I HC THI NGUYấN TRNG I HC NễNG LM H VN QUANG NGHIấN CU MT S C TNH SINH VT HC CA VI KHUN ESCHERICHIA COLI, LISTERIA MONOCYTOGENES V SALMONELLA SPP NHIM TRấN THT LN TIấU TH TI A BN THNH PH LNG SN, XUT BIN PHP PHềNG CHNG LUN VN THC S TH Y THI NGUYấN - 2016 S húa bi Trung tõm Hc liu HTN http://www.lrc.tnu.edu.vn I HC THI NGUYấN TRNG I HC NễNG LM H VN QUANG NGHIấN CU MT S C TNH SINH VT HC CA VI KHUN ESCHERICHIA COLI, LISTERIA MONOCYTOGENES V SALMONELLA SPP NHIM TRấN THT LN TIấU TH TI A BN THNH PH LNG SN, XUT BIN PHP PHềNG CHNG Chuyờn ngnh: TH Y Mó s: 60 64 01 01 LUN VN THC S TH Y Ngi hng dn khoa hc: PGS TS NG XUN BèNH THI NGUYấN - 2016 S húa bi Trung tõm Hc liu HTN http://www.lrc.tnu.edu.vn i LI CAM OAN Tụi xin cam oan õy l cụng trỡnh nghiờn cu cỏ nhõn tụi trc tip thc hin cựng vi cỏc ng nghip ti B mụn V sinh - Vin Thỳ y Quc gia Mu vt thu thp ti ch khu vc thnh ph Lng Sn; cỏc s liu v kt qu nghiờn cu trỡnh by Lun l trung thc, chớnh xỏc, cha c cụng b bt c cụng trỡnh no khỏc Tụi xin cam oan mi thụng tin trớch dn Lun u c ch rừ ngun gc Mi s giỳp hon thnh Lun u ó c cm n Thỏi Nguyờn, thỏng nm 2016 TC GI H Vn Quang S húa bi Trung tõm Hc liu HTN http://www.lrc.tnu.edu.vn ii LI CM N Trong thi gian thc v thc hin ti ny, tụi ó nhn c s quan tõm, ch bo, hng dn, giỳp tn tỡnh ca cỏc thy cụ giỏo, ng nghip, bn bố v s ng viờn khớch l ca gia ỡnh Nhõn dp ny tụi xin c by t lũng bit n sõu sc ti: Thy giỏo PGS TS ng Xuõn Bỡnh ó trc tip hng dn, ch bo tụi ht sc tn tỡnh sut quỏ trỡnh nghiờn cu v hon thnh Lun Tụi xin trõn trng cm n Ban Giỏm hiu, Phũng o to, Ban ch nhim khoa v cỏc thy cụ giỏo Khoa Chn nuụi Thỳ y - Trng i hc Nụng lõm - i hc Thỏi Nguyờn ó to iu kin v giỳp tụi sut quỏ trỡnh hc Tụi cng xin trõn trng cm n cỏc ch quy bỏn tht ln ti ch ụng Kinh, ch K La v ch Ging Vuụng ó to iu kin cho tụi ly mu thc hin ti Xin trõn trng cm n b mụn V sinh - Vin Thỳ y Quc gia ó giỳp tụi quỏ trỡnh xột nghim mu v thc hin ti Cui cựng Tụi xin by t lũng bit n sõu sc v s ng h, ng viờn, giỳp ca gia ỡnh, bn bố v ng nghip sut thi gian hc tp, nghiờn cu v hon thnh tt Lun ny S húa bi Trung tõm Hc liu HTN http://www.lrc.tnu.edu.vn iii MC LC LI CAM OAN i LI CM N .ii MC LC iii DANH MC CC Kí HIU VIT TT vii DANH MC BNG BIU viii DANH MC HèNH, TH x M U 1 Tớnh cp thit ca ti Mc tiờu nghiờn cu í ngha khoa hc v thc tin ca ti Chng 1: TNG QUAN TI LIU 1.1 C s khoa hc ca ti 1.1.1 Ng c thc phm (NTP) 1.1.2 Tht ti v cỏc dng h hng ca tht 1.1.3 c im sinh hc ca vi khun E