鸡源大肠杆菌中HPI和LEE毒力岛相关基因的检测

采用PCR方法检测辽宁锦州地区分离的150株鸡源大肠杆菌中耶尔森菌强毒力岛基因(HPI)和肠细胞脱落位点毒力岛(LEE),利用多重PCR方法检测HPI irp2和fyuA基因,以及LEE ler和eaeA基因.在分离的鸡源大肠杆菌中,HPI毒力岛基因检测结果为:18.7％的菌株irp2和fyuA基因扩增阳性,6.7％的菌株irp2基因阳性;LEE毒力岛基因检测结果为:15.3％的菌株ler和eaeA基因扩增阳性.结果表明,25.3％的鸡源大肠杆菌携带HPI,15.3％的鸡源大肠杆菌携带LEE.     

禽源性大肠杆菌HPI毒力岛irp2基因PCR检测方法的建立与应用

为了解临床禽源致病性大肠杆菌中高致病性毒力岛(High pathogenicity island HPI)流行情况和基因特征,根据Gen Bank已知禽源irp2基因序列,设计、合成一对特异性引物,建立irp2基因PCR检测方法,优化、确定PCR扩增特性,进行敏感性、特异性、重复性检测评价。对256份临床分离禽源大肠杆菌进行irp2基因PCR检测和基因遗传变异分析。结果显示,建立的PCR检测方法敏感性可达2.14×10~(-4)ng/μL;特异性显示与鸡沙门菌、副鸡嗜血杆菌、鸭疫里默菌、禽巴氏杆菌、鸡毒支原体、鸡新城疫病毒、禽流感病毒(H9N5)核酸均无交叉反应;重复性显示3份阳性样品重复5次检测,变异系数3.4%~5.0%。256份临床样品PCR检测irp2基因,阳性率30.6%。获得了9株分离株irp2基因序列,分析显示,与GenBank已知基因同源性高达96.2%以上。说明建立的禽源大肠杆菌HPI毒力岛irp2基因PCR检测方法具有良好的特异性、敏感性和重复性,可应用于临床致病性大肠杆菌毒力岛基因快速检测。     

东北地区鸡源大肠杆菌的毒力基因检测及PFGE分型研究

为了研究东北地区健康鸡源大肠杆菌毒力基因的携带分布以及PFGE分子分型情况,试验采用PCR技术对413株鸡源大肠杆菌的4种毒力基因进行检测,并运用XbaⅠ酶进行酶切后完成PFGE分析,利用软件分析菌株间的相关性和遗传关系。结果表明,在检测的413株菌株中,fimH毒力基因携带率为77.72%,iucD毒力基因的检出率为56.42%,强致病性毒力岛(HPI)的标志基因fyuA和irp2毒力基因携带率分别为44.07%和43.83%;29株大肠杆菌呈现出28种不同的PFGE型,每一株菌被XbaⅠ消化为14~20条带,菌株相似度为30%~100%;多数菌株携带毒力基因,且毒力基因的类型较为复杂,PFGE分型结果具有多样性和差异性。提示应加强鸡源大肠杆菌毒力基因的检测以及分子分型研究,为大肠杆菌病等防控提供参考。     

105株携带耶尔森菌强毒力岛大肠杆菌的毒力基因型和部分菌株毒力因子序列研究

为了解105株携带耶尔森菌强毒力岛(HPI)的大肠杆菌(E.coli)中相关毒力因子的流行情况和基因序列,根据GenBank中参考序列设计引物,采用PCR方法对广东地区养殖场来源的105分离株HPI+E.coli的fyuA、tsh、iucD、iss 4种毒力基因进行检测,统计基因类型;并对部分分离株的5种毒力基因(irp2和fuA、tsh、iucD、iss)进行了克隆与序列分析.结果显示105株HPI+E coli中4种毒力因子携带情况不尽一致,基因fyuA、tsh、iucD和iss的阳性率分别为55.24％、17.14％、49.52％和23.81％,105株HPI+E.coli共有13种基因型;分析表明,除iss基因与参考序列的同源性在88.0 ％～90.9％外,irp2、fyuA、tsh、iucD4种基因与GenBank中参考序列的同源性高达96％以上;广东省养殖场E.coli毒力因子基因型复杂,并以基因型irp2+ fyuA+ iucD+和仅含irp2+的菌株分离率最高,分别为17.14％和28％.     

