豫西地区兔巴氏杆菌病病原特性与PCR检测技术研究
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摘要
兔巴氏杆菌病是由多杀性巴氏杆菌引起兔的一种传染病,该病传播快、病程急、发病率与死亡率高,常给养兔业造成巨大损失。该病病原血清型复杂,不同血清型之间交互免疫效果不理想,且在不同地区流行的血清型差异较大,给防治工作带来较大难度。为了摸清豫西地区兔巴氏杆菌病的具体发生情况,并对该病做到早期快速诊断与有效预防,本研究主要从以下4方面进行了研究：
1.兔多杀性巴氏杆菌的分离与鉴定
本研究对2003～2005年间来自豫西地区的疑似巴氏杆菌病兔进行剖检与病原分离,先后共鉴定多杀性巴氏杆菌18株,分别编号为P1、P2……P18,其中P1、P2……P11来源于肉兔,P12、P13……P18来源于獭兔。血清琼脂平板上,18株分离菌的菌落所带的荧光有三类, Fo型荧光10株,Fg型荧光5株,中间型3株。药敏试验表明,18株分离菌一半以上对氟哌酸、庆大霉素、痢特灵3种常用抗菌药耐药；对强力霉素、环丙杀星的耐药菌株较少；多数菌株对先锋霉素、红霉素、卡那霉素、四环素、青霉素、氨苄青霉素中度敏感或高度敏感。毒力试验结果表明,18株分离菌中有10株(P,、P2、P5、P6、P8、P10、P11、P13、P15、P16)对仔兔的致死菌量在20cfu/只以下,毒力比较强,占分离株的55.6%。其中5株(P,、P2、P8、P1,、P16)分离菌的致死菌量在10cfu／只以下,为强毒。不同荧光型的菌株毒力不同,Fo型菌、Fg型菌对小白鼠与仔兔的毒力比较强,中间型菌毒力较弱。
2.兔多杀性巴氏杆菌分离株免疫学特性研究
本研究在病原分离鉴定的基础上,对18株巴氏杆菌分离株进行了血清学分型鉴定。采用间接血凝试验对分离株的荚膜抗原进行鉴定,结果表明18株分离菌的荚膜抗原均为A群；采用琼脂扩散试验对18株分离菌的菌体耐热抗原进行鉴定,结果表明分离株的菌体耐热抗原主要有两型：1型14株(77.8%)、3型4株(22.2%)。将P1、P2、P,、P6、P8、P10、P1,、P1,、P1,、P16毒力较强的10株巴氏杆菌分离株接种改良马丁肉汤进行培养,菌液用甲醛灭活制成灭活疫苗；将疫苗接种小白鼠进行免疫保护试验,疫苗接种后21天,分别用同源菌株、标准株C51-2、标准株C51-3攻击,其保护数在3/5以上的分别有9株、7株、4株。用这10株灭活疫苗对哈白兔接种进行免疫保护试验,疫苗接种后14天,用同源菌株攻击,保护数在3/5以上有7株；用标准株C51-2攻击,保护数在3/5以上的4株；用标准株C51-3攻击,保护数在3/5以上的3株。选用免疫保护效果较好的两个血清型巴氏杆菌分离株P,(A：1)、P2(A：3)灭活疫苗接种家兔,用兔巴氏杆菌标准株C51-2(A：1)、C51-3(A：3)分别攻毒进行交互免疫试验,结果表明1型菌P1株灭活疫苗对3型菌有一定的交互免疫作用,3型菌P2株灭活疫苗对1型菌的交互免疫作用较弱。
3.兔多杀性巴氏杆菌二价蜂胶灭活疫苗的研制
为有效控制豫西地区兔巴氏杆菌病的发生,保障养兔业的健康发展,本研究选用免疫保护效果较好的多杀性巴氏杆菌地方分离强毒株P1(A：1)、P2(A：3)为疫苗株,以改良马丁肉汤进行液体培养制备抗原,将灭活的抗原辅以具有广谱生物学活性的天然物质-蜂胶为免疫增强剂,制备了兔巴氏杆菌二价蜂胶灭活疫苗。动物免疫试验结果表明,二价蜂胶灭活疫苗安全可靠,免疫后14d可产生坚强保护力,对兔巴氏杆菌病的近期保护率明显优于二价水苗,可高达100%。以实验动物小白鼠代替本动物进行二价疫苗效力检验取得成功,本效力检验方法可降低检验成本,具有更敏感、更特异、更客观、更简便等特点。用琼脂扩散试验测定动物接种疫苗后的抗体消长规律,用攻毒法测定疫苗的免疫期与保存期。结果表明,该疫苗的免疫期为6个月以上；4℃-8℃的保存期为12个月,15℃-30℃的保存期为6个月。
4.兔多杀性巴氏杆菌PCR检测方法的建立及应用
传统的兔巴氏杆菌病实验室诊断常采用涂片染色镜检、病原分离、生化鉴定等方法,但这些方法较繁杂费时,且检出率较低。为建立本病快速特异的检测方法,本研究根据Genbank中13株多杀性巴氏杆菌的外膜蛋白(OmpH)基因序列,在其高度保守区设计了一对特异性引物,建立了兔多杀性巴氏杆菌的PCR检测方法,并对DNA的提取方法进行了比较,对PCR体系各反应条件进行了优化。本实验建立的兔多杀性巴氏杆菌的PCR扩增片段大小为526bp。兔和禽类的标准株C51-2和C48-1均能扩增出526bp的特异性目的条带,对实验室分离的18株兔巴氏杆菌也均能扩增出526bp的目的条带。而对兔大肠杆菌、链球菌、支气管败血性波氏杆菌和葡萄球菌球菌的扩增结果均为阴性。敏感性检测结果表明,当反应体系中模板DNA的量为1pg时,仍能看到较清晰的条带,说明建立的PCR方法敏感性较好,只要检测样品中有病原核酸存在,PCR反应即可顺利完成,而得到准确诊断结果。用该PCR法检测了先后从洛阳、三门峡等周边地区送检的40份疑似巴氏杆菌病兔病料,检出多杀性巴氏杆菌阳性29例,阳性率为72.5%。同时与传统的细菌检查法进行了比较。结果显示,该PCR检测法的检出率是传统的实验室细菌检查法的2.08倍,说明该PCR方法在临床诊断中具有极强的应用价值。
