鉴别显色培养基在奶牛乳房炎病原菌快速鉴定中的应用
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摘要
为研发用于奶牛乳房炎常见病原菌分离鉴定的鉴别显色培养基,并检验其与普通分离鉴定培养基辅以生化鉴定和16S rRNA核酸序列测定两种传统方法鉴定结果的一致性,笔者以生物信息学方法筛出各种属细菌的特异性酶、可作为唯一碳源发酵产酸的碳源,然后合成酶显色底物,连同碳源、酸碱指示剂制备显色培养基,将各种属标准菌株、野生型菌株涂布培养,评估菌落形态、菌落颜色及培养基基质颜色变化。采集隐性乳房炎(CMT法检测)及临床型乳房炎病例的牛奶样品(絮状物、凝块、清亮状或血乳)共计482份,用鉴别显色培养基和两种传统方法进行病原菌的分离、鉴定。鉴别显色培养基上菌落纯化后提取DNA用于16S rRNA序列扩增,产物送去测序(n=194)。以两种传统方法为参考,判定鉴别显色培养基鉴定病原菌的可靠性,用SAS 9.4的FREQ程序计算鉴别显色培养基的简单科恩κ系数。鉴别显色培养基上常见乳房炎病原菌不同种属的菌落形态和培养基基质颜色明显不同,肉眼即可辨别。鉴别显色培养基与两种传统方法鉴定结果的一致性参数分别为κ=0.70、κ=0.96。鉴别显色培养基能够作为常见奶牛乳房炎病原菌的标准鉴定方法。
The present study aimed to develop a new differential chromogenic medium for fast identification of pathogens associated with mastitis in dairy cows, and check its identification consistency with two traditional approaches including normal isolation and identification media combined with biochemical identifications and 16 S rRNA sequencing. Specific enzymes and the only carbon source that could be used to produce acid in bacteria of each genus were screened via bioinformatics tools. Synthesized chromogenic substrate of specific enzyme, carbon and acid-base indicator were mixed into basal enrichment medium to prepare differential chromogenic media. Standard strains and wild-type strains of related pathogens were inoculated on differential chromogenic media, and morphology of colonies and color changes of colony and medium that could be considered as identification standards were observed. Milk samples from mastitic quarters(samples diagnosed as subclinical mastitis by California Mastitis Test or samples of clinical mastitis with presence of flakes, clots, or serous milk; n=482) were cultured aerobically at 37 ℃ and identified using differential chromogenic media and traditional culture methods. Colonies from differential chromogenic media were purified and DNA was extracted for 16 S rRNA sequencing(n=194). The reliability of identification by differential chromogenic media was diagnosed referring to results of two traditional approaches, based on which consistency inspection parameter, namely, simple Cohen's kappa coefficient was calculated via FREQ program in SAS Version 9.4. The colony and color of medium matrix from common bovine mastitis-associated pathogens of different genus grown on differential chromogenic media, regardless of standard strains or wild-type strains, were morphologically distinct and visible to the naked eyes. Consistency inspection parameter of differential chromogenic media referred to normal isolation and identification media combined with biochemical identifications and 16 S rRNA sequencing was seperately κ=0.70 and κ=0.96. In conclusion, differential chromogenic media showed great potential in identifying common pathogens of bovine mastitis.
The present study aimed to develop a new differential chromogenic medium for fast identification of pathogens associated with mastitis in dairy cows, and check its identification consistency with two traditional approaches including normal isolation and identification media combined with biochemical identifications and 16 S rRNA sequencing. Specific enzymes and the only carbon source that could be used to produce acid in bacteria of each genus were screened via bioinformatics tools. Synthesized chromogenic substrate of specific enzyme, carbon and acid-base indicator were mixed into basal enrichment medium to prepare differential chromogenic media. Standard strains and wild-type strains of related pathogens were inoculated on differential chromogenic media, and morphology of colonies and color changes of colony and medium that could be considered as identification standards were observed. Milk samples from mastitic quarters(samples diagnosed as subclinical mastitis by California Mastitis Test or samples of clinical mastitis with presence of flakes, clots, or serous milk; n=482) were cultured aerobically at 37 ℃ and identified using differential chromogenic media and traditional culture methods. Colonies from differential chromogenic media were purified and DNA was extracted for 16 S rRNA sequencing(n=194). The reliability of identification by differential chromogenic media was diagnosed referring to results of two traditional approaches, based on which consistency inspection parameter, namely, simple Cohen's kappa coefficient was calculated via FREQ program in SAS Version 9.4. The colony and color of medium matrix from common bovine mastitis-associated pathogens of different genus grown on differential chromogenic media, regardless of standard strains or wild-type strains, were morphologically distinct and visible to the naked eyes. Consistency inspection parameter of differential chromogenic media referred to normal isolation and identification media combined with biochemical identifications and 16 S rRNA sequencing was seperately κ=0.70 and κ=0.96. In conclusion, differential chromogenic media showed great potential in identifying common pathogens of bovine mastitis.
