TY - JOUR T1 - Third dose of COVID-19 mRNA vaccine closes the gap in immune response between naïve nursing home residents and healthy adults. JF - Vaccine Y1 - 2023 A1 - Pieter Pannus A1 - Stéphanie Depickère A1 - Delphine Kemlin A1 - Daphnée Georges A1 - Sarah Houben A1 - Véronique Olislagers A1 - Alexandra Waegemans A1 - Stéphane De Craeye A1 - Antoine Francotte A1 - Félicie Chaumont A1 - Celien Van Oostveldt A1 - Heyndrickx, Leo A1 - Johan Michiels A1 - Willems, Elisabeth A1 - Emilie Dhondt A1 - Marharyta Krauchuk A1 - Marie-Noëlle Schmickler A1 - Mathieu Verbrugghe A1 - Nele Van Loon A1 - Katelijne Dierick A1 - André Matagne A1 - I Desombere A1 - Ariën, Kevin K A1 - Arnaud Marchant A1 - Maria Goossens KW - Adult KW - Antibodies, Viral KW - BNT162 Vaccine KW - Breakthrough Infections KW - COVID-19 KW - COVID-19 Vaccines KW - Humans KW - Immunity KW - nursing homes KW - Population Groups KW - RNA, Messenger KW - SARS-CoV-2 AB -

BACKGROUND: Nursing home residents, a frail and old population group, respond poorly to primary mRNA COVID-19 vaccination. A third dose has been shown to boost protection against severe disease and death in this immunosenescent population, but limited data is available on the immune responses it induces.

METHODS: In this observational cohort study, peak humoral and cellular immune responses were compared 28 days after the second and third doses of the BNT162b2 mRNA COVID-19 vaccine in residents and staff members of two Belgian nursing homes. Only individuals without evidence of previous SARS-CoV-2 infection at third dose administration were included in the study. In addition, an extended cohort of residents and staff members was tested for immune responses to a third vaccine dose and was monitored for vaccine breakthrough infections in the following six months. The trial is registered on ClinicalTrials.gov (NCT04527614).

FINDINGS: All included residents (n = 85) and staff members (n = 88) were SARS-CoV-2 infection naïve at third dose administration. Historical blood samples from 28 days post second dose were available from 42 residents and 42 staff members. Magnitude and quality of humoral and cellular immune responses were strongly boosted in residents post third compared to post second dose. Increases were less pronounced in staff members than in residents. At 28 days post third dose, differences between residents and staff had become mostly insignificant. Humoral, but not cellular, responses induced by a third dose were predictive of subsequent incidence of vaccine breakthrough infection in the six months following vaccination.

INTERPRETATION: These data show that a third dose of mRNA COVID-19 vaccine largely closes the gap in humoral and cellular immune response observed after primary vaccination between NH residents and staff members but suggest that further boosting might be needed to achieve optimal protection against variants of concern in this vulnerable population group.

VL - 41 CP - 17 M3 - 10.1016/j.vaccine.2023.03.047 ER - TY - JOUR T1 - Third dose of COVID-19 mRNA vaccine closes the gap in immune response between naïve nursing home residents and healthy adults JF - Vaccine Y1 - 2023 A1 - Pieter Pannus A1 - Stéphanie Depickère A1 - Delphine Kemlin A1 - Daphnée Georges A1 - Sarah Houben A1 - Véronique Olislagers A1 - Alexandra Waegemans A1 - Stéphane De Craeye A1 - Antoine Francotte A1 - Félicie Chaumont A1 - Celien Van Oostveldt A1 - Heyndrickx, Leo A1 - Johan Michiels A1 - Willems, Elisabeth A1 - Emilie Dhondt A1 - Marharyta Krauchuk A1 - Marie-Noëlle Schmickler A1 - Mathieu Verbrugghe A1 - Nele Van Loon A1 - Katelijne Dierick A1 - André Matagne A1 - I Desombere A1 - Kevin K. Ariën A1 - Arnaud Marchant A1 - Maria Goossens VL - 41 CP - 17 M3 - 10.1016/j.vaccine.2023.03.047 ER - TY - JOUR T1 - Safety and immunogenicity of a reduced dose of the BNT162b2 mRNA COVID-19 vaccine (REDU-VAC): A single blind, randomized, non-inferiority trial JF - PLOS Global Public Health Y1 - 2022 A1 - Pieter Pannus A1 - Stéphanie Depickère A1 - Delphine Kemlin A1 - Sarah Houben A1 - Kristof Y. Neven A1 - Heyndrickx, Leo A1 - Johan Michiels A1 - Willems, Elisabeth A1 - Stéphane De Craeye A1 - Antoine Francotte A1 - Félicie Chaumont A1 - Véronique Olislagers A1 - Alexandra Waegemans A1 - Mathieu Verbrugghe A1 - Marie-Noëlle Schmickler A1 - Steven Van Gucht A1 - Katelijne Dierick A1 - Arnaud Marchant A1 - I Desombere A1 - Kevin K. Ariën A1 - Maria Goossens KW - COVID-19; fractional dose; reduced dose; mRNA vaccination; non-inferiority; SARS-CoV-2 AB -

