耶氏菌属毒力株共同血清学特性

<正> 取氏菌属细菌(鼠疫耶氏菌、假结核耶氏菌和小肠结肠炎耶氏菌)对人和动物都能引起侵袭性疾病,他们的致病性与一分子量40～48Mdal的毒力质粒有关。该质粒编码VW抗原,自凝因子和特异外膜蛋白。我们对该属细菌毒力株的共同血清学特性进行了研究,发现采用小肠结肠炎耶氏菌酶免疫斑点试验,可以把这3种细菌的毒力株和无毒力株区别开来。     

O:15型小肠结肠炎耶氏菌毒力株的发现与鉴定

从腹泻病人和猪各分离出1株O:15型小肠结肠炎耶氏菌,它们含有毒力质粒(45megadaltons)、VW抗原和Vi抗原,并与毒力因子血清发生凝集反应,在小鼠体内能大量繁殖。这些特征指出:这是两个典型的毒力株,这在该型中是少见的。     

应用毒力因子血清凝集试验和VW抗原检测耶尔森氏菌毒力

本文报告采用小肠结肠炎耶氏菌毒力因子血清凝集试验和VW抗原测定等两种方法检测65株耶氏菌的毒力,结果完全一致。46株小肠结肠炎耶氏菌(包括O∶3,O∶8,O∶9,0∶6.30等血清型)有毒力,其余18株无毒力。实验表明,毒力因子血清无血清型的特异性,在耶氏菌病研究中有很大的实用价值。     

广东省65株耶氏菌的系统鉴定与鼠疫疫源地性质的探讨

目的鉴定65株耶氏菌,了解菌株的生物学特性,抗原抗体与鼠疫菌的相关性,探讨疫源地的现状和性质。方法65株菌用3种致病性耶氏菌诊断血清,分别做玻片、试管凝集试验、生化试验、生长特性,挑选14株菌做毒力因子、毒力基因、致病性、交叉免疫原性、动物试验和血清学、药敏试验。结果 63株小肠结肠炎耶氏菌血清型42株O∶3、21株O∶9,中间型1株,假结核血清II型1株;VW+2株,VW±10株,VW-2株;14株菌F1Ag、F1Ab全阴;小肠结肠炎5株菌均有2个毒力基因(ail、ystA)阳性,VW+和VW±对小白鼠、家兔有致病性,动物感染一种耶氏菌后有交叉免疫力,抗原、活菌交叉吸收鼠疫抗血清后血清学IHA、RIHA没有交叉性,常用抗菌素敏感。结论目前动物感染小肠结肠炎菌普遍,猪感染最高,局部已爆发流行,假结核菌偏低,鼠疫处于静息期。     

我国小肠结肠炎耶氏菌毒力株的特征

本文对我国各地分离的小肠结肠炎耶氏菌的毒力特征进行测定,所有毒力株除个别外,均有VW抗原、Vi抗原和毒力因子,它们的检测结果一致。有毒株还有一条40～50MDa的质粒带,即所谓毒力质粒,但有此毒力质粒的菌株并非都是有毒株,视其有否特异性外膜蛋白(约200KDa)而定。自凝性也是检测耶氏菌毒力的一种方法,但其敏感性与特异性均不及VW抗原测定。因此测定菌株毒力,以VW抗原、Vi抗原和毒力因子测定最特异而敏感,特别后两种方法简单、易行、可靠,可在基层单位推广使用。     

O∶9型小肠结肠炎耶氏菌在吉林人畜的流行

<正> 小肠结肠炎耶氏菌分布很广,存在于动物体内、食品和外环境中,猪为该菌的主要贮存宿主,我国福建,河南等省市从腹泻患者、猪、鼠类和鸡中分离到小肠结肠炎耶氏菌[1-4]。我们自1985年1～7月先后从吉林省人民医院外科取兰尾炎手术患者兰尾56份,分离出2株小肠结肠炎耶氏菌。从长春市肉联厂采取猪鼻咽腔棉拭子涂抹物120份,分离出小肠结肠炎耶氏菌13株。首次在长春市从兰尾炎患者和猪带菌     

家蝇传播小肠结肠炎耶氏菌的作用

从养猪场食堂捕获的家蝇中分离出9株小肠结肠炎耶氏菌,均为O∶3血清群和生物3型,其中3株是从体表分离的,6株从体内分离。根据蝇的个体检查,其自然感染率在1～2％左右。在9株耶氏菌中,有7株是毒力型的菌株,其体表和体内均有带菌,可能系通过食物污染而感染,对人构成严重威胁。     