Rabbit Pasteurellosis caused by Pasteurella multocida is one of the most significant bacterial diseases of rabbits and causes considerable economic losses in large production units throughout the world. The disease is characterised by various clinical symptoms, including respiratory distress, genital infections, abscesses, otitis, and septicaemia, but infection by P. multocida can also appear without manifesting any clinical signs. Pathogenic serotypes of the disease are complex and the immune effect between different serotypes is not satisfactory, and popular serotypes are different in different areas, which brings greater difficulty in prevention and treatment work of Rabbit Pasteurellosis. In this study, in order to investigate the rabbit pasteurellosis epidemiology in western Henan and provide a scientific measures for effective prevention and treatment, the auther had done some research. The contents and results are as followes:
1. Isolation and Identification of Rabbit Pasteurella Multocida
In order to find out the rabbit pasteurellosis epidemiology in western Henan and to provide a scientific measures for effective prevention and treatment, the rabbit suspected Pasteurella multocida infection from western Henan were anatomy-checked and the Pasteurella multocida were isolated from the year2003to2005.18strains were identified and were numbered as P1, P2……P18, which the P1, P2……P11from the meat rabbit, P12, P13……P18from the Rexyabbit. According to the fluorescence that they showed on serum agar plate, The18isolates were classfied into three categories:Fo-fluorescence (10strains); Fg fluorescence(5strains) and medium type(3strains). Susceptibility testing showed more than half of strains were resistant to common antibiotics norfloxacin, gentamicin, furoxone; and only a few strains were resistantto doxycycline and ciprofloxacin; and mostly of the18strains were moderately sensitive or highly sensitive to adriamycin, erythromycin, kanamycin, tetracycline, penicillin and ampicillin. The lethal dose on newborn rabbits of ten strains (P1, P2, P5, P6, P8, P10, P11, P13, P15and P16) was below20cfu. They had stronger pathogenicity and accounted for55.6%of all18isolated strains. The lethal dose of five strains (P1、P2、P8、P13、P16) among them was below10cfu. It showed they were velogenic strains. The virulence of different fluorescent type has different toxicity, Fo-type and Fg-type had stronger toxicity to newborn rabbits and mice than the medium.
