KHOA HOC CONG NGHE
PHAIM L A P V I KHLIAiXI Clostridium batuNnum T U V ! T B l "CLIIVI CAIXl"
Nguydn Thu TdmS Nguydn Due Hien^
T6MTAT
Tir thang mudi nam 2018 den tbang tu nam 2019, 200 mdu (100 mSu chat cbiia frong rugt va 100 mdu gan) duoc lay tir cac vit bi liet mem co tai mot so huyen thupc tinh An Giang, Hau Giang va Vinb Long. MSu pban lap vi khuan Clostndium botulinum trong moi tnrong Cooked Meat Medium, moi truong thach mau va kiem tra dkc tinb sinh boa bang phuong pbap nhu6m Gram k^t hop voi ky thuat PCR cua Miia Lindstrom va Hannu Korkeaia, (2006) va Amit-Romacb et al, 2004 de phan lap vi khuan Clostridium botulinum. K^t qua cbo thay vi khuan Clostridium botulinum tren mAu ruot va gan vit co tneu chung Let m^m co la 10%. Ket qua dmh type vi khuan Clostridium botulinum tren cac mki gom 5 mautypeC (1,5%); 8 mlu type D (4,0%) va 10 mSu type E (5,0%). Vi khuan Clostridium botulinumtype C, D va E la mot trong nbung nguygn nhan gay benh ciim can tren vit o mot so dia phuong thuoc tinb An Giang. Hau Giang va Vinh Long. Ket qua kiem tra tinh nhay cam ctia 20 mSu vi kbuan Closbidium botulinum phan lap duoc voi 5 loai kbang sinh gom doxycycbne, norfloxacin, marbofloxacm, florfenicol va fosfomycme cbo tbiiy cac vi kbuan pban lap duae nay nhay boan toan (100%) voi cac khang sinh tbii' ngbiem.
Tiikh6a: Botulin, Clostndium botulinum, li$t mem cd a vit, ehuotbach.
LDATVAniSE
Vi khuan Clostndium botulinum la vi khuan yem khi, co kha nang sinh nha bao va sinh doc to botubn. Botulin la loai doc td than kmh. Dpc to botubn khi xam nhap vao co the gay uc che san sinh acetylchohne - la chat dan truydn tin hieu tir dau miit than kinh den ca, gay ra li$t co, het cac chi, nang co thi gay suy ho hap va chet. Doc td nay la mot trong nhirng nguySn nhan quan trpng gay chiing bet mem cd, het mi mat trong, liet canh a vit chay ddng a dong bang sdng Cuu Long (Ho Thi Viet Thu va Nguyen Due Hien, 2017) ma ngudi dan quen goi la vit bi "cum can". Nghien cuu nay nham tim hieu su hien dien cua Clostridium botulinum tren nhung vit hi bet mem cd tai mgt sd huygn thupc cac tinh An Giang, Hau Giang va Vmh Long.
2. VAT UEU VA PHUDIW PHAP NGHIBU CUU 2.1. vat Mil
Thudc nhudm Gram, moi tnrong TSA (Trypticase Soy Agar, Merck, Germany), cac loai dudng Glucose, Maltose, Lactose, Saccarose (Merck, Diic), mau cim (Cdng ty Nam Khoa, VN), CMM (Cooked Meat Medium) (Difco, USA), SFP Base
Bo raon Thu y, Khoa Nong nghiep, Trucmg Dai hoc Can Tha
^ChicucThi^yCanTha Email: [email protected]
(Difco, My), dung dich ddm gelatin (gelatin phosphate buffer), cdn (96°, 70°), nude mudi sinh ly, nuoc cat, cac thiet bi va dung cu can thiet dimg trong nuoi cay vi khuan Clostridium botuhnum. Bp kit b trich vi khuan gram duong (Gene JET Genomic DNA Purification Kid, Thermo Scientific). Dung cu ly trich DNA Vl khuan C. botulinum va thuc hien phan iing PCR.
