Observations of the carrier state in hamsters infected with Leptospira interrogans serotype pomona

Summary A variation betweenLeptospira interrogens serotype pomona strains 3341 and MLS acute to become established in the kidney of the hamster and to produce a temporary shedding state in the urine is described. The 3341 strain readily localized in the proximal tubules of the kidney and all infected hamsters became good shedders. Leptospiruria lasted for approximately 100 post-inoculation days. The observation of few spirochetes in the renal tubules, the low incidence of positive cultures from kidney tissue and urine and the low counts of leptospires in the urine characterized the MLS acute strain as a poor shedding strain.A MLS strain (MLS chronic) was isolated from the kidney tissue of a hamster killed on the 100th post-inoculation day. Upon subsequent inoculation into hamsters, this strain produced a shedding state similar to that of the 3341 strain. When compared with the 3341 strain, the MLS chronic strain showed a lower leptospiral count in the urine as well as a greater lag phase until the maximum count was reached.Comparative studies on the 3341, MLS acute, and chronic strains indicated that the MLS chronic strain was probably a mutant because of its rare and spontaneous occurrence and the retention of its shedding characteristic after numerous passages in hamsters and artificial media.This study was supported by a contract, Nonr-3718(02), NR 103-522, between the Office of Naval Research, Department of the Navy, and the University of Nebraska.The technical assistance of Mr. Dick Miller, Miss Rose Reynolds, Mr. James Smith and Mrs. E. Karnish are gratefully acknowledged.     

Experimental immunisation of hamsters with lipopolysaccharide antigens of Leptospira interrogans

Hamsters were immunised with leptospiral lipopolysaccharide (LPS) or the polysaccharide (PS) fraction of LPS from Leptospira interrogans serovar copenhageni and the antibody responses were measured by agglutination tests. Maximum titres were observed approximately 6 weeks after immunisation and protection against lethal challenge with the homologous strain was afforded by immunisation with as little as 2.5 micrograms of LPS or PS. All animals produced IgM agglutinins but a higher proportion of-animals immunised with PS produced IgG agglutinins than did those immunised with LPS. Immunisation of guinea-pigs with autoclaved PS showed that the preparation retained some but not all of its immunogenic activity.     

The effects of gamma-irradiation on haemopoiesis and red blood cell destruction in hamsters infected with Leptospira interrogans serovar ballum

Irradiated hamsters were infected with Leptospira interrogans serovar ballum in order to study the effects of impairment of the reticulo-macrophage system on the progression of the disease and the development of haemoglobinaemia. Infected and irradiated hamsters were compared with infected controls, irradiated controls and untreated controls. Red blood cells (RBCs) from untreated controls were biconcave disks while those from irradiated controls were echinocytes. Red blood cells from animals of both infected groups which were severely affected and moribund were in the form of pitted spherocytes and were associated with increased icteric indices and haemoglobinaemia. Red blood cells from less severely affected animals were echinocytic. Pitted spherocytes, however, were seen in some irradiated-infected hamsters before increased icteric indices and haemoglobinaemia were present. Both irradiated-infected and irradiated hamsters showed severe depletion of haemopoietic cells and lymphoid cells, with regeneration occurring in those animals which survived for longer. Red cell sequestration and erythrophagocytosis were present in infected-only hamsters. In irradiated-infected animals, severe anaemia was caused by both persistence of fixed macrophages, RBC sequestration and erythrophagocytosis, and haemorrhage due to vascular damage and renal papillary necrosis. A severe leucopaenia affected the irradiated hamsters' ability to suppress leptospiral multiplication resulting in the presence of larger numbers of organisms.     

The effects of gamma-irradiation on haemopoiesis and red blood cell destruction in hamsters infected with Leptospira interrogans serovar ballum.