2. The Immunological Characteristics Study of Rabbit Pasteurella multocida Isolates
In order to find out the rabbit Pasteurella multocida serotype prevalence in western Henan, the18isolates were typed on the serological identification. The IHA test was used to identify the capsule antigen, the results showed that18isolates were all belonged to group A capsule antigen. Agar-proliferation was used to identify the heat-resistant bacterial antigen, the results showed that18isolates were grouped two main types:14isolates were type1(77.8%) and4isolates were type3(22.2%). The P1、P2、P5、P6、P8、P10、P11、P13、 P15、P16isolates with stronger toxicity were cultivated in improved Martin broth and were treated with formaldehyde to be made inactivated vaccine. Vaccination was carried out in mice to test immunogenicity and the mice were attacked with homologous strain, standard strain C51-2and standard strain C51-3on the21st day after vaccination, respectively, and the protection rate reached60%were9,7and4isolates, respectively. The above-mentioned10isolates inactivated vaccine were selected to inoculate newborn rabbits to test immunogenicity on rabbits, and the rabbits were attacked with homologous strains, standard strain C51-2and standard strain C51-3on the14th day after vaccination, respectively, and the protection rate reached60%were7,4and3isolates, respectively.The interactive immune test was done using the P1(serotypeA:1) and P2(serotypeA:3) strains which had better immunogenicity on rabbits, the results showed that vaccine prepared with type1bacteria P1had a certain interaction immune to type3bacteria, but the vaccine prepared with type3bacteria P3had weaker interaction immune to type1bacteria.
3. Development of the Rabbit Pasteurella Multocida Bivalence propolis inactived vaccine
For the effective control of rabbit pasteurellosis in western Henan, protecting the healthy development of rabbit production industry, the Pasteurella multocida isolates of P1(serotype A:1) and P2(serotype A:3) were used as vaccine strains and were cultivated in improved Martin broth for culture of antigen. The inactivated antigen were produced into pasteurella bivalence inactivated propolis vaccine assisted by the propolis as immune-enhancer which has broad-spectrum biological activity. The results of mice immunization showed that the bivalence inactivated propolis vaccine was safe and reliable and had no obvious interference, strong immunity produced14days after immunization, and the recent protection rates for pasteurellosis was almost100%. Succeeded in using mice replacement of rabbit to test bivalence vaccine efficacy, and this method can reduce reduce test cost, and more sensitive, specific, objective and simple. Using challenge method for pasteurellosis, modified micro-aggregation test for antibody titer, to check the immunity period and storage time of this vaccine. The result showed that the immunity period is more than6months,storage time is12months at4℃～8℃;6months at15℃～30℃.
4. Establishment and application of PCR method for detecting rabbit pasteurella multocida
In order to establish a quick detection method of the rabbit pasteurellosis, a pair of specific primers was designed according to the highly conservative region of the outer membrane protein (OmpH) gene sequence of13Pasteurella multocida in Genbank. By comparing the DNA extraction methods and optimizing the PCR system and procedure, a PCR based assay for detecting rabbit pasteurella multoceda was developed. The size of PCR amplified fragment was526bp. A specific fragment of526bp could be amplified with either the rabbit and poultry standard strains of C51-2and C48-1or the18laboratory separation strains from rabbits. But the amplification results were negative with the rabbit E. coli, Streptococcus, B.bronchiseptica and Rabbit Staphylococcus aureus. The results of sensitivity detection showed that the strip could be seen clearer when the template DNA was only lpg. It suggested that the sensitivity of the PCR method is better. The PCR reaction can be completed successfully and get accurate diagnosis as long as the pathogenic nucleic acid exists in the samples.40samples from from Luoyang and Sanmenxia rabbits were examined by the developed PCR assay and72.5%were positive. Comparison of the assay with the conventional bacterioscopy revealed that the sensitivity of the PCR assay was2.08times higher than that of the bacterioscopy. The results showed that the PCR method is extremely value in clinical diagnosis.
