P26-based serodiagnosis for Bartonella spp. infection in cats

Bartonella henselae P26 has been identified as an immunodominant antigen expressed during feline infection. We used antisera from cats experimentally infected with B. henselae (n = 6), B. clarridgeiae (n = 4), or B. koehlerae (n = 2) and from a sample of naturally infected cats (B. henselae, n = 34; B. clarridgeiae, n = 1) to evaluate recombinant P26 (rP26) as a serodiagnostic antigen. Immunoblots using antisera from cats infected with B. henselae and B. clarridgeiae reacted strongly with rP26, whereas B. koehlerae antisera did not. A capture ELISA was designed to evaluate the kinetics of rP26 IgG in sera from experimentally infected cats. For B. henselae and B. clarridgeiae antisera, the kinetic profiles of reactivity were similar for rP26 capture ELISA and Bartonella spp. indirect fluorescence assay. However, for B. koehlerae antisera, reactivity in rP26 capture ELISA was consistently low. The serodiagnostic potential of rP26 capture ELISA was evaluated using sera from cats with known Bartonella sp. exposure histories. All 24 (100%) uninfected cats were seronegative, and 33 of 35 (94.3%) cats bacteremic for Bartonella spp. were seropositive. We propose that rP26-based serology can serve as a useful adjunct tool for the diagnosis of feline infection with B. henselae and B. clarridgeiae, but it may not be useful for feline infection with B. koehlerae.     

Evidence of <Emphasis Type="Italic">Babesia microti</Emphasis> infection in multi-infected <Emphasis Type="Italic">Ixodes persulcatus</Emphasis> ticks in Russia

To detect Babesia-infected Ixodes persulcatus Shulze in a suburb of St. Petersburg, Russia, 738 adult ticks were studied using Babesia specific primers and PCR techniques. The entire sample (more than 1,200 individuals) was screened for the presence of Borrelia spp., Ehrlichia spp. and tick-borne encephalitis virus (TBEV). All 7 ticks infected with Babesia microti, were also infected with other pathogens (all 7 among 417 infected ticks, zero amongst the remaining 321 naive ones (χ² = 5.25, p < 0.05). Babesia microti occurred twice with Borrelia afzelii, 3 times with Borrelia garinii, once with both, and once with both B. garinii and TBEV. The prevalence of infection with Borrelia spp. was 34.0%, with Ehrlichia spp. 6.2%, with TBEV 1.5%, and with Ba microti 0.9%. Babesia microti infection was not found in combination with Ehrlichia sp. or Borrelia burgdorferi sensu stricto. The latter pathogen (prevalence 2.6%), just like Ba. microti, was not encountered as a monoinfection. The data suggest that Ba. microti infection can only survive in I. persulcatus in combination with Borrelia spp. (7 of 7 infections). The disease in humans is more severe and longer-lasting when more than one pathogen is involved. Our observations show that the well known St. Petersburg focus of tick-borne encephalitis and Lyme disease is also a focus of ehrlichiosis and babesiosis. This revised version was published online in July 2006 with corrections to the Cover Date.     

Babesia microti: Molecular and antigenic characterizations of a novel 94-kDa protein (BmP94)

A novel gene, BmP94, encoding 94-kDa protein of Babesia microti was identified by immunoscreening of the cDNA expression library. The full-length of BmP94 was expressed in Escherichia coli (rBmP94), which resulted in insoluble form with low yield, and the truncated hydrophilic C-terminus region of the gene was expressed as a soluble protein (rBmP94/CT) with improved productivity. Antiserum raised against rBmP94/CT recognized the 94-kDa native protein in the parasite extract by Western blot analysis. Next, an ELISA using rBmP94/CT was evaluated for diagnostic use, and it demonstrated high sensitivity and specificity when tested with the sera from mice experimentally infected with B. microti and closely related parasites. Moreover, the immunoprotective property of rBmP94/CT as a subunit vaccine was evaluated in BALB/c mice against a B. microti challenge, but no significant protection was observed. Our data suggest that the immunodominant antigen BmP94 could be a promising candidate for diagnostic use for human babesiosis.     

