羊源弗氏檸檬酸桿菌的分離鑒定及其生物學(xué)特性研究
發(fā)布時間：2019-02-11 17:54
【摘要】：2014年河南省某規(guī)�；驁龅男∥埠虼罅克劳觥２⊙蛑饕憩F(xiàn)為可視黏膜發(fā)紺,然后突然倒地,四肢震顫,發(fā)出痛苦低沉鳴叫聲,常于發(fā)病后8-24 h突然死亡,病死率100%。為了確定導(dǎo)致小尾寒羊細(xì)菌性感染死亡的病原。對3只不同時間發(fā)病死亡的小尾寒羊進(jìn)行系統(tǒng)剖檢,均呈現(xiàn)典型的敗血癥性病理變化。主要表現(xiàn)為腹腔有少量積液,肺臟、心肌、大腸、小腸和腎出血,肝、脾腫大并嚴(yán)重出血,腦輕度水腫。并從各組織臟器的病料中分離到3株細(xì)菌,命名為:LK-1、LK-2和LK-3。常規(guī)生化鑒定結(jié)果表明3株分離菌與弗氏檸檬酸桿菌的生化特性相符。全自動微生物鑒定儀的鑒定表明3株分離菌均為弗氏檸檬酸桿菌,可能性超過99%。16s r RNA基因的比對分析表明與三者同源性最高的均為NCBI數(shù)據(jù)庫已發(fā)布的弗氏檸檬酸桿菌的16S r RNA基因序列,其同源性均在99%以上。系統(tǒng)發(fā)育樹表明LK-1株與日本弗氏檸檬酸桿菌NBRC16624株親緣關(guān)系最近。以上鑒定結(jié)果均表明3株分離菌為弗氏檸檬酸桿菌。為了進(jìn)一步確定弗氏檸檬酸桿菌是否為小尾寒羊死亡的致病細(xì)菌。用LK-1株進(jìn)行回歸試驗,經(jīng)人工感染LK-1株的健康小尾寒羊,可復(fù)制與自然發(fā)病羊相似的臨床癥狀和敗血癥病理變化,且從感染病羊各組織臟器中再次分離到相同的弗氏檸檬酸桿菌。回歸試驗證實了弗氏檸檬酸桿菌對健康小尾寒羊具有較強(qiáng)的致病性,表明弗氏檸檬酸桿菌是該小尾寒羊致死性病例的病原菌。取自然發(fā)病羊和人工感染羊的各組織臟器進(jìn)行固定并制作病理切片,病理結(jié)果表明自然發(fā)病羊和人工感染羊的病理變化相同,肺、脾、心和腦病理變化最嚴(yán)重。均表現(xiàn)為肺泡壁毛細(xì)血管充血、出血,肺泡內(nèi)充滿紅細(xì)胞和纖維素性滲出物。脾臟組織內(nèi)嚴(yán)重出血,脾小梁結(jié)構(gòu)變小甚至看不到脾小梁,有大量紅細(xì)胞浸潤。心肌纖維斷裂、間質(zhì)增寬、排列紊亂并有紅細(xì)胞和炎性細(xì)胞浸潤。腦組織輕度水腫,血管周圍有炎性細(xì)胞浸潤。小鼠毒力試驗結(jié)果表明:3株細(xì)菌的LD50分別為5.0×107 CFU、6.0×107 CFU和5.8×107 CFU。藥敏試驗結(jié)果表明,3株弗氏檸檬酸桿菌均對慶大霉素等12種藥物高度敏感。小鼠免疫保護(hù)試驗結(jié)果表明傳統(tǒng)的白油疫苗和新型GEL疫苗均能提供70%的保護(hù)率,并且兩者可以誘導(dǎo)相同的Ig G抗體水平。根據(jù)藥敏和小鼠免疫保護(hù)試驗結(jié)果指導(dǎo)該養(yǎng)殖場用慶大霉素注射易感染羊群、用生石灰對地面消毒和LK-1株GEL佐劑滅活疫苗免疫所有成年羊和斷奶羊羔,取得了良好的治療和預(yù)防效果。本文在國內(nèi)外首次報道了弗氏檸檬酸桿菌對小尾寒羊具有致病性。并為該病的致病機(jī)理、綜合防控和高效疫苗的研制、開發(fā)奠定了基礎(chǔ)。
[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.
【學(xué)位授予單位】：河南科技大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S852.61
本文編號：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.
【學(xué)位授予單位】：河南科技大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S852.61
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