Fractional dosing of COVID-19 vaccines could accelerate vaccination rates in low-income countries. Dose-finding studies of the mRNA vaccine BNT162b2 (Pfizer-BioNTech) suggest that a fractional dose induces comparable antibody responses to the full dose in people <55 years. Here, we report the safety and immunogenicity of a fractional dose regimen of the BNT162b2 vaccine. REDU-VAC is a participant-blinded, randomised, phase 4, non-inferiority study. Adults 18–55 years old, either previously infected or infection naïve, were randomly assigned to receive 20μg/20μg (fractional dose) or 30μg/30μg (full dose) of BNT162b2. The primary endpoint was the geometric mean ratio (GMR) of SARS-CoV-2 anti-RBD IgG titres at 28 days post second dose between the reduced and full dose regimens. The reduced dose was considered non-inferior to the full dose if the lower limit of the two-sided 95% CI of the GMR was >0.67. Primary analysis was done on the per-protocol population, including infection naïve participants only. 145 participants were enrolled and randomized, were mostly female (69.5%), of European origin (95%), with a mean age of 40.4 years (SD 7.9). At 28 days post second dose, the geometric mean titre (GMT) of anti-RBD IgG of the reduced dose regimen (1,705 BAU/mL) was not non-inferior to the full dose regimen (2,387 BAU/mL), with a GMR of 0.714 (two-sided 95% CI 0.540–0.944). No serious adverse events occurred. While non-inferiority of the reduced dose regimen was not demonstrated, the anti-RBD IgG titre was only moderately lower than that of the full dose regimen and, importantly, still markedly higher than the reported antibody response to the licensed adenoviral vector vaccines. These data suggest that reduced doses of the BNT162b2 mRNA vaccine may offer additional benefit as compared to the vaccines currently in use in most low and middle-income countries, warranting larger immunogenicity and effectiveness trials.

VL - 2 CP - 12 M3 - 10.1371/journal.pgph.0001308 ER - TY - JOUR T1 - Performance of five rapid serological tests in mild-diseased subjects using finger prick blood for exposure assessment to SARS-CoV-2. JF - J Clin Virol Y1 - 2021 A1 - David Triest A1 - Geebelen, Laurence A1 - Robby De Pauw A1 - Stéphane De Craeye A1 - Alexandra Vodolazkaia A1 - Mathieu Verbrugghe A1 - Koen Magerman A1 - Lara-Lauren Robben A1 - Pieter Pannus A1 - Kristof Neven A1 - Dirk Ramaekers A1 - Steven Van Gucht A1 - Katelijne Dierick A1 - Nele Van Loon A1 - Maria Goossens A1 - I Desombere KW - Antibodies, Viral KW - COVID-19 KW - Humans KW - SARS-CoV-2 KW - Sensitivity and Specificity KW - Seroepidemiologic Studies KW - Serologic Tests AB -

OBJECTIVES: Assess the performance of five SARS-CoV-2 rapid serological tests (RST) using finger prick (FP) blood on-site to evaluate their usability for exposure assessment in population-based seroprevalence studies.

STUDY DESIGN: Since cross-reactivity with common cold human coronaviruses occurs, serological testing includes a risk of false-positive results. Therefore, the selected cohort for RST-validation was based on combined immunoassay (presence of specific antibodies) and RT-qPCR (presence of SARS-CoV-2) data. RST-performance for FP blood and serum was assessed by performing each RST in two groups, namely SARSCoV- 2 positive (n=108) and negative healthcare workers (n=89). Differences in accuracy and positive and negative predictive values (PPV, NPV) were calculated for a range (1-50%) of SARS-CoV-2 prevalence estimates.