coli 1.1.4 c im sinh hc ca vi khun Salmonella 11 1.1.5 c im sinh hc ca vi khun L monocytogenes gõy ụ nhim tht 14 1.2 Tỡnh hỡnh nghiờn cu v ngoi nc 16 1.2.1 Tỡnh hỡnh nghiờn cu nc 16 1.2.2 Tỡnh hỡnh nghiờn cu trờn th gii 19 Chng 2: I TNG, PHM VI, NI DUNG V PHNG PHP NGHIấN CU 25 2.1 i tng v vt liu nghiờn cu 25 2.1.1 i tng nghiờn cu 25 2.1.2 Vt liu, húa cht v dng c nghiờn cu 25 2.2 a im, thi gian nghiờn cu 25 S húa bi Trung tõm Hc liu HTN http://www.lrc.tnu.edu.vn iv 2.3 Ni dung nghiờn cu 26 2.3.1 Kho sỏt tỡnh hỡnh git m v tiờu th tht ln ti khu ch nghiờn cu trờn a bn TP Lng Sn 26 2.3.2 Nghiờn cu v ch tiờu tng s vi khun hiu khớ nhim trờn tht ln ti 26 2.3.3 Kho sỏt ch tiờu vi khun E coli, Salmonella spp v Listeria monocytogenes nhim trờn tht ln bỏn ti ch 26 2.3.4 Xỏc nh mc nhim vi khun E coli, Salmonella spp., L monocytogenes trờn tht ln theo thi gian ly mu 26 2.3.5 Giỏm nh mt s c tớnh sinh vt, húa hc ca vi khun E coli, Salmonella spp., L monocytogenes phõn lp c 26 2.3.6 Xỏc nh c lc ca cỏc chng vi khun E coli, Salmonella spp., L monocytogenes phõn lp c 26 2.3.7 Xỏc nh yu t gõy bnh ca cỏc chng E coli, Salmonella spp., L monocytogenes phõn lp c 26 2.3.8 Xỏc nh tớnh mn cm vi mt s loi khỏng sinh v húa dc ca cỏc chng E coli, Salmonella spp., L monocytogenes phõn lp c 26 2.3.9 xut mt s bin phỏp khng ch 26 2.4 Phng phỏp nghiờn cu 26 2.4.1 Phng phỏp iu tra 26 2.4.2 Phng phỏp ly mu 26 2.4.3 Phng phỏp xỏc nh ch tiờu tng s vi khun hiu khớ cú tht ti 27 2.4.4 Phng phỏp xỏc nh ch tiờu vi khun E coli tht ln 27 2.4.5 Phng phỏp phỏt hin Salmonella 27 2.4.6 Cỏc phng phỏp phỏt hin Listeria monocytogenes 30 2.4.7 Phng phỏp xỏc nh c lc ca vi khun E coli, Salmonella v Listeria phõn lp c 31 2.4.8 Phng phỏp xỏc nh tớnh mn cm vi mt s loi khỏng sinh ca vi khun E coli, Salmonella v Listeria phõn lp c 31 S húa bi Trung tõm Hc liu HTN http://www.lrc.tnu.edu.vn v 2.4.9 Phng phỏp xỏc nh c t ng rut (Enterotoxin) bng phng phỏp khuch tỏn da th 32 2.5 Phng phỏp x lý s liu 33 Chng 3: KT QU NGHIấN CU V THO LUN 34 3.1 Kho sỏt tỡnh hỡnh git m v tiờu th tht ln ti khu ch nghiờn cu trờn a bn TP Lng Sn 34 3.2 Nghiờn cu v ch tiờu tng s vi khun hiu khớ nhim trờn tht ln ti 35 3.3 Kho sỏt ch tiờu vi khun E coli, Salmonella spp v L monocytogenes nhim trờn tht ln bỏn ti ch 38 3.3.1 Xỏc nh ch tiờu vi khun E coli, Salmonella spp., Listeria monocytogenes trờn tht ln bỏn ti cỏc ch nghiờn cu 38 3.3.2 Xỏc nh ch tiờu vi khun E coli, Salmonella spp., Listeria