scholarly journals Low-Dose Priming before Vaccination with the Phase I Chloroform-Methanol Residue Vaccine against Q Fever Enhances Humoral and Cellular Immune Responses to Coxiella burnetii

2008 ◽  
Vol 15 (10) ◽  
pp. 1505-1512 ◽  
Author(s):  
David M. Waag ◽  
Marilyn J. England ◽  
Christopher R. Bolt ◽  
Jim C. Williams
Keyword(s):  
Phase I ◽  
Coxiella Burnetii ◽  
Booster Dose ◽  
Q Fever ◽  
Vaccine Dose ◽  
Controlled Study ◽  
Enzyme Linked Immunosorbent Assay ◽  
Dependent Manner ◽  
Chloroform Methanol

ABSTRACT Although the phase I Coxiella burnetii cellular vaccine is completely efficacious in humans, adverse local and systemic reactions may develop if immune individuals are inadvertently vaccinated. The phase I chloroform-methanol residue (CMRI) vaccine was developed as a potentially safer alternative. Human volunteers with no evidence of previous exposure to C. burnetii received a subcutaneous vaccination with the CMRI vaccine in phase I studies under protocol IND 3516 to evaluate the safety and immunogenicity of the vaccine. This clinical trial tested escalating doses of the CMRI vaccine, ranging from 0.3 to 60 μg, followed by a booster dose of 30 μg, in a placebo-controlled study. Although priming doses of the CMRI vaccine did not induce a specific antibody detectable by enzyme-linked immunosorbent assay, booster vaccination stimulated the production of significant levels of anti-C. burnetii antibody. Peripheral blood cells (PBCs) of vaccinees responded to C. burnetii cellular antigen in vitro in a vaccine dose-dependent manner. After the booster dose, PBCs were activated by recall antigen in vitro, regardless of the priming dose. These findings suggest that vaccination with the CMRI vaccine can effectively prime the immune system to mount significant anamnestic responses after infection.

scholarly journals Candidate Antigens for Q Fever Serodiagnosis Revealed by Immunoscreening of a Coxiella burnetii Protein Microarray

2008 ◽  
Vol 15 (12) ◽  
pp. 1771-1779 ◽  
Author(s):  
Paul A. Beare ◽  
Chen Chen ◽  
Timo Bouman ◽  
Jozelyn Pablo ◽  
Berkay Unal ◽  
...  
Keyword(s):  
Coxiella Burnetii ◽  
Q Fever ◽  
Protein Microarray ◽  
Protein S ◽  
In Vitro Transcription ◽  
Cross Reactivity ◽  
Open Reading Frames ◽  
Enzyme Linked Immunosorbent Assay ◽  
Translation System

ABSTRACT Q fever is a widespread zoonosis caused by Coxiella burnetii. Diagnosis of Q fever is usually based on serological testing of patient serum. The diagnostic antigen of test kits is formalin-fixed phase I and phase II organisms of the Nine Mile reference strain. Deficiencies of this antigen include (i) potential for cross-reactivity with other pathogens; (ii) an inability to distinguish between C. burnetii strains; and (iii) a need to propagate and purify C. burnetii, a difficult and potentially hazardous process. Consequently, there is a need for sensitive and specific serodiagnostic tests utilizing defined antigens, such as recombinant C. burnetii protein(s). Here we describe the use of a C. burnetii protein microarray to comprehensively identify immunodominant antigens recognized by antibody in the context of human C. burnetii infection or vaccination. Transcriptionally active PCR products corresponding to 1,988 C. burnetii open reading frames (ORFs) were generated. Full-length proteins were successfully synthesized from 75% of the ORFs by using an Escherichia coli-based in vitro transcription and translation system (IVTT). Nitrocellulose microarrays were spotted with crude IVTT lysates and probed with sera from acute Q fever patients and individuals vaccinated with Q-Vax. Immune sera strongly reacted with approximately 50 C. burnetii proteins, including previously identified immunogens, an ankyrin repeat-domain containing protein, and multiple hypothetical proteins. Recombinant protein corresponding to selected array-reactive antigens was generated, and the immunoreactivity was confirmed by enzyme-linked immunosorbent assay. This sensitive and high-throughput method for identifying immunoreactive C. burnetii proteins will aid in the development of Q fever serodiagnostic tests based on recombinant antigen.

