scholarly journals Serological Response of Shiga Toxin-Producing Escherichia coli Type III Secreted Proteins in Sera from Vaccinated Rabbits, Naturally Infected Cattle, and Humans

2011 ◽  
Vol 18 (7) ◽  
pp. 1052-1057 ◽  
Author(s):  
David J. Asper ◽  
Mohamed A. Karmali ◽  
Hugh Townsend ◽  
Dragan Rogan ◽  
Andrew A. Potter
Keyword(s):  
Escherichia Coli ◽  
Shiga Toxin ◽  
Cross Reactivity ◽  
Secreted Proteins ◽  
Enzyme Linked Immunosorbent Assay ◽  
Serological Response ◽  
Infected Cattle ◽  
Serum Samples ◽  
Content Type ◽  
Type Iii

ABSTRACTEscherichia coliO157:H7 is an important zoonotic pathogen, causing hemolytic uremic syndrome (HUS). The colonization of cattle and human hosts is mediated through the action of effectors secreted via a type III secretion system (T3SS). The structural genes for the T3SS and many of the secreted effectors are located on a pathogenicity island called the locus of enterocyte effacement (LEE). We cloned and expressed the genes coding for 66 effectors and purified each to measure the cross-reactivity of type III secreted proteins from Shiga toxin-producingEscherichia coli(STEC) serotypes. These included 37 LEE-encoded proteins and 29 non-LEE effectors. The serological response against each protein was measured by Western blot analysis and enzyme-linked immunosorbent assay (ELISA) using sera from rabbits immunized with type III secreted proteins (T3SPs) from four STEC serotypes, experimentally infected cattle, and human sera from six HUS patients. Twenty proteins were recognized by at least one of the STEC T3SP-vaccinated rabbits by Western blotting. Several structural proteins (EspA, EspB, and EspD) and a number of effectors (Tir, NleA, and TccP) were recognized by O26-, O103-, O111-, and O157-specific sera. Sera from experimentally infected cattle and HUS patients were tested using an ELISA against each of the proteins. Tir, EspB, EspD, EspA, and NleA were recognized by the majority of the samples tested. A number of other proteins also were recognized by individual serum samples. Overall, proteins such as Tir, EspB, EspD, NleA, and EspA were highly immunogenic in vaccinated and naturally infected subjects and could be candidates for a cross-protective STEC vaccine.

scholarly journals Subtractive Phage Display Selection from Canine Visceral Leishmaniasis Identifies Novel Epitopes That Mimic Leishmania infantum Antigens with Potential Serodiagnosis Applications

2013 ◽  
Vol 21 (1) ◽  
pp. 96-106 ◽  
Author(s):  
Lourena E. Costa ◽  
Mayara I. S. Lima ◽  
Miguel A. Chávez-Fumagalli ◽  
Daniel Menezes-Souza ◽  
Vivian T. Martins ◽  
...  
Keyword(s):  
Visceral Leishmaniasis ◽  
Phage Display ◽  
Leishmania Infantum ◽  
Cross Reactivity ◽  
Enzyme Linked Immunosorbent Assay ◽  
Serum Samples ◽  
Phage Display Technology ◽  
Predictive Values ◽  
Content Type ◽  
Phage Elisa

