不同品系小鼠肝螺杆菌感染实验模型的建立及病理特征分析

目的 应用肝螺杆菌感染不同品系小鼠,观察肝螺杆菌的定植状况及病理特征,以期获得稳定的动物模型.方法 SPF级雄性BABL/c Cr、SCID/Cr、C57BL/6 Cr小鼠接种肝螺杆菌(H.hepaticus,Hh)标准菌株ATCC51450菌液0.2 ml(1×108 CUF/ml),连续3次,每次间隔48 h,对照组灌饲等量的PBS.于末次接种Hh后第4周、8周及16周处死小鼠,取出小鼠食管、胃、空肠、回肠、盲肠、结肠、肝脏及胰腺组织,行病理组织学检查、微需氧菌分离培养鉴定及Hh特异性16SrRNA基因扩增.结果 BALB/c Cr小鼠和SCID/ Cr小鼠接种Hh后4周、8周及16周盲肠定植率均为8/8,4周、8周及16周结肠定植率分别为(4/8、5/8、5/8)和(3/8、6/8、5/8),16周回肠及空肠定植率均为1/8,8周、16周肝脏定植率分别为(2/8、3/8)和(2/8、2/8);C57BL/6 Cr小鼠接种Hh后4周、8周16周盲肠定植率分别为1/8、2/8和2/8,8周、16周结肠定植率分别为1/8、2/8.Hh感染的BALB/c Cr和SCID/Cr小鼠肝脏、盲肠及结肠炎症改变较C57BL／6 Cr小鼠更为明显(P＜0.01),且随着感染时间的延长病理组织学评分逐渐增加(P值分别＜0.05和0.01),其中结肠及肝脏组织中Hh定植者的组织学评分明显高于无Hh定植者(P＜0.05),盲肠组织的病理组织学评分随着Hh定植密度的增加而增加(P＜0.05).结论 不同品系小鼠对Hh的易感性不同；BALB/cCr、SCID/Cr小鼠接种Hh后可获得较好的Hh感染模型.     

一种检测肝螺杆菌套式PCR方法的建立及临床应用

目的:建立一种敏感性高、特异性强的检测肝螺杆菌(Helicobacter hepaticus,H.hepaticus)的套式PCR方法并对其临床应用进行探讨.方法:本研究基于9种H.hepaticus亚种的16S rRNA基因设计筛选出1套套式引物(2条内引物、2条外引物),优化反应条件后,通过模拟粪便和胆汁标本、相关细菌、动物模型标本和临床标本的检测对该套式PCR方法的敏感性、特异性进行评估.结果:在模拟粪便和胆汁标本中,该方法检测H.hepaticus的最低检出浓度达到102C F U/m L.幽门螺杆菌、胆螺杆菌、空肠弯曲菌、大肠埃希菌、粪肠球菌、铜绿假单胞菌、金黄色葡萄球菌检测结果均为阴性.本研究建立了3例感染肝螺杆菌的SPF级Balb/c小鼠模型.该方法在3例模型的粪便和盲肠标本及2例模型的肝脏标本中检测出H.hepaticus.10例胆石病患者中,该法在2例患者的胆囊胆汁、胆囊黏膜和粪便标本中检测出有H.hepaticus临床标本的检测中,PCR检测方法、医疗干预和标本的类型及处理可能都会影响检测的结果.结论:本研究建立的套式P C R方法,敏感性高、特异性强,可以用于小鼠和人的H.hepaticus感染.H.hepaticus感染可能和人胆石病有关.PCR检测方法、医疗干预和标本的类型及处理可能都会影响临床标本的检测结果.     

