Prevalence and Characteristics of Campylobacter Species Isolated from Gallbladder of Slaughtered Sheep in Van , ( Eastern ) Turkey

Ekin I .H. , K. Gürtürk, A. Arslan, B. Boynukara: Prevalence and Characteristics of Campylobacter Species Isolated from Gallbladder of Slaughtered Sheep in Van, (Eastern) Turkey. Acta Vet. Brno 2006, 75: 145-149. To determine the prevalence of campylobacter species in gallbladder of sheep in Van, (Eastern) Turkey, a total of 220 gallbladder samples from healthy slaughtered sheep were examined bacteriologically in October 2000 and 2002. Of the 110 samples examined each year, 27 (24.6%) and 24 (21.8%) campylobacter strains were isolated, respectively. Of the 27 campylobacter strains isolated in the year 2000, 14 (51.9%) were identified as C. jejuni, 7 (25.9%) C. fetus, 3 (11.1%) C. coli and 3 (11.1%) C. lari. Similar results were obtained in the study performed in 2002, but C. lari could not be isolated. Growth and biochemical characteristics of all identified Campylobacter species with some exceptions were typical of each species. Six of 13 examined C. fetus strains grew well at both 25 °C and 42 °C in thioglycollate medium and on blood agar. C. jejuni strains differed from C. coli only by Na-hippurate hydrolysis test. Results of the present study revealed that C. jejuni is the most common campylobacter species isolated from gallbladders of sheep. The thermophilic campylobacters in significant proportions may cause contamination of carcass during slaughter and transmission of the food-borne pathogens to humans. Prevalence, contamination, food-borne pathogens, Campylobacter species, bacteriology, identification, phenotype, strain, biochemistry Campylobacters are important pathogens of humans and animals. C. fetus has been recognized as a cause of septic abortion, infectious infertility and diarrhoea in cattle and sheep (Varga et al. 1990; Gürtürk et al. 2000; Mannering et al. 2004). Thermophilic campylobacters such as C. jejuni, C. coli are also known as causal agents of abortions in sheep (Penner 1988; Gürtürk et al. 2002; Butzler 2004). C. jejuni and C. coli were found to be commensal in the rumens and small intestines of healthy calves and adult cattle. They cause acute gastroenteritis in human and enteritis in animals. C. jejuni is recognized as a causative agent of avian hepatitis of fowl (Butzler 2004; Tangvatcharin et al. 2005). Nowadays, campylobacter species are a leading cause of food-borne diseases in the industrialized world. C. jejuni and C. coli are the most frequent species isolated from humans. The presence of campylobacter species in intestines and gallbladder of sheep may cause contamination of carcass during slaughtering and transmission of these foodborne pathogens to human beings by consumption of contaminated and low-heated sheep meat, meat products, liver or by direct contact with infected animals (Penner 1988; Butz le r 2004; Blaser et al. 1983). Campylobacter species have been isolated from the intestines and gallbladders of apparently healthy sheep (Diker 1985; Wokatsch and Bockemühl 1988; Raj i et al. 2000; Yazic ioglu 2000). Previous studies (Diker 1985; Yazic ioglu 2000; Er tas et al. 2003) indicated that the prevalence of Campylobacter spp. in the gallbladder of slaughtered sheep varied from 52.8% to 66% in some regions of Turkey. ACTA VET. BRNO 2006, 75: 145–149 Address for correspondence: Dr. Ismail Hakki EKIN Dept. of Microbiology, Faculty of Veterinary Medicine University of Yuzuncu Yil 65080 Kampus-Van, Turkey Phone: +90-432-2251128 Fax:: +90-432-2251127 GSM: +90-532-4957220 E-mail: ihekin@yyu.edu.tr http://www.vfu.cz/acta-vet/actavet.htm The present study was performed to determine the prevalence of campylobacter species isolated from gallbladders of slaughtered sheep in Van, (Eastern)Turkey and to evaluate the biochemical characteristics for identifying campylobacter species. Materials and Methods