Dia khang sinh: doxycycbne, marbofloxacm, norfloxacin, florfenicol, fosfomycm (Oxoid, Anh).
2.2. Mlu va phirong phap lay mlu
- Mau. Chat chiia trong rupt gia - doan tiep giap vdi manh trang va mlu gan lay tir nhiing con vit voi cac trigu chiing nhu: ligt co, bgt cban, ligt canh thu thap bi vit chay dong tai mot sd huyen thupc cac tinh Hau Giang, An Giang va Vinh Long
Phuang pbap lay mau: Tien hanh thu thap nhting vit cd tneu chung cua benh tai mdt sd huyen thupc cac tinh Hau Giang, An Giang va Vinh Long.
Chpn lay nhiing vit hon 2 thang tuoi co trieu chiing dac tnmg nhu cd bi mem, liet mi mat, ddi khi bet canh hoac cban, sau do hiiy tuy. Mo lay toan bo dudng tieu hoa cua vit tif hSu den hau mdn (cot day tai hai vi tri cdt de giii mdi trudng ydm khi ben trong dudng tieu hda). Ddi vdi mlu gan, nhe tay lay toan bo la gan vit ra. Cac mlu rudt va gan cho vao tiii nilon vo triing, ep het khong khi, cdt chat, mdi mlu
NONG NGHIEP VA PHAT TRIEN NONG THON - KY 2 - THANG 1 /2020 53
dupe dat vao bii rieng va ghi chu code mau, cho vao thiing tru lanh mang v^ phdng thi nghiem.
2.3. Phuang phap nghign ciiu
2.3.1. Phuong pbap pban lap vi khuan Clostridium botulinum tii tdt billet mem cd
Dung tam bong vo triing nhting vao chat chiia trong mlu rupt hoac dilng tam bong vo trimg khuay sau vao ben trong gan. Phet true tiep tam bong vua lay mlu len dia thach man, ria cay mlu, sau dd dem li ygm khi (37X fa-ong 24 gib).
Chpn nhiing khuan lac mau trang due, to, nhay, be mat kho, khong bdng, cd kba nang dung huydt, dudng kinh khoang tu 0,5-1 cm. Nhupm Gram, quan sat tieu ban dudi kinh hien vi voi vat kinh lOOX de xem hinh dang, tinh chat bit mau, binh dang nha bao, dp tbuan nhat ciia vi khuan. Clostridium spp. la true khuan hinh que thing hoac hoi cong, bat mau Gram duong, thing, hai dau trdn, kicb thudc chidu rong 0,9 - 1 pm, chigu dai 4 - 8 pm. Nba bao cd hinh tning dai voi nhieu binh dang: dang diii trdng va dang khong din (tao bao tu ovale d' tam hoac nim d' Bang 2. Trinh tu nucleotide cijia cap doan m6i
mpt diu vi khuan. Sau khi nhupm Gram, vi khuan Clostridium botulinum dupe kiem b-a dac imb sinh hda theo Nguygn Nhu Thanh, 1997 gdm CTlucose {+), Maltose (+), Lactose (-), Saccarose (-) va 'ndole
(-)
Vl khuan Closdidium spp. sau khi duoc thu sinh hda se duoc tang sinh tren moi bordng thach mau de tien hanh ly b-ich DNA. DNA ciia vi kbuan dupe ly trich theo phuong pbap ciia bo kit ly trich GeneJET Genomic DNA Purificabon K0721, K0722 (Thermo Scientific). Chu binh nhiet va dien di trong ky thuat PCR dupe thuc hien theo Miia Lindstrom, (2001).