引文
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[2] HAGNESTAM-NIELSEN C,?STERGAARD S.Economic impact of clinical mastitis in a dairy herd assessed by stochastic simulation using different methods to model yieldlosses[J].Animal,2009,3(2):315-328.
[3] POL M,RUEGG P L.Treatment practices and quantification of antimicrobial drug usage in conventional and organic dairy farms in Wisconsin[J].J Dairy Sci,2007,90(1):249-261.
[4] SCHERPENZEEL C G M,HOGEVEEN H,MAAS L,et al.Economic optimization of selective dry cow treatment[J].J Dairy Sci,2018,101(2):1530-1539.
[5] OLDE RIEKERINK R G M,BARKEMA H W,KELTON D F,et al.Incidence rate of clinical mastitis on Canadian dairy farms[J].J Dairy Sci,2008,91(4):1366-1377.
[6] GAO J,BARKEMA H W,ZHANG L M,et al.Incidence of clinical mastitis and distribution of pathogens on large Chinese dairy farms[J].J Dairy Sci,2017,100(6):4797-4806.
[7] ROBERSON J R.Treatment of clinicalmastitis[J].Vet Clin North Am Food Anim Pract,2012,28(2):271-288.
[8] SUOJALA L,KAARTINEN L,PY?R?L? S.Treatment for bovine Escherichia coli mastitis—an evidence-based approach[J].J Vet Pharmacol Ther,2013,36(6):521-531.
[9] LAGO A,GODDEN S M,BEY R,et al.The selective treatment of clinical mastitis based on on-farm culture results:I.Effects on antibiotic use,milk withholding time,and short-term clinical and bacteriological outcomes[J].J Dairy Sci,2011,94(9):4441-4456.
[10] ROYSTER E,GODDEN S,GOULART D,et al.Evaluation of the Minnesota easy culturesystem II Bi-plate and Tri-plate for identification of common mastitis pathogens in milk[J].J Dairy Sci,2014,97(6):3648-3659.
[11] DOWN P M,BRADLEY A J,BREEN J E,et al.Factors affecting the cost-effectiveness of on-farm culture prior to the treatment of clinical mastitis in dairycows[J].Prev Vet Med,2017,145:91-99.
[12] Office International Des Epizooties.The OIE strategy on antimicrobial resistance and the prudent use of antimicrobials[EB/OL].(2016)[2017-07-26].http://www.uvhvvr.gov.si/fileadmin/uvhvvr.gov.si/pageuploads/DELOVNA_PODROCJA/Zdravila/OIE-AMR strategy.pdf.
[13] World Health Organization.Global action plan on antimicrobial resistance[EB/OL].(2015)[2017-07-26].http://www.who.int/antimicrobial-resistance/publications/global-action-plan/en/.
[14] KALCHAYANAND N,ARTHUR T M,BOSILEVAC J M,et al.Chromogenic agar medium for detection and isolation of Escherichia coli serogroups O26,O45,O103,O111,O121,and O145 from fresh beef and cattle feces[J].J Food Prot,2013,76(2):192-199.
[15] National Mastitis Council.Guidelines on normal and abnormal raw milk based on somatic cell counts and signs of clinical mastitis[R].Madison,U.S.:NMC Inc.,2001.
[16] DOHOO I,MARTIN W,STRYHN H.Veterinary epidemiologicresearch[M].2nd ed.Charlottetown:VER Inc,2009:91-134.
[17] CYR L,FRANCIS K.Measures of clinical agreement for nominal and categorical data:the kappa coefficient[J].Comput Biol Med,1992,22(4):239-246.
[18] GANDA E K,BISINOTTO R S,DECTER D H,et al.Evaluation of an on-farm culture system (Accumast) for fast identification of milk pathogens associated with clinical mastitis in dairy cows[J].PLoS One,2016,11(5):e0155314.
[19] CHA E,KRISTENSEN A R,HERTL J A,et al.Optimal insemination and replacement decisions to minimize the cost of pathogen-specific clinical mastitis in dairy cows[J].J Dairy Sci,2014,97(4):2101-2117.