耶尔森氏菌Yop1蛋白的表达及其鉴别诊断意义

利用Ｙｏｐ１－ＭｃＡｂ－ＲＩＨＡ（反向间接血球凝集试验）检测全国１７个生态型１０５株鼠疫耶氏菌（Ｙｅｒｓｉｎｉａ ｐｅｓｔｉｓ）强毒株和４株弱毒标准株、６株不同血清型的假结核耶氏菌,４株小肠结肠炎耶氏菌以及３株其它肠道菌。结果显示,假结核和小肠结肠炎耶氏菌呈现较高滴度的阳性反应（滴度１：２＾７－１：２＾１０）,其余均为阴性,提示：（１）Ｙｏｐ１蛋白为耶尔森氏菌所特有;（２）鼠疫耶氏菌中突变的ｙｏｐ     

小肠结肠炎耶氏菌诊断噬菌体(J+H)溶菌谱的进一步研究

从猪便分离的两株小肠结肠炎耶氏菌噬菌体,在临用时混合为J+H噬菌体,能裂解小肠结肠炎耶氏菌48个血清型标准株中的42个型;裂解498个地方株中的97％;裂解志贺氏菌属的0.5％,艾希氏菌属的2.2％,但不能裂解沙门氏菌属和枸橼酸杆菌属;裂解26株假结核耶氏菌中的1株。J+H混合噬菌体具有高度敏感性和特异性,是一种很理想的诊断噬菌体,很适用于菌株的初步鉴定。     

冷冻食品中小肠结肠炎耶尔森氏菌的分离和鉴定

<正> 小肠结肠炎耶尔森氏菌广泛分布于自然界,是一种人畜共患的病原菌。食品和饮水受到耶氏菌的污染是爆发胃肠炎型结肠炎耶氏菌病的重要原因,已从各种食品中分离到本菌,但目前国内尚未见从冷冻食品中分离出耶氏菌的报导。本文报告从557份冷冻食品中分出26株耶氏菌,其血清群和人体分离株相符,具有流行病学意义。     

Yersinia enterocolitica, a primary model for bacterial invasiveness

Yersinia enterocolitica is now the species of Yersinia most frequently isolated from human and animal infections. The species includes pathogens and ubiquitous strains. Among the human pathogens, those isolated in America are more virulent than those isolated elsewhere, especially in Europe and Japan, and these isolates differ biochemically and serologically. The relation between Y. enterocolitica and Y. pestis only became obvious in 1980 with the discovery that at 37 degrees C Y. enterocolitica requires Ca++, a phenotype described in the 1960s for Y. pestis. This requirement as well as virulence is dependent on a 70-kilobase plasmid found later in Y. pseudotuberculosis and Y. pestis. Thus, many bacteriologists elected Y. enterocolitica as a model for bacterial invasiveness. However, studies with non-American strains were impeded by the lack of an inexpensive, simple animal test, a difficulty now circumvented by supplying an appropriate siderophore to the bacteria. Ca++ dependence can be viewed as a transition between free growth and protection against the immune system. In the latter phase, Y. enterocolitica synthesizes and releases large amounts of six plasmid-encoded outer membrane proteins. Most of these are under the control of the plasmid region governing Ca++ dependence. Mutants in this region either lose the Ca++ requirement at 37 degrees C or become unable to grow at 37 degrees C irrespective of the Ca++ concentration. The complex events leading to Ca++ dependence is still not understood. Virulence in Y. enterocolitica also depends on chromosomal genes: the endocytosis in intestinal epithelial cells seems not to be encoded by the pYV plasmid. Studies of Y. pseudotuberculosis suggest that this property depends on a single chromosomal locus, the study of which might be particularly important in the understanding of the first step in infection.     

A hypervariable N-terminal region of Yersinia LcrV determines Toll-like receptor 2-mediated IL-10 induction and mouse virulence

The virulence antigen LcrV of Yersinia enterocolitica O:8 induces IL-10 in macrophages via Toll-like receptor 2 (TLR2). The TLR2-active region of LcrV is localized within its N-terminal amino acids (aa) 31-57. Sequencing of codons 25-92 of the lcrV gene from 59 strains of the three pathogenic Yersinia species revealed a hypervariable hotspot within aa 40-61. According to these sequence differences, seven LcrV groups were identified, with Y. pestis and Y. pseudotuberculosis represented in group I and the other six distributed within Y. enterocolitica. By testing LcrV sequence-derived synthetic oligopeptides of all seven LcrV groups in CD14/TLR2-transfected human embryonic kidney 293 cells, we found the highest TLR2 activity with a peptide derived from group IV comprising exclusively Y. enterocolitica O:8 strains. These findings were verified in murine peritoneal macrophages by using recombinant LcrV truncates representing aa 1-130 from different Yersinia spp. By systematically replacing charged aa residues by glutamine in synthetic oligopeptides, we show that the K42Q substitution leads to abrogation of TLR2 activity in both in vitro cell systems. This K42Q substitution was introduced in the lcrV gene from Y. enterocolitica O:8 WA-C(pYV), resulting in WA-C(pYVLcrV(K42Q)), which turned out to be less virulent for C57BL/6 mice than the parental strain. This difference in virulence was not observed in TLR2(-/-) or IL-10(-/-) mice, proving that LcrV contributes to virulence by TLR2-mediated IL-10 induction. LcrV is a defined bacterial virulence factor shown to target the TLR system for evasion of the host's immune response.     