Minimizing the risk of occupational Q fever exposure: A protocol for ensuring Coxiella burnetii–negative pregnant ewes are used for medical research

2020 ◽  
pp. 002367722096562
Author(s):  
Laura A Galganski ◽  
Benjamin A Keller ◽  
Connor Long ◽  
Kaeli J Yamashiro ◽  
Mennatalla S Hegazi ◽  
...  
Keyword(s):  
Amniotic Fluid ◽  
Phase I ◽  
Phase Ii ◽  
Coxiella Burnetii ◽  
Q Fever ◽  
Buffy Coat ◽  
Ovis Aries ◽  
Enzyme Linked Immunosorbent Assay ◽  
Research Facility ◽  
Increased Risk

Q fever is a worldwide zoonosis caused by Coxiella burnetii that can lead to abortion, endocarditis, and death in humans. Researchers utilizing parturient domestic ruminants, including sheep, have an increased risk of occupational exposure. This study evaluated the effectiveness of our screening protocol in eliminating C. burnetii–positive sheep from our facility. From August 2010 to May 2018, all ewes ( N  =  306) and select lambs ( N  =  272; ovis aries) were screened twice for C. burnetii utilizing a serum Phase I and Phase II antibody immunofluorescence assay (IFA). The first screen was performed by the vendor prior to breeding, and the second screen was performed on arrival to the research facility. Ewes that were positive on arrival screening were quarantined and retested using repeat IFA serology, enzyme-linked immunosorbent assay, buffy coat polymerase chain reaction (PCR), and amniotic fluid PCR. The overall individual seroprevalence of C. burnetii in the flocks tested by the vendor was 14.2%. Ewes with negative Phase I and Phase II IFA results were selected for transport to the research facility. Upon arrival to the facility, two (0.7%) ewes had positive Phase I IFA results. Repeat testing demonstrated seropositivity in one of these two ewes, though amniotic fluid PCR was negative in both. The repeat seropositive ewe was euthanized prior to use in a research protocol. No Q fever was reported among husbandry, laboratory or veterinary staff during the study period. Serologic testing for C. burnetii with IFA prior to transport and following arrival to a research facility limits potential exposure to research staff.

scholarly journals Validation of a Novel Commercial ELISA Test for the Detection of Antibodies against Coxiella burnetii

Pathogens ◽  
2020 ◽  
Vol 9 (12) ◽  
pp. 1075
Author(s):  
Salvatore Ledda ◽  
Cinzia Santucciu ◽  
Valentina Chisu ◽  
Giovanna Masala
Keyword(s):  
Diagnostic Accuracy ◽  
Phase Ii ◽  
Coxiella Burnetii ◽  
Q Fever ◽  
Animal Health ◽  
Elisa Test ◽  
Clinical Diagnostics ◽  
Complex Life Cycle ◽  
Enzyme Linked Immunosorbent Assay ◽  
Specificity And Sensitivity

Q fever is a zoonosis caused by Coxiella burnetii, a Gram-negative pathogen with a complex life cycle and a high impact on public and animal health all over the world. The symptoms are indistinguishable from those belonging to other diseases, and the disease could be symptomless. For these reasons, reliable laboratory tests are essential for an accurate diagnosis. The aim of this study was to validate a novel enzyme-linked immunosorbent assay (ELISA) test, named the Chorus Q Fever Phase II IgG and IgM Kit (DIESSE, Diagnostica Senese S.p.A), which is performed by an instrument named Chorus, a new device in medical diagnostics. This diagnostic test is employed for the detection of antibodies against C. burnetii Phase II antigens in acute disease. Our validation protocol was performed according to the Italian Accreditation Body (ACCREDIA) (Regulation UNI CEI EN ISO/IEC 17025:2018 and 17043:2010), OIE (World Organization for Animal Health), and Statement for Reporting Studies of Diagnostic Accuracy (STARD). Operator performance was evaluated along with the analytical specificity and sensitivity (ASp and ASe) and diagnostic accuracy of the kit, with parameters such as diagnostic specificity and sensitivity (DSp and DSe) and positive and negative predictive values (PPV and NPV), in addition to the repeatability. According to the evaluated parameters, the diagnostic ELISA test was shown to be suitable for validation and commercialization as a screening method in human sera and a valid support for clinical diagnostics.