monocytogenes nhim trờn tht ln theo thi gian ly mu ngy 40 3.4 Xỏc nh mc nhim vi khun Escherichia coli, Salmonella spp., Listeria monocytogenes trờn tht ln ti cỏc ch nghiờn cu 43 3.4.1 Xỏc nh mc ụ nhim ca vi khun E coli trờn tht ln 43 3.4.2 Xỏc nh mc ụ nhim ca vi khun Salmonella spp trờn tht ln 45 3.4.3 Xỏc nh mc ụ nhim ca vi khun L monocytogenes trờn tht ln 47 3.5 Giỏm nh mt s c tớnh sinh vt, húa hc ca vi khun Escherichia coli, Salmonella spp., L monocytogenes phõn lp c 49 3.5.1 Giỏm nh mt s c tớnh sinh vt, húa hc ca cỏc chng vi khun Escherichia coli phõn lp c 49 3.5.2 Giỏm nh mt s c tớnh sinh vt, húa hc ca vi khun Salmonella spp phõn lp c 50 3.5.3 Giỏm nh mt s c tớnh sinh vt, húa hc ca vi khun Listeria monocytogenes phõn lp c 51 3.6 Xỏc nh c lc ca cỏc chng vi khun E coli, Salmonella spp., L monocytogenes phõn lp c 52 S húa bi Trung tõm Hc liu HTN http://www.lrc.tnu.edu.vn vi 3.7 Xỏc nh yu t gõy bnh ca cỏc chng E coli, Salmonella spp., L monocytogenes phõn lp c 54 3.7.1 Xỏc nh kh nng sn sinh c t ng rut (Enterotoxin) ca vi khun E coli, Salmonella spp bng Phn ng khuch tỏn da th 54 3.7.2 Xỏc nh ADN mang gen mó húa yu t gõy bnh Listeriolysin (hly A) ca vi khun Listeria monocytogenes bng phn ng PCR 55 3.8 Xỏc nh tớnh mn cm vi mt s loi khỏng sinh v húa dc ca cỏc chng E coli, Salmonella spp., L monocytogenes phõn lp c 56 3.8.1 Xỏc nh tớnh mn cm vi mt s loi khỏng sinh v húa dc ca cỏc chng E coli phõn lp c 57 3.8.2 Xỏc nh tớnh mn cm vi mt s loi khỏng sinh v húa dc ca cỏc chng Salmonella spp phõn lp c 59 3.8.3 Xỏc nh tớnh mn cm vi mt s loi khỏng sinh v húa dc ca cỏc chng L monocytogenes phõn lp c 61 3.9 xut mt s bin phỏp khng ch tỡnh trng ụ nhim vi sinh vt tht ln bỏn ti ch trờn a bn thnh ph Lng Sn 63 3.9.1 Trong hot ng git m ti cỏc c s git m ln 63 3.9.2 Cỏc gii phỏp qun lý 63 3.9.3 Cỏc gii phỏp kinh t - xó hi 63 KT LUN V NGH 65 Kt lun 65 ngh 66 TI LIU THAM KHO 67 PH LC NH 77 S húa bi Trung tõm Hc liu HTN http://www.lrc.tnu.edu.vn vii DANH MC CC Kí HIU VIT TT - : n % : T l phn trm Cs : Cng s E coli : Escherichia coli L monocytogenes : Listeria monocytogenes NTP : Ng c thc phm Nxb : Nh xut bn TCVN : Tiờu chun Vit Nam QCVN : Quy chun Vit Nam S húa bi Trung tõm Hc liu HTN http://www.lrc.tnu.edu.vn viii DANH MC BNG BIU Bng 1.1: ỏnh giỏ kt qu cm quan tht (Nguyn Vnh Phc, 1976 Bng 1.2 c lc ca cỏc chng E coli (Sabra A., 2002) 10 Bng 1.3 c im sinh húa ca mt s loi Listeria (TCVN 7700-1:2007 15 Bng 2.1 Bng phõn