Outer surface protein E (OspE) mediates Borrelia burgdorferi sensu stricto strain-specific complement evasion in the eastern fence lizard, Sceloporus undulatus.

In North America, Lyme disease is primarily caused by the spirochetal bacterium Borrelia burgdorferi sensu stricto (Bb), which is transmitted between multiple vertebrate hosts and ixodid ticks, and is a model commonly used to study host-pathogen interactions. While Bb is consistently observed in its mammalian and avian reservoirs, the bacterium is rarely isolated from North American reptiles. Two closely related lizard species, the eastern fence lizard (Sceloporus undulatus) and the western fence lizard (Sceloporus occidentalis), are examples of reptiles parasitized by Ixodes ticks. Vertebrates are known to generate complement as an innate defense mechanism, which can be activated before Bb disseminate to distal tissues. Complement from western fence lizards has proven lethal against one Bb strain, implying the role of complement in making those lizards unable to serve as hosts to Bb. However, Bb DNA is occasionally identified in distal tissues of field-collected eastern fence lizards, suggesting some Bb strains may overcome complement-mediated clearance in these lizards. These findings raise questions regarding the role of complement and its impact on Bb interactions with North American lizards. In this study, we found Bb seropositivity in a small population of wild-caught eastern fence lizards and observed Bb strain-specific survivability in lizard sera. We also found that a Bb outer surface protein, OspE, from Bb strains viable in sera, promotes lizard serum survivability and binds to a complement inhibitor, factor H, from eastern fence lizards. Our data thus identify bacterial and host determinants of eastern fence lizard complement evasion, providing insights into the role of complement influencing Bb interactions with North American lizards.

Prevalence of Rh and K phenotypes among blood donors from different ethnicities in Samtah (Southwestern Region) Saudi Arabia.

INTRODUCTION: Rh and Kell blood group systems are amongst the most important blood group systems; being highly immunogenic after ABO system. The aim of this study was to evaluate the frequencies of Rh antigens, haplotypes and K antigen among blood donors belonging to various ethnicities in Samtah, Jazan, Saudi Arabia. METHODS: This study was conducted during January 2019 and August 2020 at Samtah General Hospital, Samtah. Records of all blood donors recruited during this period were included for data acquisition. A total of 4977 blood donors' records were reviewed and data were analysed. A total of 3863 donors' results were considered in the final analysis. RESULTS: In comparison to Saudi blood donors, C antigen was less frequent in Sudanese donors (69.7% and 34.0%), the c antigen was less frequent in Indian (79.2% and 59.3%) and Philippine (79.2% and 40.0%) donors and more frequent in Sudanese (79.2% and 97.9%) donors, the E antigen was less frequent in Yemini (27.0% and 19.5%) and the e antigen was more frequent in Yemini (96.7% and 99.2%) donors. The DcE haplotype was less frequent (3.1% and 0.7%) and the ce haplotype was more frequent (4.3% and 7.6%) in Yemini donors. The K antigen was less frequent in Pakistani (11.9% and 4.1%; p = .041) and Indian (11.9% and 1.9%; p = .023) donors. CONCLUSION: Rh and K antigens showed marked variations in their frequencies among blood donors of different ethnicities. Utilization of blood from various ethnicities warrant extended phenotyping of Rh and K antigens to avoid the risk of alloimmunization in multiply transfused patients.

Association between serum vitamin D levels and Helicobacter pylori cytotoxic-associated gene A seropositivity: a cross-sectional study in US adults from NHANES III.

