A.O. Vet. Fak. Derg. 34: (2) 207-215 1987
ISOLATION OF CAMPYLOBACTER JEJUNI, CAMPYLOBACTER COLI AND CAMPYLOBACTER LARIDIS FROM INTESTINE OF BROILERS
K. Serdar Dikerı Nejat Aydın2
Mustafa Arda4
Hakan Yardınıcı3
Broiler barsaklarından Campylobacter jejuni, Campylobacter coli ve Campylo-bacter laridis izolasyonu
Özet: Term~rilik kampilobakterlerin izolasyonu amac?yla
ı
50 adet tavuk barsalı incelendi. Ileum mukozasından ekim yapılan Preston selektif besimleri, 42 0(; de mikroaer~rilik atmoşferde 48 saat inkiibe edildi. Morfo-lojik olarak Camp)'lobacter şeklinde qyrılan suşların identifikasyonları, üreme, tolerans ve b£yokim)iasal testler ile )'apıldı.r:.
laridis nalidiksik aside dirençli olması, C.jejuni pozitif hippurat testi,r:.
coli negatif hippurat testi ile ayrıldı.incelenen toplam
ı.')o
adet ö'rne/!,intümünden termofilik kampilobakterler izole edildi. Bu suşlardan % 42. 7sic'.
jejuni, % 5ı
.1 ü C. coli ve % 2.7 siC. laridis olarak identif£Ye edildiler. Bu tüderin h:.ola.f)'onu, Türk£Ye' de
tavuk-lardan ilk ke:: bildirilmektedir.
Sunınıary: In this stud)', intestines
if ı
50 poultT)! were examined toisolate the thermophilic camp)'lobacters. Samples taken from ileum were cultured on Preston selective medium and incubated at 42 oCin microaerophilic atmosp-here for 48 hours. The fiather dijJerentiatio/l of stmins identified as Camp)'-lobacter sp. was done b)' growth, tolerance and biochemical tests.
All samples investigated were found positive for campylobacters. The distribution of C.jejuni, C.coli and C.laridis among these isolates was 42.7, 5
ı
.3 and 2.7 per cent, respectively. This report presents the first isolation of these three species from poultry in Turkey.i Dr., A.C. Vetc'riner Fakültesi, Mikrobiyoloji Anabilim Dalı, Ankara. 2 Doç. Dr., A.O. Veteriner Fakültesi, Mikrobiyoloji Anabilim Dalı, Ankara. 3 Ar. GÖL, A.C. Veteriner Fakültesi, Mikrobiyoloji Anabilim Dalı, Ankara. 4 Prof. Dr., A.O. Veteriner Fakülte,i, Mikrobiyoloji Anabilim Dalı, Ankara.
208 K.S. DİKER-N. AYDIN-H. YARDlMCT-M. ARDA
Introductıon
Bacteria bclonging to the genııs Campylobaetcr have attracted con-siderable interest during the past decade due to an increasing fre-quency ofisolations from animal and human sources. The most widcly isolated species of this genus is Camp)'lobaetcr jl'jııni. Other two species of this group called "thermophilic campylobacters" which shO\v mi-nor phenotype differences from each other are Camp)'lobaetcr eoli and
Campylobaeter laridis. Thermophilic campylobacters have been isolated
from intestinal tracts of wild and domestic mammals and birds (6,9, 17), and considered as a part of normal intestinal flora of poul tr)'
(17). Recent studies showed that the prevalence of these organisms in the feces ofbirds ranged from 40 to 100 per cent (20,30). The mic-roorganism spreads through the flock from one or mort' sources and continues to survive in a high percentage of poultry, Iıoth at the broo-der facility and at the proct'ssing plant. Recent studies have confir-med that campylobacter can readily calanize the gastrointestinal tract of young chicks (14). In the past, it had alsa been suggested that bac-teria, possibly similar to C. jejııni, have been implicated in infectious hepatitis of chickens (I).