scholarly journals Contributions of lipopolysaccharide and the type IVB secretion system to Coxiella burnetii vaccine efficacy and reactogenicity

npj Vaccines ◽  
2021 ◽  
Vol 6 (1) ◽  
Author(s):  
Carrie M. Long ◽  
Paul A. Beare ◽  
Diane C. Cockrell ◽  
Jonathan Fintzi ◽  
Mahelat Tesfamariam ◽  
...  
Keyword(s):  
Phase I ◽  
Coxiella Burnetii ◽  
Vaccine Efficacy ◽  
Q Fever ◽  
Secretion System ◽  
Cell Vaccine ◽  
Post Vaccination ◽  
Whole Cell Vaccine ◽  
Type Ivb Secretion ◽  
Type Ivb Secretion System

AbstractCoxiella burnetii is the bacterial causative agent of the zoonosis Q fever. The current human Q fever vaccine, Q-VAX®, is a fixed, whole cell vaccine (WCV) licensed solely for use in Australia. C. burnetii WCV administration is associated with a dermal hypersensitivity reaction in people with pre-existing immunity to C. burnetii, limiting wider use. Consequently, a less reactogenic vaccine is needed. Here, we investigated contributions of the C. burnetii Dot/Icm type IVB secretion system (T4BSS) and lipopolysaccharide (LPS) in protection and reactogenicity of fixed WCVs. A 32.5 kb region containing 23 dot/icm genes was deleted in the virulent Nine Mile phase I (NMI) strain and the resulting mutant was evaluated in guinea pig models of C. burnetii infection, vaccination-challenge, and post-vaccination hypersensitivity. The NMI ∆dot/icm strain was avirulent, protective as a WCV against a robust C. burnetii challenge, and displayed potentially altered reactogenicity compared to NMI. Nine Mile phase II (NMII) strains of C. burnetii that produce rough LPS, were similarly tested. NMI was significantly more protective than NMII as a WCV; however, both vaccines exhibited similar reactogenicity. Collectively, our results indicate that, like phase I LPS, the T4BSS is required for full virulence by C. burnetii. Conversely, unlike phase I LPS, the T4BSS is not required for vaccine-induced protection. LPS length does not appear to contribute to reactogenicity while the T4BSS may contribute to this response. NMI ∆dot/icm represents an avirulent phase I strain with full vaccine efficacy, illustrating the potential of genetically modified C. burnetii as improved WCVs.

scholarly journals Effect ofTityus serrulatusvenom on cytokine production and the activity of murine macrophages

2002 ◽  
Vol 11 (1) ◽  
pp. 23-31 ◽  
Author(s):  
Vera L. Petricevich
Keyword(s):  
Cytokine Production ◽  
Peritoneal Macrophages ◽  
Interferon Γ ◽  
Enzyme Linked Immunosorbent Assay ◽  
No Production ◽  
Dependent Manner ◽  
Immune Functions ◽  
Macrophage Activity ◽  
Ifn Γ