OBJECTIVE: To assess the association of serum vitamin D (VD) levels and Helicobacter pylori (H. pylori) cytotoxic-associated gene A (CagA) seropositivity, and further explore potential effect modifiers in this association. DESIGN: Cross-sectional study. SETTING: Data from phase I of the National Health and Nutrition Examination Survey (NHANES III, 1988-1991) led by the Center for Disease Control and Prevention. PARTICIPANTS: A total of 3512 US adults (≥20 years) with both serum VD levels and H. pylori CagA antibody data from NHANES III were included in the analysis. METHODS: VD deficiency was defined as serum 25(OH)D concentrations<20 ng/mL. Logistic regression models were used to assess the association of serum VD levels and H. pylori CagA seropositivity (VD-Hp CagA+), and stratification analyses were used to explore potential effect modifiers. RESULTS: There was no significant association of VD-Hp CagA+ in the general population. But serum 25(OH)D concentrations were associated with H. pylori CagA+ in non-Hispanic whites (adjusted OR=1.02, 95% CI: 1.00 to 1.03), other races/ethnicities (adjusted OR=1.08, 95% CI: 1.01 to 1.06), populations born in other countries (adjusted OR=1.09, 95% CI: 1.04 to 1.15) or occasional drinkers (adjusted OR=0.93, 95% CI: 0.88 to 0.99). VD deficiency was associated with H. pylori CagA+ in non-Hispanic whites (adjusted OR=0.69, 95% CI: 0.53 to 0.92), populations born in other countries (adjusted OR=0.47, 95% CI: 0.25 to 0.89), non-drinkers (adjusted OR=0.80, 95% CI: 0.65 to 0.99), occasional drinkers (adjusted OR=2.53, 95% CI: 1.06 to 6.05), population with first quartile level of serum ferritin (adjusted OR=0.70, 95% CI: 0.51 to 0.96) or fourth quartile level of serum folate (adjusted OR=0.63, 95% CI: 0.46 to 0.87). CONCLUSIONS: Racial/ethnic differences and different serum ferritin or serum folate levels may be effect modifiers for the association of VD-Hp CagA+.

Rapid Detection of Carbapenemase-producing Enterobacteriaceae From Blood Culture Bottles of Known CPE Carriers: Real-world Experience.

We used a rapid antigen test for the detection of carbapenemases directly from positive blood culture bottles of pediatric hemato-oncologic patients, known carriers of carbapenemase-producing enterobacteriaceae. Resistance mechanism was detected within 15 minutes of observing Gram-negative bacilli from a positive bottle, leading to treatment modification. This simple-to-use, inexpensive assay shortens the interval between empiric to tailored antimicrobial therapy.

An age-stratified serosurvey against purified Salmonella enterica serovar Typhi antigens in the Lao People´s Democratic Republic.

The epidemiology of typhoid fever in Lao People`s Democratic Republic is poorly defined. Estimating the burden of typhoid fever in endemic countries is complex due to the cost and limitations of population-based surveillance; serological approaches may be a more cost-effective alternative. ELISAs were performed on 937 serum samples (317 children and 620 adults) from across Lao PDR to measure IgG antibody titers against Vi polysaccharide and the experimental protein antigens, CdtB and HlyE. We measured the significance of the differences between antibody titers in adults and children and fitted models to assess the relationship between age and antibody titers. The median IgG titres of both anti-HylE and CdtB were significantly higher in children compared to adults (anti-HylE; 351.7 ELISA Units (EU) vs 198.1 EU, respectively; p<0.0001 and anti-CdtB; 52.6 vs 12.9 EU; p<0.0001). Conversely, the median anti-Vi IgG titer was significantly higher in adults than children (11.3 vs 3.0 U/ml; p<0.0001). A non-linear trend line fitted to the anti-CdtB and anti-HlyE IgG data identified a peak in antibody concentration in children <5 years of age. We identified elevated titers of anti-HlyE and anti-CdtB IgG in the serum of children residing in Lao PDR in comparison to adults. These antigens are associated with seroconversion after typhoid fever and may be a superior measure of disease burden than anti-Vi IgG. This approach is scalable and may be developed to assess the burden of typhoid fever in countries where the disease may be endemic, and evidence is required for the introduction of typhoid vaccines.

An electrochemical aptasensor for Mycobacterium tuberculosis ESAT-6 antigen detection using bimetallic organic framework.