bit cỏc loi Listeria 30 Bng 3.1 Thi gian hot ng, ỡnh hỡnh tiờu th tht ln ta i khu ch ln trờn a bn TP Lng Sn 34 Bng 3.2 Kt qu xỏc nh ch tiờu tng s VKHK nhim trờn tht ln 36 Bng 3.3 Tỡnh hỡnh nhim khun E coli, Salmonella spp., Listeria monocytogenes trờn tht ln 38 Bng 3.4 Nhim khun E coli, Salmonella spp., Listeria monocytogenes trờn tht ln theo thi gian ly mu ngy 41 Bng 3.5 Mc nhim E coli trờn tht ln 43 Bng 3.6 Mc nhim Salmonella spp trờn tht ln 45 Bng 3.7 Mc nhim Listeria monocytogenes trờn tht ln 47 Bng 3.8 c tớnh sinh vt, húa hc ca vi khun E coli phõn lp c 49 Bng 3.9 c tớnh sinh vt, húa hc ca cỏc chng vi khun Salmonella spp phõn lp c 51 Bng 3.10 c tớnh sinh vt, húa hc ca cỏc chng vi khun L monocytogenes phõn lp c 52 Bng 3.11 c lc ca vi khun E coli, Salmonella spp v Listeria monocytogenes phõn lp c 53 Bng 3.12 Kh nng sn sinh c t ng rut (Enterotoxin) ca vi khun E coli, Salmonella spp 54 Bng 3.13 Xỏc nh ADN mang gen mó húa sn sinh Listeriolysin (hly A) ca vi khun Listeria monocytogenes phõn lp c 55 Bng 3.14 Kt qu th tớnh mn cm vi mt s loi khỏng sinh v húa dc ca cỏc chng vi khun E coli phõn lp c 57 S húa bi Trung tõm Hc liu HTN http://www.lrc.tnu.edu.vn 65 KT LUN V NGH Kt lun - Khi lng tht ln git tht trung bỡnh ti ch (ụng Kinh, K La v Ging Vuụng) t 14,63 - 35,05 con/ngy, tng ng 1,14 - 2,87 tn tht; tng lng tht ln c tiờu th ti ch l khong tn/ngy; cỏc quy bỏn tht ln u c kim dch thỳ y nhng ch yu l v cm quan - Cú 53,70% mu tht ln cú ch tiờu tng s VKHK khụng t tiờu chun k thut (theo TCVN 7046-2002) - Tht ln bỏn ti ch khu vc thnh ph Lng Sn cú t 40 - 50% s mu khụng t tiờu chun v sinh v ch tiờu E coli; t 16,6 - 26,6% v ch tiờu Salmonella spp.; t 10 - 16,6% v ch tiờu L monocytogenes T l cỏc vi khun nhim trờn tht ln bỏn ti ch cú s khỏc theo thi gian ly mu ngy v cú xu hng tng mc ụ nhim - Cỏc chng vi khun E coli, Salmonella spp., L monocytogenes phõn lp c cú c tớnh sinh vt húa hc phự hp vi mụ t cỏc ti liu kinh in - ó xỏc nh c 37,50% s chng vi khun E coli v 60,00% s chng vi khun Salmonella spp cú kh nng sn sinh c t ng rut T l chng vi khun L monocytogenes phõn lp c mang gen mó húa sn sinh yu t gõy bnh Listeriolysin chim 11,11% - Vi khun phõn lp c cú c lc mnh vi chut thớ nghim th hin qua thi gian v s lng chut cht; E coli gõy cht 91,6% chut thớ nghim; Salmonella spp gõy cht 95,0% v L monocytogenes gõy cht 93,3% chut thớ nghim 37,5% s chng E coli v 60% s chng Salmonella spp sn sinh c t ng rut (Enterotoxin) Cú 