Trinh tu cac cap mdi dupe trinh bay d bang 1 va 2 Bang 1. Trmh hi nucleotide ciia cdc cap dogn m6i xac dinh gene ciia vi k h u ^ Clostridium spp. (Amit-
Romach etal, 2004) Primer
PCR Clos5M aos780-r
Trinh tu (5'-3') AAAGGAAGATTAATACCGCATA ATCTTGCGACCGTACTCCCC
Kich thuac SP 722bp xlc dinh vi khuan C. botulinum ^ e C, D, E
Pnmer Trinh tu Gene Kich thudc
SP BoNT/C Fw TAGTAGAATCTTCAGGTGAAG
BoNT/D Fw TTAATATAGAAAATTCGGGTCA ATATGAATCnTCCATCTCTTAA
lllSbp (typeC) 887 bp (typeD)
Nakamura et . 2013 BoNT/Ef CCAAGATITTCATCCGCCTA
BoNT/Er GCTATFGATCCAAAACGGTGA
389 bp (typeE)
Lindstrom et al., 2001
2.3.2. Pbuong pbap kiem tra dp nhay cua vi khuan vdi khang sinb
Chuan bi canh tning cua vi khuan Clostridium botulinum thuan sao cho dat nong do 10^ CFU/ml (bang cich so do due vdi dng chuan Mc Farland 0,5).
Dimg que tam bdng vo tning nhiing vao canh tning, ep vIo thanh dng nghiem cho bot nuo'c rdi na ddu khap mat thach SFP. Diing kep v6 trung dat cac dia
Bang 3. Budng kinh vong vo khudn chuan ciia mdt s6 loai khing smh (CLSI, 2016) khang sinh da chon Ign mat thach, cac dia khang sinh each nhau 2,5 cm den 3,5 cm vl cIch ria ^ a thach 2 cm. Dia dupe u trong til am CO2 d' nhiet dd 37*'C. Sau 24 gid' do duong kmh vong vo khuan (mm), rdi ddi chieu vdi bang tieu chuan cua CLSI (Cbnieal and Laboratory Standards Institute) nam 2016 de dlnh gia.
STT 1 2 3 4 5
Khang sinh Doxycycline Marbofloxacm Norfloxacin Florfenicol Fosfomycin
Ham lugng (p) 30
5 10 30 200
Duong kinh vong vo khuan ( Nhay
>13
>18
>17
>19
>16
Trung binh 10-12 15-17 13-16 15-18 13-15
Tim) khang
< 9
<15
<12
<14
<12
KHOA HOC CONG NGHE
2.3.3. Phmmg phap xit Iy so lidu
Sd lieu duoc xtr ly bang phan mem Microsoft Excel 2013 va Minitab 16.0.
3. KET QUA VA T H M LUAN
3.1. ffit qua phSn Up vi khuin Clostridiam botnlinum tu vit bi li6t m ^ c6 theo dia phmmg Bang 4. Ty 16 n h i ^ vi khuIn Clostiidiwn botulinum
tfaeo dia phuong Dia di^m
An Giang Hau Giang Vinh Long Tong
Sdmiiu phan lap
72 64 64 200
Clostridium botulinum
(+) 8 7 5 20
Tyle
%
11,11"
10,94"
7,81"
10 Qua bang 4, vi khuan Clostridium botulinum chi^m 10% trong cac mlu kiem tra (20/200 mlu). Ty le nay cho thay su hien dien ciia vi khuan Clostridium botulinum tren vit tuong ddi cao. Su hien dign ciia vi khuan Clostridium botulinum trong cac Vlt ehay ddng se 11 ngudn phat tan vi khuan vao mdi trudng, tir dd cd the lay lan sang vit khac hoac ddi tupng khic.
Bang 4 cQng cbo thay cac vit thu thap a An Giang co ty le duong tmh vdi Clostridium botulinum cao nhat (11,11%); kd den la Hau Giang (10,94%) va tbap nhat 11 oVinh Long (7,81%), tuy nhidn su khac biet nay khong cd y nghia thdng ke. Dieu nly co the ly giai do 3 tinh cung thudc dia vimg dong bang sdng Ciiu Long, chiu anh huong cua dieu kien tu nhien khi hau khong khic biet nhieu. Ngoai ra, viec nuoi vit chay ddng cQng ludn thay ddi lidn tuc eac canh ddng dd cd the tan dung ngudn thiie an roi vai, dieu nay lam cho ty le hien dien cua vi khuan se khong khae nhau giiia cle dia bin khic nhau.