实验感染昆明鼠粪便微小隐孢子虫18SrRNA基因检测

目的 探讨隐孢子虫感染的基因检测方法。方法 用分离自人和牛的微小隐孢子虫各1株分别感染2组昆明小鼠,于接种后第6d、10d和14d分别收集每只动物粪便,用显微镜检查,再用Sheather's蔗糖梯度密度离心法纯化卵囊,提取总DNA,对每份DNA样本做10~0～10~(-3)稀释,然后用PCR对微小隐孢子虫18SrRNA基因进行特异性扩增。结果 接受具有活力的1×10~4个卵囊/只的36只小鼠,均获得感染;阳性样本被扩增出1条500hp的片段。隐孢子虫感染小鼠粪便样本各稀释度的PCR检测实验结果表明,PCR检测阳性率随模板DNA样本稀释倍数的增加而增高;小鼠感染后第6d、10d和14d的10~0和10~(-1)两个稀释度的PCR检测阳性率低于镜检结果,而10~(-2)和10~(-3)两个稀释度均高于镜检结果。结论 受染昆明鼠粪便内的微小隐孢子虫可通过扩增其18SrRNA基因检出。     

一种检测胆螺杆菌套式PCR方法的建立

目的 建立一种敏感性高、特异性强的检测胆螺杆菌的套式PCR方法。方法 基于17种胆螺杆菌亚种的16S rRNA 基因设计筛选出1套套式引物(2条内引物、2条外引物),优化反应条件后,通过模拟粪便标本、动物模型标本和临床标本的检测对方法的敏感性和特异性进行评估。结果 模拟粪便标本中,该方法 检测胆螺杆菌的敏感性达到10 CFU/100 μL。3例感染胆螺杆菌的SPF级BALB/c小鼠模型中,3例小鼠粪便和盲肠中均可检测到胆螺杆菌,1例肝脏标本中检测到了胆螺杆菌。10例胆石病患者中,有2例患者的胆汁、胆囊粘膜和粪便标本中检测到了胆螺杆菌。结论 本研究建立的套式PCR方法 ,敏感性高、特异性强,可用于检测胆螺杆菌的感染。     

幽门螺杆菌诊断方法准确性的影响因素

常用的幽门螺杆菌(H.pylori)诊断方法包括侵入性试验,如快速尿素酶试验(RUT)、组织学检测、培养等和非侵入性试验,如血清学试验、13C和14C,尿素呼气试验(UBT)、聚合酶链反应(PCR)等,各种方法的敏感性和特异性不一。其中组织学检测和培养被作为诊断H.pylori感染的金标准,但也存在一定的缺点。近年来一些新的检测手段不断问世,如H.pylori粪便抗原(HpSA)试验和粪便标本的PCR等。随     

Helicobacter suis感染小鼠模型的建立及其意义

目的 建立Helicobacter suis(H. suis)感染小鼠模型并探究其意义。方法 将40只雌性C57BL/6小鼠随机分为模型组和对照组,模型组给予0.5 mL猪胃黏膜匀浆液(经PCR检测确定存在H. suis)灌胃,对照组给予0.5 mL PBS溶液灌胃。灌胃1个月及3个月后,各处死两组中半数小鼠,取其胃黏膜组织,分别行PCR、HE染色检测H. suis在小鼠胃内的定植与胃黏膜淋巴滤泡形成情况。结果 灌胃所用猪胃黏膜匀浆液中存在H. suis;H. suis可以在小鼠胃内定植,并且3个月后仍存在;感染H. suis 1个月及3个月后的小鼠胃黏膜均有淋巴滤泡的形成,并且后者的淋巴滤泡较前者明显增大。结论 成功建立了H. suis感染C57BL/6小鼠模型,H. suis可以诱导胃黏膜淋巴滤泡的形成,可能在胃黏膜相关淋巴组织(MALT)淋巴瘤的发生过程中发挥重要作用。     