The purpose of this study was to investigate the effects ofTityus serrulatusvenom (TSV) on murine peritoneal macrophages evaluated in terms of activation. The effects of crude TSV were analysed by detection of cytokines, oxygen intermediate metabolites (H2O2) and nitric oxide (NO) in supernatants of peritoneal macrophages. Several functional bioassays were employed including anin vitromodel for envenomating: cytotoxicity of TSV was assessed using the lyses percentage. Tumor necrosis factor (TNF) activity was assayed by measuring its cytotoxic activity on L-929 cells, and interleukin-6 (IL-6) and interferon-γ (IFN-γ) were assayed by enzyme-linked immunosorbent assay, whereas NO levels were detected by Griess colorimetric reactions in culture supernatant of macrophages incubated with TSV and subsequently exposed to either lipopolysaccharide or IFN-γ. Incubation of macrophages with TSV increased production of IL-6 and IFN-γ in a dose-dependent manner. TNF production was not detected in supernatants treated with TSV at any concentration. The increase in IL-6 secretion was not associated with concentration-dependent cytoxicity of TSV on these cells. These data suggest that the cytotoxicity does not appear to be the main cause of an increased cytokine production by these cells. Although NO is an important effector molecule in macrophage microbicidal activity, the inducing potential of the test compounds for its release was found to be very moderate, ranging from 125 to 800 mM. Interestingly, NO levels of peritoneal macrophages were increased after IFN-γ. Moreover, NO production had an apparent effect on macrophage activity. The results obtained here also shown that the TSV induces an important elevation in H2O2release. These results combined with NO production suggest that TSV possesses significant immunomodulatory activities capable of stimulating immune functionsin vitro.

Production and utilization of monoclonal antibodies to human/rat corticotrophin-releasing factor-41

1990 ◽  
Vol 5 (2) ◽  
pp. 159-166 ◽  
Author(s):  
N. G. N. Milton ◽  
E. W. Hillhouse ◽  
S. A. Nicholson ◽  
C. H. Self ◽  
A. M. McGregor
Keyword(s):  
Monoclonal Antibodies ◽  
Enzyme Linked Immunosorbent Assay ◽  
Dependent Manner ◽  
Corticotrophin Releasing Factor ◽  
Tissue Extracts ◽  
Rat Pituitary ◽  
Hypothalamic Tissue ◽  
Dose Dependent

ABSTRACT Murine monoclonal antibodies against human/rat corticotrophin-releasing factor-41 (CRF-41) were produced and characterized for use in the immunological and biological characterization of CRF-41. Spleen cells from BALB/c mice immunized with CRF-41 conjugated to bovine γ-globulin were fused with a BALB/c-derived non-secretor X-63 myeloma line. Hybridomas were selected for CRF antibody production by enzyme-linked immunosorbent assay, and positive hybridomas cloned twice. Three monoclonal antibodies were obtained (KCHMB001, KCHMB002 and KCHMB003) and characterized as IgG1, IgG1 and IgG2a isotypes respectively, with affinity constants for rat CRF-41 of 30, 53 and 34 nmol/l respectively. All three monoclonal antibodies recognize an epitope contained between residues 34 and 41 of the human/rat sequence. The antibodies were able to neutralize the ACTH-releasing activity of rat CRF-41, applied to rat pituitary fragments in vitro, in a dose-dependent manner. Isoelectric focusing showed that KCHMB 003 detected bands of synthetic rat CRF-41 and rat [Met(O)21,38]-CRF-41 at pH 7·1 and 6·8 respectively. Use of KCHMB003 in a two-site enzyme-amplified immunoassay showed that this antibody recognizes both synthetic rat CRF-41 and immunoreactive CRF-41 in rat hypothalamic tissue extracts.

Seroprevalence of Coxiella burnetii in sheep flocks in Kaduna State, Northwestern Nigeria

2021 ◽  
Author(s):  
Shuaibu Gidado Adamu ◽  
Junaidu Kabir ◽  
Jarlath Udo Umoh ◽  
Mashood Abiola Raji
Keyword(s):  
Risk Factors ◽  
Coxiella Burnetii ◽  
Q Fever ◽  
Jugular Vein ◽  
Cross Sectional Study ◽  
Enzyme Linked Immunosorbent Assay ◽  
Sectional Study ◽  
High Seroprevalence ◽  
Cross Sectional ◽  
Significant Difference

Abstract A cross-sectional study was carried out to determine the seroprevalence and risk factors of Q fever in sheep in the northern part of Kaduna State, Nigeria. This study aimed to determine Coxiella burnetii infection and its risk factors in sheep in Kaduna State. A total of 400 blood samples consisting of 259 samples from females and 141 from males were aseptically collected from the jugular vein of sheep from flocks in Kaduna State. The sera obtained were screened for Q fever using an indirect enzyme-linked immunosorbent assay (iELISA). The obtained data were analysed to determine whether there is a relationship between sex, age, and the animals tested. The analysis revealed that 8.0% of the sera was seropositive by iELISA. There was no significant difference in Q fever seropositivity in the study area according to the sex of sheep (P > 0.05). There was a statistically significant difference (P < 0.05) in Q fever seropositivity according to the age of sheep. This study indicated a high seroprevalence of Q fever mainly among female animals and older sheep. Further studies are required to determine the epizootiology of Q fever in the study area more precisely.

