韓国の2か所のブロイラー生産農場におけるpersistent

Salmonella Enteritidis株の特徴

誌名 The journal of veterinary medical scienceISSN 09167250著者名発行元 Japanese Society of Veterinary Science巻/号 70巻10号掲載ページ p. 1031-1035発行年月 2008年10月

農林水産省 農林水産技術会議事務局筑波産学連携支援センターTsukuba Business-Academia Cooperation Support Center, Agriculture, Forestry and Fisheries Research CouncilSecretariat

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FULL PAPER Public Health

Characteristics ofPersistent Salmonella Enteritidis Strains in Two Integrated Broiler

Chicken Operations of Korea

Sang Ick KWAG1l， Dong Hwa BAE2l， Jae Keun CH03l， Hee Soo LEE4l， Bok Gyeong KU4l， Byoung Han KIM4l，

Gil-Jae CH02l and Young Ju LEE2l*

IJAnimal Health Division， Ministlア01Agriculture & Forestry， Gwacheon， 427-71久2JCollege01 Veterinary Medicine， Kyungpook National University， Daegu， 702-701， 3) Daegu Metropolitan City Research lnstitute 01 Health & Environment， Daegu， 706-732 and のNationalVeterinary Research and Quarantine Service， Ministry 01 Agriculture & Forestry， Anyang， 430-824， Republic 01 Korea

(Received 25 December 2007/Accepted 2 June 2008)

ABST貼 CT.The objectives of the study were to investigate the phenotypic and genotypic characterization of the persistent Salmonella Enteritidis (s. Enteritidis) isolates in two integrated broiler chicken operations， with attention focused mainly on the epidemiological approach. In the distribution of virulence genes， Salmonella enterotoxin (s的)， invading host cell (invA)， and Salmonella plasmid viru-lence (spvC) genes were widely distributed among the S. Enteritidis irrespective of their source of isolation， and Salmonella timbrial (sejC) and plasmid encoded timbrial (pψgenes were present in 28 and 20 S. Enteritidis strains， respectively. A total of 5 different Xbal-PFGE types were obtained from 31 S. Enteritidis isolates. Twenty-one types were divided on the basis their PFGE pattem， phage type and antimicrobial resistance pattem determined. There was a signiticant difference in phenotypic and genotypic characterization by two integrated broiler operations. Also， 8 isolates shown susceptible to all antimicrobials and 11 isolates with resistance to nalidixic acid were partly c1assitied by Xba1 PFGE pa悦emand by the phage type. KEY WORDS: chicken， Salmonella Enteritidis.

Many researchers have reported that salmonellosis out-

b閃 akswere associat疋dwith poultry meat or eggs [9， 18，29]. Vertical transfer of infections合ombreeding hens to prog明

eny has been an important aspect of the epidemiology of

Salmonella species infections within the poultry industry [1， 7，8，12，30].

Epidemiological studies are needed to identifシthesource

of an infection and implement prevention. Several typing

methods， based on phenotypes or genotypes， have been developed， but the research for an efficient procedure still continues. Serotyping， which is based on the differentiation of 0 and H antigens， is the most widely used phenotyping method. However， the complexity of the system and diffi-culty of laboratory to laboratory comparison of results limit

the application of serotyping to reference laboratories. For

the future discrimination within Salmonella Typhimurium

(S. Typhimurium) and Salmonella Enteritidis (s. Enteriti-

dis)， phage typing is the primary subtyping technique. But， phage typing frequently fails to discriminate between out-

break-related and unrelated isola臼s[17]. In recent years， many DNA-based genotyping techniques have been utilized

to delineate epidemiological relationships between various

isolates [23， 32]. But， the ∞mbination of several methods

inc¥uding phage typing， serotyping， antibiogram and genetic typing may provide a powerful discriminatory tool for the

epidemiological analysis of unrelated and related Salmo-

nella strains [14， 16]. The objectives ofthe study were ωinvestigate the pheno-

typic and genotypic characterization of the persistent Sal-

キCORRESPONDENCE下0: LEE， Y. 1.， College of Veterinary Medicine， Kyungpook National University， Daegu， 702-701， Korea. ト ma創il上:y卯ouげr咽 u@knu.ac.kr

J. Vet. Med. Sci. 70(10): 103ト1035，2008

monella isolates in two integrated broiler chicken

operations， with attention focused mainly on the epidemio・logical approach.