An Improved Antiserum for Sensitive Serologic Detection of Chickpea chlorotic dwarf virus

A Syrian chickpea isolate of Chickpea chlorotic dwarf virus (CpCDV; genus Mastrevirus, family Geminiviridae) was purified and yielded 0.6–0.8 mg of purified virus per kg of infected chickpea tissue. The purified preparations were injected into a rabbit and an antiserum of good quality was obtained and used to evaluate different serological tests for the detection of CpCDV in infected chickpea leaf tissue and extracts. CpCDV was detected in sap dilutions of 1/640 by double‐antibody sandwich enzyme‐linked immunosorbent assay (DAS‐ELISA) and dot‐blot ELISA, and in sap dilutions of 1/1280 by direct antigen‐coating (DAC)‐ELISA using CpCDV immunoglobulin G (IgG) at 0.5 μg/ml. The antiserum was also able to detect the capsid protein of CpCDV by Western blot using raw antiserum at a dilution of 1/2000. The CpCDV raw antiserum (third bleeding) produced had a titre of 1/320 000 when determined by tissue‐blot immunoassay (TBIA); whereas, coating ELISA plates with CpCDV IgG at a concentration of 0.004 μg/ml was enough to detect the virus by DAS‐ELISA in a sap dilution of 1/20 using an enzyme conjugate at a dilution of 1/2000.     

Host distribution and pathogen infection of fleas (Siphonaptera) recovered from small mammals in Pennsylvania

The number of recognized flea‐borne pathogens has increased over the past decade. However, the true number of infections related to all flea‐borne pathogens remains unknown. To better understand the enzootic cycle of flea‐borne pathogens, fleas were sampled from small mammals trapped in central Pennsylvania. A total of 541 small mammals were trapped, with white‐footed mice (Peromyscus leucopus) and southern red‐backed voles (Myodes gapperi) accounting for over 94% of the captures. Only P. leucopus were positive for examined blood‐borne pathogens, with 47 (18.1%) and ten (4.8%) positive for Anaplasma phagocytophilum and Babesia microti, respectively. In addition, 61 fleas were collected from small mammals and tested for pathogens. Orchopeas leucopus was the most common flea and Bartonella vinsonii subspecies arupensis, B. microti, and a Rickettsia felis‐like bacterium were detected in various flea samples. To the best of our knowledge, this is the first report of B. microti DNA detected from a flea and the first report of a R. felis‐like bacterium from rodent fleas in eastern North America. This study provides evidence of emerging pathogens found in fleas, but further investigation is required to resolve the ecology of flea‐borne disease transmission cycles.     

Expression of Coat Protein of an Ordinary Strain of Potato virus Y in Escherichia coli and Production of Polyclonal Antibodies for Diagnosis

The coat protein gene (CP) of an ordinary strain of Potato virus Y (PVY^O) was cloned into the expression vector, pET‐28a(+). The insert was sequenced and analysis showed that the CP gene was in frame with intact N‐terminal 6X histidine tags. An approximately 35 kDa recombinant fusion protein was observed in inclusion bodies of induced Escherichia coli BL21 cells. This fusion protein was purified and used as antigen to raise polyclonal antibodies in rabbits. In Western blot and dot blot immuno‐binding assay (DIBA), both PVY^O‐CP IgG and PVY^O IgG strongly reacted with the recombinant CP. The PVY^O‐CP IgG could detect PVY^O in infected samples up to 1 : 3200 dilutions. A PVY^O‐CP ELISA kit was prepared and compared with conventional ELISA kit based on purified virus particles (PVY^O ELISA kit). The PVY^O‐CP ELISA kit consistently detected the PVY^O in DAS‐ELISA of field samples and was as effective as PVY^O ELISA kit.     

Characterization of secreted recombinant <Emphasis Type="Italic">Toxoplasma gondii</Emphasis> surface antigen 2 (SAG2) heterologously expressed by the yeast <Emphasis Type="Italic">Pichia pastoris</Emphasis>

The surface antigen 2 (SAG2) gene of the protozoan parasite, Toxoplasma gondii, was cloned and extracellularly expressed in the yeast Pichia pastoris. The effectiveness of the secreted recombinant SAG2 (rSAG2-S) as a serodiagnosis reagent was assessed by western blots and ELISA. In the western blot assay, rSAG2-S reacted with all Toxoplasma-antibody positive human serum samples but not with Toxoplasma-negative samples. In the ELISA, rSAG2-S yielded sensitivity rates ranging from 80% (IgG negative, IgM positive) to 100% (IgG positive, IgM negative). In vivo experiments showed that serum from mice immunized with rSAG2-S reacted specifically with the native SAG2 of T. gondii. These mice were protected when challenged with live cells of T. gondii.     