Studies throughout the world have implicated C. jejlllli as the causative agent of acute gastroenteritis in men in 3 to i4 per cent of cases requiring medical attention (3, 18). Several investigators have reported that poultry products contaminated with C. jejuni are an important source of transmission (12, 15). Recent studies have revealed isolation rates of C. jejuni from all parts of processed chickens that reached to 83 per cent (26). Campylobactcrs have also been detected in fresh and frozen chicken at market levcl, and theyare ablc to survive for extended periods in frozen or refrigerated meat (ı O, 13).
The present study was undertaken to assess the occurrence of thermophilic campylobactcrs in the intestines of broilcr chickcns.
Materials and Methods
Animals: A total of 150 chickens from 2 poultry flocks were
in-. vestigatedin-. Ranch A was a broiler brooder flock having 2000 animals, aged 45 weeks. Ranch B had 5000 animals, aged 8 weeks. Whole in-testinal tracts of 100 chickens from ranch A and 50 chickens from
ISOLATION OF CAMPYLOBACTER JEJUN!. .. 209
ranch B were colIeeted during slaughtering and transported to the laboratOl-y in ice box within 4 hours.
jltedia: Campylobacter blood agar supplemented with Preston
antibiotic combination (4) was used for the isolation of thermophilic campylobacters. This meelium contained folIowing ingredients per liter of distilIed water: blood agar base 1'\0. 2 (Oxoid), 40 g; polymyxin B, 5000 iU; rifampicin, iO mg; trim(~thoprim lactate, iO mg; actidione,
i00 mg and 7
%
elefibrinated sheep blood. Bioehemical and growth tests were performed in nutrient broth No. 2 (Oxoid) containing 0.16%
agar and on MuelIer-Hinton agar (Oxoid). The isolates were subcultured in Thiol Medium (Difco) and preserved in BruceIla broth (Difco) at -- 70 oC.Intestinal samphng and isolation: When the intestines were sampled,
the extemal surface of ikum was seared with a hot spatula prior to incision of ileum waIl with a sterilc scalpeI. Then a steril swab intro-duced into the intestinal lumen to eoIlect sample. Swabs eontaminated with intestinal eontent were seedeel on Campylobaeter seleetive agar plates. Plates were incubateel in anaerobic dessieators without eatalyst and microaerophilic condition was obtained by using Gas Generating Kit (Oxoid). Dessieator was hcld at 42 oC for 48 hours. After incu-bation, any growth on plates observed anel colonies similar to the typi-cal morphology of campylobaeters were sclected for further examina-tion.
Characteri<.ation: Suspected colonies were stained with Gram
sta-ining method and investigated for motility under dark field iIlumuna-tion. Biochemical tests (oxielasc, catalase, nitrate reduction, selenite reduction), tolcrance tests (3.5
%
sodium ehloride, i%
glycine, 30 mcg jml cephalothin) and growth tests (25 oC, 30.5 oC, 45.5 oc) were us cd for the identification of thermophilic campylobaeters. Ther-mophilie campylobaeters were further divided into three speeies by hippurate hydrolysis test and nalidixie acid (30 megımı)
suseeptibility test.The biochemical, tolerance and growth tests used for the difIeren-tiation of thermophilic campylobaeters from each other and other campylobaeters are shown in Tablc I.
210 KoS. DİKER-No AYDIN-Ho YARDIMCI-Mo ARDA
Table io Plıenotype charaeteristics of Campylobaeter species"
Co jejuni Co coli Co laridis Co fetus C. sputonım Others'
---
.• -0 ~--- ---_._ ..--- --- --- __ o----Oxidase -1- ...- -i- ~-
-1-Catahse -i- -.- -i- ..
-Xitrate -i- .. -i-
--
---H,.S -i- -i- d .. Hippurate -i- 0••0
--
- -25 'C - ...- .~.- i --42 oC i--
-"-- d oı i Glycine ; -j" '0' d cl /0 o: 305 ~aCI -- -- - - di
/0 Nalidi"İe aeidb S S R R d Cephalotlıinb R R R S S -i-:1. -J -I ., RS S a symbols: "-, 90%,or more of the strains are posİtive: - '0 90%or more of tlıe sırainsare negative: d, iıto 98 '~{,oftlıe straiııs are positİ"c: :le, some spccics are positive: R, resis-tant: S, semiti"e: RS, some species are resistanl.