A label-free electrochemical aptasensor is reported for sensitive detection of the 6-kDa early secreted antigenic target (ESAT-6). For the first time, the bimetallic organic framework (b-MOF) of Zr-MOF-on-Ce-MOF was decorated with nitrogen-doped graphene (NG) and applied as the matrix for electroactive toluidine blue (Tb) to form the NG@Zr-MOF-on-Ce-MOF@Tb nanohybrid. The prepared nanohybrid with excellent hydrophilicity, dispersibility, and large specific surface exhibited significant electrochemical response. This nanohybrid could be directly used for anchoring ESAT-6 binding aptamers (EBA) through the interaction between the 5'-phosphate group (PO43-) of EBA and Zr4+ of Zr-MOF. The signal response before and after incubating the ESAT-6 antigen has been evaluated by cyclic voltammetry at a scan rate of 100 mV s-1 from - 0.7 to 0.3 V (vs. SCE). Under optimal conditions, the proposed aptasensor displayed a wide linear range from 100 fg mL-1 to 10 ng mL-1 with a limit of detection (LOD) of 12 fg mL-1. The developed method showed good reproducibility with a relative standard deviation (RSD) of 2.27%. The aptasensor showed favorable results in the analysis of the real samples. With these merits, the aptasensor has exceptional potential as a diagnostic tool for tuberculosis in clinical practice.

Detection and quantification of Mycobacterium tuberculosis antigen CFP10 in serum and urine for the rapid diagnosis of active tuberculosis disease.

Outside of the ongoing COVID-19 pandemic, tuberculosis is the leading cause of infectious disease mortality globally. Currently, there is no commercially available point-of-care diagnostic that is rapid, inexpensive, and highly sensitive for the diagnosis of active tuberculosis disease. Here we describe the development and optimization of a novel, highly sensitive prototype bioelectronic tuberculosis antigen (BETA) assay to detect tuberculosis-specific antigen, CFP10, in small-volume serum and urine samples. In this proof-of-concept study we evaluated the performance of the BETA assay using clinical specimens collected from presumptive tuberculosis patients from three independent cohorts. Circulating CFP10 antigen was detected in ALL serum (n = 19) and urine (n = 3) samples from bacteriologically confirmed tuberculosis patients who were untreated or had less than one week of treatment at time of serum collection, successfully identifying all culture positive tuberculosis patients. No CFP10 antigen was detected in serum (n = 7) or urine (n = 6) samples from individuals who were determined to be negative for tuberculosis disease. Additionally, antigen quantification using the BETA assay of paired serum samples collected from tuberculosis patients (n = 8) both before and after treatment initiation, indicate consistently declining within-person levels of CFP10 antigen during treatment. This novel, low-cost assay demonstrates potential as a rapid, non-sputum-based, point-of-care tool for the diagnosis of tuberculosis disease.

Identification of a Novel Antigen for Serological Diagnosis of Scrub Typhus.

Scrub typhus is an acute infectious disease caused by the bacterium Orientia tsutsugamushi, which is widely distributed in northern, southern, and eastern Asia. Early diagnosis is essential because the average case fatality rate is usually >10% but can be as high as 45% if antimicrobial treatment is delayed. Although an O. tsutsugamushi 56-kDa type-specific antigen (TSA) is commonly used for serological diagnosis of scrub typhus, the 56-kDa TSA shows variations among O. tsutsugamushi strains, which may lead to poor diagnostic results. Therefore, the discovery of new antigenic proteins may improve diagnostic accuracy. In this study, we identified an O. tsutsugamushi 27 kDa antigen through an immunoinformatic approach and verified its diagnostic potential using patient samples. Compared with the O. tsutsugamushi 56-kDa antigen, the new 27-kDa antigen showed better diagnostic specificity with similar diagnostic sensitivity. Therefore, the O. tsutsugamushi 27-kDa antigen shows potential as a novel serological diagnostic antigen for scrub typhus, providing higher diagnostic accuracy for O. tsutsugamushi than the 56-kDa antigen.

The Performance of Immunoassays to Measure Antibodies to the Chlamydia trachomatis Antigen Pgp3 in Different Epidemiological Settings for Trachoma.