禽致病性大肠杆菌中耶尔森菌强毒力岛的分子流行病学调查

为了解耶尔森菌强毒力岛（HPI）在禽致病性大肠杆菌（APEC）中的流行情况，根据HPI结构基因irp2和fyuA参考序列设计了引物，用PCR方法和斑点杂交法对从江苏等地分离的APEC基因组进行了扩增和检测，并对E．coli NTJC040406菌株相关基因进行了克隆和序列分析。结果表明，216株APEC中有44．9％的菌株携带有HPI，序列分析表明相关基因与GenBank中参考序列的同源性高达98％以上。提示HPI在APEC中广泛存在，经进一步分析，发现分离菌株是否携带HPI与O78等特定血清型有一定的相关性。     

鸭源致病性大肠杆菌的血清型鉴定及其相关毒力基因分析

自规模化养鸭场患典型大肠杆菌败血症雏鸭分离的282株致病性大肠杆菌（E.coli）中鉴定出210株（包含37种血清型）,其中O93、O78、O92、O76占43.8%（92/210）为优势血清型,O46、O32＆O93混合型、O60＆O93混合型为首次从鸭群中分离到。应用PCR结合核酸序列测定对210株致病性E.coli（鸭大肠杆菌病分离株）和28株自健康雏鸭泄殖腔拭子分离的E.coli（临床健康鸭大肠杆菌分离株）进行包括强毒力岛（HPI）中的鼠疫菌素受体基因（fyuA）和铁调节蛋白基因（irp2）、Ⅰ型菌毛必需蛋白基因（fimC）、P型菌毛结构基因（papA）和血清耐受基因（iss）检测,结果表明：fyuAi、rp2、fimC、papA和iss基因在鸭大肠杆菌病分离株的携带率分别为41.0%、43.3%、92.9%、97.6%和96.7%,在临床健康鸭大肠杆菌分离株的携带率分别为21.4%、25.0%、92.9%、100%和92.9%,患病鸭和临床健康鸭大肠杆菌分离株iss、fimC和papA携带率差异不显著（P〉0.05）,但papA的携带率均显著高于其他宿主（鸡、猪和人）源E.coli;HPI毒力岛在鸭源E.coli中分布较广,其携带率表现为鸭大肠杆菌病分离株极显著高于临床健康鸭大肠杆菌分离株。HPI毒力岛的携带率与菌株的致病性呈明显的正相关,与O78等特定的血清型有一定的关系。鸭大肠杆菌病分离株有37.6%（79/210）同时携带fyuAi、rp2、fimCi、ss和papA基因,极显著高于健康鸭大肠杆菌分离株的14.3%（4/28）（P〈0.01）。     

犊牛腹泻源大肠杆菌耐药情况及HPI相关基因的检测

本研究旨在明确犊牛腹泻源性大肠杆菌的耐药情况、强毒力岛(HPI)标志基因及相关基因的携带情况,以及大肠杆菌分离株与HPI携带的关系。采集犊牛病理性腹泻样本158份,采用麦康凯培养基和伊红美蓝培养基进行筛选,镜检符合大肠杆菌形态的进行VITEK 2 Compact生化鉴定和PCR鉴定,采用K-B纸片法对大肠杆菌分离株进行药敏试验,应用PCR方法进行分离株HPI相关基因携带情况的检测。结果显示,共分离得到75株大肠杆菌,分离株对阿莫西林、哌拉西林、氨苄西林、头孢唑啉、氟苯尼考、恩诺沙星、四环素的耐药率均90%,对头孢氨苄、头孢拉定、头孢曲松、环丙沙星、诺氟沙星和氧氟沙星的耐药率均≥60.00%,对阿米卡星较敏感,耐药率为9.33%;75株大肠杆菌全部耐3种以上药物,多重耐药(≥10)的菌株占85.33%,耐药谱集中在耐14～17种药物,5株对19种药物全部耐药。HPI标志基因irp2的阳性率为100%,其他相关基因fyuA、irp3、irp5、irp8和ytbA的检出率在66.00%以上。综上所述,宁夏地区犊牛腹泻源性大肠杆菌耐药普遍,多重耐药现象严重。     