ABSTRACTVisceral leishmaniasis (VL) is a zoonotic disease that is endemic to Brazil, where dogs are the main domestic parasite reservoirs, and the percentages of infected dogs living in regions where canine VL (CVL) is endemic have ranged from 10% to 62%. Despite technological advances, some problems have been reported with CVL serodiagnosis. The present study describes a sequential subtractive selection through phage display technology from polyclonal antibodies of negative and positive sera that resulted in the identification of potential bacteriophage-fused peptides that were highly sensitive and specific to antibodies of CVL. A negative selection was performed in which phage clones were adhered to purified IgGs from healthy andTrypanosoma cruzi-infected dogs to eliminate cross-reactive phages. The remaining supernatant nonadhered phages were submitted to positive selection against IgG from the blood serum of dogs that were infected withLeishmania infantum. Phage clones that adhered to purified IgGs from the CVL-infected serum samples were selected. Eighteen clones were identified and their reactivities tested by a phage enzyme-linked immunosorbent assay (phage-ELISA) against the serum samples from infected dogs (n= 31) compared to those from vaccinated dogs (n= 21), experimentally infected dogs with cross-reactive parasites (n= 23), and healthy controls (n= 17). Eight clones presented sensitivity, specificity, and positive and negative predictive values of 100%, and they showed no cross-reactivity withT. cruzi- orEhrlichia canis-infected dogs or with dogs vaccinated with two different commercial CVL vaccines in Brazil. Our study identified eight mimotopes ofL. infantumantigens with 100% accuracy for CVL serodiagnosis. The use of these mimotopes by phage-ELISA proved to be an excellent assay that was reproducible, simple, fast, and inexpensive, and it can be applied in CVL-monitoring programs.

scholarly journals New Skin Test for Detection of Bovine Tuberculosis on the Basis of Antigen-Displaying Polyester Inclusions Produced by Recombinant Escherichia coli

2014 ◽  
Vol 80 (8) ◽  
pp. 2526-2535 ◽  
Author(s):  
Shuxiong Chen ◽  
Natalie A. Parlane ◽  
Jason Lee ◽  
D. Neil Wedlock ◽  
Bryce M. Buddle ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Skin Test ◽  
Skin Testing ◽  
Cross Reactivity ◽  
Pha Synthase ◽  
Enzyme Linked Immunosorbent Assay ◽  
Content Type ◽  
Flight Mass Spectrometry ◽  
Environmental Mycobacteria ◽  
Test Specificity

ABSTRACTThe tuberculin skin test for diagnosing tuberculosis (TB) in cattle lacks specificity if animals are sensitized to environmental mycobacteria, as some antigens in purified protein derivative (PPD) prepared fromMycobacterium bovisare present in nonpathogenic mycobacteria. Three immunodominant TB antigens, ESAT6, CFP10, and Rv3615c, are present in members of the pathogenicMycobacterium tuberculosiscomplex but absent from the majority of environmental mycobacteria. These TB antigens have the potential to enhance skin test specificity. To increase their immunogenicity, these antigens were displayed on polyester beads by translationally fusing them to a polyhydroxyalkanoate (PHA) synthase which mediated formation of antigen-displaying inclusions in recombinantEscherichia coli. The most common form of these inclusions is poly(3-hydroxybutyric acid) (PHB). The respective fusion proteins displayed on these PHB inclusions (beads) were identified using tryptic peptide fingerprinting analysis in combination with matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS). The surface exposure and accessibility of antigens were assessed by enzyme-linked immunosorbent assay (ELISA). Polyester beads displaying all three TB antigens showed greater reactivity with TB antigen-specific antibody than did beads displaying only one TB antigen. This was neither due to cross-reactivity of antibodies with the other two antigens nor due to differences in protein expression levels between beads displaying single or three TB antigens. The triple-antigen-displaying polyester beads were used for skin testing of cattle and detected all cattle experimentally infected withM. boviswith no false-positive reactions observed in those sensitized to environmental mycobacteria. The results suggested applicability of TB antigen-displaying polyester inclusions as diagnostic reagents for distinguishing TB-infected from noninfected animals.

scholarly journals Seroreactive Profiling of Filoviruses in Chinese Bats Reveals Extensive Infection of Diverse Viruses

2020 ◽  
Vol 94 (7) ◽  
Author(s):  
Chang Zhang ◽  
Zhongyi Wang ◽  
Jianqiu Cai ◽  
Xiaomin Yan ◽  
Fuqiang Zhang ◽  
...  
Keyword(s):  
Southern China ◽  
Hot Spot ◽  
Emerging Infectious Diseases ◽  
Neutralization Assay ◽  
Cross Reactivity ◽  
Enzyme Linked Immunosorbent Assay ◽  
Antigenic Relationship ◽  
Serum Samples ◽  
Content Type ◽  
Fruit Bat