Campylobacters are important pathogens of humans and animals.C. fetus has been recognized as a cause of septic abortion, infectious infertility and diarrhoea in cattle and sheep (Varga et al. 1990;Gürtürk et al. 2000;Mannering et al. 2004).Thermophilic campylobacters such as C. jejuni, C. coli are also known as causal agents of abortions in sheep (Penner 1988;Gürtürk et al. 2002;Butzler 2004).C. jejuni and C. coli were found to be commensal in the rumens and small intestines of healthy calves and adult cattle.They cause acute gastroenteritis in human and enteritis in animals.C. jejuni is recognized as a causative agent of avian hepatitis of fowl (Butzler 2004;Tangvatcharin et al. 2005).
Nowadays, campylobacter species are a leading cause of food-borne diseases in the industrialized world.C. jejuni and C. coli are the most frequent species isolated from humans.The presence of campylobacter species in intestines and gallbladder of sheep may cause contamination of carcass during slaughtering and transmission of these foodborne pathogens to human beings by consumption of contaminated and low-heated sheep meat, meat products, liver or by direct contact with infected animals (Penner 1988;Butzler 2004;Blaser et al. 1983).Campylobacter species have been isolated from the intestines and gallbladders of apparently healthy sheep (Diker 1985;Wokatsch and Bockemühl 1988;Raji et al. 2000;Yazicioglu 2000).Previous studies (Diker 1985;Yazicioglu 2000;Ertas et al. 2003) indicated that the prevalence of Campylobacter spp. in the gallbladder of slaughtered sheep varied from 52.8% to 66% in some regions of Turkey.
The present study was performed to determine the prevalence of campylobacter species isolated from gallbladders of slaughtered sheep in Van, (Eastern)Turkey and to evaluate the biochemical characteristics for identifying campylobacter species.

Gallbladder samples
In October 2000 and 2002, 110 gallbladder samples for each year were collected from slaughtered sheep older than 1 year in Van,Turkey and were examined bacteriologically within 2 h of slaugther.

Preliminary identification
Preliminary identification of campylobacter species was based on phenotypic characteristics; such as colony appearance, Gram staining, microscopic morphology, motility, catalase and oxidase reaction, nitrate reduction and fermentation of glucose (Holt et al. 1994;Nachamkin 1999).

Results
A total of 27 (24.6%)and 24 (21.8%)campylobacter strains were isolated from gallbladders of slaughtered sheep in the years 2000 and 2002, respectively.All isolates were found to be Gram negative, motile, curved rods, catalase and oxidase positive, reducing nitrate, not utilizing glucose and growing microaerobically but not aerobically at 37 °C.Of the 27 strains isolated in the year 2000, 14 (51.9%) were identified as C. jejuni, 7 (25.9%)C. fetus, 3 (11.1%)C. coli and 3 (11.1%)C. lari.Similar results have obtained, but C. lari could not be isolated in the year 2002 (Table 1).
Further growth and biochemical characteristics of all examined campylobacter strains were typical of each species with some exceptions.The C. coli could be distinguished only by sodium-hippurate test from those of C. jejuni.Six C. fetus strains grew well at both 25 °C and 42 °C in thioglycollate medium and on blood agar.Except for C. fetus strains, 11 of 14 C. jejuni strains and all C. coli strains grew also in medium with presence of 0.04% TTC (Table 2).