狐狸源大肠杆菌毒力岛基因HPI、LEE的检测

为检测从狐狸体内分离的大肠杆菌的毒力岛基因和毒力,对从病死狐狸的肝脏、肺脏等器官分离的细菌进行了生化鉴定、毒力试验和毒力岛HPI(irp2、fyuA)与LEE(ler、eaeA)检测。结果表明:在所测的6株菌中有4株检测出HPI毒力岛基因,6株菌均未检测出LEE毒力岛基因,各注射菌液组小鼠均不同程度发病,且含有HPI毒力岛基因的菌株对小鼠的毒力较强,可见分离到的菌株对狐狸的健康养殖存在较大的危害。     

云南撒坝猪源大肠杆菌优势血清型强毒力岛的检测及其致病性

为研究撒坝猪大肠杆菌强毒力岛(HPI)的致病性,对撒坝猪致病性大肠杆菌进行了分离和血清型鉴定,获取优势血清型,采用PCR检测HPI,进行同源性比较,并通过耐药性试验和致病性试验对分离菌株与HPI的相关性进行分析。结果显示:分离得到101株大肠杆菌,确定血清型84株,其中O_3、O_4、O_(24)为优势血清型;HPI阳性率为88.64%,与GenBank登录的参考序列同源性比较大于99.3%;氨基糖苷类药物耐药性试验结果显示分离菌株普遍耐药,而HPI阳性株耐药性强,并检出含aac(3)-Ⅱa基因12株,含有aac(6′)-Ⅰb基因21株;致病性试验表明含irp2的菌株致病性强,而具有fyuA-irp2基因簇的菌株致病性更强。结果表明:HPI对大肠杆菌的毒力有增强作用。     

貉致病性大肠杆菌分离鉴定及HPI、LEE毒力岛检测

为了解致病性大肠杆菌引起貉腹泻的机理,采用了貉致病性大肠杆菌分离鉴定、检测毒力岛基因HPI(irp2、fyuA)与LEE(ler、eaeA),小鼠腹腔注射检测菌株毒力的方法。结果表明:从腹泻貉体内分离出血清型分别为O_(29)、O_(38)、O_(78)和O_(107)的大肠杆菌,4个血清型中有2个血清型(O_(38)、O_(78))携带有irp2和fyuA,4个血清型均未检测到ler和eaeA;4个血清型的大肠杆菌均可使小鼠不同程度发病,其中以携带irp2和fyuA的菌株毒力较强,可见该菌对貉的健康存在危害。     

Heartworm in dogs in Canada in 1984

In late December 1984, 1853 institutional veterinarians and small and mixed animal clinics across Canada were sent a questionnaire in order to assess the status of Dirofilaria immitis in Canada in 1984 and 35% of them responded. Veterinarians reported that 97,794 dogs were blood-tested to check for microfilariae and 1417 dogs (1.45% of those tested) were found with heartworm. Another 34 dogs were amicrofilaremic, but were diagnosed as having heartworm disease, to give the total number diagnosed in 1984 as 1451 (1.48%). Heartworm was reported from all provinces except Prince Edward Island and Newfoundland but most (1310) of the cases were in Ontario. In Quebec, 126 cases were reported mostly from west of Montreal.

Heartworm was found most frequently in companion dogs over three years of age maintained mainly outdoors in rural areas. About 27% of the cases were observed with clinical signs of heartworm disease and 72% had a history of not having left Canada. Southwestern Ontario continued to be the primary focus of the infection.

     

Heartworm in dogs in Canada in 1988

In late November 1988, 1581 small and mixed animal clinics and institutional veterinarians across Canada were sent a questionnaire in order to assess the status of Dirofilaria immitis in Canada in 1988, and 46% of them responded. Veterinarians reported that 181,577 dogs were blood-tested for heartworm disease and 367 dogs were found with D. immitis microfilariae. Another 60 dogs were amicrofilaremic but diagnosed with heartworm disease to give the total number of cases diagnosed in 1988 as 441 (0.24%).     

