猪葡萄球菌临床分离株感染仔猪渗出性皮炎模型的建立
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摘要
为建立研究仔猪渗出性皮炎发病机制及治疗制剂的动物模型,本研究采用PCR及BLAST序列比对方法对猪葡萄球菌437-2株毒素基因型进行鉴定,采用纸片扩散法检测其耐药性,通过不同浓度菌液人工感染仔猪试验分析该菌株的临床致病力、最小致病浓度、发病周期及组织病理学变化。结果显示,该分离菌株为ExhD毒素基因型,其毒素基因与德国分离株(GenBank登录号:AM94662.1)和俄罗斯分离株(GenBank登录号:AM950188.1)的ExhD基因同源性达99%;药敏试验结果显示,该菌株为多重耐药菌株,对青霉素、磺胺异噁唑、链霉素、阿奇霉素、红霉素、四环素、杆菌肽及诺氟沙星耐药;仔猪致病性试验结果显示,低剂量组在整个试验周期内仅注射部位表现出皮炎症状,而中、高剂量组在耳后皮下注射2 d后均出现显著的渗出性皮炎症状,病猪表现为耳后出现油皮,全身被毛粗糙,皮肤呈深褐色厚皮痂,有大片蜕皮和黄色液体渗出;感染仔猪的脏器和皮肤病理切片结果显示,低剂量组仔猪脏器无异常,耳部皮肤角质层角化过度,炎性细胞浸润,无棘皮层细胞分离,而中、高剂量组感染仔猪脏器具有一定程度的损伤,皮肤表皮棘细胞层出现明显的细胞分离。上述试验结果说明,利用猪葡萄球菌437-2株可成功建立仔猪渗出性皮炎模型,所需的最小攻菌浓度为1×10~9 CFU/mL。
In order to provide a target animal model of exudative dermatitis of piglet for exploring the pathogenesis and therapeutic reagents,in this study,the toxin type of isolated Staphylococcus hyicus 437-2 was identified by PCR and BLAST sequence alignment,its drug resistance was detected by disk diffusion method,and the clinical pathogenicity,minimum pathogenic concentration,pathogenesis and histopathological changes caused by the strain were analyzed by artificially infecting piglets with different concentrations of bacterial liquid.The results showed that the strain carried the ExhD virulence gene,which showed 99% gene sequence homology with the isolated Germany strain(GenBank accession No.:AM94662.1) and Russian isolate(GenBank accession No.:AM950188.1).The drug resistant assay displayed S.hyicus 437-2 was a multi-drug resistant strain,which showed resistance to penicillin,sulfamethoxazole,streptomycin,azithromycin,erythromycin,tetracycline,bacitracin and norfloxacin.And the pathogenicity test of S.hyicus 437-2 on piglets showed that the low dose group displayed dermatitis symptoms only at the injection site throughout the experimental period,while significant exudative dermatitis symptoms appeared in the middle and high dose groups after subcutaneous injection for 2 d,which showed detailedly as oily skin behind the ear,rough fur,a large piece of suede and yellow liquid exuded,and thick dark brown skin.The results of pathological sections showed that organs in the low dose group were not damaged,while the skin tissue displayed excessive keratinization on the ear skin,inflammatory cell infiltration,and no cell separation.Meanwhile,the piglets in the middle and high dose groups all showed a certain degree of damage,the cell epidermal layer of the skin appeared obvious cell separation.This results indicated that this strain had strong pathogenicity and could successfully replicate exudative dermatitis on piglets.In addition,this result further indicated that the minimum bacterium concentration required for the establishment of exudative dermatitis model was 1×10~9 CFU/mL.
