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 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 Specific Serodiagnosis of Canine Visceral Leishmaniasis Using Leishmania Species Ribosomal Protein Extracts

2009 ◽  
Vol 16 (12) ◽  
pp. 1774-1780 ◽  
Author(s):  
Eduardo A. F. Coelho ◽  
Laura Ramírez ◽  
Mariana A. F. Costa ◽  
Vinicio T. S. Coelho ◽  
Vivian T. Martins ◽  
...  
Keyword(s):  
Visceral Leishmaniasis ◽  
Ribosomal Proteins ◽  
High Sensitivity ◽  
Leishmania Species ◽  
Cross Reactivity ◽  
Enzyme Linked Immunosorbent Assay ◽  
Canine Visceral Leishmaniasis ◽  
Leishmania Chagasi ◽  
Serum Samples ◽  
Potential Tool

ABSTRACT In the present work, we have analyzed the antigenicity of Leishmania species ribosomal proteins (LRPs). To accomplish this, Leishmania infantum ribosomes were biochemically purified from promastigote cytosolic extracts, and their reactivities were analyzed by using the sera from dogs naturally infected with L. infantum. Since antibodies reacting against different ribosomal proteins were observed in all the serum samples obtained from dogs with symptomatic visceral leishmaniasis tested, we have analyzed the potential usefulness of the LRP extracts in the development of an enzyme-linked immunosorbent assay (ELISA) for the serodiagnosis of canine visceral leishmaniasis (CVL) in an area of Brazil where visceral leishmaniasis is endemic due to infection by Leishmania chagasi. A comparative ELISA with crude soluble Leishmania chagasi antigen (SLA) and L. infantum LRPs was performed. LRP- and SLA-based ELISAs gave similar sensitivities for the diagnosis of symptomatic CVL, but the LRP extract provided a very high sensitivity for the detection of oligosymptomatic and asymptomatic dogs. In addition, an LRP-based ELISA showed a higher specificity when the sera from dogs harboring other infections were included in the analysis. The LRP antigen displayed no cross-reactivity with sera from dogs that had any of the other diseases tested, notably, Chagas' disease. Our findings suggest that LRPs are a potential tool for the diagnosis of CVL and will be particularly useful for the diagnosis of asymptomatic CVL.

scholarly journals Cross-Reactivity Using Chimeric Trypanosoma cruzi Antigens: Diagnostic Performance in Settings Where Chagas Disease and American Cutaneous or Visceral Leishmaniasis Are Coendemic

2019 ◽  
Vol 57 (8) ◽  
Author(s):  
Ramona Tavares Daltro ◽  
Leonardo Maia Leony ◽  
Natália Erdens Maron Freitas ◽  
Ângelo Antônio Oliveira Silva ◽  
Emily Ferreira Santos ◽  
...  
Keyword(s):  
Visceral Leishmaniasis ◽  
Chagas Disease ◽  
Diagnostic Performance ◽  
Latent Class ◽  
Cross Reactivity ◽  
Chimeric Proteins ◽  
Serum Samples ◽  
Perfect Agreement ◽  
Content Type ◽  
Chronic Chagas Disease