Irradiated hamsters were infected with Leptospira interrogans serovar ballum in order to study the effects of impairment of the reticulo-macrophage system on the progression of the disease and the development of haemoglobinaemia. Infected and irradiated hamsters were compared with infected controls, irradiated controls and untreated controls. Red blood cells (RBCs) from untreated controls were biconcave disks while those from irradiated controls were echinocytes. Red blood cells from animals of both infected groups which were severely affected and moribund were in the form of pitted spherocytes and were associated with increased icteric indices and haemoglobinaemia. Red blood cells from less severely affected animals were echinocytic. Pitted spherocytes, however, were seen in some irradiated-infected hamsters before increased icteric indices and haemoglobinaemia were present. Both irradiated-infected and irradiated hamsters showed severe depletion of haemopoietic cells and lymphoid cells, with regeneration occurring in those animals which survived for longer. Red cell sequestration and erythrophagocytosis were present in infected-only hamsters. In irradiated-infected animals, severe anaemia was caused by both persistence of fixed macrophages, RBC sequestration and erythrophagocytosis, and haemorrhage due to vascular damage and renal papillary necrosis. A severe leucopaenia affected the irradiated hamsters'' ability to suppress leptospiral multiplication resulting in the presence of larger numbers of organisms.     

Protein and antigen profiles of prevalent serovars of Leptospira interrogans.

Whole-cell and detergent-soluble proteins, enriched for outer membrane antigens, of the Leptospira interrogans serovars present in commercially available pentavalent vaccines (hardjo, pomona, icterohaemorrhagiae, grippotyphosa, and canicola) were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting (immunoblotting). Protein and antigenic profiles of these serovars, representing several serogroups, were compared with similar profiles of the most common North American bovine pathogen, serovar hardjo type hardjo bovis. The reference strain of serovar balcanica and a hardjoprajitno bovine field isolate (serovar hardjo) were also assayed. Coomassie blue-stained gels revealed extensive protein similarities, and Western blots demonstrated antigenic relatedness throughout the low- and high-molecular-weight regions. Possible quantitative differences in protein expression among the strains were noted, as were similarities in the protein profiles of type hardjo bovis and the balcanica reference strain. A cocktail composed of these homologous antigens may serve as an efficacious subunit vaccine for leptospirosis.     

Hematologic and biochemical alterations in Wistar rats experimentally infected by Leptospira interrogans

The aim of this study was to evaluate hematologic and biochemical alterations in Wistar rats experimentally infected by eight pathogenic serovars of Leptospira interrogans. Animals were divided in nine groups, six rats each, resulting in eight groups infected and one as the negative control group. Serovars L. hardjo (group A), L. wolffi (B), L. grippotyphosa (C), L. grippotyphosa canicola (D), L. Icterohaemorrhagiae (E), L. bratislava (F), L. pomona (G), L. butembo (H) were utilized in the experiment. Group I was the negative control. The dose-infection was 10⁸ leptospires/mL and the control of infection was carried out through the microscopic observation of leptospires in urine and through the leptospires recovery in culture medium using blood of the infected rats. After 15?days post-infection all rats were euthanatized for blood collection. Hematocrit, total erythrocyte, hemoglobin, total leukocyte, alkaline phosphatase (ALP), alanine aminotransferase (ALT), urea, and creatinine were evaluated. Reduction of the hematocrit, hemoglobin, and total erythrocytes were observed in groups A, B, and G when compared to group I. Total leukocytes had a decrease in groups B and C. Increased levels of ALP (group A, D, and F), ALT (group E, G, xand H), urea (C, D, F, and G), and creatinine (A, C, D, F, and H) were observed when compared to the control group. At necropsy rodents of groups A, D, F, and G showed enlarged spleen and jaundice of viscera and musculature. The final results showed differences between serovars to hematologic and biochemical parameters, but these variations had particular oscillation showing that each serovar of L. interrogans tested may cause alterations in different organs or even systems, independently.     