ABSTRACT Southern China is a hot spot of emerging infectious diseases, in which diverse species of bats dwell, a large group of flying mammals considered natural reservoirs for zoonotic viruses. Recently, divergent filoviruses (FiVs) have been identified in bats within this region, which pose a potential risk to public health, but the true infection situation in bats remains largely unclear. Here, 689 archived bat serum samples were analyzed by enzyme-linked immunosorbent assay (ELISA), Western blotting, and neutralization assay to investigate the seroprevalence and cross-reactivity of four divergent FiVs and two other viruses (rabies virus and Tuhoko pararubulavirus 1) of different families within the order Mononegavirales. Results showed no cross-antigenicity between FiVs and other mononegaviruses but different cross-reactivity among the FiVs themselves. The total FiV seroreactive rate was 36.3% (250/689), with infection by the indigenous Chinese FiV DH04 or an antigenically related one being the most widely and the most highly prevalent. Further viral metagenomic analysis of fruit bat tissues also identified the gene sequence of a novel FiV. These results indicate the likely prevalence of other so far unidentified FiVs within the Chinese bat population, with frugivorous Rousettus leschenaultii and Eonycteris spelaea bats and insectivorous Myotis horsfieldii and Miniopterus schreibersii bats being their major reservoirs. IMPORTANCE Bats are natural hosts of many FiVs, from which diverse FiVs were serologically or virologically detected in Africa, Europe, and East Asia. Recently, very divergent FiVs were identified in the Chinese bat population, but their antigenic relationship with other known FiVs remains unknown. Here, we conducted serological characterization and investigation of Chinese indigenous FiVs and prototypes of other viruses in bats. Results indicated that Chinese indigenous FiVs are antigenically distant to other FiVs, and infection of novel or multiple FiVs occurred in Chinese bats, with FiV DH04 or an antigenically related one being the most widely and the most highly prevalent. Additionally, besides Rousettus leschenaultii and Eonycteris spelaea bats, the insectivorous Myotis horsfieldii and M. schreibersii bats are highly preferential hosts of FiVs. Seroreactive and viral metagenomic results indicated that more as yet unknown bat-borne FiVs circulate in Southern China, and to uncover them further, investigation and timely surveillance is needed.

scholarly journals Chimeric Protein Designed by Genome-Scale Immunoinformatics Enhances Serodiagnosis of Bovine Neosporosis

2020 ◽  
Vol 58 (7) ◽  
Author(s):  
Higor Sette Pereira ◽  
Ludmila Tavares e Almeida ◽  
Vitória Fernandes ◽  
Renato Lima Senra ◽  
Patrícia Pereira Fontes ◽  
...  
Keyword(s):  
Neospora Caninum ◽  
Leukemia Virus ◽  
Clinical Signs ◽  
Chimeric Protein ◽  
Cross Reactivity ◽  
Enzyme Linked Immunosorbent Assay ◽  
Serum Samples ◽  
Noninvasive Test ◽  
Content Type ◽  
Bovine Neosporosis

ABSTRACT Neosporosis has become a concern since it is associated with abortion in cattle. Currently, in situ diagnosis is determined through anamnesis, evaluation of the history, and perception of the clinical signs of the herd. There is no practical and noninvasive test adapted to a large number of samples, which represents a gap for the use of new approaches that provide information about infections and the risks of herds. Here, we performed a search in the Neospora caninum genome by linear B-cell epitopes using immunoinformatic tools aiming to develop a chimeric protein with high potential to bind specifically to antibodies from infected cattle samples. An enzyme-linked immunosorbent assay with the new chimeric antigen was developed and tested with sera from natural field N. caninum-infected bovines. The cross-reactivity of the new antigen was also evaluated using sera from bovines infected by other abortive pathogens, including Trypanosoma vivax, Leptospira sp., Mycobacterium bovis, and Brucella abortus, and enzootic bovine leucosis caused by bovine leukemia virus, as well as with samples of animals infected with Toxoplasma gondii. The assay using the chimeric protein showed 96.6% ± 3.4% of sensitivity in comparison to healthy animal sera. Meanwhile, in relation to false-positive results provided by cross-reactivity with others pathogens, the specificity value was 97.0% ± 2.9%. In conclusion, immunoinformatic tools provide an efficient platform to build an accurate protein to diagnose bovine neosporosis based on serum samples.