ABSTRACT Chimeric T. cruzi antigens have been proposed as a diagnostic tool for chronic Chagas disease (CD) in both settings where Chagas disease is endemic and those where it is not endemic. Antibody response varies in accordance to each T. cruzi strain, presenting challenges to the use of antigens lacking demonstrated cross-reactivity with Leishmania spp. Our group expressed four chimeric proteins (IBMP-8.1, IBMP-8.2, IBMP-8.3, and IBMP-8.4) and previously assessed their diagnostic performance to determine cross-reactivity with Leishmania spp. Here, we validated our findings using serum samples from different Brazilian geographic areas reporting endemic Chagas disease, endemic visceral or American cutaneous leishmaniasis (ACL), or both. Overall, 829 serum samples were evaluated using commercial and IBMP enzyme-linked immunosorbent assays. Due to the absence of a reference assay to diagnosis CD, latent class analysis (LCA) was performed through the use of a statistical model. The incidence of cross-reactivity for ACL-positive samples varied from 0.35% (IBMP-8.3) to 0.70% (IBMP-8.1 and IBMP-8.2). Regarding visceral leishmaniasis (VL)-positive samples, the IBMP-8.2 and IBMP-8.3 antigens cross-reacted with six (3.49%) and with only one sample (0.58%), respectively. No cross-reactivity with either ACL or VL was observed for the IBMP-8.4 antigen. Similarly, no cross-reactions were found when VL-positive samples were assayed with IBMP-8.1. The agreement among the results obtained using IBMP antigens ranged from 97.3% for IBMP-8.2 and 99% for IBMP-8.1 and IBMP-8.3 to 100% for IBMP-8.4, demonstrating almost perfect agreement with LCA. Accordingly, in light of the negligible cross-reactivity with both ACL and VL, we suggest the use of IBMP antigens in regions where T. cruzi and Leishmania spp. are coendemic.

scholarly journals Development of an Enzyme-Linked Immunosorbent Assay for Serodiagnosis of Ringworm Infection in Cattle

2013 ◽  
Vol 20 (8) ◽  
pp. 1150-1154 ◽  
Author(s):  
Elena Tatiana Băguţ ◽  
Ludivine Cambier ◽  
Marie-Pierre Heinen ◽  
Vasile Cozma ◽  
Michel Monod ◽  
...  
Keyword(s):  
Positive Predictive Value ◽  
Negative Predictive Value ◽  
Immunosorbent Assay ◽  
Predictive Value ◽  
Skin Lesions ◽  
Enzyme Linked Immunosorbent Assay ◽  
Serum Samples ◽  
Predictive Values ◽  
Content Type ◽  
Ringworm Infection

ABSTRACTThe aim of this study was to develop an in-house enzyme-linked immunosorbent assay (ELISA) for the serological diagnosis of ringworm infection in cattle. We used available recombinant forms ofTrichophyton rubrumdipeptidyl peptidase V (TruDppV) andT. rubrumleucin aminopeptidase 2 (TruLap2), which are 98% identical toTrichophyton verrucosumorthologues. Field serum samples from 135 cattle with ringworm infection, as confirmed by direct microscopy, fluorescence microscopy, and PCR, and from 55 cattle without any apparent skin lesions or history of ringworm infection that served as negative controls were used. Sensitivities, specificities, and positive and negative predictive values were determined to evaluate the diagnostic value of our ELISA. Overall, the ELISAs based on recombinant TruDppV and TruLap2 discriminated well between infected animals and healthy controls. Highly significant differences (P< 0.0001, Mann-Whitney U test) were noted between optical density values obtained when sera from infected versus control cattle were tested. The ELISA developed for the detection of specific antibodies against DppV gave 89.6% sensitivity, 92.7% specificity, a 96.8% positive predictive value, and a 78.4% negative predictive value. The recombinant TruLap2-based ELISA displayed 88.1% sensitivity, 90.9% specificity, a 95.9% positive predictive value, and a 75.7% negative predictive value. To the best of our knowledge, this is the first ELISA based on recombinant antigens for assessing immune responses to ringworm infection in cattle; it is particularly suitable for epidemiological studies and also for the evaluation of vaccines and/or vaccination procedures.

scholarly journals Evaluation of a Prototype Flow Cytometry Test for Serodiagnosis of Canine Visceral Leishmaniasis

2013 ◽  
Vol 20 (12) ◽  
pp. 1792-1798 ◽  
Author(s):  
Henrique Gama Ker ◽  
Wendel Coura-Vital ◽  
Rodrigo Dian de Oliveira Aguiar-Soares ◽  
Bruno Mendes Roatt ◽  
Nádia das Dores Moreira ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Visceral Leishmaniasis ◽  
Cross Reactivity ◽  
Leishmania Braziliensis ◽  
Canine Visceral Leishmaniasis ◽  
Predictive Values ◽  
Content Type ◽  
Prototype Test ◽  
Serological Reactivity ◽  
Positive Results

