A. O. Vet. Fak. Derg. 34 (3) : 570-576, 1987
LOCATION OF THERMOPIllLIC CAMPYLOBACTER SPP IN VARIOUS PARTS
OF CmCKEN INTESTINES
K. Serdar Diker1 Hakan Yardımcı2 Nejat Aydın3
Tavuk barsaklaruun çeşitli bölümlerinde tennofillk Carnpylobacter türlerinin yerleşirni
Özet: Tavuk barsaklarının duodenum, jeJunum, ileum ve eaeeum bölüm-lerinde termofilik Campylobaeter türlerinin dağılımı ince/endi. Selektif izolas-yon yöntemi ile incelenen 80 tavuğun
%
86. 3'ünün bir urya daha fazıa barsak bölümünde Campylobaeter' lerin varlığı saptandı. Duodenum, JeJunum, ileum ve eaeeum'dan Campylobaeter izolasyon oranları sırasıyla%
52.5,%
63.8,%
78.8 ve%
81.3 olarak belirlendi. Seksen tavukda bulunan toplam 69Campylobaeter suşunun
%
50. 7'si C. JeJuni,%
49. 3' ii C. eoli olarak identifiyeedildi. Caeeum ve ileum'un, duodenum ue jeJunum'a göre daha fazıa sayıda Campylobaeter hücresi içerdiği bulundu. Buna karşın, kalm barsaktan yapılan ekimlerde, diğer bölümlere kıyasla daha fazıa sayıda veyoğunlukta kontaminant mikroorganizma ürediği gözlendi.
Summary: The distribution of thermophilie Campylobaeter spp. in duodenum, JeJunum, ileum and eaeeum of ehieken intestines was investigated. Of 80 ehiekens examined by seleetiue isolation teehnique, 69 (86. 3 %) were found to be harboured Campylobaeter sp in one or more parts of their intestines. Isolation rates of Campylobaeter sp from duodenum, JeJunum, ileum and eaeeum were 52.5, 63.8, 78.8 and 81.3 per cent, respeetively. Of 69 strains of
Campylobrıeter sp. isolated from 80 ehiekens, 35 (50. 7 %) were identified dS C. JeJuni and 34 (49.3 %) as C. oli .lt was found that eaeeum and ileum
ear-ried eamp..:vlobactersin large numbers relative to duodenum and JeJunum. Lower part of intestine eontained more eontaminant mieroorganisms whieh grew oıı se/eetive medium.
ıDr.; 2 Res. Assist.;
3 Doç. Dr. Department of Microbiology, Faculty of Veterinary Medicine, Ankara University, Dışkapı, 061
ıo
Ankara, TURKEY.LOCATlON OF THERMOPHILIC CAMPYLOBACTER... Sil
Introduction
The importancc of animal origins in the cpidcmiology of Campy-lobacter infeetions is bascd on the fact that many mammalian and avian speeies earry eampylobaeters as part of their intcstinal mierof-lora (2, 7). Camp)'lobaeter jejl/ni and C. eoli, known as therrnophilic campylobaeters, are the common inhabitants of the gut of healthy poultry and wild birds (5, 2 I). These microorganisms often seem to be harbouı'ed in the large intestine of broilcr ehickens (I 2, i5, 18). Reeent studies suggest that contami~lation may be nearly universal (6, iO, 17). The organism spreads through the floek from one or 1110re sourees. Potcntial sources for entr)' of campylobacters into a floek incIude infeetion of newborn chieks from old cr birds, contaminated feed, water or litter (8, 9, 17). Infection is usually without obvious signs ofillness and long term earriage is liTquently present (8, 12). During the process of slaughtering, C. jej/ıııi and C. eoli sprcaus li'om the intestinal content to the careasses (4, lG, 20).
Campylobactcr eolonization in the intestinal traet of young ehicks takes plaee about two weeks after hatching (9, 17). Experimental studies have showed that resistanel' of. young specif'ie pathogen free ehieks to C. jejuni has been substantially increased by early exposure
to native gut mieroflora (19).
Disease eonditions eaused by Campylobaeter sp. are not dear in poultry. A vibrio-like organism, now corisidered to be C. j~juni, has be en isolated from ehiekens that showed dcgeneratiye liver changes and depressed egg produetion (11). This condition, termed avian vibrionie hepatitis, has been li"Cquently eneountered during the 1950's
and 1960's in USA and Europe (I, 3), but is now diagnosed kss fre- . quently. Recently, it has becn demonstrated that law lcvel mortality and diarrhoea follow infeetion of 3 days old ehieks with C. .iej/mi (I 3).
The present study was undertakl'n to compare the loeation ol thermophi1ie Campylobaeter spp. in various parts of chicken intcstine.