* Ot lıers : Co fecalis, Co hyoinlestinalis, C. coııcisus, C. einaedi, Co fenneliae, Co pylo. ridiso
Results
Thermophilic eampylobaeters wcre isolated from aıı (I 00 %) of 150 intestinal sampIcs of broiler ehiekens. Of 150 isolates identified, 5i.3
%
v••..ere C. eoli, 42. 7%
were C. jejuni and 2o7%
were C. lal'idis(Table 2). Five strains identificd as Campylobatcr sp eould not sur-vıve while subculturing bclare further identification. Sin ce they grew
Tablc 20 Isolation of thermoplıilie eampylobaeters from poulıry
Specie; Co jejuııi Co coli Co laridis
i
Unidentified Ranclı A (n' 100) 43 (43.0°;.) 52 (5200%) 3 ( 3,0%) 2 ( 200%) Imestinal samples Ranch B (n = :ı9) :21 (4200°;,) 25 ('iO.O~o) i ( 2 ,Oo~) 3 ( u.OOo) Total (II -- 150) G4 (42o7%) 77 ('il 03%) 4 ( 2.7%) :'ı ( 303%)at 42 oC, they were thermophilie eampylobaeters. The prevalcnce of C. jqjuni, C. eoli and C. lal'idis were found similar in eaeh flock. Iso-lated strains showed typical clıaraeteristies of thermophilic eampylo-baeters. Phenotypc chareeteristies of thermophilic eaınpylobactcrs isolated are shown in Table 3. All strains of C. jejuıli Iıydrolysed hip-puratc, but C. eoli did not. Aıı C. laridis strains were resistant to nali-dixie acid but other two strairıs were not.
ISOLATION OF CAMPYLOBACTER JEJUNI. .. 211
Tablc 3. Plıenotype clıaracteristics of C. jejuni, C. coli and C. laridis isolated from poultry (%)"
I
C. jeJuni C. coli C. laridisTests (n - 64) (n~. 77) (n =4)
-g~~~:~~~--
---I-:~~:
ğ----:~~~.
-
--':gff-Catalase 100.0 100.0 100.0 Nitratc reduction 100.0 100.0 100.0 H,S production 100.0 100.0 100.0 ii ippurat lıydrolvsis 100. O O. O O. O ~~ vC 0.0 0.0 0.0 Gro\\'ılı at 30.5 oc 7.8 70.1 75.0 45.5 oC 53.1 36.3 2:).0 Tolerans to ıO~ Glvcine 100.0 97.4 100.0Nalidixie aeidc' O.Ob 0.0 100.0
Ceplıalotlıinc 100.0 100.0 100.0
a =. Percentage of positive resulı, b Percentage of rcsistant strains, e, 30 mcg{mI.
Discussion and Condusion
In this study, thcrmophilic campylobacters were isolated from
i00 per cent of animals. This isolation rate is one of the highest in the world. In a previous study, prevalence of C. jejuni in ceca of broilers was also 100 per cent (30).
C. jejııııi has been frequently isolatcd from the intestinal tract and feces of healthy poultry throughout the world. This microorganism has been recovered from 74 % of rectal swabs from broilers in Aust-ralia (23), LO
%
of Norwegian poultry (21), 72 % from broilers in Sweden (I 1.) and 4i % from broilcrs in Israel (20). In the present study the incidence of C. coli, C. jejuni and C. laridis was 51 .3, 42.7 and 2.7 per cent, respectivcly. The present results indicate that thcr-mophilic campylobacters, particularly C. coli and C. jejuni, are also widespread among poultry population in Turkey.It is known that there are several species of Campylobacter and most attention has been focused on C. jejuni due to its contribution to many infections. Infections due to C. coli which is closcly related to C. jejııni, appear to share many clinical and epidemiological charac-teristics. In this study, the prevalence of C. coli was very high when comparing with the results of other surveys. Only a few report indi-cate the presencc of C. coli and C. laridis in domestic poultry (12, 20). The cause of this situation is most likdy the old classification of campy-lobacters in these previous studies. Befon~ new classification of
ther-212 K.S. DİKER-N. AYDIN-H. YARDıMCı-M. ARDA
mophilic campylobacters, C. evli strains had bcen considered as tlıe hippurate negative biotype of C.jejuni (I i), and C. laridü haCı been named as naIidixic acid resistant thermophilie campylobaeter (KAR TC). Althouglı the strains similar to C. cvli anel C. laridis wc re isolateel in tlıe most of previous works, all these strains were nameel as
C.jejııni. Reeendy, hippurate negative strains of C.jejııni w'ere
trans-ferred to C. evli anel NAR TC strains to C. laridis (2, I.')). As a result of new nomenclator, reeent reports indicating the presence of C. evli
anel C. laridis have been seen in literature (12, 20). Likewise, C.jejııııi
had becn previously isolateel from eattk anel slıccp, (5, 6) anel from human with gastrocnteritis (I 9, 29) in Turkey. Since they hael becn conelucted bclüre new classifieation, many strains of thesc studies were named as C.jejııııi, instead of C. evli anel C. laridis. In any ease C. evli
predominated in this study while C.jejııni predominatcd in other stu-e1ies from all over the world.