[20] ROYSTER E,WAGNER S.Treatment of mastitis incattle[J].Vet Clin North Am Food Anim Pract,2015,31(1):17-46.
[21] HERTL J A,SCHUKKEN Y H,WELCOME F L,et al.Pathogen-specific effects on milk yield in repeated clinical mastitis episodes in Holstein dairy cows[J].J Dairy Sci,2014,97(3):1465-1480.
[22] OLIVEIRA L,RUEGG P L.Treatments of clinical mastitis occurring in cows on 51 largedairy herds in Wisconsin[J].J Dairy Sci,2014,97(9):5426-5436.
[23] ZHANG L M,GAO J,BARKEMA H W,et al.Virulence gene profiles:alpha-hemolysin and clonal diversity in Staphylococcus aureus isolates from bovine clinical mastitis in China[J].BMC Vet Res,2018,14(1):63.
[24] BOSS R,NASKOVA J,STEINER A,et al.Mastitis diagnostics:quantitative PCR for Staphylococcus aureus genotype B in bulk tank milk[J].J Dairy Sci,2011,94(1):128-137.
[25] VIORA L,GRAHAM E M,MELLOR D J,et al.Evaluation of a culture-based pathogen identification kit for bacterial causes of bovine mastitis[J].Vet Rec,2014,175(4):89.
[26] BOSS R,COSANDEY A,LUINI M,et al.Bovine Staphylococcus aureus:Subtyping,evolution,and zoonotic transfer[J].J Dairy Sci,2016,99(1):515-528.
[27] DA COSTA L B,RAJALA-SCHULTZ P J,SCHUENEMANN G M.Management practices associated with presence of Staphylococcus aureus in bulk tank milk from Ohio dairy herds[J].J Dairy Sci,2016,99(2):1364-1373.
[28] TOMAZI T,GON?ALVES J L,BARREIRO J R,et al.Bovine subclinical intramammary infection caused by coagulase-negative staphylococci increases somatic cell count but has no effect on milk yield or composition[J].J Dairy Sci,2015,98(5):3071-3078.
[29] BOERLIN P,KUHNERT P,HüSSY D,et al.Methods for identification of Staphylococcus aureus isolates in cases of bovine mastitis[J].J Clin Microbiol,2003,41(2):767-771.
[30] ERSKINE R J,BARTLETT P C,VANLENTE J L,et al.Efficacy of systemic ceftiofur as a therapy for severe clinical mastitis in dairy cattle[J].J Dairy Sci,2002,85(10):2571-2575.
[2] HAGNESTAM-NIELSEN C,?STERGAARD S.Economic impact of clinical mastitis in a dairy herd assessed by stochastic simulation using different methods to model yieldlosses[J].Animal,2009,3(2):315-328.
[3] POL M,RUEGG P L.Treatment practices and quantification of antimicrobial drug usage in conventional and organic dairy farms in Wisconsin[J].J Dairy Sci,2007,90(1):249-261.
[4] SCHERPENZEEL C G M,HOGEVEEN H,MAAS L,et al.Economic optimization of selective dry cow treatment[J].J Dairy Sci,2018,101(2):1530-1539.
[5] OLDE RIEKERINK R G M,BARKEMA H W,KELTON D F,et al.Incidence rate of clinical mastitis on Canadian dairy farms[J].J Dairy Sci,2008,91(4):1366-1377.
[6] GAO J,BARKEMA H W,ZHANG L M,et al.Incidence of clinical mastitis and distribution of pathogens on large Chinese dairy farms[J].J Dairy Sci,2017,100(6):4797-4806.
[7] ROBERSON J R.Treatment of clinicalmastitis[J].Vet Clin North Am Food Anim Pract,2012,28(2):271-288.
[8] SUOJALA L,KAARTINEN L,PY?R?L? S.Treatment for bovine Escherichia coli mastitis—an evidence-based approach[J].J Vet Pharmacol Ther,2013,36(6):521-531.
[9] LAGO A,GODDEN S M,BEY R,et al.The selective treatment of clinical mastitis based on on-farm culture results:I.Effects on antibiotic use,milk withholding time,and short-term clinical and bacteriological outcomes[J].J Dairy Sci,2011,94(9):4441-4456.
[10] ROYSTER E,GODDEN S,GOULART D,et al.Evaluation of the Minnesota easy culturesystem II Bi-plate and Tri-plate for identification of common mastitis pathogens in milk[J].J Dairy Sci,2014,97(6):3648-3659.