海尔曼螺杆菌感染小鼠的病理学研究

目的 建立海尔曼螺杆菌(Hh)感染小鼠模型,以了解Hh感染小鼠的病理学特点。方法 50只Ⅱ级Balb／c小鼠随机分为实验组(30只),实验组小鼠采用由Hh感染患者转归的小鼠胃粘膜组织磨碎后直接灌喂方法建立Hh感染小鼠模型,而对照组不作相应处理。于灌喂后第1、2、4、8、及16周分批处死小鼠,每批处死实验组小鼠6只、对照组小鼠4只。取胃粘膜组织分别进行细菌培养、涂片Gram染色、快速尿素酶试验(RUT)、组织切片Warthin-Starry银染、组织病理学检查。结果 Hh容易在Balb／c小鼠胃内定植,成功率达到100％。在感染第4周始,部分Hh感染小鼠胃粘膜可见少量淋巴细胞、中性粒细胞和嗜酸性粒细胞浸润,至感染第16周,全部感染小鼠胃粘膜均可见不同程度的淋巴细胞、浆细胞浸润,部分小鼠伴有中性粒细胞浸润,而对照组小鼠未见有明显的炎细胞浸润。结论 Hh可长期稳定定植于小鼠胃内,并可引起慢性活动性胃炎,可用于Hh的致病性研究。     

幽门螺杆菌致肝脏病变的2年动物实验观察

目的 探讨幽门螺杆菌(H.pylori)经口接种长期感染后,是否可到达肝脏并作为独立的致病因素导致小鼠肝脏病变,甚至肝癌的发生.方法 H.pylori悉尼株(SS1)经口接种C57BL/6小鼠,24个月时处死.检测小鼠胃、肝组织内细菌定植情况与病理改变;提取肝组织内细菌DNA,巢式PCR扩增小鼠肝组织H.pylori 16S rRNA基因,PCR产物测序,并与胃黏膜分离的H.pylori、接种H.pylori SS1的16S rRNA PCR产物测序结果进行同源性比较.结果 接种组12只小鼠在胃黏膜检测到H.pylori,11只出现胃组织病理改变,其中1只发生胃淋巴瘤.接种组7只在肝脏检测到H.pylori,6只出现肝脏病理变化,其中1只轻微炎症;3只炎症伴纤维化病变;2只炎症伴纤维化及异型性增生.接种组肝脏H.pylori染色阳性且出现肝脏病理改变小鼠,胃H.pylori染色均呈阳性.接种组肝脏H.pylori染色阴性小鼠和(或)胃H.pylori染色阴性小鼠,以及正常对照组小鼠均未发现肝脏病理改变.肝组织中扩增的H.pylori 16S rRNA PCR产物测序结果经序列分析后显示:与胃黏膜分离培养细菌、接种细菌同源性为100%.结论 经口接种H.pylori 2年长期感染后,除导致小鼠胃组织病变外,部分小鼠胃组织感染细菌可到达肝脏,引起肝脏炎症,甚至肝硬化、癌前病变的发生.     

瘦素在猪螺杆菌感染后胃黏膜相关淋巴样组织形成中的作用

目的研究瘦素在猪螺杆菌(Helicobacter suis,H.suis)感染后胃黏膜相关淋巴样组织(mucosa-associated lymphoid tissue,MALT)形成中的作用及其可能的机制。方法 C57BL/6野生型小鼠、瘦素缺陷(Ob/Ob)小鼠各20只随机分为以下四组:正常小鼠未感染组(WT组)、正常小鼠H.suis感染组(WT+HS组)、Ob/Ob小鼠未感染组(Ob组)、Ob/Ob小鼠H.suis感染组(Ob+HS组),每组10只,感染12周后处死,收集小鼠血清与胃组织。采用HE染色检测各组小鼠胃黏膜淋巴滤泡的数量与大小;酶联免疫吸附测定(ELISA)检测各组小鼠血清中瘦素的浓度;实时荧光定量PCR检测各组小鼠胃黏膜中瘦素及其受体Ob-R、炎症因子IFN-γ及趋化因子CXCL13 mRNA表达水平。结果正常小鼠感染H.suis后可见胃黏膜淋巴滤泡形成;与WT+HS组相比,Ob+HS组小鼠血清及胃黏膜中均未检测到瘦素的表达,胃黏膜中未观察到淋巴滤泡形成,且IFN-γ与CXCL13 mRNA表达水平明显降低;体外实验结果发现,瘦素可刺激淋巴瘤B细胞分泌表达IFN-γ。结论瘦素可能通过促进H.suis感染后胃黏膜IFN-γ的表达参与了胃MALT的形成。     