RESULTS: The OrientGene showed overall acceptable performance, with sensitivities of 94.4% and 100%, and specificities of 96.6% and 94.4%, using FP blood and serum, respectively. Although three RST reach optimal specificities (100%), the OrientGene clearly outperforms in sensitivity. At a SARS-CoV-2 prevalence rate of 40%, this RST outperforms the other tests in NPV (96.3%) and reaches comparable PPV (94.9%). Although the specificity of the Covid-Presto is excellent when using FP blood or serum (100% and 97.8%, respectively), its sensitivity decreases when using FP blood (76.9%) compared to serum (98.1%).

CONCLUSIONS: Performances of the evaluated RST differ largely. Only one out of five RST (OrientGene) had acceptable sensitivity and specificity using FP blood. Therefore, the latter could be used for seroprevalence studies in a high-prevalence situation. The OrientGene, which measures anti-RBD antibodies, can be valuable after vaccination as well.

VL - 142 M3 - 10.1016/j.jcv.2021.104897 ER - TY - JOUR T1 - Poor antibody response to BioNTech/Pfizer COVID-19 vaccination in SARS-CoV-2 naïve residents of nursing homes. JF - Clin Infect Dis Y1 - 2021 A1 - Pieter Pannus A1 - Kristof Y Neven A1 - Stéphane De Craeye A1 - Heyndrickx, Leo A1 - Sara Vande Kerckhove A1 - Daphnée Georges A1 - Johan Michiels A1 - Antoine Francotte A1 - Marc Van den Bulcke A1 - Maan Zrein A1 - Steven Van Gucht A1 - Marie-Noëlle Schmickler A1 - Mathieu Verbrugghe A1 - André Matagne A1 - Isabelle Thomas A1 - Katelijne Dierick A1 - Joshua A Weiner A1 - Margaret E Ackerman A1 - Stanislas Goriely A1 - Maria Goossens A1 - Ariën, Kevin K A1 - I Desombere A1 - Arnaud Marchant AB -

BACKGROUND: Residents of nursing homes (NH) are at high risk of COVID-19 related morbidity and death and may respond poorly to vaccination because of old age and frequent comorbidities.

METHODS: Seventy-eight residents and 106 staff members, naïve or previously infected with SARS-CoV-2, were recruited in NH in Belgium before immunization with two doses of 30µg BNT162b2 mRNA vaccine at day 0 and day 21. Binding antibodies (Ab) to SARS-CoV-2 receptor binding domain (RBD), spike domains S1 and S2, RBD Ab avidity, and neutralizing Ab against SARS-CoV-2 wild type and B.1.351 were assessed at days 0, 21, 28, and 49.

RESULTS: SARS-CoV-2 naïve residents had lower Ab responses to BNT162b2 mRNA vaccination than naïve staff. These poor responses involved lower levels of IgG to all spike domains, lower avidity of RBD IgG, and lower levels of Ab neutralizing the vaccine strain. No naïve resident had detectable neutralizing Ab to the B.1.351 variant. In contrast, SARS-CoV-2 infected residents had high responses to mRNA vaccination, with Ab levels comparable to infected staff. Cluster analysis revealed that poor vaccine responders not only included naïve residents but also naïve staff, emphasizing the heterogeneity of responses to mRNA vaccination in the general population.

CONCLUSIONS: The poor Ab responses to mRNA vaccination observed in infection naïve residents and in some naïve staff members of NH suggest suboptimal protection against breakthrough infection, especially with variants of concern. These data support the administration of a third dose of mRNA vaccine to further improve protection of NH residents against COVID-19.

M3 - 10.1093/cid/ciab998 ER - TY - JOUR T1 - Pork as a source of transmission of Toxoplasma gondii to humans: a parasite burden study in pig tissues after infection with different strains of Toxoplasma gondii as a function of time and different parasite stages. JF - Int J Parasitol Y1 - 2018 A1 - I. Gisbert Algaba A1 - Bavo Verhaegen A1 - Jennes, Malgorzata A1 - Mizanur Rahman A1 - Wim Coucke A1 - Cox, Eric A1 - Dorny, Pierre A1 - Katelijne Dierick A1 - Stéphane De Craeye AB -