Proteomic analysis of Leptospira interrogans shed in urine of chronically infected hosts

Leptospirosis is a global zoonotic disease. The causative agent, pathogenic Leptospira species, survives in the renal tubules of chronically infected hosts, from where leptospires are shed via urine into the environment. Infection of new hosts can present as an array of acute and chronic disease processes reflecting variations in host-pathogen interactions. The present study was designed to reproduce the carrier phase of infection in Rattus norvegicus, thus facilitating shedding of leptospires in urine. Leptospires shed in urine were collected for proteomic analysis because these organisms reflect a naturally virulent form of Leptospira associated with infection of new hosts. Experimentally infected rats remained clinically asymptomatic but shed leptospires in urine for several months at concentrations of up to 10⁷ leptospires/ml of urine. Proteomic analysis of rat urine-isolated leptospires compared to in vitro-cultivated leptospires confirmed differential protein and antigen expression, as demonstrated by two-dimensional gel electrophoresis and immunoblotting. Furthermore, while serum from chronically infected rats reacted with many antigens of in vitro-cultivated Leptospira, few antigens of rat urine-isolated Leptospira were reactive. Results confirm that differential protein expression by Leptospira during chronic infection facilitates its persistence in the presence of a specific host antibody response.     

Studies on the kidneys of pregnant hamsters infected with Leptospira canicola.

An electron microscopic study on the histopathological changes in the kidneys of pregnant hamsters resulting from infection with Leptospira canicola was undertaken. The experiment revealed damage to the glomeruli as well as the tubules. The severity of these lesions increased with the progress of the disease and examination of kidney sections showed leptospires in the tissue. It is suggested that the stress of pregnancy has some influence on the severity of the lesions seen in this disease.     

感染细胞内问号钩端螺旋体吞噬泡形成及其与溶酶体的融合

目的了解问号钩端螺旋体（简称钩体）感染宿主细胞后吞噬泡形成、吞噬泡与溶酶体融合及感染细胞超微结构改变。方法用问号钩体黄疸出血群赖型56601株感染小鼠单核巨噬样细胞j774A．1和猴肾成纤维细胞Cos-7。采用透射电镜观察J774A．1细胞和Cos-7细胞胞内钩体吞噬泡的形成及感染细胞超微结构的改变。采用免疫双荧光染色法和激光共聚焦显微镜，观察含钩体吞噬泡与溶酶体融合情况。结果问号钩体56601株感染J774A．1细胞30min、感染Cos-7细胞15min即可在胞浆中发现膜包绕的含钩体吞噬泡，吞噬泡内钩体均保持原有生理弯曲。56601株问号钩体感染Cos-7细胞2h后，钩体吞噬泡膜开始消失，但未发现j774A．1细胞内钩体吞噬泡膜消失的现象。J774A．1细胞内钩体吞噬泡与溶酶体发生共区域化，表明吞噬泡与溶酶体发生融合。J774A．1细胞感染钩体后出现染色质浓缩形成的凋亡小体样结构、细胞空泡变性、线粒体肿胀等超微结构病变，但Cos-7细胞感染钩体后其超微结构基本正常。结论问号钩体感染J774A．1细胞和Cos-7细胞后可迅速形成吞噬泡并可能与钩体Ⅲ型分泌系统产物有关。J774A．1细胞内钩体吞噬泡可与溶酶体发生融合。不同细胞的钩体吞噬泡膜消失及超微结构改变有明显差异。     

Serology and cytokine profiles in patients infected with the newly discovered Bundibugyo ebolavirus

A new species of Ebolavirus, Bundibugyo ebolavirus, was discovered in an outbreak in western Uganda in November 2007. To study the correlation between fatal infection and immune response in Bundibugyo ebolavirus infection, viral antigen, antibodies, and 17 soluble factors important for innate immunity were examined in 44 patient samples. Using Luminex assays, we found that fatal infection was associated with high levels of viral antigen, low levels of pro-inflammatory cytokines, such as IL-1α, IL-1β, IL-6, TNF-α, and high levels of immunosuppressor cytokines like IL-10. Also, acute infected patients died in spite of generating high levels of antibodies against the virus. Thus, our results imply that disease severity in these patients is not due to the multi-organ failure and septic shock caused by a flood of inflammatory cytokines, as seen in infections with other Ebolavirus species.     