The souree of eampylobaetcr eontamination and the cause of high incidence of eampylobacters have been investigated by several workers. Montrose et aL. (I 6), infCeted the SPI' chiekens with Campy-lobacter by using (~xperimentalIy cantaminated litter anel showed that litter was an important souree of caınpylobaeters. In the present study, high isolation rate may Iw related to !itter eontamination. Shane et aL. (22) demonstrated in a series of cxperiments that flies eould transmit the C. jejll1li to SPF elıickens under controlled labora-tory conditions. Sinee the present study was eonducteel in ",inter season during whieh flies do not appear, this source eould not be the eause of transmission of campylobacters to broilcrs.
Studies on tlıe incidence of Caınpylobacter speeies in broilcr flocks monitored from hatelıing to slaughter have slıown that these mieroorganisms colonize the intestinal tracts of birds at the age of
4-8 weeks (I 7, 27, 28). In the previous studies, the laek of isolation of campylobaeters from eggs of hellS which are fecal exereter of this agent (8) and the laek of attempt to transmit infcetion verticalIy (23) support these findings. In the pn~sent study,animals were 8 and 15
weeks of age and these ages are the most susccptible period for tlıe eolonization of eampylobacters in the intestine of poultry.
Another possiblc reason of higlı prevalcnee of eampylobacters in this study was the colIeetion of speeimcns from mueosal surfaee of intestine. When comparing the isolation studies in \vhieh both mueosal
ISOLATION OF CAMPYLOBACTER JEJUNI.,. 213
swabs and faeces were used as inoeulum, it has been deterınined that isolation rate (rom mucosal spceimens was always higher than those £i'om faeces (7). This can be attributed the afrinity of mierooqpnism to mueosal surlacc, partieularly to mucus layer.
The most important aspeet of high prevalcnce of campylobaeters in poultry is the possibility of transmission to human. The intestinal eontent of broilcrs always contaminates the processing plant during slaughtering. Kinde et aL. (I 3) reported that C. jejzıııi was found in 83
%
of ehieken wings analysed on the day of arrival at supcrmarkets. Other studies on the same subject in whieh various body parts of broiler carcasses "ere exarnined [or eampylobacters at slaughtn-house or at the supermarket kvcl have showed that isolation rates [rom carcasses were as high as intestines (12, 26, 30). Many faod.bome outbreaks of eampylobaeter enteritis in men dul' to undereooked broi-ler have been reported (3, ıo, IS). Shankcr et aL. (24) (aund that most of ehieken and human isolates from same area were of identical bioty-pes. In Turkey, limited numbcrs of paper indicates Campylobaetn infCetion in human (I 9, 29). 1\lost likcly, thermophilie eampylobaeters from poultry cause enterie infeetion in human in Turkey.hırther studies on the distribution of eampylobaetcrs among poultry and men should be performcd and the epidemiological as-peets of eampylobaeter infections should be dc,termined. On the other hand, despite the faet that eampylobacters can survive in the intestine of poultry as normal flora such a high prevalenee, i t is necessary to undel'stand in which eonditions this agent causes the disease caııed baeterial infCetious hepatitis or whieh mcchanism proteet the animals from this disesase.
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