Clinical and immunological effect of intravesical interleukin-2 on superficial bladder cancer

TheBamHI Z EBV replication activator (ZEBRA) protein is involved in the switch from latency to productive cycle of Epstein-Barr virus. A recombinant ZEBRA protein was synthesized and assessed in enzymelinked immunosorbent assay (ELISA) for serum IgG response in nasopharyngeal carcinoma (NPC) patients. In 100 NPC serum samples that were positive for IgA to the EBV viral capsid antigen (VCA), 75% had IgG anti-ZEBRA antibodies. In contrast, only 3/83 (3.6%) serum samples from healthy donors and 2/50 (4%) from other cancers were positive for IgG to ZEBRA. Interestingly, in a selected group of 100 NPC sera negative for IgA to VCA, 25% contained IgG anti-ZEBRA antibodies. This suggests that the ELISA for IgG anti-ZEBRA may also identify earlier cases of NPC not detected by the conventional immunofluorescence test for IgA to VCA.     

The distribution of poplar mosaic virus in hybrid poplars and virus detection by ELISA

Purified poplar mosaic virus (PMV) at a concentration of 8 ng/ml was readily detected by enzyme-linked immunosorbent assay (ELISA). Bioassay in Nicotiana megalosiphon was more sensitive (detecting 1–4 ng/ml) and latex flocculation less sensitive (c. 25 ng/ml) than ELISA assays. While the foliar sap of fresh, naturally-infected poplars (e.g. Populus. euramericana cv. Robusta) was not infective at dilutions greater than 2 . 10–², ELISA easily detected PMV antigen when sap was diluted 4 . 10–³ in buffer or when one part of infected tissue was triturated with 99 parts healthy leaf. Furthermore, although sap from poplar leaves stored at -20 °C for 6 months was not infective, PMV was still detectable in ELISA tests. PMV antigen in poplar leaves was not all pelleted after centrifugation for 2.5 h at 130 000 g yet parallel tests using unbuffered sap from systemically infected Nicotiana megalosiphon foliage showed that infectivity was restricted to the pellet. In poplar foliage, the concentration of PMV antigen was generally greatest where symptoms were most obvious; least antigen was detected in the overwintering leaves located at the bases of long shoots. In winter, when root and inner bark tissue in the trunk was an erratic source of PMV, the virus was readily detected in buds, the concentration being greatest in the bases, including the meristem, of terminal buds. Propagation from single node cuttings of P. euramericana cv. Regenerata allowed the selection of clones that consistently showed either ‘severe’ or ‘mild’ foliar symptoms. The associated virus isolates also infected another poplar clone causing symptoms characteristic of their source. ELISA consistently detected less PMV antigen in field-grown cv. Regenerata than in cv. Robusta foliage, but this was reversed when the associated virus isolates were propagated in Nicotiana glutinosa at 24 °C. During 6 yr, 21 out of 127 poplars at a site in Western England, became infected with PMV. By contrast, in Eastern England, none of 46 were infected. The aphids Pterocomma populea and Myzus persicae did not transmit PMV.     

Detection of bluetongue virus group-specific antigen using monoclonal antibody based sandwich ELISA

A monoclonal antibody (MAb) specific for the bluetongue virus (BTV) group specific antigen (VP7) was characterized for its reactivity with purified virus and recombinant BTV VP7 (rVP7) protein and its suitability for use in the sandwich ELISA. The MAb, designated as 5B5 was specific to VP7 and belongs to IgG2a subclass and was selected for the development of the sELISA in this study. The MAb had a titer of 1:25 with BTV and 1:2 with the rVP7 protein. The sELISA is based on capturing of BTV antigen with VP7 specific MAb followed by detection using BTV polyclonal antiserum raised in rabbits. The assay was evaluated with six cell culture adapted serotypes of BTV that have been isolated from India, 1, 2, 15, 17, 18 and 23. The assay could detect BTV antigen as early as day 8 in blood. It was also successfully applied for the detection of BTV group specific antigen in clinical samples of blood, washed RBCs, buffy coat and plasma. A total of 102 field samples from animals, suspected of being infected with BTV, were tested and 29.42% were positive. The blood samples were also amplified in cell culture which improved the sensitivity of the assay. Results confirmed that the sELISA is rapid and specific.     