In order to provide a target animal model of exudative dermatitis of piglet for exploring the pathogenesis and therapeutic reagents,in this study,the toxin type of isolated Staphylococcus hyicus 437-2 was identified by PCR and BLAST sequence alignment,its drug resistance was detected by disk diffusion method,and the clinical pathogenicity,minimum pathogenic concentration,pathogenesis and histopathological changes caused by the strain were analyzed by artificially infecting piglets with different concentrations of bacterial liquid.The results showed that the strain carried the ExhD virulence gene,which showed 99% gene sequence homology with the isolated Germany strain(GenBank accession No.:AM94662.1) and Russian isolate(GenBank accession No.:AM950188.1).The drug resistant assay displayed S.hyicus 437-2 was a multi-drug resistant strain,which showed resistance to penicillin,sulfamethoxazole,streptomycin,azithromycin,erythromycin,tetracycline,bacitracin and norfloxacin.And the pathogenicity test of S.hyicus 437-2 on piglets showed that the low dose group displayed dermatitis symptoms only at the injection site throughout the experimental period,while significant exudative dermatitis symptoms appeared in the middle and high dose groups after subcutaneous injection for 2 d,which showed detailedly as oily skin behind the ear,rough fur,a large piece of suede and yellow liquid exuded,and thick dark brown skin.The results of pathological sections showed that organs in the low dose group were not damaged,while the skin tissue displayed excessive keratinization on the ear skin,inflammatory cell infiltration,and no cell separation.Meanwhile,the piglets in the middle and high dose groups all showed a certain degree of damage,the cell epidermal layer of the skin appeared obvious cell separation.This results indicated that this strain had strong pathogenicity and could successfully replicate exudative dermatitis on piglets.In addition,this result further indicated that the minimum bacterium concentration required for the establishment of exudative dermatitis model was 1×10~9 CFU/mL.
引文
[1] 李艳,张乐宜,宋长绪.1株猪葡萄球菌强毒株的分离鉴定及致病性试验[J].中国兽医学报,2015,35(9):1456-1462.LI Y,ZHANG L Y,SONG C X.Isolation and identification of a high virulent Staphylococcus hyicus and its pathogenic to piglets[J].Chinese Journal of Veterinary Science,2015,35(9):1457-1462.(in Chinese)
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[6] PETER A,LARS OLE A.Cloning and sequence analysis of genes encoding Staphylococcus hyicus exfoliative toxin types A,B,C,and D[J].Journal of Bacteriology,2004,186(6):1833-1837.
[7] FUDABA Y,NISHIFUJI K,ANDRESEN L O,et al.Staphylococcus hyicus exfoliative toxins selectively digest porcine desmoglein 1[J].Microbial Pathogenesis,2005,39(5):171-176.
[8] 杨鼎,郝永清,宋长绪.猪葡萄球菌脱落毒素研究进展[J].动物医学进展,2009,30(4):60-64.YANG D,HAO Y Q,SONG C X.Advance in exfoliative toxin of Staphylococcus hyicus[J].Progress in Veterinary Medicine,2009,30(4):60-64.(in Chinese)
[9] KHALILI H,SOLTANI R,NEGAHBAN S,et al.Reliability of disk diffusion test results for the antimicrobial susceptibility testing of nosocomial Gram-positive microorganisms:Is E-test method better?[J].Iranian Journal of Pharmaceutical Research Ijpr,2012,11(2):559-563.
[10] ANDRESEN L O,AHRENS P.A multiplex PCR for detection of genes encoding exfoliative toxins from Staphylococcus hyicus[J].Journal of Applied Microbiology,2010,96(6):1265-1270.
[11] LU L,HE K,NI Y,et al.Exudative epidermitis of piglets caused by non-toxigenic Staphylococcus sciuri[J].Veterinary Microbiology,2017,199:79-84.
[12] CHEN S,WANG Y,CHEN F,et al.A highly pathogenic strain of Staphylococcus sciuri caused fatal exudative epidermitis in piglets[J].PLoS One,2007,2(1):e147.
[13] ANDRESEN L O,AHRENS P,DAUGAARD L,et al.Exudative epidermitis in pigs caused by toxigenic Staphylococcus chromogenes[J].Veterinary Microbiology,2005,105(3):291-300.
[14] 王明月,王新平,郭昌明,等.吉林省某猪场新发猪葡萄球菌引起的化脓性肺炎[J].中国兽医学报,2016,36(9):1577-1581.WANG M Y,WANG X P,GUO C M,et al.Rare infection characterized with suppurative pneumonia by Staphylococcus hyicus[J].Chinese Journal of Veterinary Science,2016,35(9):1577-1581.(in Chinese)
[15] FOSTER A P.Staphylococcal skin disease in live-stock[J].Veterinary Dermatology,2012,23(4):342-351.
[16] JEONGHWA P,FRIENDSHIP R M,ZVONIMIR P,et al.An investigation of exudative epidermitis (greasy pig disease) and antimicrobial resistance patterns of Staphylococcus hyicus and Staphylococcus aureus isolated from clinical cases[J].Canadian Veterinary Journal-revue Veterinaire Canadienne,2013,54(2):139-144.
[17] AARESTRUP F M,JENSEN L B.Trends in antimicrobial susceptibility in relation to antimicrobial usage and presence of resistance genes in Staphylococcus hyicus isolated from exudative epidermitis in pigs[J].Veterinary Microbiology,2002,89(1):83-94.