11,1% s chng L monocytogenes mang gen mó húa sn sinh yu t gõy dung huyt Listeriolysin (hly A) - Vi khun E coli mn cm mnh vi ceftazidime (65,0%), mn cm trung bỡnh vi trimethoprim/sunfamethoxazole, gentamicin v spectinomycin; 100% ó khỏng li amoxicilline v 90,0% khỏng streptomycin S húa bi Trung tõm Hc liu HTN http://www.lrc.tnu.edu.vn 66 - Vi khun Salmonella ó khỏng li hu ht cỏc loi khỏng sinh (streptomycin 100,%, trimethoprim/sunfamethoxazole 85,0%, amoxicilline 80,0% v spectinomycin 70,0%) Mn cm trung bỡnh vi gentamicin 80,0% v ceftazidime 30,0% - Vi khun L monocytogenes mn cm mnh vi loi khỏng sinh thụng thng (amoxicilline 100%, erythrommycin 100%) v 55,0% mn cm trung bỡnh vi streptomycin xut mt s bin phỏp nhm khụng ch tỡnh trng ụ nhim vi sinh vt vo thõn tht nhm m bo v sinh an ton thc phm ngh Do kinh phớ cú hn nờn kt qu nghiờn cu ca ti cũn hn ch Vỡ vy, chỳng tụi ngh cn tip tc cú nghiờn cu tip theo trờn phm vi rng hn, tng thờm mt s ch tiờu, c th nh sau: Tip tc cú cỏc nghiờn cu v s ụ nhim vi khun E coli, L monocytogenes v Salmonella phõn lp c t tht ln Xỏc nh cỏc yu t nguy c, cỏc ngun lõy nhim vi khun vo tht ln qua trỡnh git m, phõn phi, lu thụng, by bỏn, t dng c, trang thit b, qun ỏo bo h v tay ngi tham gia git m, ngi kinh doanh tht Nghiờn cu thờm v c tớnh sinh hc, yu t c lc, tớnh mn cm vi khỏng sinh v húa dc ca cỏc chng vi khun khun E coli, L monocytogenes v Salmonella phõn lp c t tht ln S húa bi Trung tõm Hc liu HTN http://www.lrc.tnu.edu.vn 67 TI LIU THAM KHO I TING VIT Ngc nh (2011), Bnh vi khun Listeria monocytogenes - Nguy him i vi ph n mang thai, Hc vin quõn y Lờ Huy Chớnh (2007), Vi sinh vt y hc, Nh xut bn Y hc Cc V sinh an ton thc phm - B Y t (2009), S liu cỏc v ng c 2009 Cc V sinh an ton thc phm - B Y t (2010), S liu ng c nm 2010 Cc V sinh an ton thc phm - B Y t (2011), S liu ng c nm 2011 Cc V sinh an ton thc phm - B Y t (2012), S v ng c thc phm nm 2012 Cc V sinh an ton thc phm - B Y t (2013), S v ng c thc phm nm 2013 Cc V sinh an ton thc phm - B Y t (2014), S v ng c thc phm nm 2014 Cc V sinh an ton thc phm - B Y t (2015), S v ng c thc phm nm 2015 10 Bớch Du (2012), Nghiờn cu mt s c im sinh hc ca vi khun Escherichia coli trờn tht ln bỏn ti thnh ph Thỏi Nguyờn, Lun Vn thc s Cụng ngh sinh hc, Thỏi Nguyờn, tr 53 - 55 11 H Th Anh o (2005), Kho sỏt tỡnh trng ụ nhim Salmonella, E coli, Campylobacter thc phm nguyờn liu ti bp n trng mm non H Ni nm 2004, Bỏo cỏo ti nghiờn cu khoa hc Vin Dinh dng 