scholarly journals Evaluation of the Diagnostic Potential of Recombinant Coxiella burnetii Com1 in an ELISA for the Diagnosis of Q Fever in Sheep, Goats and Cattle

2020 ◽  
Vol 8 (8) ◽  
pp. 1235 ◽  
Author(s):  
Mareike Stellfeld ◽  
Claudia Gerlach ◽  
Ina-Gabriele Richter ◽  
Peter Miethe ◽  
Dominika Fahlbusch ◽  
...  
Keyword(s):  
Sensitivity And Specificity ◽  
Coxiella Burnetii ◽  
Q Fever ◽  
Roc Curves ◽  
Diagnostic Tools ◽  
Enzyme Linked Immunosorbent Assay ◽  
Domestic Ruminants ◽  
Diagnostic Potential ◽  
Assay Performance ◽  
Higher Sensitivity

Coxiella burnetii is the causative agent of Q fever, a zoonosis infecting domestic ruminants and humans. Currently used routine diagnostic tools offer limited sensitivity and specificity and symptomless infected animals may be missed. Therefore, diagnostic tools of higher sensitivity and specificity must be developed. For this purpose, the C. burnetii outer membrane protein Com1 was cloned and expressed in Escherichia coli. The His-tagged recombinant protein was purified and used in an indirect enzyme-linked immunosorbent assay (ELISA). Assay performance was tested with more than 400 positive and negative sera from sheep, goats and cattle from 36 locations. Calculation of sensitivity and specificity was undertaken using receiver operating characteristic (ROC) curves. The sensitivities and specificities for sheep were 85% and 68% (optical density at 450nm, OD450 cut-off value 0.32), for goats 94% and 77% (OD450 cut-off value 0.23) and for cattle 71% and 70% (OD450 cut-off value 0.18), respectively. These results correspond to excellent, outstanding and acceptable discrimination of positive and negative sera. In summary, recombinant Com1 can provide a basis for more sensitive and specific diagnostic tools in veterinary medicine.

scholarly journals The Effect of pH on Antibiotic Efficacy against Coxiella burnetii in Axenic Media

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Cody B. Smith ◽  
Charles Evavold ◽  
Gilbert J. Kersh
Keyword(s):  
Coxiella Burnetii ◽  
Antimicrobial Agents ◽  
Q Fever ◽  
Antimicrobial Treatment ◽  
Etiologic Agent ◽  
Growth And Survival ◽  
Intracellular Compartments ◽  
The Impact

AbstractCoxiella burnetii, the etiologic agent of Q fever, replicates in an intracellular phagolysosome with pH between 4 and 5. The impact of this low pH environment on antimicrobial treatment is not well understood. An in vitro system for testing antibiotic susceptibility of C. burnetii in axenic media was set up to evaluate the impact of pH on C. burnetii growth and survival in the presence and absence of antimicrobial agents. The data show that C. burnetii does not grow in axenic media at pH 6.0 or higher, but the organisms remain viable. At pH of 4.75, 5.25, and 5.75 moxifloxacin, doxycycline, and rifampin are effective at preventing growth of C. burnetii in axenic media, with moxifloxacin and doxycycline being bacteriostatic and rifampin having bactericidal activity. The efficacy of doxycycline and moxifloxacin improved at higher pH, whereas rifampin activity was pH independent. Hydroxychloroquine is thought to inhibit growth of C. burnetii in vivo by raising the pH of typically acidic intracellular compartments. It had no direct bactericidal or bacteriostatic activity on C. burnetii in axenic media, suggesting that raising pH of acidic intracellular compartments is its primary mechanism of action in vivo. The data suggest that doxycycline and hydroxychloroquine are primarily independent bacteriostatic agents.

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