scholarly journals Limitations of the BP26 Protein-Based Indirect Enzyme-Linked Immunosorbent Assay for Diagnosis of Brucellosis

2013 ◽  
Vol 20 (9) ◽  
pp. 1410-1417 ◽  
Author(s):  
Ting Xin ◽  
Hongjun Yang ◽  
Nan Wang ◽  
Fang Wang ◽  
Peng Zhao ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Beef Cattle ◽  
Yersinia Enterocolitica ◽  
Immunosorbent Assay ◽  
Indirect Elisa ◽  
Cross Reactivity ◽  
Infected Sheep ◽  
Enzyme Linked Immunosorbent Assay ◽  
Content Type ◽  
Experimental Animals

ABSTRACTBrucellosis is a serious zoonosis that occurs worldwide, and its diagnosis is typically based on the detection of antibodies againstBrucellalipopolysaccharide (LPS). However, the specificity of the LPS-based test is compromised by cross-reactivity withEscherichia coliO157:H7 andYersinia enterocoliticaO:9. Also, diagnosis based on the LPS test cannot differentiate between vaccinated and infected individuals. The detection of the 26-kDa cytosoluble protein (BP26) antibody is considered an alternative that circumvents these drawbacks because it is exclusively expressed by infectiousBrucella. A BP26-based enzyme-linked immunosorbent assay (ELISA) has been tried for the diagnosis ofBrucella-infected animals and humans, but a few results showed that BP26 couldn't react with allBrucella-positive sera. In order to explore whether different animals could produce antibodies against BP26 after being infected with variousBrucellaspecies, we infected sheep, goats, and beef cattle with common virulent referenceBrucellaspecies. All sera were collected from the experimental animals and tested using both LPS-based ELISAs and BP26-based ELISAs. The results showed that allBrucella-infected individuals could produce high levels of antibodies against LPS, but onlyB. melitensis16M- andB. melitensisM28-infected sheep andB. melitensis16M- andB. abortus2308-infected goats could produce antibodies against BP26. Therefore, we concluded that the BP26-based indirect ELISA (i-ELISA) showed bothBrucellaspecies and host specificity, which obviously limits its reliability as a substitute for the traditional LPS-based ELISA for the detection of brucellosis.

scholarly journals Recombinant Antigen-Based Enzyme-Linked Immunosorbent Assay for Diagnosis of Baylisascaris procyonis Larva Migrans

2011 ◽  
Vol 18 (10) ◽  
pp. 1650-1655 ◽  
Author(s):  
Sriveny Dangoudoubiyam ◽  
Ramesh Vemulapalli ◽  
Momar Ndao ◽  
Kevin R. Kazacos
Keyword(s):  
Immunosorbent Assay ◽  
Cross Reactivity ◽  
Larva Migrans ◽  
Enzyme Linked Immunosorbent Assay ◽  
Parasitic Infections ◽  
Serum Samples ◽  
Western Blot Assay ◽  
Human Patient ◽  
Content Type ◽  
Baylisascaris Procyonis