MATERIALS AND METHODS

Bacterial isolates: A total of 31 s位ainsof S. Enteritidis

isolated合ombreeder farms， hatcheries， commercial broiler farms， and slaughter houses of 2 integrated broiler compa・

nies were investigated for this study (Table 1). Two typical

colonies picked at different sample sites of various sources

were serotyped by slide and tube agglutination for 0 and H

antiserum according to the latest version of the Kauffmann

and White scheme [24]. If two colonies showed the same

serotypes and antimicrobial susceptible pattem， only one colony was randomly chosen and inc¥uded in this study.

Detection 0/ sejC， pef， s的，invA and spvC genes: For the detection of virulence genes by polymerase chain reaction

(PCR)， bacterial cells合omthe ovemight cultures were sus-

Table 1. Salmonella enteritidis used in this study

Company code

A

B

Total

Source

8reeder farm Hatchery Commercial broiler farm Chicken slaughter house

Hatchery Commercial broiler farm Chicken slaughter house

NO.of isolates

3 5 5 4

7 2 5

Total

17

14

31

1032 S. 1. KWANG ET AL

pended in 350μ1 distilled water and boiled at 1000C for 10 min. After boiling， the cell suspensions were used for PCR. The oligonucieotide primers for PCR were synthesized according to the published reports [4， 19]. The PCR mixture consisted of 5μ1 of 10 x PCR amplification buffer (Promega， Madison， WI， U.S.A.)， 1.5 mM MgCI2， 200μM (each) of primer pairs， 1.25 U of Taq polymerase (Promega)， 2μ1 of cell suspension， and distilled water that was combined to make a total volume of 50μ1. PCR reac-tions perforrned using a GeneAmp 9700 PCR system (Per・

kin-Elmer， Applied Biosystems Division， Foster city， CA， U.S.A.) and the parameters for the amplification cycles were done according to the reports [4， 19]. After the last cycle， the mixture was mixed with agarose gel sample buffer with tracking dye， and electrophoresis in a 2% agarose gel. After staining with 'ethidium bromide， the amplified DNA fragments in the gel were visualized and photographed under UV illumination.

Phage typing: AII S. Enteritidis tested were phage-typed with 16 bacteriophages at the National Veterinary Research and Quarantine Service (Anyang， Republic ofKorea). Stan-dard phages were obtained from the Laboratory of Enteric Pathogens， Public Health Laboratory Service (PHLS)， in England. Briefly， cultures incubated for 24 hr on agar plates were inoculated into 3 ml of a phage broth. After incubation for 2 hr with vigorous shalくing，the broth was poured onto a phage agar plate. After the excess broth was removed from the plate， 16 typing phages were spotted onto an agar plate with a micropipette. The dried plates were incubated over-night， and the phage Iysis pattem of each culture was com-pared with the published pattems. Strains showing a pattem that did not conforrn to any recognized phage type were des-ignated as “reacted but did not conforrn" (RDNC). Strains that did not react with any of the standard typing phages were designated as “untypable" (UT)

Antimicrobial susceptibility testing: AII S. Enteritidis tested were investigated for their antimicrobial resistance by the agar disk diffusion test using the following disks (Difco， Becton Dickinson， NJ， U.S.A.): ampicillin (Am， 10 μg)， amoxicillinlclavulanic acid (Amc， 20110μg)， cephalothin (Cf，30μg)， cefoxitin (Fox， 30μg)， cefotaxime (Ctx， 30μg)， kanamycin (K， 30μg)， streptomycin (S， 10 μg)， gentamicin (Gm， 10μg)， amikacin (An， 30μg)， nalidixic acid (Na， 30 μg)， ciprof