Heartworm in dogs in Canada in 1985

In late December 1985, 1485 institutional veterinarians and small and mixed animal clinics across Canada were sent a questionnaire in order to assess the status of Dirofilaria immitis in Canada in 1985 and 44% of them responded. Veterinarians reported that 137,300 dogs were blood-tested to check for microfilariae and 1210 dogs were found with heartworm. Another 36 dogs were amicrofilaremic but diagnosed with heartworm disease to give the total number diagnosed in 1985 as 1247 (0.91%).

Heartworm was reported from all provinces except Prince Edward Island, Newfoundland and Saskatchewan but most (1126) of the cases were in Ontario. Southwestern Ontario continued to be the primary focus of the infection in Canada. From Quebec, 91 cases were reported mostly from and around Montreal. From Manitoba, 19 cases were reported from Winnipeg and surrounding areas. Heartworm was found most frequently in companion dogs over three years of age maintained mainly outdoors in rural areas. About 28% of the cases were observed with clinical signs of heartworm disease and 78% had a history of not having left Canada.

     

Heartworm in dogs in Canada in 1989

In late November 1989, 1732 clinics and institutional veterinarians were sent a questionnaire to assess the status of Dirofilaria immitis, and 51.7% responded. Of 247,716 dogs tested, 394 had D. immitis microfilariae and 51 were amicrofilaremic for a total of 445 cases and heartworm prevalence of 0.17%. Most (408) of these dogs had no preventive medication and the prevalence among dogs tested and unprotected was 1.01%. That prevalence was considerably higher in endemic areas. Thirty-seven dogs with heartworm had preventive medication. Heartworm was most frequent in companion dogs over three years of age maintained outdoors in rural areas. About 75% of the cases had never left Canada, 26% had clinical signs and 125 were not treated.

Heartworm was reported from British Columbia, Manitoba, Ontario, Quebec, Nova Scotia and Newfoundland, but 383 cases were in Ontario. South-western Ontario was the primary focus of infection. There were 33 cases in Quebec and 24 in Manitoba, mainly found in and around Metropolitan Montreal and Winnipeg respectively.

     

Heartworm in dogs in Canada in 1987

In late November 1987, 1246 institutional veterinarians and small and mixed animal clinics across Canada were sent a questionnaire in order to assess the status of Dirofilaria immitis in Canada in 1987, and 50% of them responded. Veterinarians reported that 165,428 dogs were blood tested for heartworm disease and 511 dogs were found with D. immitis microfilariae. Another 78 dogs were amicrofilaremic but diagnosed with heartworm disease to give the total number of cases diagnosed in 1987 as 589 (0.35%).     

Heartworm in dogs in Canada in 1986

In late December 1986, 1224 institutional veterinarians and small and mixed animal clinics across Canada were sent a questionnaire in order to assess the status of Dirofilaria immitis in Canada in 1986; 46% of them responded. Veterinarians reported that 150,989 dogs were blood-tested for microfilariae and 869 dogs were found with heartworm. Another 65 dogs were amicrofilaremic but diagnosed with heartworm disease and one was found with heartworm at necropsy to give the total number diagnosed in 1986 as 935 (0.62%).

Heartworm was reported from Manitoba, New Brunswick, Ontario and Quebec, but most (810) of the cases were from Ontario. South-western Ontario continued to be the primary focus of the infection in Canada. There were 103 cases reported from Quebec, mostly from and around Montreal, and 21 cases from Manitoba, from Winnipeg and surrounding areas. Heartworm was found most frequently in companion dogs over three years of age maintained mainly outdoors in rural areas. About 33% of the cases were observed with clinical signs of heartworm disease and 81% had a history of not having left Canada.

     

Heartworm in dogs in Canada in 1983

In late December 1983, 2 800 veterinarians across Canada were sent a questionnaire in order to assess the status of heartworm disease in Canada in 1983 and 26% of them responded. Veterinarians reported that 59 504 dogs were blood-tested to check for microfilariae and 771 dogs (1.30% of those tested) were found with Dirofilaria immitis. Heartworm disease was diagnosed in all provinces except New Brunswick and Newfoundland but most (733) of the cases were in Ontario.

Heartworm disease was found most frequently in companion dogs over three years of age maintained mainly outdoors in rural areas. About 31% of the cases were observed with clinical signs of heartworm disease and 64% had a history of not having left Canada. Southwestern Ontario continues to be the focus of the infection and most of the dogs there had not left the province previously.