羊源弗氏柠檬酸杆菌的分离鉴定及其生物学特性研究
发布时间：2019-02-11 17:54
【摘要】：2014年河南省某规模化羊场的小尾寒羊大量死亡。病羊主要表现为可视黏膜发绀,然后突然倒地,四肢震颤,发出痛苦低沉鸣叫声,常于发病后8-24 h突然死亡,病死率100%。为了确定导致小尾寒羊细菌性感染死亡的病原。对3只不同时间发病死亡的小尾寒羊进行系统剖检,均呈现典型的败血症性病理变化。主要表现为腹腔有少量积液,肺脏、心肌、大肠、小肠和肾出血,肝、脾肿大并严重出血,脑轻度水肿。并从各组织脏器的病料中分离到3株细菌,命名为:LK-1、LK-2和LK-3。常规生化鉴定结果表明3株分离菌与弗氏柠檬酸杆菌的生化特性相符。全自动微生物鉴定仪的鉴定表明3株分离菌均为弗氏柠檬酸杆菌,可能性超过99%。16s r RNA基因的比对分析表明与三者同源性最高的均为NCBI数据库已发布的弗氏柠檬酸杆菌的16S r RNA基因序列,其同源性均在99%以上。系统发育树表明LK-1株与日本弗氏柠檬酸杆菌NBRC16624株亲缘关系最近。以上鉴定结果均表明3株分离菌为弗氏柠檬酸杆菌。为了进一步确定弗氏柠檬酸杆菌是否为小尾寒羊死亡的致病细菌。用LK-1株进行回归试验,经人工感染LK-1株的健康小尾寒羊,可复制与自然发病羊相似的临床症状和败血症病理变化,且从感染病羊各组织脏器中再次分离到相同的弗氏柠檬酸杆菌。回归试验证实了弗氏柠檬酸杆菌对健康小尾寒羊具有较强的致病性,表明弗氏柠檬酸杆菌是该小尾寒羊致死性病例的病原菌。取自然发病羊和人工感染羊的各组织脏器进行固定并制作病理切片,病理结果表明自然发病羊和人工感染羊的病理变化相同,肺、脾、心和脑病理变化最严重。均表现为肺泡壁毛细血管充血、出血,肺泡内充满红细胞和纤维素性渗出物。脾脏组织内严重出血,脾小梁结构变小甚至看不到脾小梁,有大量红细胞浸润。心肌纤维断裂、间质增宽、排列紊乱并有红细胞和炎性细胞浸润。脑组织轻度水肿,血管周围有炎性细胞浸润。小鼠毒力试验结果表明:3株细菌的LD50分别为5.0×107 CFU、6.0×107 CFU和5.8×107 CFU。药敏试验结果表明,3株弗氏柠檬酸杆菌均对庆大霉素等12种药物高度敏感。小鼠免疫保护试验结果表明传统的白油疫苗和新型GEL疫苗均能提供70%的保护率,并且两者可以诱导相同的Ig G抗体水平。根据药敏和小鼠免疫保护试验结果指导该养殖场用庆大霉素注射易感染羊群、用生石灰对地面消毒和LK-1株GEL佐剂灭活疫苗免疫所有成年羊和断奶羊羔,取得了良好的治疗和预防效果。本文在国内外首次报道了弗氏柠檬酸杆菌对小尾寒羊具有致病性。并为该病的致病机理、综合防控和高效疫苗的研制、开发奠定了基础。
[Abstract]:Small tail Han sheep died in a large scale sheep farm in Henan province in 2014. The main manifestations of the disease were cyanosis of the visible mucosa, then sudden fall to the ground, tremor of the limbs, low and painful chirping, which often died 8 to 24 hours after the onset of the disease, with a mortality of 100. To determine the cause of death from bacterial infection in small tail Han sheep. Three small tail Han sheep which died at different time showed typical septicemia pathological changes. The main manifestations were a small amount of effusion in the abdominal cavity, lung, myocardium, large intestine, small intestine and kidney hemorrhage, liver and spleen swelling and severe hemorrhage, mild cerebral edema. Three strains of bacteria were isolated from tissues and organs, named LK-1,LK-2 and LK-3.. The results of routine biochemical identification showed that the biochemical characteristics of the three isolates were consistent with those of Citrate flexneri. The automatic microbiological identification instrument showed that the three isolates were all strains of Citrobacter flexneri. The comparison analysis of the probability of more than 990.16s r RNA gene showed that the 16s r RNA gene sequence of Citrobacter flexneri published in NCBI database had the highest homology, and its homology was above 99%. Phylogenetic tree showed that LK-1 strain had the closest relationship with NBRC16624 strain. All the above identification results showed that the three isolates were Citrobacter flexneri. In order to further determine whether Citrate flexneri is the death of small tail Han sheep pathogenic bacteria. The regression test of LK-1 strain showed that healthy small tail Han sheep infected artificially with LK-1 strain could replicate the clinical symptoms and pathological changes of septicemia similar to those of naturally occurring sheep. The same strain of Citrobacter flexneri was isolated from the tissues and organs of infected sheep. The regression test confirmed the strong pathogenicity of Citrate flexneri to healthy small tail Han sheep, which indicated that Citrate flexneri was the pathogenic bacteria in the fatal case of small tail Han sheep. The tissues and organs of naturally occurring sheep and artificially infected sheep were fixed and pathological sections were made. The pathological results showed that the pathological changes of the naturally infected sheep and artificially infected sheep were the same, and the pathological changes of lung, spleen, heart and brain were the most serious. The alveolar wall was characterized by capillary congestion, hemorrhage, red blood cells and cellulose exudates in the alveoli. The splenic trabecular structure became smaller and the splenic trabeculae could not be seen, and a large number of red blood cells infiltrated. Myocardial fiber rupture, interstitial widening, disorder and infiltration of red blood cells and inflammatory cells. Mild edema of brain tissue and infiltration of inflammatory cells around blood vessels. The virulence test showed that the LD50 of the three strains were 5.0 脳 10 ~ 7 CFU,6.0 脳 10 ~ 7 CFU and 5.8 脳 10 ~ 7 CFU., respectively. The results of drug sensitivity test showed that all three strains of Citrate flexneri were highly sensitive to gentamicin and other 12 drugs. The results of mouse immune protection test showed that both the traditional white oil vaccine and the new GEL vaccine could provide 70% protection, and both could induce the same level of Ig G antibody. According to the results of drug sensitivity test and mouse immune protection test, the farm was immunized with gentamicin to infect sheep, ground disinfection with quicklime and LK-1 strain GEL adjuvant inactivated vaccine to immunize all adult sheep and weaned lambs. Good therapeutic and preventive effects have been achieved. The pathogenicity of Citrate flexneri to small tail Han sheep was reported for the first time at home and abroad. It also lays a foundation for the research and development of the disease's pathogenic mechanism, comprehensive prevention and control and high efficiency vaccine.