bioMerieux Vitek colorimeter. Immediately， 400μlof

adjusted cell suspension was transferred to 1.5 ml micro-centrifuge tubes with 20μ1 of proteinase K (20 mg/ml stock)， subsequently mixed with 400μ1 of melted 1 % SeaKem Gold (Cambrex， East Rutherford， NJ， U.S.Aよ1%SDS agarose prepared with TE Buffer (10 mM Tris:1 mM EDT A， pH 8.0)， and pipetted into disposable plug moulds. Three plugs were transferred to 50 ml polypropylene screw-tubes with 5 ml of cell Iysis buffer (50 mM Tris:50 mM EDT A， pH 8.0 with 1 % sarcosyl) and 25μl of proteinase K (20 mg/ml stock) and incubated at 540C in a shaker water bath and agitated for 2 hr. Afterwards， the plugs were washed twice with 15 ml of sterile water and three more times with TE Buffer at 500C for 15 min. Chromosomal DNA was digested with 50 U of XbaI (Promega). PFGE was performed on a CHEF Mapper XA system (Bio-Rad Laboratories， Richmond， CA， U.S.A.) in 0.5X Tris-Borate-EDTA buffer (Bio司 RadLaboratories) with recirculation at 140C. Pulse times ramped合om2.2 to 63.8 sec during an 18 hr run at 6.0 V/cm. After electrophoresis， the gels were stained in 2μg of aqueous ethidium bromide (Sigma-Ald-rich， St. Louis， MO， U.S.A.) per milliliter for 15 min and were photographed using 300 nm UV light.

Similarity of patterns: The similarity of PFGE pattems was calculated by means of computer-based similarity and c1ustering programs (BioNumerics 3.0， Applied Maths， Bio-sistematica， Devon， UK). Dice coefficient was used for similarity calculation and the similarity matrix was expressed graphically by an unweighted average linkage (UPGMA). The relatedness ofthe PFGE profiles of Salmo-nella isolates was estimated based on the presence or absence of shared bands.

RESULTS

A rapid and simple method， PCR assay， was carried out for the detection of均配，pef， stn， invA， and spvC genes in 31 S. Enteritidis isolates (Table 2). The stn gene， invA gene and spvC gene were found in all S. Enteritidis isolates tested without a difference between two integrated broiler chicken operations. In the occurrence of Salmonella fimbriae gene， Sぞfand pef， S. Enteritidis was found to ca町時陀 (90.3%)and pef(64.5%) genes partly

A total of5 different XbaI-types were obtained from 31 S. enteritidis isolates (Fig. 1). Five different types were divided into 2 c1usters， A and B. The c1uster A included pat-tern Xl， X2 and X3， and genetic similarity was over 90%. The similarity of cluster B， which included X4 and X5， was over 85%.

AII PFGE pattems， phage types and antimicrobial r巴SIS-tance pattems of the 31 S. Enteritidis isolates are summa-rized in Table 3. Eight ofthe 17 S. Enteritidis and all 14 S Enteritidis isolated from company A and company B， respectively， were resistant to one or more antimicrobial agents tested， respectively. A total of 31 S. Enteritidis strains isolated from two integrated broiler chicken opera-tions showed six phage types and one RDNC type. PTI was

1033 SALMONELLA ENTER1TlDlS IN INTEGRA TED CHJCKEN OPERA T10N

Table 2. Prevalence of virulence genes among Salmonella enleritidis isolated仕omtwo mte-grated broiler companies

No. (%) of positive for virulence gene

pef sln invA

NO.of

isolates

Company

code spvC sefC

17 (100) 14 (100)

16(94.1)") 12(85.7)

17(100) 14 (100)

17(100) 14 (100)

10(58.8) LO (71.4)

17 14

A

B

31 (50.0) 31(100) 31 (100) 20 (64.5) 28 (90.3) 31 Total

X3

Lambda ladder

A値マ，、d

tl

xxx X2

a) Number of isolates included virulence gene (%)

Xbal-type

Fig. 1. Representative XbaI restriction pattem. Dendrogram was generated by BioNumerics so仕wareshowing the relationships between pa仕emtypes

lates with resistance to Na were partly c1assified by XbaI PFGE pattem and by the phage type

Table 3. PFGE pattem， phage type and antimicrobial resistance paはemof Salmonella enleritidis obtained from two integrated broiler companies

The most frequent serovars isolated from humans in Korea were S. Enteritidis， S. Typhimurium and S. Typhi. These serovars were responsible for over 63% offood-bome illnesses recorded during 2004-2005 [13]. Recently， it has been reported that the prevalence of Salrnonella spp. in food， especially in poultry products (up to 2.2%)， is high in Korea [5]. Investigations have shown that the prevalence of S. Enteritidis infected chicken and eggs in retail outlets is 18.5% [3]， and have suggested that infected raw eggs or chickens could be a m勾orsource of Salmonellosis in Korea.