Programs to eliminate trachoma as a public health problem use prevalence of the clinical sign trachomatous inflammation-follicular (TF) in 1- to 9-year-olds in endemic districts to make decisions to begin or end mass drug administration with azithromycin. Trachomatous inflammation-follicular is used as a proxy for transmission of ocular Chlamydia trachomatis infection. Long-term monitoring of previously endemic districts for recrudescence of ocular C. trachomatis infection would benefit from a simple blood test that could be integrated with other public health programs. In this study, we evaluated multiple tests to measure antibodies against the C. trachomatis antigen Pgp3-a multiplex bead assay (MBA), an ELISA, and two versions of a lateral flow assay (LFA)-in four districts of the Amhara region of Ethiopia with varying levels of TF. Seroprevalence and seroconversion rate (SCR) results were proportional to TF prevalence by district for most tests, with the notable exception of the LFA using colloidal gold as the developing reagent. Changing the test developing reagent to black latex improved agreement between serological measures and TF prevalence and in inter-rater agreement. Seroconversion rate estimates using data derived from the LFA-gold assay were inconsistent with the shape of the age-seroprevalence curve, which did not increase in older ages. These data revealed potential complications with using SCR that will need further evaluation. Data from MBA, ELISA, and LFA with the black test line showed good agreement with each other and proportionality to TF estimates, providing further data that serology has potential utility for trachoma surveillance.

Retrospective Performance Analyses of over Two Million U.S. QuantiFERON Blood Sample Results.

Both the QuantiFERON-TB Gold Plus (QFT-Plus) and the QuantiFERON-TB Gold In-Tube (QFT-GIT) tests are interferon gamma (IFN-γ) release assays (IGRAs) intended to detect in vitro cell-mediated immune responses to Mycobacterium tuberculosis antigens. In this study, we retrospectively analyzed performance data for both the QFT-GIT and QFT-Plus test systems from over 2 million samples. QFT-Plus and QFT-GIT testing was performed as specified in the respective package inserts at 23 Quest Diagnostics sites. Blood specimens were collected from individuals in all 50 states from November 2018 through December 2019. Retrospective analyses compared the proportion of positive, indeterminate, and conversion/reversion results. The overall proportion of QFT-positive results was 7% for both the QFT-Plus and QFT-GIT. The proportion of positive results was highest for QFT-GIT (7.5%) followed by the heparin 1-tube QFT-Plus (7.2%); a lower proportion of positives was observed with the 4-tube (all four QFT tubes were used in blood collection) QFT-Plus (6.0%). The proportions of indeterminate results for the 1-tube (heparin-only tube collection) and 4-tube QFT-Plus methods were less than 1% and 4%, respectively. This study indicates a higher proportion of positive results for M. tuberculosis than data from other studies. Additionally, the proportion of indeterminate QFT results were markedly lower when the sample was transported in one lithium-heparin tube instead of direct inoculation into 4 QFT-Plus tubes at the site of blood collection. IMPORTANCE In this study, we retrospectively analyzed results from both the QFT-GIT and QFT-Plus test systems from over 2 million blood specimens. The variables analyzed were (i) QFT positivity rates among various U.S. populations, (ii) indeterminate rates among various types of blood draws and how often an indeterminate result was resolved within 30 days after the initial draw, and (iii) the association of TB1 and TB2 antigen tubes with IGRA reversion and conversion events from serial QFT testing. This is, to our knowledge, the largest QFT study representing patients from an extensive geographic coverage across the United States and U.S. territories.

Mapping the distribution and risk factors of Anaplasmataceae in wild and domestic canines in Chile and their association with Rhipicephalus sanguineus species complex lineages.