ABSTRACTBaylisascarislarva migrans is an important zoonotic disease caused byBaylisascaris procyonis, the raccoon roundworm, and is being increasingly considered in the differential diagnosis of eosinophilic meningoencephalitis in children and young adults. Although aB. procyonisexcretory-secretory (BPES) antigen-based enzyme-linked immunosorbent assay (ELISA) and a Western blot assay are useful in the immunodiagnosis of this infection, cross-reactivity remains a major problem. Recently, a recombinantB. procyonisantigen, BpRAG1, was reported for use in the development of improved serological assays for the diagnosis ofBaylisascarislarva migrans. In this study, we tested a total of 384 human patient serum samples in a BpRAG1 ELISA, including samples from 20 patients with clinicalBaylisascarislarva migrans, 137 patients with other parasitic infections (8 helminth and 4 protozoan), and 227 individuals with unknown/suspected parasitic infections. A sensitivity of 85% and a specificity of 86.9% were observed with the BpRAG1 ELISA, compared to only 39.4% specificity with the BPES ELISA. In addition, the BpRAG1 ELISA had a low degree of cross-reactivity with antibodies toToxocarainfection (25%), while the BPES antigen showed 90.6% cross-reactivity. Based on these results, the BpRAG1 antigen has a high degree of sensitivity and specificity and should be very useful and reliable in the diagnosis and seroepidemiology ofBaylisascarislarva migrans by ELISA.

scholarly journals Identification of a Highly Antigenic Region of Subtilisin-Like Serine Protease 1 for Serodiagnosis of Neospora caninum Infection

2013 ◽  
Vol 20 (10) ◽  
pp. 1617-1622 ◽  
Author(s):  
Rochelle Haidee D. Ybañez ◽  
Mohamad Alaa Terkawi ◽  
Kyohko Kameyama ◽  
Xuenan Xuan ◽  
Yoshifumi Nishikawa
Keyword(s):  
Serine Protease ◽  
Tandem Repeat ◽  
Neospora Caninum ◽  
Single Copy ◽  
Enzyme Linked Immunosorbent Assay ◽  
Infected Cattle ◽  
Serum Samples ◽  
Caninum Infection ◽  
Content Type ◽  
Field Samples

ABSTRACTNeospora caninumis an apicomplexan parasite that causes abortion in cattle; hence, accurate diagnosis of this pathogen is important to the cattle farming industry. Our previous proteomics and immunoscreening analyses revealed that theN. caninumsubtilisin-like serine protease 1 (NcSUB1) has potential as a serodiagnostic tool forNeospora. Consequently, we expressed two fragments containing five NcSUB1 tandem repeat copies covering amino acids (aa) 524 to 843 (NcSUB1t) and 555 to 679 (NcSUB1tr) to identify the antigenic regions. The serodiagnostic performances of NcSUB1t and NcSUB1tr were compared with that of N54, which contains a single copy of the repeats (aa 649 to 784), and with the truncated NcSAG1 (NcSAG1t), which lacks a signal peptide and C-terminal hydrophobic regions, as a positive reference. Serum samples fromN. caninumexperimentally infected cattle and mice and cattle from a farm with confirmed cases ofNeosporaabortion were tested by enzyme-linked immunosorbent assay (ELISA) with the four antigens. In theN. caninumexperimentally infected cattle, the highest IgG1 antibody titers were detected against NcSUB1t, while specific IgG1 antibodies were detectable from 16 days postinfection (dpi), with levels peaking at 36 dpi for all of the antigens. On the other hand, the levels of anti-NcSUB1 IgG2 antibodies were lower than those of anti-SAG1t IgG2 antibodies. The ELISA with NcSUB1t and NcSUB1tr had good sensitivity (94.59 to 95.95%) and specificity (80 to 100%) with bovine serum field samples compared to NcSAG1t and showed no cross-reactions with sera fromToxoplasma gondiiexperimentally infected mice. Moreover, IgG antibodies against NcSUB1t were detected during parturition in the NcSAG1t antibody-positive cattle, and NcSUB1t-specific antibody transfer was observed from a mother to her calf. Our results show that the NcSUB1 tandem repeat is potentially useful for serodiagnosis ofN. caninum.

scholarly journals trans-Sialidase Neutralizing Antibody Detection in Trypanosoma cruzi-Infected Domestic Reservoirs