Proinflammatory and anti-inflammatory cytokine profiles in psoriasis: use as laboratory biomarkers and disease predictors

Inflammation Research - The objectives of this study were to delineate the pro and anti-inflammatory cytokine profiles of psoriasis and cytokine profile models that externally validate the...     

Early expression of cytokine mRNA in mice infected with Listeria monocytogenes.

Protective immunity first becomes evident at 3 to 4 days after inoculation of mice with a sublethal dose of Listeria monocytogenes. Recent evidence suggests that production of gamma interferon (IFN-gamma) occurs earlier (within the first 24 h of infection). The purpose of this study was to define better the sequence of cytokine mRNA expression during the early stages of L. monocytogenes infection. Cytokine mRNA expression was detected by polymerase chain reaction-assisted amplification of RNA extracted from the spleen cells of individual mice euthanized at 0.5 to 120 h after L. monocytogenes challenge. By using this method, mRNAs for tumor necrosis factor alpha, interleukin-1 alpha (IL-1 alpha), IL-2, IL-4, IL-5, and IFN-gamma were detected in RNA from the spleen cells of uninfected mice. The intensity of the bands for IFN-gamma, however, was increased greatly at 16 h after intravenous injection of 5 x 10(4) CFU (nearly 1 50% lethal dose) of L. monocytogenes. IL-6 and granulocyte-macrophage colony-stimulating factor mRNAs were not detected in spleen cell RNA from uninfected mice but were induced within 30 and 60 min, respectively, after inoculation with L. monocytogenes. Increased amounts of mRNAs for IFN-gamma, IL-6, and granulocyte-macrophage colony-stimulating factor were detected after injection of viable, but not killed, L. monocytogenes. IL-3 mRNA was not detected at any time in RNA extracted from the spleen cells of uninfected or L. monocytogenes infected mice. These results suggest that infection with L. monocytogenes elicits a detectable cytokine mRNA response within the first few hours of infection.     

问号钩端螺旋体鞘磷脂酶类溶血素基因功能分析及其感染细胞后转录水平变化

目的 了解问号钩端螺旋体(简称钩体)鞘磷脂酶类溶血素基因sph1～sph4产物溶血活性及其感染细胞后转录水平的变化.方法 以致病性问号钩体黄疸出血群赖型赖株、波摩那群波摩那型罗株和非致病性双曲钩体三宝垄群Patoc型Patoc Ⅰ株基因组DNA为模板,采用PCR扩增全长sph1～spl4基因片段,扩增产物T-A克隆后测序.构建sph1～sph4基因原核表达系统,采用SDS-PAGE检测目的 重组蛋白rSph1～rSph4的表达情况,Ni-NTA亲和层析柱提纯rSph1～rSpM.采用绵羊血平板对rSph1～rSph4溶血活性进行鉴定,实时荧光定量RT-PCR检测问号钩体赖株感染J774A.1细胞前后sph1～sph4基因转录水平的变化.结果 问号钩体赖株和罗株基因组DNA中均能扩增sph1～sph4基因,双曲钩体Patoc Ⅰ株则否.与报道的相应基因序列比较,所克隆的sph1～sph4基因核苷酸序列相似性均为100%.所构建的原核表达系统能分别表达目的蕈组蛋白rSph1～rSph4.rSph1～rSph4均有溶血活性,其中以rSph2溶血活性最强.问号钩体赖株感染J774A.1细胞后,sph1～sph4基因转录水平均上调,其中sph2和sph4基因mRNA水平上调更为明显.结论 sph1～sph4基因仅存在于致病性问号钩体中,其表达产物有溶血活性.问号钩体赖株感染细胞后sph1～sph4基因转录水平的上调,提示此类鞘磷脂酶类溶血素可能在问号钩体感染宿主过程中有重要作用.     