3.2. K^t qui su hi$n di$n ciia vi khuan Clostriditim botulinum trgn cac mlu hgnh pham ciia vit
Qua bang 5 eho thay su hien dien cua vi khuan Clostridium botulinum trong ruot vit chiem ty le 13%
(13/100) va b-ong mlu gan vm ty le 7% (7/100). Ty le pbln lap vi khuan tir mlu rudt cao hon mau gan vl su sai khac nly cd y nghia vd mat thong ke. Didu nay cho thay vi khuan khu tiii b mot so co quan trong co the khi vit bi bgnh 11 cd khic nhau. Su khac biet nly phan Inh su thich nghi cua vi khuan trong dudng
tieu hda duoi. Duong tieu hda dudi 11 mdi trudng yem khi, chiia nhieu dinh duong thuan lpi cho su phat trien cua vi khuan Clostridium botulinum. Vi khuan Clostridium spp. vdn cd sin trong rupt vl chd' CO hdi gay benh (Smidi, 1987). Nghien ciiu cua Dolor (2013) chiing minh vi khuan C. botulinum tdn tai trong duong tigu hda nhung chiing hidn didn d' phan dudng tieu hda dudi nhu rudt (14/26 mlu, ehiem 54%), manh ti-|ng (16/22 mlu, chiem 73%) nhidu hon phan tren nhu da day (10/29 mlu, chiem 34%).
Ty le nhigm vi khuan Clostridium botlinum a mlu rudt vit cao hon so vdi mau gan la hoan toan phil hpp vdl cac nghidn cuu da cd trud'c day vi nguyen nhan chu yeu gay ngo dpc 11 do Clostridium botulinum xam nhap vao co the bing dudng tieu hda vl vet thuong ho. Neu vit an phai cua, dc, dong vat thiiy suih, loai nhuygn the, xac ddng vat chdt...c6 mang mam benh khi ehan tha trgn moi trudng dat, nude cd nhidm khuan Clostridium botulinum a the nghi khi vao rupt gap dieu kign thich hpp se san sinh dpc to gay ngp dpc.
Bang 5. Ty lg nhilm vi khuan Clostridium botulinum.
tii chit chiia trong rupt vl gan ciia vit Loai mau
Gan Ruot Tong
Sdmau phanlap 100 100 200
SomSu duong tinh
7 13 20
Tyle
(%)
7"
13' 10 Gbi cbu: Nhdng sd trong cung 1 cdt mang chd sd mu kbac nbau tbi khac nhau cd y nghia thdng ke (P<0,05).
3.3. K^t qua dinh type C, D, E c i a vi k h u ^ Clostridium botidintrm bing phan iing PCR trgn cle mlu
Su hien dien ciia vi khuan Clostridium botulinum type C, D, E tren vit bet mdm cd dupe ghi nhan d'bang 6.
Bang 6 cho thay su bien dien ciia vi khuan C.
botulinum type C, D, E trong mlu vit ed bieu hien liet ed, bet ehan, bdt canh. Trong dd, C. botuhnum type E chiem ty lg cao nhat (5%), ke den 11 type D (4%) va type C 11 1,5%. Tuy nhign su khac biet nly khdng cd y nghia thdng ke. Mac du ty Id cua cac type C, D va E chua cao nhung ket qui nly cho thay cd su hien dien cua cac lype ddc td eiia vi khuan C.