1.兔多杀性巴氏杆菌的分离与鉴定
本研究对2003～2005年间来自豫西地区的疑似巴氏杆菌病兔进行剖检与病原分离,先后共鉴定多杀性巴氏杆菌18株,分别编号为P1、P2……P18,其中P1、P2……P11来源于肉兔,P12、P13……P18来源于獭兔。血清琼脂平板上,18株分离菌的菌落所带的荧光有三类, Fo型荧光10株,Fg型荧光5株,中间型3株。药敏试验表明,18株分离菌一半以上对氟哌酸、庆大霉素、痢特灵3种常用抗菌药耐药；对强力霉素、环丙杀星的耐药菌株较少；多数菌株对先锋霉素、红霉素、卡那霉素、四环素、青霉素、氨苄青霉素中度敏感或高度敏感。毒力试验结果表明,18株分离菌中有10株(P,、P2、P5、P6、P8、P10、P11、P13、P15、P16)对仔兔的致死菌量在20cfu/只以下,毒力比较强,占分离株的55.6%。其中5株(P,、P2、P8、P1,、P16)分离菌的致死菌量在10cfu／只以下,为强毒。不同荧光型的菌株毒力不同,Fo型菌、Fg型菌对小白鼠与仔兔的毒力比较强,中间型菌毒力较弱。
2.兔多杀性巴氏杆菌分离株免疫学特性研究
本研究在病原分离鉴定的基础上,对18株巴氏杆菌分离株进行了血清学分型鉴定。采用间接血凝试验对分离株的荚膜抗原进行鉴定,结果表明18株分离菌的荚膜抗原均为A群；采用琼脂扩散试验对18株分离菌的菌体耐热抗原进行鉴定,结果表明分离株的菌体耐热抗原主要有两型：1型14株(77.8%)、3型4株(22.2%)。将P1、P2、P,、P6、P8、P10、P1,、P1,、P1,、P16毒力较强的10株巴氏杆菌分离株接种改良马丁肉汤进行培养,菌液用甲醛灭活制成灭活疫苗；将疫苗接种小白鼠进行免疫保护试验,疫苗接种后21天,分别用同源菌株、标准株C51-2、标准株C51-3攻击,其保护数在3/5以上的分别有9株、7株、4株。用这10株灭活疫苗对哈白兔接种进行免疫保护试验,疫苗接种后14天,用同源菌株攻击,保护数在3/5以上有7株；用标准株C51-2攻击,保护数在3/5以上的4株；用标准株C51-3攻击,保护数在3/5以上的3株。选用免疫保护效果较好的两个血清型巴氏杆菌分离株P,(A：1)、P2(A：3)灭活疫苗接种家兔,用兔巴氏杆菌标准株C51-2(A：1)、C51-3(A：3)分别攻毒进行交互免疫试验,结果表明1型菌P1株灭活疫苗对3型菌有一定的交互免疫作用,3型菌P2株灭活疫苗对1型菌的交互免疫作用较弱。
3.兔多杀性巴氏杆菌二价蜂胶灭活疫苗的研制
为有效控制豫西地区兔巴氏杆菌病的发生,保障养兔业的健康发展,本研究选用免疫保护效果较好的多杀性巴氏杆菌地方分离强毒株P1(A：1)、P2(A：3)为疫苗株,以改良马丁肉汤进行液体培养制备抗原,将灭活的抗原辅以具有广谱生物学活性的天然物质-蜂胶为免疫增强剂,制备了兔巴氏杆菌二价蜂胶灭活疫苗。动物免疫试验结果表明,二价蜂胶灭活疫苗安全可靠,免疫后14d可产生坚强保护力,对兔巴氏杆菌病的近期保护率明显优于二价水苗,可高达100%。以实验动物小白鼠代替本动物进行二价疫苗效力检验取得成功,本效力检验方法可降低检验成本,具有更敏感、更特异、更客观、更简便等特点。用琼脂扩散试验测定动物接种疫苗后的抗体消长规律,用攻毒法测定疫苗的免疫期与保存期。结果表明,该疫苗的免疫期为6个月以上；4℃-8℃的保存期为12个月,15℃-30℃的保存期为6个月。
4.兔多杀性巴氏杆菌PCR检测方法的建立及应用
传统的兔巴氏杆菌病实验室诊断常采用涂片染色镜检、病原分离、生化鉴定等方法,但这些方法较繁杂费时,且检出率较低。为建立本病快速特异的检测方法,本研究根据Genbank中13株多杀性巴氏杆菌的外膜蛋白(OmpH)基因序列,在其高度保守区设计了一对特异性引物,建立了兔多杀性巴氏杆菌的PCR检测方法,并对DNA的提取方法进行了比较,对PCR体系各反应条件进行了优化。本实验建立的兔多杀性巴氏杆菌的PCR扩增片段大小为526bp。兔和禽类的标准株C51-2和C48-1均能扩增出526bp的特异性目的条带,对实验室分离的18株兔巴氏杆菌也均能扩增出526bp的目的条带。而对兔大肠杆菌、链球菌、支气管败血性波氏杆菌和葡萄球菌球菌的扩增结果均为阴性。敏感性检测结果表明,当反应体系中模板DNA的量为1pg时,仍能看到较清晰的条带,说明建立的PCR方法敏感性较好,只要检测样品中有病原核酸存在,PCR反应即可顺利完成,而得到准确诊断结果。用该PCR法检测了先后从洛阳、三门峡等周边地区送检的40份疑似巴氏杆菌病兔病料,检出多杀性巴氏杆菌阳性29例,阳性率为72.5%。同时与传统的细菌检查法进行了比较。结果显示,该PCR检测法的检出率是传统的实验室细菌检查法的2.08倍,说明该PCR方法在临床诊断中具有极强的应用价值。