幽门螺杆菌ureI核酸疫苗免疫活性的初步研究

目的 观察幽门螺杆菌ureI核酸疫苗诱导C57BL/6小鼠产生体液免疫应答及细胞免疫应答水平.方法 重组质粒pcDNA3.1(+)/ureI、空质粒pcDNA3.1(+)及PBS分别肌注免疫C57BL/6小鼠.ELISA法检测小鼠血清中特异性IgG抗体水平以及脾淋巴细胞培养上清中IL-4和IFN-γ含量,MTT比色法检测脾淋巴细胞增殖反应,PCR和免疫荧光组化法检测ureI基因和蛋白表达.结果 pcDNA3.1(+)/ureI能够诱导小鼠产生特异性IgG抗体,该组小鼠脾淋巴细胞经UreI重组蛋白刺激后,其刺激指数和培养上清中IL-4、IFN-γ含量明显升高; ureI基因可在小鼠肌细胞中有效表达.结论 pcDNA3.1(+)/ureI核酸疫苗能刺激C57BL/6小鼠产生较强的体液免疫应答和细胞免疫应答,为进一步研究该疫苗的免疫保护作用奠定了基础.     

肝螺杆菌多重PCR检测方法的建立及应用

目的建立肝螺杆菌的多重PCR检测方法,对中国动物肝螺杆菌进行检测。方法以毒力基因flaB基因、ureA基因和cdtB基因和cdtC基因作为靶基因,建立检测肝螺杆菌的多重PCR方法。对肝螺杆菌、空肠弯曲菌、幽门螺杆菌、沙门氏菌、志贺氏菌、小肠结肠炎耶尔森菌、大肠埃希氏菌和铜绿假单胞杆菌抽提的DNA进行多重PCR扩增。应用本研究建立的多重PCR检测方法对482只动物(其中:海南30只猴、江苏34只小鼠和北京32只猴、30头猪、66只犬、13只兔、29只豚鼠、69只大鼠、213只小鼠)进行检测,对扩增出的阳性结果进行测序。同时,对所有样本采用选择性培养基进行肝螺杆菌培养以作对照。结果肝螺杆菌能扩增出各自的特异性条带,而其他参考菌株均未扩增出条带,这表明该方法具有较强的特异性。482份样本中检出43份肝螺杆菌阳性样本,阳性率8.92%(43/482),其中:2只犬(3.03%,2/66)、1只兔(7.69%,1/13)、5只大鼠(7.25%,5/69)和35只小鼠(16.43%,35/247)均扩增出肝螺杆菌毒力基因片段。结果显示,肝螺杆菌flaB基因、ureA基因和cdtB基因和cdtC基因序列与GenBank中的肝螺杆菌ATCC51449的相应基因序列核苷酸同源性高达99%。用选择性培养基能培养出肝螺杆菌。结论中国动物中的犬、兔、大鼠和小鼠均能检出肝螺杆菌flaB基因、ureA基因、cdtB基因和cdtC基因。建立的多重PCR检测方法可作为肝螺杆菌大规模检测的新技术,这为在中国开展肝螺杆菌流行病学调查提供了有效科学工具。     

慢性乙型肝炎患者肝组织中螺杆菌菌属特异性16SrRNA基因及HP特异性基因分析

目的对慢性乙型肝炎患者肝组织进行螺杆菌菌属特异性16SrRNA基因及幽门螺杆菌（HP）特异性基因检测,探讨螺杆菌在慢性乙型肝炎发生、发展中的作用。方法对56例行肝穿刺活检的慢性乙型肝炎患者的肝组织应用针对螺杆菌菌属特异性16SrRNA基因的通用引物进行基因扩增及微需氧分离培养,并对螺杆菌菌属特异性16SrRNA基因阳性者进一步应用HPcagA、vacA和glmM基因特异引物进行扩增。结果对56例慢性乙型肝炎患者的肝组织DNA应用螺杆菌菌属特异性16SrRNA基因通用引物进行扩增,共有35例患者的肝组织中发现了螺杆菌菌属特异性16SrRNA基因,其中肝硬化组（72.7%）和原发性肝癌组（87.5%）的检出率明显高于慢性肝炎组（42.3%,P〈0.05）,而慢性肝炎组中随着炎症评分的增加,检出率亦增加。对这35例螺杆菌菌属特异性16SrRNA基因阳性的肝组织DNA进一步应用HPcagA、vacA和glmM基因特异性引物进行扩增,结果证实有21例为HPDNA。所有肝组织经微需氧分离培养均未发现可疑菌落生长。结论慢性乙型肝炎患者肝组织中除存在HPDNA外,可能还存在其他螺杆菌DNA。螺杆菌在慢性乙型肝炎向肝硬化和肝癌的发展中可能发挥致病作用。     