Serological studies on cassava latent virus

Particles of cassava latent virus (CLV) were purified by a method that yielded up to 3 mg per 100 g of systemically infected Nicotiana benthamiana leaf. Specific antiserum was prepared and used for enzyme-linked immunosorbent assay (ELISA), which detected purified virus at 5 ng/ml. As estimated by ELISA, CLV antigen reached a greater concentration in leaves of N. benthamiana plants kept at 20–25 °C than in those at 15 °C or 30 °C. CLV was also detected in leaf extracts of naturally infected cassava plants kept at 25 C but its concentration was only 1–7% of that in comparable extracts from N. benthamiana. Staining sections of N. benthamiana leaves with fluorescent antibody indicated that CLV particle antigen accumulates in the nuclei of many phloem cells and of some cells in other tissues. In tests on mosaic-affected cassava plants of Angolan origin, three plants were found in which CLV could not be detected by either ELISA or immunosorbent electron microscopy, or by transmission to indicator plants. This suggests that the mosaic symptoms were caused by a pathogen other than CLV, but no such agent was detected by electron microscopy of leaf extracts. Three kinds of serological test indicated that CLV is related to bean golden mosaic virus. Evidence was also obtained of a distant relationship to beet curly top virus but none was detected to four other geminiviruses.     

Utilization of Proteinase K-Treated Cells as Lipopolysaccharide Antigens for the Serodiagnosis of Helicobacter pylori Infections

We have evaluated the use of proteinase K (PK)-treated cells isolated from Helicobacter pylori as lipopolysaccharide (LPS) antigens in an immunoblot assay and an enzyme-linked immunosorbent assay (ELISA) for the serodiagnosis of H. pylori infection. The sera from patients with chronic gastritis, gastric ulcer, duodenal ulcer or gastric cancer, and from healthy adults with or without H. pylori infection were assayed with three commercial serodiagnostic kits (HM-CAP, Helico-G, and G.A.P. II) and novel methods relying on the use of PK-treated cells. The PK-treated cells used in these assays were selected on the basis of their possibility to possess a common epitope in the O-polysaccharides of H. pylori, which is known to be highly immunogenic in humans. Of the sera from these patients, 71-94% were positive with the commercial kits, 97% with immunoblot assay, and 90% with ELISA. On the other hand, of the healthy adults infected with H. pylori, 72-97% were positive with the commercial kits, 86% with immunoblot assay, and 72% with ELISA. PK-treated cells that did not contain the common epitope were unsuitable as an antigen for immunoblot assay or ELISA. Furthermore, the reactivity of these sera reacted specifically with H. pylori PK-treated cells but not with LPSs from other gram-negative bacteria, such as Campylobacter, Proteus, Bordetella, and Salmonella. These results demonstrate that the serological assays relying on the use of H. pylori PK-treated cells possessing a highly antigenic epitope are potentially useful as a serodiagnostic test for H. pylori infection.     

Production and Characterization of Monoclonal Antibodies Against a Highly Immunogenic Fraction of Entamoeba histolytica (NIH:200) and Their Application in the Detection of Current Amoebic Infection

Six monoclonal antibodies (MAbs) were produced against a highly immunogenic fraction derived by the chromatographic separation of the soluble preparation of axenic Entamoeba histolytica (strain NIH:200) trophozoites. Isotype characterization of the six MAbs revealed that four belonged to the IgM class and one each to the IgG1 and the IgG2a subclasses. The immunoreactivity patterns and the specificity of the MAbs with homologous and heterologous antigens were analyzed by the enzyme-linked immunotransfer blot technique and by the enzyme-linked immunosorbent assay. The MAbs reacted intensely with isolates of E. histolytica (strain NIH:200 as well as a local isolate MX1) but showed no reactivity with Entamoeba coli, Iodamoeba butschlii, Endolimax nana, Entamoeba hartmanni, free-living amoeba (Acanthamoeba harticolus) and other enteric parasites. Using the IgG1 MAb as a detecting antibody, a polyclonal-monoclonal antibody-based enzyme-linked immunosorbent assay was developed for the detection of E. histolytica antigens in stool samples of infected patients. The detection limit of the assay was 8 ng of amoebic antigen. This test was found to be specific and sensitive and yielded 100% positive results in cases with amoebiasis but did not react with controls included in the evaluation. The MAb-based enzyme-linked immunosorbent assay developed in this study will be an important test for the diagnosis of E. histolytica in the feces of infected humans; however, the limitation of the test is the inability to discriminate the pathogenic status of the amoeba detected in the stool.     