Spontaneous Yersinia enterocolitica septicemia in a patient with iron overload

Yersinia enterocolitica septicemia is described in a patient with transfusional iron overload and a myelodysplastic syndrome. The organism was biotype 1 serotype 0:5,27 and carried a virulence-encoding plasmid. It was calcium-dependent, autoagglutinating and virulent to orally challenged mice, but not resistant to the bacteriocidal activity of serum. The patient had depressed neutrophil chemotaxis and bactericidal activity. In this case, both host and microbial factors were present to select out this particular bacteremic disease. Patients with iron overload states should be recognized as compromised hosts and potentially susceptible to spontaneous sepsis due to Y enterocolitica.     

Interaction of Yersinia enterocolitica biotype 1A strains of diverse origin with cultured cells in vitro

Yersinia enterocolitica biotype 1A isolates are increasingly being associated with diarrhea. However, the mechanism of their pathogenicity is not well understood. In the present study interaction of Y. enterocolitica isolates with CHO cells, HEp-2 cells and J774 mouse macrophages was studied. Y. enterocolitica biotype 1A strains of clinical origin invaded CHO and HEp-2 cells to a significantly higher degree than non-clinical isolates. However, among non-clinical isolates, Y. enterocolitica strains of swine origin showed significantly more invasion in CHO and HEp-2 cells than water isolates. Y. enterocolitica isolates from clinical samples exhibited a greater level of survival in macrophages than isolates from non-clinical sources. It may be construed that Y. enterocolitica biotype 1A isolates of clinical and swine origin have higher virulence potential than those from other sources.     

三种致病耶尔森氏菌Hsp60基因序列的比较研究

目的　研究 3种致病耶尔森氏菌Hsp6 0基因序列的差异。方法　根据文献和基因库中的Hsp6 0基因序列设计了两对PCR引物 ,获得Hsp6 0基因的部分序列。将扩增产物纯化回收 ,克隆至 pGEM -T载体后进行测序。 结果　用CLUSTALX软件进行序列分析 ,发现 3种耶尔森氏致病菌的Hsp6 0基因序列较为保守 ,其中鼠疫耶尔森氏菌和假结核耶尔森氏菌的该基因序列在所研究范围内仅相差一个碱基。结论　 3种菌的Hsp6 0基因保守 ,不足以在此区间设计种特异性探针。小肠结肠炎耶尔森氏菌与鼠疫耶尔森氏菌和假结核耶尔森氏菌的Hsp6 0基因序列差异较大 ,易与这两个种区分开     

Clinical and microbiologic characteristics of cutaneous infection with Yersinia enterocolitica.

The clinical and microbiologic features of primary cutaneous infections by Yersinia enterocolitica are described in three children. Vesiculobullous lesions developed in two patients, and an intense granulation response followed incision and drainage. In the third child, cellulitis and abscess formation developed at the site of minor skin trauma. Y. enterocolitica isolates from the patients were extensively serologically, biochemically, and molecularly analyzed and compared with virulent Y. enterocolitica strains. The ability of the isolates to adhere to and invade eukaryotic cells was determined using in vitro assays; virulence was assessed by inoculation of suckling mice. The resulting data suggest that primary cutaneous infections by Y. enterocolitica involve strains that are as virulent as pathogenic gastrointestinal isolates.     