Study on the association of Helicobacter species with viral hepatitis-induced hepatocellular carcinoma

Helicobacter species are important pathogens and previous studies in mice suggested a link between colonization by Helicobacter hepaticus (H. hepaticus) and hepatocellular carcinoma (HCC). This study aimed at corroborating this potential link in human patients. We used a sensitive and specific Helicobacter ssp PCR assay to screen stool samples from a collective of patients with viral-induced HCC (hepatitis B or hepatitis C) and a control group for presence of Helicobacter ssp DNA. Although retrieving DNA of H. pylori and H. canadensis from stool samples of non-HCC patients, we found no evidence indicating the presence of H. hepaticus in HCC-patients with chronic hepatitis B or hepatitis C. Interestingly we found H. canadensis in a stool sample of a patient presenting with diarrhea. Taken together, our data argue against a pathogenic role of H. hepaticus in viral-induced HCC. Yet, our results do not exclude a role of H. hepaticus in those HCC cases caused by other carcinogens, such as aflatoxin. Moreover, we speculate that H. canadensis might be a novel gastrointestinal pathogen.     

Identification of Helicobacter species by 16S rDNA PCR and sequence analysis in human liver samples from patients with various etiologies of benign liver diseases

BACKGROUND/AIMS: Several reports indicated an increased prevalence of the Helicobacter species in hepatocellular cancer tissue and in liver samples infected with hepatitis viruses. The frequency of Helicobacter spp. in benign liver diseases was, however, not thoroughly investigated. METHODS: Seventy-five consecutive patients with suspected liver disease were enrolled. The indications were hepatitis B virus (n=30), C virus (n=8), B and C dual infection (n=1), nonalcoholic steatohepatitis (n=27), autoimmune hepatitis (n=3), primary biliary cirrhosis (n=1) and idiopathic elevation of liver enzymes (n=5). PCR detection of 16S recombinant RNA gene of Helicobacter spp. was performed on liver samples. PCR products of positive samples were further identified by DNA sequencing. The patients also had upper gastrointestinal endoscopy and gastric biopsy for the detection of H. pylori using histopathology and PCR. RESULTS: Helicobacter spp. DNA was detected in two out of 75 liver biopsy samples (2.6%), which were typed as H. pylori by DNA sequencing. One of these patients had chronic hepatitis C infection (man, 51 years old) and the other had nonalcoholic steatohepatitis (woman, 44 years old). Fifty-two out of 75 of the patients (69.3%) had H. pylori infection in their stomachs. CONCLUSION: We have found that H. pylori infection is much less prevalent in benign liver diseases. The presence of H. pylori in nonalcoholic steatohepatitis (NASH) patients is a novel finding and this finding should be confirmed in a larger series.     