12 Trn Th Hng Giang, Hunh Th M L (2012), Xỏc nh t l nhim v c lc ca vi khun Escherichia coli phõn lp c t tht (ln, bũ, g) mt s huyn ngoi thnh H Ni, Tp khoa hc v phỏt trin 2012 - Trng i hc Nụng nghip H Ni, 10 (2), tr 295 - 300 13 Nguyn Th Hin, Phan Th Kim, Trng Thi Ho, Lờ Th Lan Chi (2003), Vi sinh vt nhim lng thc thc phm, Nxb Nụng nghip, H Ni 14 Vn Hip (2007), Kho sỏt thc trng hot ng git m ln v trõu bũ ti huyn Quc Oai - H Tõy, Lun thc s khoa hc Nụng nghip, i hc Nụng nghip - H Ni, tr 51 - 56 S húa bi Trung tõm Hc liu HTN http://www.lrc.tnu.edu.vn 68 15 Cm Ngc Hong, Nguyn Th Thanh Thy, Nguyn Bỏ Tip (2014), ỏnh giỏ thc trng git m v ụ nhim vi khun tht ln ti cỏc c s git m thuc tnh Nam nh, Tp Khoa hc v Phỏt trin, 12 (4), tr 549 - 557 16 V Th Huyn (2012), Nghiờn cu phc hp cỏc ht nano - khỏng th nhm phỏt hin vi khun gõy ng c thc phm Listeria monocytogenes, Lun Thc s Cụng ngh Nano - Sinh hc, i hc Quc Gia H Ni 17 Nguyn Vit Khụng, Phm Th Ngc, inh Xuõn Tựng, Lapar Ma Lucila, Fred Unger, Nguyn Vit Hựng, Phm c Phỳc, Phm Th Nga, Gilbert Jeffrey v cỏc cng s (2012), ễ nhim Salmonella cỏc im git m gia cm quy mụ nh ti cỏc huyn ngoi H Ni, Tp Nụng nghip v Phỏt trin Nụng thụn, k thỏng 12, tr 60 - 67 18 Phm Th Ngc Lan, Ngụ Th Tuyt Mai (2012), Kho sỏt ụ nhim vi sinh vt mt s thc phm trờn a bn thnh ph Hu nm 2010 - 2011, Tp khoa hc, i hc Hu, 73 (4), tr 137 - 145 19 Lờ Th Lnh (2015), Nghiờn cu ch to vng nano v mt s ng dng, Lun ỏn Tin s Húa Hc, i hc Hu 20 Chu Vn Mn, o Hu H (2002), Thng kờ sinh hc, Nxb i hc Quc gia H Ni, tr 171 - 176 21 V Bỡnh Minh, Cự Hu Phỳ (1999), Kt qu phõn lp vi khun E coli v Salmonella ln mc bnh tiờu chy, xỏc nh mt s c tớnh sinh vt hoỏ hc ca cỏc chng vi khun phõn lp c, Tp Khoa hc k thut Thỳ y, Hi thỳ y Vit Nam, H Ni, tr 32 - 36 22 Phựng Vn Mch (2008), Kho sỏt thc trng hot ng git m v ụ nhim 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thực đề tài: Nghiên cứu số đặc tính sinh vật học vi khuẩn Escherichia coli, Listeria monocytogenes Salmonella spp nhiễm thịt lợn tiêu thụ địa bàn thành phố Lạng Sơn, đề xuất. .. HỌC THÁI NGUYÊN TRƯỜNG ĐẠI HỌC NÔNG LÂM HÀ VĂN QUANG NGHIÊN CỨU MỘT SỐ ĐẶC TÍNH SINH VẬT HỌC CỦA VI KHUẨN ESCHERICHIA COLI, LISTERIA MONOCYTOGENES VÀ SALMONELLA SPP NHIỄM TRÊN THỊT LỢN TIÊU THỤ... xuất biện pháp phòng chống Mục tiêu nghiên cứu - Xác định mức độ nhiễm vi khuẩn E coli, Salmonella spp. , L monocytogenes thịt lợn tiêu thụ TP Lạng Sơn số đặc điểm sinh học chúng (đặc tính sinh vật