[11] DOWN P M,BRADLEY A J,BREEN J E,et al.Factors affecting the cost-effectiveness of on-farm culture prior to the treatment of clinical mastitis in dairycows[J].Prev Vet Med,2017,145:91-99.
[12] Office International Des Epizooties.The OIE strategy on antimicrobial resistance and the prudent use of antimicrobials[EB/OL].(2016)[2017-07-26].http://www.uvhvvr.gov.si/fileadmin/uvhvvr.gov.si/pageuploads/DELOVNA_PODROCJA/Zdravila/OIE-AMR strategy.pdf.
[13] World Health Organization.Global action plan on antimicrobial resistance[EB/OL].(2015)[2017-07-26].http://www.who.int/antimicrobial-resistance/publications/global-action-plan/en/.
[14] KALCHAYANAND N,ARTHUR T M,BOSILEVAC J M,et al.Chromogenic agar medium for detection and isolation of Escherichia coli serogroups O26,O45,O103,O111,O121,and O145 from fresh beef and cattle feces[J].J Food Prot,2013,76(2):192-199.
[15] National Mastitis Council.Guidelines on normal and abnormal raw milk based on somatic cell counts and signs of clinical mastitis[R].Madison,U.S.:NMC Inc.,2001.
[16] DOHOO I,MARTIN W,STRYHN H.Veterinary epidemiologicresearch[M].2nd ed.Charlottetown:VER Inc,2009:91-134.
[17] CYR L,FRANCIS K.Measures of clinical agreement for nominal and categorical data:the kappa coefficient[J].Comput Biol Med,1992,22(4):239-246.
[18] GANDA E K,BISINOTTO R S,DECTER D H,et al.Evaluation of an on-farm culture system (Accumast) for fast identification of milk pathogens associated with clinical mastitis in dairy cows[J].PLoS One,2016,11(5):e0155314.
[19] CHA E,KRISTENSEN A R,HERTL J A,et al.Optimal insemination and replacement decisions to minimize the cost of pathogen-specific clinical mastitis in dairy cows[J].J Dairy Sci,2014,97(4):2101-2117.
[20] ROYSTER E,WAGNER S.Treatment of mastitis incattle[J].Vet Clin North Am Food Anim Pract,2015,31(1):17-46.
[21] HERTL J A,SCHUKKEN Y H,WELCOME F L,et al.Pathogen-specific effects on milk yield in repeated clinical mastitis episodes in Holstein dairy cows[J].J Dairy Sci,2014,97(3):1465-1480.
[22] OLIVEIRA L,RUEGG P L.Treatments of clinical mastitis occurring in cows on 51 largedairy herds in Wisconsin[J].J Dairy Sci,2014,97(9):5426-5436.
[23] ZHANG L M,GAO J,BARKEMA H W,et al.Virulence gene profiles:alpha-hemolysin and clonal diversity in Staphylococcus aureus isolates from bovine clinical mastitis in China[J].BMC Vet Res,2018,14(1):63.
[24] BOSS R,NASKOVA J,STEINER A,et al.Mastitis diagnostics:quantitative PCR for Staphylococcus aureus genotype B in bulk tank milk[J].J Dairy Sci,2011,94(1):128-137.
[25] VIORA L,GRAHAM E M,MELLOR D J,et al.Evaluation of a culture-based pathogen identification kit for bacterial causes of bovine mastitis[J].Vet Rec,2014,175(4):89.
[26] BOSS R,COSANDEY A,LUINI M,et al.Bovine Staphylococcus aureus:Subtyping,evolution,and zoonotic transfer[J].J Dairy Sci,2016,99(1):515-528.
[27] DA COSTA L B,RAJALA-SCHULTZ P J,SCHUENEMANN G M.Management practices associated with presence of Staphylococcus aureus in bulk tank milk from Ohio dairy herds[J].J Dairy Sci,2016,99(2):1364-1373.
[28] TOMAZI T,GON?ALVES J L,BARREIRO J R,et al.Bovine subclinical intramammary infection caused by coagulase-negative staphylococci increases somatic cell count but has no effect on milk yield or composition[J].J Dairy Sci,2015,98(5):3071-3078.
[29] BOERLIN P,KUHNERT P,HüSSY D,et al.Methods for identification of Staphylococcus aureus isolates in cases of bovine mastitis[J].J Clin Microbiol,2003,41(2):767-771.
[30] ERSKINE R J,BARTLETT P C,VANLENTE J L,et al.Efficacy of systemic ceftiofur as a therapy for severe clinical mastitis in dairy cattle[J].J Dairy Sci,2002,85(10):2571-2575.