首次从中国小鼠中分离到肝螺杆菌及其鉴定

目的对中国小鼠肝螺杆菌进行分离鉴定。方法采集中国某实验动物中心发病小鼠标本,采用细菌学分离培养、血清学试验、多重PCR检测、病理组织学检查等方法进行鉴定,并对细菌的分子生物学特征进行分析。结果分离到一株能产生多种毒素的细菌,经鉴定为肝螺杆菌(HHm0801)。用螺杆菌属16SrRNA基因特异引物和肝螺杆菌16SrRNA基因、flaA基因、flaB基因、ureA基因、cdtB基因、cdtC基因特异引物对HHm0801细菌的PCR扩增为阳性,经核苷酸序列测定分析证实上述序列与肝螺杆菌ATCC51449基因序列(GenBank登录号:NC004917)同源性高达99%。肝螺杆菌感染小鼠肝脏的肝细胞肿大,出现大小不等的空泡,部分胞核深染,局部坏死,肝小叶脂肪变性;直肠部分绒毛脱落,有少量炎性细胞,局部充血。脾脏淋巴细胞散在分布,小叶间隔不清晰,脾巨噬细胞增多。眼睛巩膜增厚,晶状体部可见深色物质沉着。结论首次从中国小鼠中分离到了肝螺杆菌,为进一步研究该菌及开展流行病学调查提供了科学依据。     

Identification of cholelithogenic enterohepatic helicobacter species and their role in murine cholesterol gallstone formation

BACKGROUND & AIMS: Helicobacter spp are common inhabitants of the hepatobiliary and gastrointestinal tracts of humans and animals and cause a variety of well-described diseases. Recent epidemiologic results suggest a possible association between enterohepatic Helicobacter spp and cholesterol cholelithiasis, chronic cholecystitis, and gallbladder cancer. To test this, we prospectively investigated the effects of Helicobacter spp infection in cholesterol gallstone pathogenesis in the highly susceptible C57L/J mouse model. METHODS: Helicobacter spp-free adult male C57L mice were infected with several different enterohepatic Helicobacter spp or left uninfected and fed either a lithogenic diet or standard mouse chow for 8 and 18 weeks. At the conclusion of the study, bile was examined microscopically and diagnostic culture and polymerase chain reaction were performed. RESULTS: Mice infected with Helicobacter bilis or coinfected with Helicobacter hepaticus and Helicobacter rodentium and fed a lithogenic diet developed cholesterol gallstones at 80% prevalence by 8 weeks compared with approximately 10% in uninfected controls. Monoinfections with H hepaticus , Helicobacter cinaedi , and H rodentium gave a cholesterol gallstone prevalence of 40%, 30%, and 20%, respectively; the latter 2 groups did not differ significantly from uninfected animals. Neither infected nor uninfected mice fed a chow diet developed cholesterol gallstones. CONCLUSIONS: These findings, along with prior epidemiologic studies, suggest that Helicobacter spp play a major role in the pathophysiology of cholesterol gallstone formation in mice and perhaps humans.     

Association between Helicobacter felis-induced gastritis and elevated glycated hemoglobin levels in a mouse model of type 1 diabetes

Helicobacter pylori infection has been described in association with increases in glycated hemoglobin (HbA(1c)) levels in patients with type 1 diabetes. The purpose of the present study was to use an animal model of Helicobacter infection to test, under controlled conditions, the hypothesis that infection is associated with high HbA(1c) levels. Diabetes was induced in C57BL/6 mice by administration of streptozotocin, and the mice were orally inoculated with H. felis. Six weeks after inoculation, infected mice (n=10) showed gastritis scores significantly greater (P=.01) than those of uninfected mice (n=10). HbA(1c) levels were significantly higher in infected mice with gastritis (11.6%; n=6) than in infected mice without gastritis (8.4%; n=4) or uninfected mice (7.6%; n=10). It was concluded that gastritis induced by H. felis is associated with increased HbA(1c) levels in the mouse model of streptozotocin-induced diabetes.     