Rabbit Pasteurellosis caused by Pasteurella multocida is one of the most significant bacterial diseases of rabbits and causes considerable economic losses in large production units throughout the world. The disease is characterised by various clinical symptoms, including respiratory distress, genital infections, abscesses, otitis, and septicaemia, but infection by P. multocida can also appear without manifesting any clinical signs. Pathogenic serotypes of the disease are complex and the immune effect between different serotypes is not satisfactory, and popular serotypes are different in different areas, which brings greater difficulty in prevention and treatment work of Rabbit Pasteurellosis. In this study, in order to investigate the rabbit pasteurellosis epidemiology in western Henan and provide a scientific measures for effective prevention and treatment, the auther had done some research. The contents and results are as followes:
1. Isolation and Identification of Rabbit Pasteurella Multocida
In order to find out the rabbit pasteurellosis epidemiology in western Henan and to provide a scientific measures for effective prevention and treatment, the rabbit suspected Pasteurella multocida infection from western Henan were anatomy-checked and the Pasteurella multocida were isolated from the year2003to2005.18strains were identified and were numbered as P1, P2……P18, which the P1, P2……P11from the meat rabbit, P12, P13……P18from the Rexyabbit. According to the fluorescence that they showed on serum agar plate, The18isolates were classfied into three categories:Fo-fluorescence (10strains); Fg fluorescence(5strains) and medium type(3strains). Susceptibility testing showed more than half of strains were resistant to common antibiotics norfloxacin, gentamicin, furoxone; and only a few strains were resistantto doxycycline and ciprofloxacin; and mostly of the18strains were moderately sensitive or highly sensitive to adriamycin, erythromycin, kanamycin, tetracycline, penicillin and ampicillin. The lethal dose on newborn rabbits of ten strains (P1, P2, P5, P6, P8, P10, P11, P13, P15and P16) was below20cfu. They had stronger pathogenicity and accounted for55.6%of all18isolated strains. The lethal dose of five strains (P1、P2、P8、P13、P16) among them was below10cfu. It showed they were velogenic strains. The virulence of different fluorescent type has different toxicity, Fo-type and Fg-type had stronger toxicity to newborn rabbits and mice than the medium.
