;Tizard, 2004, Palmgren et al, 2006Kocijan et ак, 2009). Both Salmonella and E. coli can be pathogenic for birds; however, more frequently, the birds are potential healthy carriers and play a role in the dissemination of the agents in the environment by contaminating the natural waters and animal feed (Palmgren et al., 2006;Čižek et ак, 2007). Though their role in the epidemiology of salmonellosis is still debated, several studies have confirmed the possibility that wild birds could serve as the source of human infection (Millan et al., 2004, Kruse et al., 2004. Eurasian griffon vultures (Gypsfulvus) are at the top of thefood chain andplay an essential role in the nature as theyfeed exclusively on carcasses ofdead animals. The species inhabits the regions of Southern Europe, Southwestern Asia and North Africa. During the '90s ofthe last century, the survival ofthis species was critically endangered. Decrease in Eurasian griffon vulture population is closely associated with abandoning of extensive livestock breeding in the area and thus food scarcity, poisoning (Sabočanec et al., 2005) and laying poisoned baits for large predators (Pavoković, 2005) as well as the EU Regulations on mandatory carcass disposal aimed at prevention of Bovine Spongiform Encephalopathy. Limitation of natural habitat and reduced availability of their natural ргеу in our country resulted in changes of feeding habits of Eurasian griffon vultures. The diet of these wild birds is based mainly on slaughterhouse waste from intensive farming systems. Such diet could contribute to alterations of common microflora of the digestive tract.

Sample collection
Location: Uvac River сапуоп valley with its three lakes (Uvačko, Zlatarsko and Radoinjsko Lake) as a Special Nature Reserve is protected natural asset of great importance according to the Decree of the Government of the Republic of Serbia ("Official Gazette of the RS" No. 25/06 and 110/06). It occupies some 7 500 ha between mountain Zlatar massif in the Southwest and mountain Javor in the Northeast. The estimated population of griffon vultures in this reservation is currently around 100 nesting couples, and total population (including young birds) is some 300 birds. The adult nesting birds mostly do not leave their natural habitat, whereas young birds migrate during winter months to the north (via Slovenia and Italy to the Alps), to the south (Bulgaria, Greece, Israel and Africa) and to the west (France and Spain).
www.politika.rs/rubrike/putovanja/U-carstvu-beloglavog-supa.lt.html) Sampling was carried out at Uvac natural reservation in June 2012, during early morning hours. At the moment of sampling, around 50 Eurasian griffon vultures were present at the selected location. According to observation results, the birds were in good health condition. Fifteen fresh fecal samples were collected using sterile swabs. The samples were cooled and transported to the laboratory of clinical microbiology of the Scientific Veterinary Institute "Novi Sad".

Results AND Discussion
In the '90s of the last century, the griffon vulture population in the Republic of Serbia was threatened by extinction. In the territory of Uvac-lakes, there were only seven individuals of this rare species from the Accipitridae family. The survival of this endangered species was enabled through continual delivery of slaughterhouse waste (some 50 tons per уеаг). Nowadays, Uvac is the largest colony of this unique bird species in the Balkans, and among the large ones in Europe. Diet based on animal carcasses, i.e. sheep, bovine and horses from intensive farming systems, undoubtedly contributed to the survival of griffon vultures; however, feeding slaughterhouse waste could negatively affect the health of avian scavengers and promote transmission and spread of bacteria from these sources (Blanco et al., 2006).
In this research, bacteria of the genus Salmonella were isolated from five (33.3%) fecal samples originating from Eurasian griffon vultures. All isolates fermented glucose, produced H2S and L-lysine decarboxylase. Negative reactions were obtained for Voges Proskauer and indole tests, lactose fermentation, production of (3-galactosidase (ONPG) and urease. In the national Reference Laboratory for Salmonella, Shigella, Vibrio cholerae, Yersinia enterocolitica, all isolates were serotyped as Salmonella enterica subsp. enterica ser. Veneziana (11: i: e, n, х). This Salmonella serotype was first isolated from an apparently normal Italian civilian food handler in Venice, Italy, and described as a new serotype in 1945 (Bruner et al., 1945). S. Venezianae is not listed among the 15 most frequently isolated serotypes in Serbia (www.who.int/gfn). According to the available literature, data on isolation of this serotype are scanty. In the period 1998-2008 in the U.S., only one case of laboratory-confirmed isolation of this serotype in humans was reported (CDC, 2008). S. Venezianae was isolated from mesenteric lymph nodes of one horse out of 500 examined samples taken from 100 slaughtered healthy horses (Bonardi et al., 1997). Disc diffusion test with S. Veneziana isolates did not reveal resistance against antibiotics used in the experiment.
The birds are generally considered as healthy carriers of Salmonella Bacteriological examination of fecal samples in this research revealed presence of Escherichia coli in four (26.6%) samples. Kocijan et al. (2009) performed bacteriological, mycological and parasitological analysis of fecal samples (n=10) and vomit samples (n=5) of griffon vultures from the island Cres, Croatia. Escherichia coli was the most frequent bacterial isolate in both sample categories (feces 9/10, vomit samples 2/5). In raptors, Escherichia coli can be associated with diverse pathologic lesions (bumble foot, pericarditis, hepatic abscesses, salpingitis, and egg peritonitis) (Morishita et ак, 1997). However, the virulence factors of particular E.coli strains, which are of importance in humans and mammals, do not accurately predict the relative importance of the strains for birds (Gerlach, 1994 Undoubtedly, humans play an important role in spreading pathogens among the wild bird population through contamination of waters by sewage waste, inadequate waste management and by artificial feeding of birds. Predominance of enteric bacteria and antibiotic-resistant strains within the population of griffon vultures, as well as their role in survival and spread of such bacteria in the natural environment remains unclear. Transmission of bacteria from birds to mammals requires repeated exposure and large numbers of microorganisms (Gerlah, 1994), thus the importance of Eurasian griffon vultures as a source of human and animal infection is not significant, which is due to the ,,solitary lifestyle" of these birds in inaccessible terrain. However, as feeding ecology is the principal factor that influences exposure of birds to intestinal bacteria (Benskin et al., 2009), the diet of griffon vultures based on slaughterhouse waste might have some negative impact upon health status of these birds and spreading of resistant strains of enteric bacteria over large distances by young migrating birds.