Salrnonella control in integrated broiler operations is complicated， because there are many opportunities for it to gain entry to these broiler operations and for it to be ampli-fied by the mass production of feed， and by the hatching， handling and processing facilities [11，21，28]. In this study， a total of 31 strains of S. Enteritidis isolated from breeder farms， hatcheries， commercial broiler farms， and slaughter houses of two integrated broiler operations were investi-gated to determine the phenotypic and genotypic character-ization for an epidemiological approach.

Pathogenesis of salmonel1osis depends upon a large num-ber offactors controlled by an訂 rayof genes that induce the actual virulence of Salrnonella. The current study investi-gated the distribution of five such genes， namely， Salrno-nella enterotoxin (s的)， Salrnonella fimbrial (sめ， plasmid encoded fimbrial (pφ， invading host cel1 (invA)， and Sal-rnonella plasmid virulence (spvC) genes， among S. Enteriti-dis isolated from two integrated broiler chicken operations.

The present study indicated that the stn， invA， and spvC

DISCUSSION No. of strains Company incl uded code

AAAAABBBABAAAABAABBBB

司

JE--s'』

tt

今L'I

今rh'l

つ-A斗

ti---11・'l'l

今、“，ltl'l

S Na Te

Na Am S Na Sxt Tmp Te C Na Na Na Na AmSTe

Na k

S Na NaTmp Na Na TeC Am S TeC SC Na Na

Antimicrobial resistance pattem')

Phage type

c

c

c

c

c

c

c

N

N

N

N

N

N

-

l

l比

日

間

l

7山

日

幻

問

問

m

m

m

l

m引

引

以

お

PFGE pattem

111111111A

今4ヲ&フ白今4ウ&

内，L

フ-フ白ヲ&「41d1dA斗ペJ

，、dp

コ

xxxxxxxxxxxxxxxxxxxxx a) Am， Ampicillin; S， Streptomycin; Na， Nalidixic acid; Sxt， Trimethoprim/sulfa-methoxazole; Tmp， Trimethoprim; Te， Tetracycline; C，Chloramphenicol.

the major phage type for strains from company A， whereas PT21 for strains from company B. Twenty-one types were divided into the basis of all properties determined. There was a significant difference in phenotypic and genotypic characterization by 2 integrated broiler operations. Also， 8 isolates shown susceptible to al1 antimicrobials and I1 iso-

1034 S. I. KWANG ET AL.

genes were widely distributed among the S. Enteritidis irre-spective of their source of isolation. These findings are in

agreement with earlier reports [4，19，25，26]. The S~バご andpef genes were present in 28 and 20 S. Enteritidis， respec-tively. But the prevalence of virulence genes showed no

significant differences between two integrated broiler

chicken operations

Antibiotic resistance in Salmonella spp. is a serious

health problem worldwide， and increased multiple antibiotic resistance has been reported in Salmonella isolates from

various countries including Korea [3， 6，15，31]. Therefore，

the characterization of resistance is essential to understand-

ing the epidemiological relationship of the Salmonella iso-

1ates. Eight of the 17 S. Enteritidis isolated合omcompany

A were resistant to one or more drugs tested， and one iso1ate showed multiple resistance to seven drugs. But， all ofthe S. Enteritidis isolated from company B showed resistance to

one or more drugs， and three iso1ates showed mu1tip1e resis-tance to 4 drugs.

Like the difference in antibiotic resistance， a1though in a

1imited number of iso1ates， the most frequent phage type was PT1 in S. Enteritidis iso1ated from company A， but PT21 in isolates仕omcompany B. Especially， there were no S. Enteritidis iso1ates合omcompany A that showed PT21.

The present study demonstrated that the predominant pat-

tem of antibiotic resistance and phage type showed signifi-

cant differences between two integrated broiler chicken

operatJOns.

This study also showed that both PFGE and phage typing

were capable of differentiating between S. Enteritidis

Especially， S. Enteritidis isolated in Korea have somewhat

of a difference to the PFGE pattem by origin of the inte-

grated broiler operation. Several studies indicated that

PFGE may offer an improved level of discrimination over

other genotypic typing methods such as plasmid analysis

and ribotyping for the epidemiological typing of S. Enteriti-

dis [10， 22， 27]. In this study， 21 types were distinguishable

on the basis of antimicrobial resistance pattem， phage type and PFGE pattem. The results of this study suggest that

molecular typing methods such as XbaI PFGE analysis in

addition to phage typing and antibiogram， support that a

specific discriminatory group of S. Enteritidis was detect-

able among independent chicken production lines of two

integrated broiler operations. Moreover， this

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