Discussion
In the present study, the isolation rate of campylobacter strains from gallbladders of slaughtered sheep was determined as 24.6 -21.8% in the years 2000 and 2002, respectively.These findings appeared to be lower than other reports (Diker 1985;Yazicioglu 2000;Ertas et al. 2003) in Turkey.In these reports, the prevalence of Campylobacter spp. in the gallbladders of slaughtered sheep was found to be 57.3%, 52.8% and 66%, respectively.It has been reported that the isolation rate of campylobacter species from intestines or gallbladder of lambs and calves are higher than those of sheep and cattle (Diker 1985;Giacoboni et al. 1993).The lower isolation rate obtained in this study might be due to the fact that sheep examined in this study were older than one year of age.
Previous reports (Clarck and Mounbourgh 1979; Yazicioglu 2000) indicated that C. fetus is more prevalent than C. jejuni in the gallbladders of slaughtered sheep.In contrast, the results of this study revealed that the C. jejuni was found to be predominant campylobacter species in the gallbladder of slaughtered sheep.The distribution of campylobacter species were 12.7% C. jejuni, 6.4% C. fetus and 2.7% C. coli and C. lari in the year 2000.Similar results were also obtained in 2002.The isolation rate of C. jejuni, C. fetus and C. coli were 12.7%, 5.5% and 3.6%, respectively, but C. lari could not be isolated in 2002.The differences in the occurrence and distribution of campylobacter species in gallbladder of sheep might be due to the differences in environmental and breeding conditions between areas or countries.
In  (Griffiths and Park 1990;Waino et al. 2003).The speciation was also confirmed using DNA-DNA hybridization techniques (Vandamme and Goossens 1992).Although the hippurate hydrolysis is a useful test for distinguishing C. coli and C. jejuni strains, there are some misidentifications with wild hippurate-negative C. jejuni strains (Griffiths and Park 1990; Waino et al. 2003).Totten et al. (1987) reported that the hippurate-negative campylobacter strains were identified as C. jejuni (20%), C. coli (78%) and C. lari (2%) by DNA hybridization test.Ronner et al. (2004) indicated that the C. jejuni strains, 5% from human and 10% from chicken, found to be negative with Na-hippurate test after genotyping In addition, our study indicated that the tests for growth in presence of TTC and hydrolysis of indoxyl acetate appeared to be more characteristic of C. jejuni (except to three strains) and C. coli strains, but not for C. fetus and C. lari which were negative by both tests (On 1996;H o lt et al. 1994;Nachamkin 1999).
However, six strains of C. fetus grew well at both 25 °C and 42 °C, and this property did not change after several culture passages.Because all other characteristics were typical, these thermophilic strains were identified as C. fetus (Penner 1988;H o lt 1994).The thermophilic strains of C. fetus isolated from humans (Anstead et al. 2001;W o o et al. 2002) and from aborting sheep (Varga et al. 1990) have already been reported.
The C. lari strains identified in this study were resistant to both nalidixic acid and cephalothine, negative in hippurate test and grew well at 42 °C but not at 25 °C as reported by VanDamme and Goossens (1992) and Raji et al. (2000).These strains were described as "NARTC" (nalidixic acid resistant thermophilic campylobacter) strains.C. lari is rarely isolated from the intestines of healthy sheep or cattle and did not appear to be associated with disease (Penner 1988;Butzler 2004;Diker 1985).
In conclusion, the prevalence of campylobacter species in gallbladder of slaughterhouse sheep was found to vary from 21.8% to 24.6% in Van, (Eastern) Turkey.C. jejuni are found to be the most common campylobacter species in gallbladder of sheep.Presented data showed also that the gallbladder of sheep containing thermophilic campylobacters in significant proportions may cause contamination of carcass during slaughter and transmission of the food-borne pathogens to humans.Even if the biochemical characteristics are suitable to confirm the preliminary identification of the campylobacter isolates on the species level, additional molecular biological techniques should be applied to reliable identification of the hippurate-negative campylobacter isolates.

Table 1 .
Prevalence of Campylobacter species isolated from gallbladder of sheep at slaughterhouse in the years 2000 and 2002 (Diker 1985;Yazicioglu 2000;Raji et al. 2000)ioglu 2000;Raji et al. 2000)C.coliwasalso detected in gallbladders of sheep examined in this study.C. coli are found to be common in humans and chickens but rare in sheep and cattle(Wokatsch and  Bockemühl 1988; B a e et al. 2005).On the other hand, C. jejuni and C. coli could be recognized as separate species based on the hippurate hydrolysis reaction

Table 2 .
Some characteristics of 51 Campylobacter strains isolated from gallbladder of slaughtered sheep