Toxoplasma gondii is an ubiquitous apicomplexan parasite which can infect any warm-blooded animal including humans. Humans and carnivores/omnivores can also become infected by consumption of raw or undercooked infected meat containing muscle cysts. This route of transmission is considered to account for at least 30% of human toxoplasmosis cases. To better assess the role of pork as a source of infection for humans, the parasite burden resulting from experimental infection with different parasite stages and different strains of T. gondii during the acute and chronic phases was studied. The parasite burden in different tissues was measured with a ISO 17025 validated Magnetic Capture-quantitative PCR. A high burden of infection was found in heart and lungs during the acute phase of infection and heart and brain were identified as the most parasitised tissues during the chronic phase of infection, independent of the parasite stage and the strain used. Remarkably, a higher parasite burden was measured in different tissues following infection with oocysts of a type II strain compared with a tissue cyst infection with three strains of either type II or a type I/II. However, these results could have been affected by the use of different strains and euthanasia time points. The parasite burden resulting from a tissue cyst infection was not significantly different between the two strains.

VL - 48 CP - 7 M3 - 10.1016/j.ijpara.2017.12.009 ER - TY - JOUR T1 - A more sensitive, efficient and ISO 17025 validated Magnetic Capture real time PCR method for the detection of archetypal Toxoplasma gondii strains in meat. JF - Int J Parasitol Y1 - 2017 A1 - I. Gisbert Algaba A1 - Geerts, Manon A1 - Jennes, Malgorzata A1 - Wim Coucke A1 - Opsteegh, Marieke A1 - Cox, Eric A1 - Dorny, Pierre A1 - Katelijne Dierick A1 - Stéphane De Craeye AB -

Toxoplasma gondii is a globally prevalent, zoonotic parasite of major importance to public health. Various indirect and direct methods can be used for the diagnosis of toxoplasmosis. Whereas serological tests are useful to prove contact with the parasite has occurred, the actual presence of the parasite in the tissues of a seropositive animal is not demonstrated. For this, a bioassay is still the reference method. As an alternative, various PCR methods have been developed, but due to the limited amount of sample that can be tested, combined with a low tissue cyst density, those have proved to be insufficiently sensitive. A major improvement of the sensitivity was achieved with magnetic capture-based DNA extraction. By combining the hybridization of specific, biotinylated probes with the capture of those probes with streptavidin-coated paramagnetic beads, T. gondii DNA can selectively be "fished out" from a large volume of meat lysate. Still, several studies showed an insufficient sensitivity compared with the mouse bioassay. Here we present a method that is more sensitive (99% limit of detection: 65.4 tachyzoites per 100g of meat), economical and reliable (ISO 17025 validated) by adding a non-competitive PCR inhibition control (co-capture of cellular r18S) and making the release of the target DNA from the streptavidin-coated paramagnetic beads UV-dependent. The presented results demonstrate the potential of the modified Magnetic Capture real time PCR as a full alternative to the mouse bioassay for the screening of various types of tissues and meat, with the additional advantage of being quantitative.

U1 - http://www.ncbi.nlm.nih.gov/pubmed/28694187?dopt=Abstract M3 - 10.1016/j.ijpara.2017.05.005 ER - TY - JOUR T1 - Strain- and Dose-Dependent Reduction of Toxoplasma gondii Burden in Pigs Is Associated with Interferon-Gamma Production by CD8(+) Lymphocytes in a Heterologous Challenge Model. JF - Front Cell Infect Microbiol Y1 - 2017 A1 - Jennes, Malgorzata A1 - Stéphane De Craeye A1 - Devriendt, Bert A1 - Katelijne Dierick A1 - Dorny, Pierre A1 - Cox, Eric AB -

Toxoplasma gondii is a worldwide prevalent parasite of humans and animals. The global infection burden exceeds yearly one million disability-adjusted life years (DALY's) in infected individuals. Therefore, effective preventive measures should be taken to decrease the risk of infection in humans. Although human toxoplasmosis is predominantly foodborne by ingestion of tissue cysts in meat from domestic animals such as pigs, the incidence risk is difficult to estimate due to the lack of screening of animals for infection and insights in location and persistence of the parasite in the tissues. Hence, experimental infections in pigs can provide more information on the risk for zoonosis based on the parasite burden in meat products intended for human consumption and on the immune responses induced by infection. In the present study, homo- and heterologous infection experiments with two distinct T. gondii strains (IPB-LR and IPB-Gangji) were performed. The humoral and cellular immune responses, the presence of viable parasites and the parasite load in edible meat samples were evaluated. In homologous infection experiments the parasite persistence was clearly strain-dependent and inversely correlated with the infection dose. The results strongly indicate a change in the amount of parasite DNA and viable cysts in porcine tissues over time. Heterologous challenge infections demonstrated that IPB-G strain could considerably reduce the parasite burden in the subsequent IPB-LR infection. A strong, however, not protective humoral response was observed against GRA7 and TLA antigens upon inoculation with both strains. The in vitro IFN-γ production by TLA-stimulated PBMCs was correlated with the infection dose and predominantly brought about by CD3(+)CD4(-)CD8α(bright) T-lymphocytes. The described adaptive cellular and humoral immune responses in pigs are in line with the induced or natural infections in mice and humans. Previous studies underscored the heterogeneity of T. gondii strains and the corresponding virulence factors. These findings suggest the potential of the IPB-G strain to elicit a partially protective immune response and to reduce the parasite burden upon a challenge infection. The IPB-G strain could be used as a promising tool in limiting the number of viable parasites in edible tissues and, hence, in lowering the risk for human toxoplasmosis.