Anaplasma platys and Ehrlichia canis are members of the Anaplasmataceae family that cause disease in dogs and are mainly transmitted by Rhipicephalus sanguineus species group ticks. We performed a cross-sectional study on these pathogens across six bioclimatic regions of Chile, including 719 free-ranging rural dogs, 132 Andean foxes (Lycalopex culpaeus), and 82 South American gray foxes (Lycalopex griseus). Dog and fox blood samples were first screened for DNA of Anaplasmataceae followed by two Ehrlichia-specific protocols. Antibodies against Anaplasma sp. and E. canis were assessed by immunofluorescence in dogs. Ectoparasites were collected and identified, with the determination of the lineages of the Rhipicephalus sanguineus species group by molecular and phylogenetic analyses. Finally, potential risk factors for infection were investigated across the different bioclimatic regions and host species. All DNA amplicons obtained from the screening protocol corresponded to Anaplasma platys. The occurrence of both A. platys DNA and antibodies was confirmed in all six bioclimatic regions, except for regions at high altitude and/or without either R. sanguineus species group lineage present. Dogs infested with R. sanguineus ticks were significantly more prone to be infected and exposed to Anaplasma spp. Prevalence of DNA was significantly higher in juvenile (19%) than in adult dogs (9%), whereas the opposite was found for seroprevalence (19% versus 35%, respectively). Overall prevalence of A. platys DNA was higher in dogs (11%) than in foxes (4%), probably owing to markedly lower tick infestations in the foxes. Ehrlichia canis DNA was not detected in any sample, and antibodies against this pathogen were detected only in four dogs, in areas with both R. sanguineus lineages present. Free-ranging dogs in Chile could be favoring the maintenance of A. platys in all areas suitable for its tick vector. Although apparently infrequent, spillovers from dogs to foxes may be taking place and should be considered in management plans in Chile.

A primary Chlamydia trachomatis genital infection of rhesus macaques identifies new immunodominant B-cell antigens.

To identify immunodominant antigens that elicit a humoral immune response following a primary and a secondary genital infection, rhesus monkeys were inoculated cervically with Chlamydia trachomatis serovar D. Serum samples were collected and probed with a protein microarray expressing 864/894 (96.4%) of the open reading frames of the C. trachomatis serovar D genome. The antibody response to the primary infection was analyzed in 72 serum samples from 12 inoculated monkeys. The following criteria were utilized to identify immunodominant antigens: proteins found to be recognized by at least 75% (9/12) of the infected monkeys with at least 15% elevations in signal intensity from week 0 to week 8 post infection. All infected monkeys developed Chlamydia specific serum antibodies. Eight proteins satisfied the selection criteria for immunodominant antigens: CT242 (OmpH-like protein), CT541 (mip), CT681 (ompA), CT381 (artJ), CT443 (omcB), CT119 (incA), CT486 (fliY), and CT110 (groEL). Of these, three antigens, CT119, CT486 and CT381, were not previously identified as immunodominant antigens using non-human primate sera. Following the secondary infection, the antibody responses to the eight immunodominant antigens were analyzed and found to be quite different in intensity and duration to the primary infection. In conclusion, these eight immunodominant antigens can now be tested for their ability to identify individuals with a primary C. trachomatis genital infection and to design vaccine strategies to protect against a primary infection with this pathogen.

Alfalfa Plants (Medicago sativa L.) Expressing the 85B (MAP1609c) Antigen of Mycobacterium avium subsp. paratuberculosis Elicit Long-Lasting Immunity in Mice.

Mycobacterium avium subsp. paratuberculosis (MAP) is the etiological agent of Paratuberculosis, a contagious, untreatable, and chronic granulomatous enteritis that results in diarrhea, emaciation, and death in farmed ruminants (i.e., cattle, sheep, and goats). In this study, the Ag85B antigen from MAP was expressed in transgenic alfalfa as an attractive vaccine candidate. Agrobacterium-mediated transformation allowed the rescue of 56 putative transformed plants and transgenesis was confirmed in 19 lines by detection of the Ag85B gene (MAP1609c) by PCR. Line number 20 showed the highest Ag85B expression [840 ng Ag85B per gram of dry weight leaf tissue, 0.062% Total Soluble Protein (TSP)]. Antigenicity of the plant-made Ag85B was evidenced by its reactivity with a panel of sera from naturally MAP-infected animals, whereas immunogenicity was assessed in mice immunized by either oral or subcutaneous routes. The plant-made Ag85B antigen elicited humoral responses by the oral route when co-administered with cholera toxin as adjuvant; significant levels of anti-85B antibodies were induced in serum (IgG) and feces (IgA). Long-lasting immunity was evidenced at day 180 days post-first oral immunization. The obtained alfalfa lines expressing Ag85B constitute the first model of a plant-based vaccine targeting MAP. The initial immunogenicity assessment conducted in this study opens the path for a detailed characterization of the properties of this vaccine candidate.