botulinum. Tir diy, vai trd gay bgnh het mem co cua NONG NGHIEP VA PHAT TRIEN NONG THON - KY 2 - THANG 1 /2020 55
KHOA HOC CdNG NGHE
C. botijlinum type C, D, E tren vit da dupe khing td than kinh botulin (BoNTs), phd bien nhat cua lo^
dinh. Theo Anza etal. (2014), khao sit tinh hinh C. C/D. Type E cd su xuit hien phd bien tiong mdi botulinum gay benh ngp dpc gia elm d- mien Nam vl trudng, eae vung dat ngap nuoc. Vi kbuan L.
mien Bac chau Au thi ngd ddc gia cam la mot benh botiilinum type E tdn tai rat phd bi^n tt-ong (ia* ^^
bai liet gay ra bdi vi khuan C. botulinum san xuat doe nhidu nhit la d tren luu vuc Gohannsen etal., 1963).
Bang 6. Tyie nhilm C^toAipfM? type C.D.E trdn mlu gan/niQt dugc xac dmh falngkythuatPCR Loai
Mau Gan Ru6t Tong
Soman kiem tra 100 100 200
TypeC Duong
tinh 1 2 3
Tyle%
1,00 2,00 1,5'
Type D Duong
tinh 3 5 8
Tyle%
3,00 5,00 4,0"
TypeE Duong tinh
3 7 11
Tyle%
3,00 7,00 5,0- 3.4. K^t qua kilm tra dp nhay cam cua vi khuan
Clostridium botulinum phan l^p duoc vdi mdt so loai khing sinh
Ket qua khao sit tinh nhay elm ddi vdi khing sinh vi khuIn Clostridium botuhnum phan lap bi vit bi bet mem cd dupe trinh bay b bang 7.
STT 1 2 3 4 5
Bang 7. K^t qua khang sinh d6 cua vi khuan Clostridium botulinum phan lap dupe Khang sinh
Doxycycline Marbofloxacin Norfloxacin Florfenicol Fosfomycin
Soman KT
20 20 20 20 20
Nhay 20 20 20 20 20
Tyle(%) 100 100 100 100 100
TB 0 0 0 0 0
Tyle(%) 0 0 0 0 0
Khang 0 0 0 0 0
Tyle
(%)
0 0 0 0 0 Kdt qua bing 7 cho thay 100% (20/20) ehiing vi
khuan Clostiidium botulinum phan lap dupe nhay cam holn toln vdi 5 loai khing sinh: doxycyclme, norfloxacm, marbofloxacm, florfenicol, fosfomycin.
Ket qua nly phii hpp vdi nghien ciiu cua Nguygn Diic Hien (2012), C. botulinum nhay cam hoan toan vdi norfloxacm, cephalexin, fosfomycin, eetiofur, kbing vdi ampieillin va amoxicilHn (80%). Cd the ehung vi khuan nay tdn tai trong mpt moi trudng tu nhien each biet, cd lap vda quln the vi khuan khac cu tni trgn nguoi vl cac dong vat nuoi, do vay ehua hi lay nhigm nhung gen de khang khang smh dang luu hanh kha pho bidn trong quln the vi khuan a khu vuc DBSCL. Nhimg thong tin ve tinh nhay cam va de khang ddi vdi timg loai vi khuan Clostndium botulinum cu the la nhung sd lieu hiiu ich cho nbiing ngudi lim cdng tac dieu tri.
4. KFT LUAN
Vi khuan Clostndium botulinum hign dien ti*ong phin vl gan cua vit cd trieu chting bet mem cd, bet canh, liet ehan 11 k h i cao (10%), trong dd vi khuan pbln lap bi rupt cao bon tii gan. Clostndium botulinum type E la type phd bien trong cic mlu thu
thap dupe. Cac vi khuIn phan lap dupe nhay hoan toln voi khing sinh doxycycbne, norfloxacin, marbofloxacin, florfenicol, fosfomycin
TAI LEU THAM KHAO
1. Hd Thi Viet Thu, Nguyin Diic Hien (2017).
Giao trinh Benh tmyen nhiim gia sue gia cam. NXB Dai hpe Cin Tho, ti-ang 101-111.