问号钩端螺旋体铁离子调节蛋白A的免疫原性及保守性研究

目的克隆表达和鉴定问号钩端螺旋体（L．interrogans，简称钩体）黄疸出血群赖型赖株中铁离子调节蛋白A（IRPA），研究IRPA的免疫原性和在不同钩体菌种中的保守性，探讨其在致病和疫苗研究中的意义。方法生物信息学软件分析预测IRPA的特征。构建原核表达质粒pQE31-IRPA，经IPTC诱导后用SDS-PAGE及Western blot鉴定表达情况。用表达的重组蛋白免疫BALB／c小鼠，Western blot检测其免疫原性和在不同血清型钩体中的保守性。ELISA和Western blot检测钩体全菌兔抗血清中的IRPA抗体。结果生物信息学预测结果显示，IRPA是可能位于外膜的脂蛋白，在钩体内有多个蛋白可能与之相互作用。成功克隆表达了重组质粒pQE31-IRPA，重组蛋白能刺激BALB／c小鼠产生抗体（效价为1：32000），并能与相应抗体反应，具有良好的免疫原性。在15株不同血清型的问号钩体及1株双曲钩体（L．biflex）中均可检测到重组蛋白的表达，并在钩体全菌兔抗血清中检测到其抗体。结论IRPA具有良好的免疫原性和保守性，为进一步研究其在钩体病中的作用机制及其作为候选基因疫苗的研究奠定了基础。     

Serum cytokine profiles associated with clinical presentation in Vietnamese infected with hepatitis B virus.

BACKGROUND/OBJECTIVES: An ineffective cytokine response is thought to be one of the reasons for the failure to suppress hepatitis B virus (HBV) replication and to eliminate the virus. We investigated the serum levels of interleukin (IL)-6, IL-10, IL-12, and interferon (IFN)-gamma in HBV-infected Vietnamese patients to determine whether they were related to the outcome of HBV infection. STUDY DESIGN: Samples from a total of 154 HBV-infected patients with well-characterised clinical profiles and 56 healthy controls were assessed. RESULTS: Serum IL-6 levels, which were inversely correlated with transaminase levels, were highest in patients with liver cirrhosis (LC) and hepatocellular carcinoma (HCC) and lowest in those with either asymptomatic (ASYM), acute or chronic HBV, and thus, represented the best marker of HBV-related clinical progression. Compared with the healthy control group, serum IL-12 was uniformly elevated in all HBV-infected patients apart from those with ASYM infections, implying no impairment of production of this cytokine in HBV-infected individuals. Serum IL-10 and IFN-gamma levels, however, were uniformly low and showed no association with clinical presentation. Cytokine profiles were not influenced by the presence of hepatitis B e antigen (HbeAg). CONCLUSIONS: Serum IL-6 and IL-12 but not IL-10 and IFN-gamma are associated with the clinical presentation in HBV-infected Vietnamese patients.     

Integrative analysis of microRNA and mRNA expression profiles in MARC-145 cells infected with PRRSV

Virus Genes - Porcine reproductive and respiratory syndrome virus (PRRSV) causes tremendous economic losses to the swine industry worldwide. miRNAs are crucial regulators of gene expression and a...     

Reaction of monoclonal antibodies with species specific determinants in Leptospira interrogans outer envelope

A set of 24 monoclonal antibodies (MABs) was produced against an outer envelope preparation from Leptospira interrogans serovar copenhageni. The MABs reacted in enzyme immunoassay with species-specific determinants of an antigen in the leptospiral outer envelope (OE) of pathogenic but not of saprophytic species of Leptospira. The MABs did not agglutinate whole leptospires, nor could they opsonise homologous leptospires for phagocytosis by mouse macrophages or protect new-born guinea-pigs against lethal infection. The MABs reacted by Western blotting with a 35 x 10(3)-mol-wt band in OE separated on SDS-polyacrylamide gels, and also reacted with other bands to a lesser extent. The determinants to which the MABs were directed were localised in the leptospiral OE by immunogold labelling techniques.     

Bim, a new serovar of Leptospira interrogans isolated from a dog in Barbados.

A new serovar of Leptospira interrogans was isolated from a dog in Barbados. The proposed name of the new serovar is bim, and the designated type strain is 1051. The serogroup of the new serovar is the Autumnalis serogroup. The new serovar was subsequently isolated from six patients with leptospirosis in Barbados.