[18] WEGENER H C,WATTS J L,SALMON S A,et al.Antimicrobial susceptibility of Staphylococcus hyicus isolated from exudative epidermitis in pigs[J].Journal of Clinical Microbiology,2015,32(3):793-795.
[19] 李淑红,王京仁,成钢,等.猪大肠杆菌和葡萄球菌的分离鉴定及耐药性分析[J].中国畜牧兽医,2012,39(8):195-198.LI S H,WANG J R,CHENG G,et al.Isolation,identification and analysis of drug resistance on E.coli and Staphylococcus in pigs[J].China Animal Husbandry & Veterinary Medicine,2012,39(8):195-198.(in Chinese)
[20] DHAND A,SAKOULAS G.Daptomycin in combination with other antibiotics for the treatment of complicated methicillin-resistant Staphylococcus aureus bacteremia[J].Clinical Therapeutics,2014,36(10):1303-1316.
[21] LI Z,WANG X,XIAO W,et al.Research advances on plectasin and its derivatives as new potential antimicrobial candidates[J].Process Biochemistry,2017,56:62-70.
[22] VAILLANCOURT K,LEBEL G,YI L,et al.In vitro antibacterial activity of plant essential oils against Staphylococcus hyicus and Staphylococcus aureus,the causative agents of exudative epidermitis in pigs[J].Archives of Microbiology,2018,200(7):1001-1007.
[23] ARSENAKIS I,BOYEN F,HAESEBROUCK F,et al.Autogenous vaccination reduces antimicrobial usage and mortality rates in a herd facing severe exudative epidermitis outbreaks in weaned pigs[J].Veterinary Record,2018,182(26):744.
[24] 睢艳平,徐镔蕊,王勇,等.仔猪渗出性皮炎的实验室诊断[J].中国畜牧兽医,2006,33(10):13-16.JU Y P,XU B R,WANG Y et al.Laboratory diagnosis of exudative dermatitis in piglets[J].China Animal Husbandry & Veterinary Medicine,2006,33(10):13-16.(in Chinese)
[25] LADHANI S.Understanding the mechanism of action of the exfoliative toxins of Staphylococcus aureus[J].FEMS Immunology & Medical Microbiology,2003,39(2):181-189.
[26] PATEL G K,FINLAY A Y.Staphylococcal scalded skin syndrome:Diagnosis and management[J].American Journal of Clinical Dermatology,2010,123(6):505-507.
[2] 王忠山.猪渗出性皮炎的诊断与防治[J].猪业科学,2014,31(5):46-47.WANG Z S.Diagnosis and prevention of porcine exudative epidermitis[J].Swine Industry Science,2014,31(5):46-47.(in Chinese)
[3] TANABE T,SATO H,SATO H,et al.Correlation between occurrence of exudative epidermitis and exfoliative toxin-producing ability of Staphylococcus hyicus[J].Veterinary Microbiology,1996,48(1-2):9-17.
[4] SATO H,TANABE T,KURAMOTO M,et al.Isolation of exfoliative toxin from Staphylococcus hyicus subsp.hyicus and its exfoliative activity in the piglet[J].Veterinary Microbiology,1991,27(3-4):263-275.
[5] ANDRESEN L O.Differentiation and distribution of three types of exfoliative toxin produced by Staphylococcus hyicus from pigs with exudative epidermitis[J].Pathogens & Disease,2013,20(4):301-310.
[6] PETER A,LARS OLE A.Cloning and sequence analysis of genes encoding Staphylococcus hyicus exfoliative toxin types A,B,C,and D[J].Journal of Bacteriology,2004,186(6):1833-1837.
[7] FUDABA Y,NISHIFUJI K,ANDRESEN L O,et al.Staphylococcus hyicus exfoliative toxins selectively digest porcine desmoglein 1[J].Microbial Pathogenesis,2005,39(5):171-176.
[8] 杨鼎,郝永清,宋长绪.猪葡萄球菌脱落毒素研究进展[J].动物医学进展,2009,30(4):60-64.YANG D,HAO Y Q,SONG C X.Advance in exfoliative toxin of Staphylococcus hyicus[J].Progress in Veterinary Medicine,2009,30(4):60-64.(in Chinese)
[9] KHALILI H,SOLTANI R,NEGAHBAN S,et al.Reliability of disk diffusion test results for the antimicrobial susceptibility testing of nosocomial Gram-positive microorganisms:Is E-test method better?[J].Iranian Journal of Pharmaceutical Research Ijpr,2012,11(2):559-563.