ABSTRACTDiagnosing canine visceral leishmaniasis (CVL) is a critical challenge since conventional immunoserological tests still present some deficiencies. The current study evaluated a prototype flow cytometry serology test, using antigens and fluorescent antibodies that had been stored for 1 year at 4°C, on a broad range of serum samples. Noninfected control dogs andLeishmania infantum-infected dogs were tested, and the prototype test showed excellent performance in differentiating these groups with high sensitivity, specificity, positive and negative predictive values, and accuracy (100% in all analyses). When the CVL group was evaluated according to the dogs' clinical status, the prototype test showed outstanding accuracy in all groups with positive serology (asymptomatic II, oligosymptomatic, and symptomatic). However, in dogs which had positive results by PCR-restriction fragment length polymorphism (RFLP) but negative results by conventional serology (asymptomatic I), serological reactivity was not observed. Additionally, sera from 40 dogs immunized with different vaccines (Leishmune, Leish-Tec, or LBSap) did not present serological reactivity in the prototype test. Eighty-eight dogs infected with other pathogens (Trypanosoma cruzi,Leishmania braziliensis,Ehrlichia canis, andBabesia canis) were used to determine cross-reactivity and specificity, and the prototype test performed well, particularly in dogs infected withB. canisandE. canis(100% and 93.3% specificities, respectively). In conclusion, our data reinforce the potential of the prototype test for use as a commercial kit and highlight its outstanding performance even after storage for 1 year at 4°C. Moreover, the prototype test efficiently provided accurate CVL serodiagnosis with an absence of false-positive results in vaccinated dogs and minor cross-reactivity against other canine pathogens.

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 Identification and Diagnostic Utility of Leishmania infantum Proteins Found in Urine Samples from Patients with Visceral Leishmaniasis

2012 ◽  
Vol 19 (6) ◽  
pp. 935-943 ◽  
Author(s):  
Claudia Abeijon ◽  
Suely S. Kashino ◽  
Fernando O. Silva ◽  
Dorcas L. Costa ◽  
Ricardo T. Fujiwara ◽  
...  
Keyword(s):  
Visceral Leishmaniasis ◽  
Leishmania Infantum ◽  
Antigen Detection ◽  
Enzyme Linked Immunosorbent Assay ◽  
Accession Number ◽  
Content Type ◽  
Link Type ◽  
Detection Assay ◽  
Ncbi Accession ◽  
Ncbi Accession Number

ABSTRACTDespite the clear need to control visceral leishmaniasis (VL), the existing diagnostic tests have serious shortcomings. Here, we introduce an innovative approach to directly identifyLeishmania infantumantigens producedin vivoin humans with VL. We combined reverse-phase high-performance liquid chromatography (RP-HPLC) with mass spectrometry and categorized three distinctL. infantumproteins presumably produced in bone marrow/spleen/liver and excreted in the urine of patients with VL. The genes coding for these proteins (L. infantumiron superoxide dismutase, NCBI accession numberXP_001467866.1;L. infantumtryparedoxin, NCBI accession numberXP_001466642.1; andL. infantumnuclear transport factor 2, NCBI accession numberXP_001463738.1) were cloned, and the recombinant molecules were produced inEscherichia coli. Antibodies to these proteins were produced in rabbits and chickens and were used to develop a capture enzyme-linked immunosorbent assay (ELISA) designed to detect theseL. infantumantigens in the urine of VL patients. Specificity of the antibodies was confirmed by a Western blot analysis using both recombinant proteins and whole parasite extract. Importantly, a urinary antigen detection assay assembled with pairs of antibodies specific for each of these antigens identified 17 of 19 patients with VL. These results indicate that an improved antigen detection assay based onL. infantumproteins present in the urine of patients with VL may represent an important new strategy for the development of a specific and accurate diagnostic test that has the potential to both distinguish active VL from asymptomatic infection and serve as an important tool to monitor therapy efficacy.

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 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.

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