Materials and Methods
Samplingand isolation: Wholc intestines of 80 broilcr ehiekens from
8 diırerent floeks were eollectcd during slaughtcring(Ten broiler ehiekens had been randomly sdectcd fi'om caclı [lock). Speeimens takcn from the following parts of eaeh iutestine \Yere used as
inocu-572 K. SERDAR DiKER - HAKAN YARDıMCı - NEJAT AYDIN
lum: duadenum, jejunum, ileum and caecum. Outer surface of selec-ted part of intestine was disinfecselec-ted and a longitudinal incision was made. Then a steril swab introduced into lumen to collect intestinal content.
Samplcs were cultured directly for Campylobaeter sp. on sclective medium comprising B100d Agar Base no. 2 (Oxoid) containing 5-7
%
defibrinated sheep blood and Preston Selective Supplement (Oxoid). The inoculated mcdia were incubated at 42 oC 101' 2 days under re-duced oxygen and increased carbon dioxide tensioıı (5 '10 O2, LO%
CO2 and 85
%
Hı).Tdentification and evaıııation: Typical swarmıng colonies of ther-mophilic Campylobaeter sp. wc re examined for cell morphology ,and motility by phase-contrast microscopy. Where possible, onlyone single colony was taken from the selective medium for subculture onto shecp blood agar. Suspect colonies were further confirmed as C. jejuni or C. eoli by the following tests: oxidase and catalase produc-tion, nitrate and sodium selenite reduction, H2S production, resi
s-tance to cephalathin (30 mcg), sensitivity to nalidixie acid (30 mcg), tolerance for i
%
glycin but failure to grow at 25oc.
C. jejuni was differentiated from C. eoli by its ability to hydrolyse lıippurate. Referen-ce strains of C. jejuni and C. eoli were uscd as controls in each test.Observations on the growth of Campylobacter were made and arbitrarily recorded, scoring from a ICw colonies observed to almost confluent growtlı of a large number of colonics on the agar (O = no growth, i = 1-5 colonies, 2 = 6-25 colonies, 3 = 26-100 colonies, 4
= <
101 colonics). All growths on selective media other than Campylobacter \Vere considered as contamination and scored in a similar manncı'.Results
Of 80 ehickens cxamined, 69 (86.3 %) werc found to be carrier of Campylobaeter sp. in one or more parts of their intestincs. Isolation rates of Campylobaeter sp. from various parts of chieken intcstines were as follows: 52.5
%
(42/80) from duodenuın, 63.8%
(51/80) from jejunum, 78.8 % (63/80) from ileum and SI.3 % (65/80) fromcaecum (Table I).
LOCATION OF THERMOPHILIC CAMPYLOBACTER... 573
Tablc 1. Thc distribution of C. jejımi and C. coli in different parts of "hi"ken intest;ncs. "'umbel' of positi"e samplcs
Duadenuma .J~.iuntım;ı Caeeum3
21 21 C. jcjuni C. coli _.--'--'._'-_.~ ---".---_. / 23 33 3S 2n 30 30
::~~s~f---
~
~~2~:li:
)--I~~~~i~1O
~--.'(-:~~~,~)-
ı- (::~;:;--_.-.a = Eighty samples from eaeh part
Thc distribution of C.j,jııni and C. eoli in differcnt parts of intcs-tines is alsa shown in Table 1. Of 69 w-ains of Campylobaeter sp. isola-ted, 35 (50.7 %) wcrc idcntificd as C.jejll1li and 34 (49.:-3 %) wcre identified as C. coli. In the intestine of a chicken, C. (ali was isolated from duodcnum and C.jejuni \Vas isolatcd from jejunum, ileum and caecum.
The growth of campylobacters on sdcctive media ,yas scored from Grade 1 to Grade 4 according to thc number of colonies. Average growth scores of Campylobacter colonies from each part of intestine were calculatcd by adding all grades~togeher and dividing by the number of Campylobactcr positivc samples. Averagescores of campy-lobacters from duadenum, jejunum, i1cum and caecum were 1.90, 2.43, 3. i 1 and 3.33, respectivdy (Tabil' 2).
Tablc 2. The growth of C'nmIJylobac1cr Sı}. on sclecti,'c medi;ı. :'\ umbel' of straiIls
Duodelltım .Jejunum Ileum Caecum
--- ----_.._--- ...--- ---_.-._- ---_ .. _--Grade3 1 Grade 2 Grade 3 Grade 4. 23 II .j. 7 18 10 li 17 5 12 17 29 4 9 13 39 ---_.-- --- --- --_.---
---i
Average score 1. 90 2.43 3. 11 . 3.33aKey: O =no growth, 1 ~ 1 - 5 colonies, 2 = G.- 25 colonies, 3 ~ 26 -- 100 colon;es, 4 :> 100 eolonics.