Suppressive effect of Sl/Sld mouse embryo-derived fibroblast cell lines on diffusible factor-dependent proliferation of mast cells

Two modes of mast cell growth are present, one dependent on diffusible growth factors (interleukins [IL] 3 and 4) and another dependent on contact with fibroblasts. The 3T3 fibroblast cell lines derived from WCB6F1-+/+ mouse embryos supported the proliferation of cultured mast cells (CMC), whereas the 3T3 fibroblast cell lines from WCB6F1-Sl/Sld mouse embryos did not. To investigate the relationship between growth factor-dependent and fibroblast-dependent growths of mast cells, we cocultured CMC and 3T3 fibroblasts in the presence of diffusible growth factors. WCB6F1-+/+ mouse embryo-derived 3T3 cells did not affect the growth factor-dependent proliferation of CMC, but WCB6F1-Sl/Sld mouse embryo-derived 3T3 cells significantly suppressed the proliferation. Close cell-to-cell contact was necessary for the suppression. The NWS1 fibroblast cell line was established from the spleen cells of an adult WBB6F1-+/+ mouse. Although the NWS1 cell line had no supporting effect on the proliferation of CMC in the absence of diffusible growth factors, it did not suppress the proliferation of CMC induced by the growth factors. The present result suggests that a product of mutant Sl genes may be involved in the suppressive activity of WCB6F1-Sl/Sld mouse embryo-derived 3T3 cells.     

Experimental intestinal infection of rats by Yersinia enterocolitica 0:3. A follow-up study with specific antibodies to the virulence plasmid specified antigens

Rats were infected by intragastric inoculation of Yersinia enterocolitica 0:3 grown at room temperature. The events of the first 6 days of the infection were followed by staining sections of small intestine using an immunoperoxidase method. The specific antibodies used were either rabbit antibodies to antigens of Y. enterocolitica, to the temperature-inducible antigens specified by the virulence plasmid (pYV) of Y. enterocolitica, or monoclonal antibody to the pYV-specified autoagglutination protein P1. In the course of the infection Y. enterocolitica organisms were detectable in the terminal ileum already 1 h after the challenge. By 1 h the pYV-specified temperature-inducible antigens were expressed in Y. enterocolitica both in the lumen of the intestine and in the intestinal tissues, which indicates that the activation of pYV in vivo was very rapid. Y. enterocolitica organisms which had penetrated into the lamina propria of the villi were rapidly removed by phagocytic cells. Later organisms were located intracellularly in the lamina propria, in Peyer's patches and in the regional lymph nodes. The terminal ileum was the most severely affected part of the small intestine.     

Cancer-derived VEGF plays no role in malignant ascites formation in the mouse

AIM: Vascular endothelial growth factor (VEGF) is a potent mediator of peritoneal fluid accumulation following tumor progression. This study investigated the role of VEGF secreted by cancerous cells in the formation of malignant ascites. METHODS: VEGF expression was eliminated by knockdown in the pancreas cancer cell-line PancO2 using vector-based short-hairpin type RNA interference (RNAi). Malignant ascites formation in the mouse was analyzed by intraperitoneal injection of PancO2 cells expressing VEGF or with expression knockdown. RESULTS: The VEGF knockdown PancO2 cell was successfully established. Knockdown of VEGF did not affect cancer cell proliferation in vitro or in vivo. The volume of ascites following peritoneal expansion of the tumor in VEGF knockdown cells and control cells did not differ statistically in this in vivo study. Moreover, the VEGF concentration in the ascites did not differ statistically. CONCLUSION: Malignant ascites formation might be mediated by VEGF production in noncancerous tissues, such as stromal compartments. An anti-VEGF strategy against malignant ascites could be applied to various tumors regardless of whether they secrete VEGF.     

Gamma delta T cells differentiate into a functional but nonproliferative state during a normal immune response.

To obtain a homogeneous population of gamma delta T cells to investigate their role in an immune response, we have made a scid mouse doubly transgenic for rearranged gamma and delta genes. The receptor (KN6) encoded by these genes is specific for the major histocompatibility complex class I protein encoded by the T22b gene. This mouse contains high levels of transgenic gamma delta T cells in the spleen and thymus and no other T lymphocytes. Immunization of these KN6-scid (H-2d, TLd) mice with 10(7) C57BL/6J (abbreviated B6) (H-2b, TLb) spleen cells resulted in proliferation and activation of the gamma delta T cells in spleen and clearing of the allogeneic B6 lymphocytes. Subsequently, the majority of activated cells died by apoptosis and the remaining cells were anergic with regard to proliferation. The anergic cells did not respond to restimulation by B6 spleen cells in vitro or in vivo, and addition of exogenous interleukin 2 failed to restore the response to B6 cells. Cytotoxicity, a property of KN6+ cells during a primary stimulation, was no longer detectable in the proliferatively anergic cells. However, B6 spleen cells injected into mice primed 12 days previously were cleared with a much greater efficiency than on primary challenge and in an antigen-specific manner. We conclude that after exposure to antigen, gamma delta T cells rapidly proliferate into blasts; the majority of the blasts rapidly die, with the nonproliferating cells remaining in a highly active state for several weeks and able to initiate elimination of lymphoid cells bearing the TLb epitope.