Point-of-care detection of tuberculosis using magnetoresistive biosensing chip.

In this paper, a highly sensitive and specific technique based on the principle of giant magnetoresistance (GMR) has been proposed for the early stage Tuberculosis (TB) diagnostics. This GMR biosensing assay employs monoclonal antibodies against M. tuberculosis specific ESAT-6 antigen with the use of magnetic nanoparticles (MNPs) as labels. MNPs bind to the GMR sensor in presence of ESAT-6 and the binding is proportional to the ESAT-6 protein concentration leading to the change in overall resistance of GMR sensor. GMR biosensor simulation showed that ESAT-6 concentration can be detected in the range of pg/mL in comparison to the other transduction techniques available for ESAT-6 detection and further, the signal strength increased with the increase in the concentration. This work has shown that the GMR biosensing strategy is pertinent for the TB detection at the primitive phases when compared with other magnetic techniques used for TB diagnostics.

A glycoengineered antigen exploiting a conserved protein O-glycosylation pathway in the Burkholderia genus for detection of glanders infections.

We recently described a protein O-glycosylation pathway conserved in all species of the Burkholderia genus that results in the synthesis and incorporation of a trisaccharide glycan to membrane-exported proteins. Here, we exploited this system to construct and evaluate a diagnostic tool for glanders. Burkholderia mallei, the causative agent of glanders, is a highly infectious and fatal zoonotic pathogen that infects horses, mules, donkeys, and occasionally humans. A highly sensitive and specific diagnostic tool is crucial for the control, elimination, and eradication of B. mallei infections. We constructed plasmids carrying synthetic genes encoding a modified, previously unannotated Burkholderia glycoprotein containing three glycosylation sequons fused to the cholera toxin B-subunit. The resulting proteins were glycosylated in the B. cenocepacia K56-2 parental strain, but not in glycosylation-deficient mutants, as determined by SDS-PAGE and fluorescent lectin blots. One of these glycoproteins was used as an antigen in ELISA and western blots to screen a panel of serum samples collected from glanders-infected and healthy horses, which were previously investigated by complement fixation test and indirect ELISA based on a semi-purified fraction of B. mallei. We show that ELISA and western blot assays based on our glycoprotein antigen provide 100% specificity, with a sensitivity greater than 88%. The glycoprotein antigen was recognized by serum samples collected from patients infected with B. pseudomallei, B. mallei, B. multivorans, and B. cenocepacia. Our results indicate that protein O-glycosylation in Burkholderia can be exploited as a biomarker for diagnosis of Burkholderia-associated infections.

Clindamycin Protects Nonhuman Primates Against Inhalational Anthrax But Does Not Enhance Reduction of Circulating Toxin Levels When Combined With Ciprofloxacin.

BACKGROUND: Inhalational anthrax is rare and clinical experience limited. Expert guidelines recommend treatment with combination antibiotics including protein synthesis-inhibitors to decrease toxin production and increase survival, although evidence is lacking. METHODS: Rhesus macaques exposed to an aerosol of Bacillus anthracis spores were treated with ciprofloxacin, clindamycin, or ciprofloxacin + clindamycin after becoming bacteremic. Circulating anthrax lethal factor and protective antigen were quantitated pretreatment and 1.5 and 12 hours after beginning antibiotics. RESULTS: In the clindamycin group, 8 of 11 (73%) survived demonstrating its efficacy for the first time in inhalational anthrax, compared to 9 of 9 (100%) with ciprofloxacin, and 8 of 11 (73%) with ciprofloxacin + clindamycin. These differences were not statistically significant. There were no significant differences between groups in lethal factor or protective antigen levels from pretreatment to 12 hours after starting antibiotics. Animals that died after clindamycin had a greater incidence of meningitis compared to those given ciprofloxacin or ciprofloxacin + clindamycin, but numbers of animals were very low and no definitive conclusion could be reached. CONCLUSION: Treatment of inhalational anthrax with clindamycin was as effective as ciprofloxacin in the nonhuman primate. Addition of clindamycin to ciprofloxacin did not enhance reduction of circulating toxin levels.