<Emphasis Type="Italic">Blastomyces Dermatitidis</Emphasis> Antigen Detection in Urine Specimens from Dogs with Blastomycosis Using a Competitive Binding Inhibition ELISA

A competitive binding inhibition enzyme linked immunosorbent assay (ELISA) was used to detect Blastomyces dermatitidis antigens in urine specimens from dogs with blastomycosis. Sera from rabbits immunized with B. dermatitidis killed whole yeast cells were used as the primary antibody in the competitive ELISA. This initial study was performed to determine if B. dermatitidis antigen detection was possible and to test the efficacy of the rabbit sera as a primary antibody. An indirect ELISA was also performed to compare antigen detection in urine to antibody detection in the sera of the infected dogs. The results indicate 100% (36/36 specimens) detection of both antigen and antibody. Cross reactivity with Histoplasma capsulatum, as well as non-specific binding with the normal urine specimens, was observed with the competitive binding inhibition ELISA.     

Rapid enzyme-linked immunosorbent assay (ELISA) for detection of IgG antibodies to Candida albicans

A rapid enzyme-linked immunosorbent assay (ELISA) where the performance time was shortened to 4h was compared with counter-immunoelectrophoresis (CIE) and a standard ELISA procedure for the detection of IgG antibodies to Candida albicans in 61 patients with suspected invasive candidosis. Using a C. albicans cytoplasmic antigen the rapid ELISA compared well with CIE and the standard ELISA. Seventeen sera that reacted with two concentrations of C. albicans antigen in CIE were also positive in both forms of ELISA. Four sera that were CIE-negative were positive in the standard ELISA and three were also positive in the rapid ELISA. The rapid ELISA provides a sensitive and reproducible test for routine serological investigation of different forms of candidosis.     

不同人类免疫缺陷病毒感染途径群体中戊型肝炎病毒的流行情况

本研究旨在了解不同人类免疫缺陷病毒(human immunodeficiency virus,HIV)感染途径群体中戊型肝炎病毒(hepatitis E virus,HEV)抗体情况,探讨HEV疫苗接种的必要性。采集HIV感染者的血清或血浆,利用酶联免疫吸附试验(enzyme-linked immunosorbent assay,ELISA)检测HEV IgG抗体、IgM抗体及抗原,荧光定量聚合酶链反应(polymerase chain reaction,PCR)检测HEV核酸,Roche高纯化HIV-1核酸定量检测试剂盒(PCR荧光法)检测HIV感染者的HIV载量。比较分析不同HIV感染途径群体中HEV流行率的差别。结果显示,HIV感染者中HEV IgG抗体的阳性率为37.4%,静脉吸毒、成分献血和传播途径不明HIV感染群体的HEV IgG抗体阳性率分别为49.3%、39.5%和30.4%。HEV核酸荧光PCR检测结果均为阴性。3种HIV感染群体之间HEV IgG抗体阳性率差异无统计学意义(χ~2=2.978,P0.05)。HEV IgG阳性与阴性感染者之间HIV载量差异无统计学意义(P0.05)。结果提示,为保护HIV感染者免受HEV感染,应考虑接种HEV疫苗。     

Studies on beet western yellows virus in oilseed rape (Brassica napus ssp. oleifera) and sugar beet (Beta vulgaris)

Oilseed rape (Brassica napus L. ssp. oleifera) was studied as a potential overwintering host for the sugar-beet yellowing viruses, beet yellows virus (BYV) and beet mild yellowing virus (BMYV), and their principal vector, Myzus persicae. In spring 1982, plants infected with a virus which reacted positively in enzyme-linked immunosorbent assay (ELISA) with BMYV antibody globulin were found in oilseed-rape crops; none of the plants contained virus which reacted with BYV antibody globulin. This virus was subsequently identified as beet western yellows virus (BWYV). No leaf symptoms could be consistently associated with infection of oilseed rape, but the virus was reliably detected by sampling any leaf on an infected oilseed-rape plant. Some isolates from oilseed rape did infect sugar beet in glasshouse tests, but the proportions of inoculated plants which became infected were low. Apparently there is therefore little danger of much direct transmission of BWYV by M. persicae from oilseed rape to sugar beet in spring. BWYV was introduced to and spread within oilseed-rape crops in autumn by M. persicae, and autumn-sown oilseed rape proved to be a potentially important overwintering host for M. persicae. In a survey of 80 autumn-sown crops of oilseed rape in East Anglia, northern England and Scotland in spring 1983, 78 were shown to be extensively infected with BWYV. Experimental plots of oilseed rape with 100% BWYV-infection yielded approximately 13.4% less oil than plots with 18% virus infection, the result of a decrease in both seed yield and oil content.