【学位授予单位】：河南科技大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.61
本文编号：2419967
[Abstract]:Small tail Han sheep died in a large scale sheep farm in Henan province in 2014. The main manifestations of the disease were cyanosis of the visible mucosa, then sudden fall to the ground, tremor of the limbs, low and painful chirping, which often died 8 to 24 hours after the onset of the disease, with a mortality of 100. To determine the cause of death from bacterial infection in small tail Han sheep. Three small tail Han sheep which died at different time showed typical septicemia pathological changes. The main manifestations were a small amount of effusion in the abdominal cavity, lung, myocardium, large intestine, small intestine and kidney hemorrhage, liver and spleen swelling and severe hemorrhage, mild cerebral edema. Three strains of bacteria were isolated from tissues and organs, named LK-1,LK-2 and LK-3.. The results of routine biochemical identification showed that the biochemical characteristics of the three isolates were consistent with those of Citrate flexneri. The automatic microbiological identification instrument showed that the three isolates were all strains of Citrobacter flexneri. The comparison analysis of the probability of more than 990.16s r RNA gene showed that the 16s r RNA gene sequence of Citrobacter flexneri published in NCBI database had the highest homology, and its homology was above 99%. Phylogenetic tree showed that LK-1 strain had the closest relationship with NBRC16624 strain. All the above identification results showed that the three isolates were Citrobacter flexneri. In order to further determine whether Citrate flexneri is the death of small tail Han sheep pathogenic bacteria. The regression test of LK-1 strain showed that healthy small tail Han sheep infected artificially with LK-1 strain could replicate the clinical symptoms and pathological changes of septicemia similar to those of naturally occurring sheep. The same strain of Citrobacter flexneri was isolated from the tissues and organs of infected sheep. The regression test confirmed the strong pathogenicity of Citrate flexneri to healthy small tail Han sheep, which indicated that Citrate flexneri was the pathogenic bacteria in the fatal case of small tail Han sheep. The tissues and organs of naturally occurring sheep and artificially infected sheep were fixed and pathological sections were made. The pathological results showed that the pathological changes of the naturally infected sheep and artificially infected sheep were the same, and the pathological changes of lung, spleen, heart and brain were the most serious. The alveolar wall was characterized by capillary congestion, hemorrhage, red blood cells and cellulose exudates in the alveoli. The splenic trabecular structure became smaller and the splenic trabeculae could not be seen, and a large number of red blood cells infiltrated. Myocardial fiber rupture, interstitial widening, disorder and infiltration of red blood cells and inflammatory cells. Mild edema of brain tissue and infiltration of inflammatory cells around blood vessels. The virulence test showed that the LD50 of the three strains were 5.0 脳 10 ~ 7 CFU,6.0 脳 10 ~ 7 CFU and 5.8 脳 10 ~ 7 CFU., respectively. The results of drug sensitivity test showed that all three strains of Citrate flexneri were highly sensitive to gentamicin and other 12 drugs. The results of mouse immune protection test showed that both the traditional white oil vaccine and the new GEL vaccine could provide 70% protection, and both could induce the same level of Ig G antibody. According to the results of drug sensitivity test and mouse immune protection test, the farm was immunized with gentamicin to infect sheep, ground disinfection with quicklime and LK-1 strain GEL adjuvant inactivated vaccine to immunize all adult sheep and weaned lambs. Good therapeutic and preventive effects have been achieved. The pathogenicity of Citrate flexneri to small tail Han sheep was reported for the first time at home and abroad. It also lays a foundation for the research and development of the disease's pathogenic mechanism, comprehensive prevention and control and high efficiency vaccine.
【学位授予单位】：河南科技大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.61
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