VL - 7 U1 - http://www.ncbi.nlm.nih.gov/pubmed/28642841?dopt=Abstract M3 - 10.3389/fcimb.2017.00232 ER - TY - JOUR T1 - Molecular Analysis of Rising Fluoroquinolone Resistance in Belgian Non-Invasive Streptococcus pneumoniae Isolates (1995-2014). JF - PLoS One Y1 - 2016 A1 - Pieter-Jan Ceyssens A1 - Van Bambeke, Françoise A1 - Wesley Mattheus A1 - Sophie Bertrand A1 - Fux, Frédéric A1 - Van Bossuyt, Eddie A1 - Damée, Sabrina A1 - Nyssen, Henry-Jean A1 - Stéphane De Craeye A1 - Verhaegen, Jan A1 - Tulkens, Paul M A1 - Vanhoof, Raymond KW - Amino Acid Substitution KW - ATP-Binding Cassette Transporters KW - Bacterial Proteins KW - Belgium KW - DNA Gyrase KW - Drug Resistance, Bacterial KW - Female KW - Fluoroquinolones KW - Humans KW - Longitudinal Studies KW - Male KW - Mutation, Missense KW - Pneumococcal Infections KW - Streptococcus pneumoniae AB -

We present the results of a longitudinal surveillance study (1995-2014) on fluoroquinolone resistance (FQ-R) among Belgian non-invasive Streptococcus pneumoniae isolates (n = 5,602). For many years, the switch to respiratory fluoroquinolones for the treatment of (a)typical pneumonia had no impact on FQ-R levels. However, since 2011 we observed a significant decrease in susceptibility towards ciprofloxacin, ofloxacin and levofloxacin with peaks of 9.0%, 6.6% and 3.1% resistant isolates, respectively. Resistance to moxifloxacin arised sporadically, and remained <1% throughout the entire study period. We observed classical topoisomerase mutations in gyrA (n = 25), parC (n = 46) and parE (n = 3) in varying combinations, arguing against clonal expansion of FQ-R. The impact of recombination with co-habiting commensal streptococci on FQ-R remains marginal (10.4%). Notably, we observed that a rare combination of DNA Gyrase mutations (GyrA_S81L/GyrB_P454S) suffices for high-level moxifloxacin resistance, contrasting current model. Interestingly, 85/422 pneumococcal strains display MICCIP values which were lowered by at least four dilutions by reserpine, pointing at involvement of efflux pumps in FQ-R. In contrast to susceptible strains, isolates resistant to ciprofloxacin significantly overexpressed the ABC pump PatAB in comparison to reference strain S. pneumoniae ATCC 49619, but this could only be linked to disruptive terminator mutations in a fraction of these. Conversely, no difference in expression of the Major Facilitator PmrA, unaffected by reserpine, was noted between susceptible and resistant S. pneumoniae strains. Finally, we observed that four isolates displayed intermediate to high-level ciprofloxacin resistance without any known molecular resistance mechanism. Focusing future molecular studies on these isolates, which are also commonly found in other studies, might greatly assist in the battle against rising pneumococcal drug resistance.

VL - 11 CP - 5 U1 - http://www.ncbi.nlm.nih.gov/pubmed/27227336?dopt=Abstract M3 - 10.1371/journal.pone.0154816 ER - TY - JOUR T1 - Interferon-gamma expression and infectivity of Toxoplasma infected tissues in experimentally infected sheep in comparison with pigs. JF - Vet Parasitol Y1 - 2015 A1 - Verhelst, D A1 - Stéphane De Craeye A1 - