我国鸡群鸡杆菌的首次分离鉴定及生物学特性初步研究
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摘要
鸡杆菌属(Gallibacterium)是Christensen等2003年以16S rRNA基因序列分析为基础建议在巴氏杆菌科单独成立的一个新属。2009年Bisgaard等明确该属包括Gallibacterium anatis、Actinobacillus salpingitidis、Gallibacterium melopsittaci和Gallibacterium trehalosifermentans 4个种,其代表种是Gallibacterium anatis。该菌有溶血型和非溶血型两个表型。国内对该菌的研究甚少。为进一步了解我国部分地区鸡场不同鸡群中鸡杆菌的感染情况,本实验对河南、山西、山东等地区的30个鸡群进行了鸡杆菌的分离鉴定。30个鸡群包括15个输卵管炎患病鸡群、2个SPF鸡群、6个健康蛋鸡群、5个商品公鸡群、2个种公鸡群。同时为了解鸡杆菌的微生物学特性,以便开展其分子流行病学、分子遗传学及发病机制的研究,本试验对部分鸡杆菌分离株的培养特性、生化特性、药物敏感性及致病性进行了初步研究,并得到如下结果:
1.对107只来自河南、山西的15个鸡群的输卵管炎患病鸡的10种组织脏器(心脏、肝脏、脾脏、肺脏、气管、十二指肠、输卵管、卵巢、泄殖腔、上腭裂)进行鸡杆菌的分离鉴定。通过形态学检查、鉴别培养、特异性PCR鉴定和16SrRNA基因序列测定等方法,最终分离鉴定到鸡杆菌76株,同时得到大肠杆菌180株。鸡杆菌的感染率达42.9%。说明河南、山西两地输卵管炎患病鸡群中普遍存在鸡杆菌的感染。其中,有11只具有典型临床及病理变化的鸡仅分离出鸡杆菌一种细菌,这说明鸡杆菌具有一定的致病性,可能在鸡的输卵管炎中扮演一定的角色。但是感染鸡杆菌的46只鸡中,有35只鸡同时感染大肠杆菌(占鸡杆菌阳性鸡的76.1%),而在临床上,大肠杆菌也是引起鸡输卵管炎的重要病原菌之一,因此,鸡杆菌在鸡输卵管炎中扮演什么样的角色,到底是继发、原发或是协同仍需进一步的研究证实。
2.通过形态学检查、鉴别培养、特异性PCR鉴定等方法,对来自河南、山东、山西的2个SPF鸡群的38只SPF鸡、6个临床健康鸡群的33只健康鸡进行了鸡杆菌的分离鉴定。从38只SPF鸡未分离出鸡杆菌,从临床健康鸡群中分离出鸡杆菌10株;临床健康鸡群中鸡杆菌的感染率达27.3%,说明临床健康蛋鸡群存在鸡杆菌的感染,但是与输卵管炎患病鸡群相比,鸡杆菌的感染率降低了15.6%,说明输卵管炎是鸡杆菌感染的重要临床特征之一。
3.对来自河南省5个商品公鸡群的20只商品公鸡和两个种鸡群的9只商品种公鸡的8种组织脏器(心脏、肝脏、脾脏、肺脏、气管、十二指肠、泄殖腔、上腭裂)进行鸡杆菌的分离鉴定。从20只商品公鸡中未分出鸡杆菌,从9只商品种公鸡中分离出鸡杆菌8株。这表明,鸡杆菌可以感染种公鸡。8只鸡杆菌分离株中,其中1株不具有溶血性,可能会对鸡杆菌致病性的研究有一定价值。
4.选择不同区域、不同宿主脏器的鸡杆菌分离株64株,进行培养特性、生化特性、药物敏感性研究。鸡杆菌在绵羊血、鸡血、鸭血琼脂平板上均生长良好;但是,在SS琼脂平板均不生长。通过在不同PH值的LB液体培养基中的培养,确定了鸡杆菌最适生长PH值范围是7.0~8.0。将在绵羊血培养基上划线的鸡杆菌,放于不同温度条件下培养,发现鸡杆菌在31~40℃生长良好,且在此范围内,温度越低溶血性越明显。此外,各个菌株对温度和PH敏感度存在差异。生化试验结果表明,本研究分离的鸡杆菌的某些生化特性上与国外报道的存在差异。药敏实验结果表明,大多数鸡杆菌分离株对庆大霉素、链霉素、氨苄西林、青霉素、强力霉素、诺氟沙星及磺胺类药物等具有不同程度的耐药性,耐药率在44%~96.9%之间,给该菌的控制带来了难度。
5.首次人工复制出鸡杆菌所致的SPF鸡病例。用从输卵管囊肿自然病例中分离的鸡杆菌、大肠杆菌分别或混合对112日龄开产前和164日龄、184日龄开产后SPF蛋鸡进行人工感染。对开产前的SPF蛋鸡人工感染结果显示:单一鸡杆菌感染组和鸡杆菌-大肠杆菌混合感染组与对照组相比,平均开产日龄分别推迟7 d和13 d;攻毒后50 d内,产蛋量分别下降了69.7%和87.8%,劣质蛋分别增加了70%和75%。单一大肠杆菌感染组产蛋量和产蛋质量与对照组相比没有明显差异。对开产后的SPF蛋鸡感染结果显示:大肠杆菌感染组和鸡杆菌-大肠杆菌混合感染组均出现死亡,而鸡杆菌感染组没有肉眼可见临床症状,只是与对照组相比,产蛋量下降了33%,劣质蛋增加了23%。112日龄鸡于感染后50 d内仍可带菌排菌。112日龄鸡于感染后50 d内、164日龄和184日龄鸡于感染后16 d内,鸡只出现输卵管的充血等炎症,但均未出现输卵管囊肿。证实了鸡杆菌具有致病性,主要影响鸡的开产日龄、产蛋量和产蛋质量,只是在短期内,对鸡体造成的病理损伤不明显。并且鸡杆菌与大肠杆菌混合感染时,致病性更强。同居感染试验证实鸡杆菌2 d即可感染整群,但是从感染鸡杆菌的SPF鸡所产的42枚鸡蛋中未检测到鸡杆菌,表明鸡杆菌的主要传播途径是水平传播。
In 2003, the genus Gallibacterium was established within the family of Pasteurellaceae by Christensen et al. based on 16S rRNA gene sequences. In 2009, Bisgaard resported that the genus Gallibacterium currently includes four species: Gallibacterium anatis, Actinobacillus salpingitidis, Gallibacterium melopsittaci and Gallibacterium trehalosifermentans. And the Gallibacterium anatis is the representative species. Two biovars are described within Gallibacterium anatis, a haemolytic biovar haemolytica and a non-haemolytic, biovar anatis. Now a little research on the Gallibacterium anatis was resported in domestic. To further understand the infection of Gallibacterium anatis in parts of farms in China, thirty flocks from Henan, Shanxi, Shandong provinces were investigated for the occurrence of Gallibacterium anatis, in which fifteen layer flocks with sapingitis, two SPF flocks, six healthy layer flocks, five rooster flocks and two breeder roosters were included. Meantime, in order to carry out the molecular epidemiology, molecular genetics and pathogenesis of Gallibacterium anatis, the partial characteristic of Gallibacterium anatis isolates were studied, including cultural characteristics, biochemical properties, drug resistance and pathogenicity. The results were as follows:
1. One hundred and seven layers from fifteen sapingitis flocks in Henan and Shanxi provinces were submitted for necropsy and the following organs were examined for bacteria: choana, trachea, lung, heart, liver, spleen, ovary, oviduct, duodenum and cloaca. Seventy-six Gallibacterium anatis strains and one hundred and eighty E.coli strains were isolated by morphological observation, differential cultivation, PCR and sequence analysis. And the infection rate of Gallibacterium anatis in the sapingitis flocks was up to 42.9%, indicating that Gallibacterium anatis was prevalence in the sapingitis flocks of Henan and Shanxi provinces. Among the one hundred and seven layers with sapingitis, eleven layers were infected Gallibacterium anatis singly, which illustrated that Gallibacterium anatis participated in pathological processes of salpingitis. Among the forty-five layers infected Gallibacterium anatis, thirty-five layers were infected E.coli simultaneously. Thus, the exact role of Gallibacterium anatis played in salpingitis, primarily, secondary or opportunist needed to be proved by subsequent researches.
2. By morphological observation, differential cultivation, PCR, ten Gallibacterium anatis strains were isolated from ten tissues of thirty-three clinically healthy layers from six different farms in Henan and Shanxi provinces. No Gallibacterium anatis was isolated from thirty-eight SPF chickens in Shandong province. And the infection rate of Gallibacterium anatis in clinically healthy layers was up to 27.3%, indicating the existence of clinically healthy layer flocks. But the rate reduced by 15.6% compared with salpingitis fowls, which indicated that chicken salpingitis was an important clinical feature of Gallibacterium anatis.
3. Twenty roosters from five different farms and nine breeders from another two farms in Henan province were submitted for necropsy and the following organs were examined for bacteria: choana, trachea, lung, heart, liver, spleen, duodenum and cloaca. By morphological observation, differential cultivation, PCR, no Gallibacterium anatis was isolated from tewnty roosters. However, eight Gallibacterium anatis strains were isolated from nine breeders, showing that the breeders chould infect Gallibacterium anatis. One of the eight Gallibacterium anatis isloations wasγ-hemolytic on sheep blood agar plates, which may be useful to the pathogenic study of Gallibacterium anatis.
4. Sixty-four Gallibacterium anatis isolates from different flocks were selected to study the cultural characteristics, biochemical properties, drug resistance. The results showed Gallibacterium anatis on sheep/ chicken/duck blood agar plates growed well. However, no growth on SS agar plates. After the culture of Gallibacterium anatis in different PH value of LB broth, the optimal PH value range for growth was determined at 7.0～8.0. The culture of Gallibacterium anatis on sheep blood agar plates in different temperatures indicated that Gallibacterium anatis growed well at 31～40℃, and in this range, the lower the temperature the more obvious hemolytic. In addition, each strain differences in sensitivity to temperature and PH. Known the cultural characteristics was useful to improve the rate of isolation and separation efficiency. Biochemical test results showed that Gallibacterium anatis isolates were different in some biochemical characteristics. The Gallibacterium anatis isolates had different levels of drug resistance on gentamicin, streptomycin, ampicillin, penicillin, doxycycline, norfloxacin, and sulfa drugs, and the resistance rate ranged from 44% to 96.9%, which had brought difficulty to the control of Gallibacterium anatis.
5. It is the first successful reproduction of Gallibacterium anatis infection in SPF layer. SPF 112-day-old, 164-day-old and 184-day-old hens were experimentally infected with Gallibacterium anatis and /or Escherichia coli which were isolated from the hens with salpingitis. Results showed that, compared to uninfected control group, mean age at first egg of 112-day-old hens in Gallibacterium anatis and Gallibacterium anatis/ Escherichia coli infection group were delayed 7 and 13 days; egg production in 50 days after infection were decreased by 69.7% and 87.8%; and the rate of poor-quality eggs were increased by 70% and 75% respectively. However, there were no significant differences in egg production and clinical situation between hens infected with E.coli only and uninfected control. SPF 164-day-old hens infected with E.coli and Gallibacterium anatis/E.coli were dead, while the hens infected with Gallibacterium anatis only were healthy clinically, except for the egg production was dropped by 33%, the rate of the poor-quality eggs was increased by 23% compared to the control group. The Gallibacterium anatis could be detected and shed from the 112-day-old hens until 50 days after infection and from the 164-day-old and 184-day-old hens until 16 days after infection respectively. Salpingitis appeared in SPF hens infected with Gallibacterium anatis during 16 to 50 days experimental period. However, we didn’t observe typical oviduct cysts. The results confirmed the pathogenicity of Gallibacterium anatis. The infection of Gallibacterium anatis had serious impacts on the age at first egg, egg production and egg quality. While the pathology damage was not obviously in short time. But E.coli infection could enhance the pathogenicity of Gallibacterium anatis. The cohabitation infection experiment of 184-day-old hens confirmed that Gallibacterium anatis could be transmitted laterally, and the whole flock got infected in two days. But no Gallibacterium anatis were isolated from the eggs layed from the SPF hens infected with Gallibacterium anatis, which indicated that Gallibacterium anatis was spread from flock to flock mainly by horizontal transmission through infected birds instead of vertical transmission by eggs.
1.对107只来自河南、山西的15个鸡群的输卵管炎患病鸡的10种组织脏器(心脏、肝脏、脾脏、肺脏、气管、十二指肠、输卵管、卵巢、泄殖腔、上腭裂)进行鸡杆菌的分离鉴定。通过形态学检查、鉴别培养、特异性PCR鉴定和16SrRNA基因序列测定等方法,最终分离鉴定到鸡杆菌76株,同时得到大肠杆菌180株。鸡杆菌的感染率达42.9%。说明河南、山西两地输卵管炎患病鸡群中普遍存在鸡杆菌的感染。其中,有11只具有典型临床及病理变化的鸡仅分离出鸡杆菌一种细菌,这说明鸡杆菌具有一定的致病性,可能在鸡的输卵管炎中扮演一定的角色。但是感染鸡杆菌的46只鸡中,有35只鸡同时感染大肠杆菌(占鸡杆菌阳性鸡的76.1%),而在临床上,大肠杆菌也是引起鸡输卵管炎的重要病原菌之一,因此,鸡杆菌在鸡输卵管炎中扮演什么样的角色,到底是继发、原发或是协同仍需进一步的研究证实。
2.通过形态学检查、鉴别培养、特异性PCR鉴定等方法,对来自河南、山东、山西的2个SPF鸡群的38只SPF鸡、6个临床健康鸡群的33只健康鸡进行了鸡杆菌的分离鉴定。从38只SPF鸡未分离出鸡杆菌,从临床健康鸡群中分离出鸡杆菌10株;临床健康鸡群中鸡杆菌的感染率达27.3%,说明临床健康蛋鸡群存在鸡杆菌的感染,但是与输卵管炎患病鸡群相比,鸡杆菌的感染率降低了15.6%,说明输卵管炎是鸡杆菌感染的重要临床特征之一。
3.对来自河南省5个商品公鸡群的20只商品公鸡和两个种鸡群的9只商品种公鸡的8种组织脏器(心脏、肝脏、脾脏、肺脏、气管、十二指肠、泄殖腔、上腭裂)进行鸡杆菌的分离鉴定。从20只商品公鸡中未分出鸡杆菌,从9只商品种公鸡中分离出鸡杆菌8株。这表明,鸡杆菌可以感染种公鸡。8只鸡杆菌分离株中,其中1株不具有溶血性,可能会对鸡杆菌致病性的研究有一定价值。
4.选择不同区域、不同宿主脏器的鸡杆菌分离株64株,进行培养特性、生化特性、药物敏感性研究。鸡杆菌在绵羊血、鸡血、鸭血琼脂平板上均生长良好;但是,在SS琼脂平板均不生长。通过在不同PH值的LB液体培养基中的培养,确定了鸡杆菌最适生长PH值范围是7.0~8.0。将在绵羊血培养基上划线的鸡杆菌,放于不同温度条件下培养,发现鸡杆菌在31~40℃生长良好,且在此范围内,温度越低溶血性越明显。此外,各个菌株对温度和PH敏感度存在差异。生化试验结果表明,本研究分离的鸡杆菌的某些生化特性上与国外报道的存在差异。药敏实验结果表明,大多数鸡杆菌分离株对庆大霉素、链霉素、氨苄西林、青霉素、强力霉素、诺氟沙星及磺胺类药物等具有不同程度的耐药性,耐药率在44%~96.9%之间,给该菌的控制带来了难度。
5.首次人工复制出鸡杆菌所致的SPF鸡病例。用从输卵管囊肿自然病例中分离的鸡杆菌、大肠杆菌分别或混合对112日龄开产前和164日龄、184日龄开产后SPF蛋鸡进行人工感染。对开产前的SPF蛋鸡人工感染结果显示:单一鸡杆菌感染组和鸡杆菌-大肠杆菌混合感染组与对照组相比,平均开产日龄分别推迟7 d和13 d;攻毒后50 d内,产蛋量分别下降了69.7%和87.8%,劣质蛋分别增加了70%和75%。单一大肠杆菌感染组产蛋量和产蛋质量与对照组相比没有明显差异。对开产后的SPF蛋鸡感染结果显示:大肠杆菌感染组和鸡杆菌-大肠杆菌混合感染组均出现死亡,而鸡杆菌感染组没有肉眼可见临床症状,只是与对照组相比,产蛋量下降了33%,劣质蛋增加了23%。112日龄鸡于感染后50 d内仍可带菌排菌。112日龄鸡于感染后50 d内、164日龄和184日龄鸡于感染后16 d内,鸡只出现输卵管的充血等炎症,但均未出现输卵管囊肿。证实了鸡杆菌具有致病性,主要影响鸡的开产日龄、产蛋量和产蛋质量,只是在短期内,对鸡体造成的病理损伤不明显。并且鸡杆菌与大肠杆菌混合感染时,致病性更强。同居感染试验证实鸡杆菌2 d即可感染整群,但是从感染鸡杆菌的SPF鸡所产的42枚鸡蛋中未检测到鸡杆菌,表明鸡杆菌的主要传播途径是水平传播。
In 2003, the genus Gallibacterium was established within the family of Pasteurellaceae by Christensen et al. based on 16S rRNA gene sequences. In 2009, Bisgaard resported that the genus Gallibacterium currently includes four species: Gallibacterium anatis, Actinobacillus salpingitidis, Gallibacterium melopsittaci and Gallibacterium trehalosifermentans. And the Gallibacterium anatis is the representative species. Two biovars are described within Gallibacterium anatis, a haemolytic biovar haemolytica and a non-haemolytic, biovar anatis. Now a little research on the Gallibacterium anatis was resported in domestic. To further understand the infection of Gallibacterium anatis in parts of farms in China, thirty flocks from Henan, Shanxi, Shandong provinces were investigated for the occurrence of Gallibacterium anatis, in which fifteen layer flocks with sapingitis, two SPF flocks, six healthy layer flocks, five rooster flocks and two breeder roosters were included. Meantime, in order to carry out the molecular epidemiology, molecular genetics and pathogenesis of Gallibacterium anatis, the partial characteristic of Gallibacterium anatis isolates were studied, including cultural characteristics, biochemical properties, drug resistance and pathogenicity. The results were as follows:
1. One hundred and seven layers from fifteen sapingitis flocks in Henan and Shanxi provinces were submitted for necropsy and the following organs were examined for bacteria: choana, trachea, lung, heart, liver, spleen, ovary, oviduct, duodenum and cloaca. Seventy-six Gallibacterium anatis strains and one hundred and eighty E.coli strains were isolated by morphological observation, differential cultivation, PCR and sequence analysis. And the infection rate of Gallibacterium anatis in the sapingitis flocks was up to 42.9%, indicating that Gallibacterium anatis was prevalence in the sapingitis flocks of Henan and Shanxi provinces. Among the one hundred and seven layers with sapingitis, eleven layers were infected Gallibacterium anatis singly, which illustrated that Gallibacterium anatis participated in pathological processes of salpingitis. Among the forty-five layers infected Gallibacterium anatis, thirty-five layers were infected E.coli simultaneously. Thus, the exact role of Gallibacterium anatis played in salpingitis, primarily, secondary or opportunist needed to be proved by subsequent researches.