[10] ANDRESEN L O,AHRENS P.A multiplex PCR for detection of genes encoding exfoliative toxins from Staphylococcus hyicus[J].Journal of Applied Microbiology,2010,96(6):1265-1270.
[11] LU L,HE K,NI Y,et al.Exudative epidermitis of piglets caused by non-toxigenic Staphylococcus sciuri[J].Veterinary Microbiology,2017,199:79-84.
[12] CHEN S,WANG Y,CHEN F,et al.A highly pathogenic strain of Staphylococcus sciuri caused fatal exudative epidermitis in piglets[J].PLoS One,2007,2(1):e147.
[13] ANDRESEN L O,AHRENS P,DAUGAARD L,et al.Exudative epidermitis in pigs caused by toxigenic Staphylococcus chromogenes[J].Veterinary Microbiology,2005,105(3):291-300.
[14] 王明月,王新平,郭昌明,等.吉林省某猪场新发猪葡萄球菌引起的化脓性肺炎[J].中国兽医学报,2016,36(9):1577-1581.WANG M Y,WANG X P,GUO C M,et al.Rare infection characterized with suppurative pneumonia by Staphylococcus hyicus[J].Chinese Journal of Veterinary Science,2016,35(9):1577-1581.(in Chinese)
[15] FOSTER A P.Staphylococcal skin disease in live-stock[J].Veterinary Dermatology,2012,23(4):342-351.
[16] JEONGHWA P,FRIENDSHIP R M,ZVONIMIR P,et al.An investigation of exudative epidermitis (greasy pig disease) and antimicrobial resistance patterns of Staphylococcus hyicus and Staphylococcus aureus isolated from clinical cases[J].Canadian Veterinary Journal-revue Veterinaire Canadienne,2013,54(2):139-144.
[17] AARESTRUP F M,JENSEN L B.Trends in antimicrobial susceptibility in relation to antimicrobial usage and presence of resistance genes in Staphylococcus hyicus isolated from exudative epidermitis in pigs[J].Veterinary Microbiology,2002,89(1):83-94.
[18] WEGENER H C,WATTS J L,SALMON S A,et al.Antimicrobial susceptibility of Staphylococcus hyicus isolated from exudative epidermitis in pigs[J].Journal of Clinical Microbiology,2015,32(3):793-795.
[19] 李淑红,王京仁,成钢,等.猪大肠杆菌和葡萄球菌的分离鉴定及耐药性分析[J].中国畜牧兽医,2012,39(8):195-198.LI S H,WANG J R,CHENG G,et al.Isolation,identification and analysis of drug resistance on E.coli and Staphylococcus in pigs[J].China Animal Husbandry & Veterinary Medicine,2012,39(8):195-198.(in Chinese)
[20] DHAND A,SAKOULAS G.Daptomycin in combination with other antibiotics for the treatment of complicated methicillin-resistant Staphylococcus aureus bacteremia[J].Clinical Therapeutics,2014,36(10):1303-1316.
[21] LI Z,WANG X,XIAO W,et al.Research advances on plectasin and its derivatives as new potential antimicrobial candidates[J].Process Biochemistry,2017,56:62-70.
[22] VAILLANCOURT K,LEBEL G,YI L,et al.In vitro antibacterial activity of plant essential oils against Staphylococcus hyicus and Staphylococcus aureus,the causative agents of exudative epidermitis in pigs[J].Archives of Microbiology,2018,200(7):1001-1007.
[23] ARSENAKIS I,BOYEN F,HAESEBROUCK F,et al.Autogenous vaccination reduces antimicrobial usage and mortality rates in a herd facing severe exudative epidermitis outbreaks in weaned pigs[J].Veterinary Record,2018,182(26):744.
[24] 睢艳平,徐镔蕊,王勇,等.仔猪渗出性皮炎的实验室诊断[J].中国畜牧兽医,2006,33(10):13-16.JU Y P,XU B R,WANG Y et al.Laboratory diagnosis of exudative dermatitis in piglets[J].China Animal Husbandry & Veterinary Medicine,2006,33(10):13-16.(in Chinese)
[25] LADHANI S.Understanding the mechanism of action of the exfoliative toxins of Staphylococcus aureus[J].FEMS Immunology & Medical Microbiology,2003,39(2):181-189.
[26] PATEL G K,FINLAY A Y.Staphylococcal scalded skin syndrome:Diagnosis and management[J].American Journal of Clinical Dermatology,2010,123(6):505-507.
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