2. The Immunological Characteristics Study of Rabbit Pasteurella multocida Isolates
In order to find out the rabbit Pasteurella multocida serotype prevalence in western Henan, the18isolates were typed on the serological identification. The IHA test was used to identify the capsule antigen, the results showed that18isolates were all belonged to group A capsule antigen. Agar-proliferation was used to identify the heat-resistant bacterial antigen, the results showed that18isolates were grouped two main types:14isolates were type1(77.8%) and4isolates were type3(22.2%). The P1、P2、P5、P6、P8、P10、P11、P13、 P15、P16isolates with stronger toxicity were cultivated in improved Martin broth and were treated with formaldehyde to be made inactivated vaccine. Vaccination was carried out in mice to test immunogenicity and the mice were attacked with homologous strain, standard strain C51-2and standard strain C51-3on the21st day after vaccination, respectively, and the protection rate reached60%were9,7and4isolates, respectively. The above-mentioned10isolates inactivated vaccine were selected to inoculate newborn rabbits to test immunogenicity on rabbits, and the rabbits were attacked with homologous strains, standard strain C51-2and standard strain C51-3on the14th day after vaccination, respectively, and the protection rate reached60%were7,4and3isolates, respectively.The interactive immune test was done using the P1(serotypeA:1) and P2(serotypeA:3) strains which had better immunogenicity on rabbits, the results showed that vaccine prepared with type1bacteria P1had a certain interaction immune to type3bacteria, but the vaccine prepared with type3bacteria P3had weaker interaction immune to type1bacteria.
3. Development of the Rabbit Pasteurella Multocida Bivalence propolis inactived vaccine
For the effective control of rabbit pasteurellosis in western Henan, protecting the healthy development of rabbit production industry, the Pasteurella multocida isolates of P1(serotype A:1) and P2(serotype A:3) were used as vaccine strains and were cultivated in improved Martin broth for culture of antigen. The inactivated antigen were produced into pasteurella bivalence inactivated propolis vaccine assisted by the propolis as immune-enhancer which has broad-spectrum biological activity. The results of mice immunization showed that the bivalence inactivated propolis vaccine was safe and reliable and had no obvious interference, strong immunity produced14days after immunization, and the recent protection rates for pasteurellosis was almost100%. Succeeded in using mice replacement of rabbit to test bivalence vaccine efficacy, and this method can reduce reduce test cost, and more sensitive, specific, objective and simple. Using challenge method for pasteurellosis, modified micro-aggregation test for antibody titer, to check the immunity period and storage time of this vaccine. The result showed that the immunity period is more than6months,storage time is12months at4℃～8℃;6months at15℃～30℃.
4. Establishment and application of PCR method for detecting rabbit pasteurella multocida
In order to establish a quick detection method of the rabbit pasteurellosis, a pair of specific primers was designed according to the highly conservative region of the outer membrane protein (OmpH) gene sequence of13Pasteurella multocida in Genbank. By comparing the DNA extraction methods and optimizing the PCR system and procedure, a PCR based assay for detecting rabbit pasteurella multoceda was developed. The size of PCR amplified fragment was526bp. A specific fragment of526bp could be amplified with either the rabbit and poultry standard strains of C51-2and C48-1or the18laboratory separation strains from rabbits. But the amplification results were negative with the rabbit E. coli, Streptococcus, B.bronchiseptica and Rabbit Staphylococcus aureus. The results of sensitivity detection showed that the strip could be seen clearer when the template DNA was only lpg. It suggested that the sensitivity of the PCR method is better. The PCR reaction can be completed successfully and get accurate diagnosis as long as the pathogenic nucleic acid exists in the samples.40samples from from Luoyang and Sanmenxia rabbits were examined by the developed PCR assay and72.5%were positive. Comparison of the assay with the conventional bacterioscopy revealed that the sensitivity of the PCR assay was2.08times higher than that of the bacterioscopy. The results showed that the PCR method is extremely value in clinical diagnosis.
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