2. By morphological observation, differential cultivation, PCR, ten Gallibacterium anatis strains were isolated from ten tissues of thirty-three clinically healthy layers from six different farms in Henan and Shanxi provinces. No Gallibacterium anatis was isolated from thirty-eight SPF chickens in Shandong province. And the infection rate of Gallibacterium anatis in clinically healthy layers was up to 27.3%, indicating the existence of clinically healthy layer flocks. But the rate reduced by 15.6% compared with salpingitis fowls, which indicated that chicken salpingitis was an important clinical feature of Gallibacterium anatis.
3. Twenty roosters from five different farms and nine breeders from another two farms in Henan province were submitted for necropsy and the following organs were examined for bacteria: choana, trachea, lung, heart, liver, spleen, duodenum and cloaca. By morphological observation, differential cultivation, PCR, no Gallibacterium anatis was isolated from tewnty roosters. However, eight Gallibacterium anatis strains were isolated from nine breeders, showing that the breeders chould infect Gallibacterium anatis. One of the eight Gallibacterium anatis isloations wasγ-hemolytic on sheep blood agar plates, which may be useful to the pathogenic study of Gallibacterium anatis.
4. Sixty-four Gallibacterium anatis isolates from different flocks were selected to study the cultural characteristics, biochemical properties, drug resistance. The results showed Gallibacterium anatis on sheep/ chicken/duck blood agar plates growed well. However, no growth on SS agar plates. After the culture of Gallibacterium anatis in different PH value of LB broth, the optimal PH value range for growth was determined at 7.0～8.0. The culture of Gallibacterium anatis on sheep blood agar plates in different temperatures indicated that Gallibacterium anatis growed well at 31～40℃, and in this range, the lower the temperature the more obvious hemolytic. In addition, each strain differences in sensitivity to temperature and PH. Known the cultural characteristics was useful to improve the rate of isolation and separation efficiency. Biochemical test results showed that Gallibacterium anatis isolates were different in some biochemical characteristics. The Gallibacterium anatis isolates had different levels of drug resistance on gentamicin, streptomycin, ampicillin, penicillin, doxycycline, norfloxacin, and sulfa drugs, and the resistance rate ranged from 44% to 96.9%, which had brought difficulty to the control of Gallibacterium anatis.
5. It is the first successful reproduction of Gallibacterium anatis infection in SPF layer. SPF 112-day-old, 164-day-old and 184-day-old hens were experimentally infected with Gallibacterium anatis and /or Escherichia coli which were isolated from the hens with salpingitis. Results showed that, compared to uninfected control group, mean age at first egg of 112-day-old hens in Gallibacterium anatis and Gallibacterium anatis/ Escherichia coli infection group were delayed 7 and 13 days; egg production in 50 days after infection were decreased by 69.7% and 87.8%; and the rate of poor-quality eggs were increased by 70% and 75% respectively. However, there were no significant differences in egg production and clinical situation between hens infected with E.coli only and uninfected control. SPF 164-day-old hens infected with E.coli and Gallibacterium anatis/E.coli were dead, while the hens infected with Gallibacterium anatis only were healthy clinically, except for the egg production was dropped by 33%, the rate of the poor-quality eggs was increased by 23% compared to the control group. The Gallibacterium anatis could be detected and shed from the 112-day-old hens until 50 days after infection and from the 164-day-old and 184-day-old hens until 16 days after infection respectively. Salpingitis appeared in SPF hens infected with Gallibacterium anatis during 16 to 50 days experimental period. However, we didn’t observe typical oviduct cysts. The results confirmed the pathogenicity of Gallibacterium anatis. The infection of Gallibacterium anatis had serious impacts on the age at first egg, egg production and egg quality. While the pathology damage was not obviously in short time. But E.coli infection could enhance the pathogenicity of Gallibacterium anatis. The cohabitation infection experiment of 184-day-old hens confirmed that Gallibacterium anatis could be transmitted laterally, and the whole flock got infected in two days. But no Gallibacterium anatis were isolated from the eggs layed from the SPF hens infected with Gallibacterium anatis, which indicated that Gallibacterium anatis was spread from flock to flock mainly by horizontal transmission through infected birds instead of vertical transmission by eggs.
引文
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