Development of an in-house capture ELISA: An attempt to detect CagA antigen in sera of Helicobacter pylori infected patients.

The CagA protein one of the key virulence factors of Helicobacter pylori plays an important role in the pathogenesis of peptic ulcer diseases. Unfortunately the cagA gene status can only be determined by PCR while serology is an alternative approach to detect antigens or antibodies. Our aim is to detect the CagA antigen in sera of infected subjects by the development of an in-house capture ELISA test. Gastric antral biopsies and serum samples were collected from 63 patients. PCR was used to determine the cagA status. Our previously developed recombinant CagA protein and monoclonal antibody were used for setting up the capture ELISA test. H. pylori positive [(38 gastritis, 14 duodenal ulcers (DU), 11 gastric ulcer (GU)] patients were determined by PCR. The cagA gene was detected in 21 (55%) of gastritis, 11 (78%) of DU and 7 (60%) of GU patients. The reagents used in setting up the capture ELISA test following optimization displayed high performance. This study showed that our developed in-house capture ELISA has the potential to detect the CagA antigen at very low concentrations even though not detected in our H. pylori infected patients sera but we are also intended to use it in saliva and stool samples.

Analysis of recombinant proteins for Q fever diagnostics.

Serology is essential for Q fever diagnostics, a disease caused by the bacterial pathogen Coxiella burnetii. The gold standard test is an immunofluorescence assay utilizing whole cell antigens, which are both dangerous and laborious to produce. Complexities of the antigen coupled with the subjective nature of the assay lead to decreased uniformity of test results and underscore the need for improved methodologies. Thirty-three C. burnetii proteins, previously identified as immunoreactive, were screened for reactivity to naturally infected goat serum. Based on reactivity, 10 proteins were analyzed in a secondary screen against human serum from healthy donors. Assay sensitivity and specificity ranged from 21 to 71% and 90 to 100%, respectively. Three promising antigens were identified based on receiver operating characteristic curve analysis (CBU_1718, CBU_0307, and CBU_1398). Five multiplex assays failed to outperform the individual proteins, with sensitivities and specificities ranging from 29 to 57% and 90 to 100%, respectively. Truncating the top antigen, CBU_1718, had no effect on specificity (90%); yet sensitivity decreased dramatically (71% to 21%). Through this study, we have expanded the subset of C. burnetii immunoreactive proteins validated by enzyme-linked immunosorbent assay and demonstrate the effect of novel antigen combinations and protein truncations on assay performance.

Identification of secreted and cellular antigens of Staphylococcus aureus causing dairy sheep mastitis and their potential for vaccine development.

Staphylococcus aureus is the leading cause of clinical mastitis and is associated with persistent subclinical infections in ewes, significantly compromising the quality and quantity of milk productions. To date, vaccines intended for use in sheep have been mainly focused on biofilm production traits, but many S. aureus pathogenic isolates do not produce biofilm, including those circulating in Sardinia, one of the leading sheep milk producers in Europe. The aim of this work was to identify suitable immunodominant, alternative candidates to biofilm components for vaccine and diagnostic development. An immunoproteomics study was carried out by testing sera from naturally infected sheep with a prevalent S. aureus lineage against cellular and secreted antigens, followed by tandem mass spectrometry identification of the most prominent immunogens. Four cellular and three secreted S. aureus antigens elicited a strong humoral host immune response. The four cellular antigens were the housekeeping proteins pyruvate kinase, elongation Factor Tu, dihydrolipoyl dehydrogenase, and alpha-keto acid dehydrogenase. The three secreted antigens were the bifunctional autolysin (Atl) and the two components of the Panton-Valentine leukocidin, lukF-PV/lukM, demonstrating the carriage of prophage phiPV83 in a sheep isolate and the strong response of the sheep host against them. In consideration of the key role played by these secreted proteins in S. aureus replication and immune evasion, these antigens may represent suitable candidates for developing vaccines eliciting a more successful immunological protection in areas where non-biofilm forming Staphylococcus spp. are the most widespread intramammary pathogens.