Induction of colitis by a CD4+ T cell clone specific for a bacterial epitope

It is now well established that the intestinal flora plays an important role in the pathogenesis of inflammatory bowel disease (IBD). However, whether bacteria serve as the sole target of the immune response in this process or whether they act indirectly by triggering an anti-self response is still unclear. We have previously shown that specific pathogen-free IL-10-deficient (IL-10 KO) mice develop a T helper (Th1)-cytokine associated colitis after experimental infection with Helicobacter hepaticus. We here show that H. hepaticus Ag (SHelAg)-specific CD4+ Th1 clones transfer disease to H. hepaticus-infected T cell-deficient RAG KO hosts. Importantly, uninfected recipients of the SHelAg-specific clones did not develop intestinal inflammation, and a control Schistosoma mansoni-specific Th1 clone did not induce colitis upon transfer to infected RAG KO mice. The disease-inducing T cell clones recognized antigen(s) (Ag) specifically expressed by certain Helicobacter species as they responded when stimulated in vitro with H. hepaticus and Helicobacter typhlonius Ag, but not when cultured with Ag preparations from Helicobacter pylori, various non-helicobacter bacteria, or with cecal bacterial lysate from uninfected mice. Characterization of the Ag specificity of one of the clones showed that it reacts uniquely with a 15-mer peptide epitope on the flagellar hook protein (FlgE) of H. hepaticus presented by I-Ab. Together, our results demonstrate that colitis can be induced by clonal T cell populations that are highly specific for target Ag on intestinal bacteria, suggesting that an aberrant T cell response directed against gut flora is sufficient to trigger IBD.     

Serum antibodies to enterohepatic Helicobacter spp. in patients with chronic liver diseases and in a population with high prevalence of H. pylori infection

BACKGROUND: Enteric Helicobacter species might be a risk factor for chronic liver and biliary tract diseases. AIMS: To analyse serum antibody levels to three enteric Helicobacter species in patients with various biliary tract and chronic liver diseases and compare results with corresponding parameters for an adult population group, known to have a high prevalence of Helicobacter pylori infection, and with healthy blood donors, to explore a possible association of enteric Helicobacter with chronic liver diseases. SUBJECTS: Sera of 90 patients with various chronic liver diseases, 121 Estonian adult persons and 68 blood donors were analysed. METHODS: Sera, previously tested for H. pylori were analysed for IgG to Helicobacter hepaticus, Helicobacter bilis and Helicobacter pullorum. ELISA was initially used for screening and exclusion of negative cases. Sera with positive ELISA results were further analysed by immunoblot. To remove cross-reactive antibodies between H. pylori and the enteric species, sera were pre-absorbed with lysed H. pylori cells. RESULTS: Liver patients showed a significantly higher seroprevalence to H. hepaticus and H. bilis, compared with the adult population group (p=0.0001 and 0.04, respectively), and to H. hepaticus, compared with blood donors (p=0.01). Patients with autoimmune hepatitis showed no significant antibody reactivity to the enteric Helicobacter spp. in contrast to patients with other chronic liver diseases. CONCLUSION: Patients with chronic liver diseases, except autoimmune hepatitis patients, showed increased antibody levels to H. bilis/H. hepaticus compared with the population and blood donors indicating a possible role of enteric Helicobacter in the natural course of chronic liver diseases. Immunoblot seems to be a promising method for serodiagnosis of infections with these fastidious pathogens.     

The complete genome sequence of the carcinogenic bacterium Helicobacter hepaticus

Helicobacter hepaticus causes chronic hepatitis and liver cancer in mice. It is the prototype enterohepatic Helicobacter species and a close relative of Helicobacter pylori, also a recognized carcinogen. Here we report the complete genome sequence of H. hepaticus ATCC51449. H. hepaticus has a circular chromosome of 1,799,146 base pairs, predicted to encode 1,875 proteins. A total of 938, 953, and 821 proteins have orthologs in H. pylori, Campylobacter jejuni, and both pathogens, respectively. H. hepaticus lacks orthologs of most known H. pylori virulence factors, including adhesins, the VacA cytotoxin, and almost all cag pathogenicity island proteins, but has orthologs of the C. jejuni adhesin PEB1 and the cytolethal distending toxin (CDT). The genome contains a 71-kb genomic island (HHGI1) and several genomic islets whose G+C content differs from the rest of the genome. HHGI1 encodes three basic components of a type IV secretion system and other virulence protein homologs, suggesting a role of HHGI1 in pathogenicity. The genomic variability of H. hepaticus was assessed by comparing the genomes of 12 H. hepaticus strains with the sequenced genome by microarray hybridization. Although five strains, including all those known to have caused liver disease, were indistinguishable from ATCC51449, other strains lacked between 85 and 229 genes, including large parts of HHGI1, demonstrating extensive variation of genome content within the species.