Calonies of contaminant microorganisms were alsa .obscrved on selective media. Contamination rates of cultures inoculated with the samples from duadenum, jejunum, ileum and caecum were 28.8 %,41.3 %,62.5
%
and 73.8%,
respectively (Tablc 3). Growtlı of contaminated microarganisms was alsa scored according to thenul11-SU K. SERDAR DIKER - HAKAN YARDıMCı -- NEJAT AYDIN
bel' of colonies and average scor<~swere ealculated. Average scon~s of contamination of cu!tures sampkd from duodcnıım, j<-;junıım, ileum and caccıım were i .56, 1.5'1,2.04 and 2.74, respectivcly (Tablc 3).
Table 3. The distribution and gro"'th or conlaminanl microorganisıns on seleeıive rncdi;ı.
Duodenum .Jejunum Ileum Caeeum
j
J
Grade" ı Grade 2 Grade 3 Grade 1 ı7 2 i :in
1 7 o 13 H IL 2 .') 23 ı3 18 ~--._---- _._-. ----_.-.--- -.- .-"--- -.---."---- ._---Total 23 33 .'iD :,9 ~~~_) ~28.8) __ ~~.3) __1--~~2~~~)---
__
(~i~)--I
Average scorc 1.:)(j ı.51- 2.o,ı 2.7+
aKey: () ~ no gro",ıh, ı - i - :)colonies, 2 -- G- 2:; colonic.', 3 = 2()--IOO colonics, 4 - > 101 calonics.
Discussion and Condusion
In reccnt years, numeroııs workers have described the pres-ence of
r:.
jejııııi and C. eoli in poultry (6, 7, 21), most attention having been paid to processed broilers in whieh a high incidence has been demonstratcd (4, 16). In the most of these works, cloacal swabs, caecal eontcnt and liteces have been used as the sampling sites of intestinal tracL Studies in which distribution of Campylobaetersp.
in variolis parts of intestine has been compared are rather scarce. In the present study, sample sizes were smail relative to the sizes of flocks being monitored, bııt judging from our previous experiences using similar sampk size, they were su/Iicient to indicate the trends of infection in nods. Camp]lobaeter sp. \Yere usually either present orabsent in all or most of the birds in any [lock. This high incidcnce has been express ed by severalother workers as a result of almost universal distribution of thermophilic Campylobaeter sp. in poultry (I 2, 17).
This study has showed the variability of Carnpylobacter colo-nization among various parts of chieken in testine. Lower part of intestİne seems to be predominant sİte of Campylobaetcr colonization. Frequency of campylobaeters İn caeeum and İleum was very close to eaeh other. But, CamAl'lobaeter sp. was Icss frequendy isolated from smail intestine. Soerjadİ ct al (I 8) and Oosterom et al (I O) have also repor-ted gradual distribution of C.jej/lııi in ehieken iııte.stine. High
inciclcn-LOCATfON OF THERMOPHTUC CAMPYLOBACTER... 575
cc of Camp)'lobaeter sp. İn caeeum reported by many \Yorkers (9, 20) also earl'ciates wİth our results. Average scores of Campylobaeter eolonİes from eaceum and ilcum were higher than those of upper parts of intestİne. it means that thc incidence of CamlJ)%bacter .IP. is rclated to the numbcr of eampylobaeter eeııs in eaeh part of intestine. This suggcstion has been canfirmed by the findings of Soeıjadi ct al (18). it ,,'as of interest to determine the isolation of C. eoli (i-om duodenum and the isolation of C. Je:jııııi from other parts of intestine in a ehİeken. It ea;l be suggested that difıcrent strains of Camp)'lobacter sp. can eo-lonize the various parts of intestine of a ehieken. And if it is praetieal to identify eaeh eolony grown on seleetive medİa, this phenomenon may be deteeted more fregucntll'.
Very large numbers of eontaminant mieroorganisms were obser-ved on seleetive media onto which samples from caecum had been inoculated. This is not surprising since İt has been shmvn by Salanitro ct al (14-) that lower parts of chieken intestine ,partieularly caecum, contain large numbers of bacteria as comparing with the upper parts. For this reason, ileum can be considered as an alternative sampling site for the isolation of Camp],lobaeter sp., since its contaminant bacteria content is small relative to the bacterial content of caecum.
In this study, [om chickcns earrying Camp)'lobar:ter Si). in their smaıı intestine but not in caecum havc been determined. Examina-tion o[individual cloacal swabs or caecal content from arandam samp-le o[ birds is eonsidered as an effcctive method by several authors (6, 18). This is true lor surveys with large samplc size, but when saınpling an individual chieken for culturing CamjJ);fobac!cr sp., it must
be kept in mind that campylobaeters, evcn in smaıı numbers, may be calanize only in sınai in testine.
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