2. Nguygn Nhu Thanh (1997). Vi smh vat thuy.
NXB Nong nghiep Ha Npi.
3. Nguygn Diic Hidn (2012). Phan l§p vl xac dinb tinh nhay cam khang smh eua vi khuan Clostiidium botulinum bi vit vl moi truong ehan tha tai thinh phd Cin Tho. Tap chi Khoa hpe 2012:22c, page 64-71.
4. Amit-Romaeh E, Sklan D and Uni Z. (2004).
Microflora ecology of die chicken intestine using 16S ribosomal DNA primers. Poultiy Science 83: 1093- 1098.
5. Anza I, Vidal D and Mateo (2014). / e v insight m tbe epidemiology of avian botiibsm ahn-uks:
necrophagous fUes as vectors of Clostridium botiilinum type C/D. 6(6):738-43.
NONG NGHIEP VA PHAT TRIEN N 6 N G THON - KY 2 - THANG 1 /2020
KHOA HOC CONG NGHE
6. Burgen. S. V, Dickeen & Zatman. J. (1949). 9. Johnson AL, McAdams SC, Whitlock RH The action of botuluium toxin on the neuromuscular (2010). Type A botiihsm m horses in the United junction. JoumalofPbysiologylQ9,10-24. States: a review of the past ten years (1998-2008). J
7. Lindstrom M. (2001). Multiplex PCR assay for Vet Diagn Invest; 22:165-173.
detection and identification of Clostridium botuhnum 10. Johannsen (1963^. Clostiidium botuhnum m types A, B, E, and F in food and fecal material Sweden and tbe adjacentwateiv. Bacterial. 26:43-47.
Enviromental Microbiology. Pp: 5694-5699. ^1 ^^^.^ ^ j ^ ^ ^ ^ ^ ^ ^^^^^ ^j^gg^ ^.^^^^^^
8. Nakamura K, Kohda T, Seto Y, Mukamoto M, (Field Manual of Wildlife Diseases). Other Kozaki S. (2013). Improved detection methods by Ptibhcations in Zoonotics and Wildlife Disease. Paper genetic and immunological techniques for botulinum 16.
C/D and D/C mosaic neurotoxins. Pp:881-890.
ISOLATION OF Clostridium botiiSnum'FROU UMBERNECK-DUCKS Nguyen Tliu Tam, Ngi^en Due Hien Summary
From October 2018 to april 2019,200 samples (100 intestine samples and 100 liver samples) wewe collected from limbemeck ducks in some district of An Giang, Hau Giang and Kien Giang provme. All samples were cultured on Cooked -Meat-medium, blood agar and PCR test by Miia lindstrom and Hannu Korkeaia (2006) and Amit-Romach et al., (2004) to identify Clostndium botulinum. Tbe result showed tbat, 10%
(20/200) samples were possitive with Clostiidiwn botulinum. By PCR method, Clostridium botulinum type C, type D and type E was 1.5%; 4.0%; 5.0%, respectively. Tbe result showed (hat Clostridium botuhnum type C, D, E was main causes of limbemeck duck m An Giang, Hau Giang and Kien Giang provme. The result of antibiotic sensitivity test of 20 Clostiidium botnlinum isolates against 5 antibiotics including doxycycline, norfloxacin, marbofloxacin, florfenicol and fosfomycme showed that all isolates of these bacterial ii were fully sensitive (100%) to tested anUbloties.
Keywords: Botulin, Clostridium botulinum, duck, limbemeck.
Ngudd phan hifin: PGS.TS. Cu HiJU Phii Ngly nh$n bii: 4/11/2019 Ngly thdng qua phan bi6n: 5/12/2019 Ngly duygt dang: 12/12/2019
NONG NGHIEP VA PHAT TRIEN NONG THON - KY 2 - THANG 1 /2020