2011 ◽  
Vol 18 (6) ◽  
pp. 984-989 ◽  
Author(s):  
Paula A. Sartor ◽  
Martha V. Cardinal ◽  
Marcela M. Orozco ◽  
Ricardo E. Gürtler ◽  
M. Susana Leguizamón
Keyword(s):  
Visceral Leishmaniasis ◽  
Trypanosoma Cruzi ◽  
Neutralizing Antibodies ◽  
Cross Reactivity ◽  
Antibody Detection ◽  
Enzyme Linked Immunosorbent Assay ◽  
Trypanosoma Rangeli ◽  
Serum Samples ◽  
Dipstick Test ◽  
Content Type

ABSTRACTThe detection ofTrypanosoma cruziinfection in domestic dogs and cats is relevant to evaluating human transmission risks and the effectiveness of insecticide spraying campaigns. However, the serological assays routinely used are associated with cross-reactivity in sera from mammals infected withLeishmaniaspp. We used atrans-sialidase inhibition assay (TIA) forT. cruzidiagnosis in serum samples from 199 dogs and 57 cats from areas where these types of infections are endemic. TIA is based on the antibody neutralization of recombinanttrans-sialidase, an enzyme that is not detected in the coendemicLeishmaniaspecies orTrypanosoma rangeliparasites.T. cruziinfection was also evaluated by conventional serology (CS) (indirect immunofluorescence, indirect hemagglutination, enzyme-linked immunosorbent assay, and immunochromatographic dipstick test) and xenodiagnosis. Sera from 30 dogs and 15 cats from areas where these organisms are not endemic and 5 dogs with visceral leishmaniasis were found to be nonreactive by TIA and CS. Samples from dogs and cats demonstrated 91 and 95% copositivities between TIA and CS, whereas the conegativities were 98 and 97%, respectively. Sera from xenodiagnosis-positive dogs and cats also reacted by TIA (copositivities of 97 and 83%, respectively). TIA was reactive in three CS-negative samples and was able to resolve results in two cat serum samples that were CS inconclusive. Our study is the first to describe the development oftrans-sialidase neutralizing antibodies in naturally infected dogs and cats. High CS conegativity and the absence oftrans-sialidase neutralization in dog sera from areas where leishmaniasis is not endemic and from dogs with visceral leishmaniasis support TIA specificity. The TIA may be a useful tool forT. cruzidetection in the main domestic reservoirs.

scholarly journals Characterization of the Mode of Action of Aurodox, a Type III Secretion System Inhibitor fromStreptomyces goldiniensis

2018 ◽  
Vol 87 (2) ◽  
Author(s):  
Rebecca E. McHugh ◽  
Nicky O’Boyle ◽  
James P. R. Connolly ◽  
Paul A. Hoskisson ◽  
Andrew J. Roe
Keyword(s):  
Escherichia Coli ◽  
Shiga Toxin ◽  
Type Iii Secretion ◽  
Secretion System ◽  
Transcriptional Level ◽  
Secretion Systems ◽  
Content Type ◽  
Type Iii ◽  
Type Iii Secretion Systems

ABSTRACTRecent work has demonstrated that the polyketide natural product Aurodox fromStreptomyces goldiniensisis able to block the pathogenesis of the murine pathogenCitrobacter rodentium. In this work, we aimed to gain a better understanding of the mechanism of action of the compound. We show that Aurodox downregulates the expression of the type III secretion systems of enteropathogenic and enterohemorrhagicEscherichia coli. Furthermore, we have used transcriptomic analysis to show that Aurodox inhibits the expression at the transcriptional level by repressing the master regulator,ler. Our data support a model in which Aurodox acts upstream oflerand not directly on the secretion system itself. Finally, we have shown that Aurodox, unlike some traditional antibiotics, does not induce expression of RecA, which is essential for the production of Shiga toxin. We propose that these properties nominate Aurodox as a promising antivirulence therapy for the treatment of these infections.

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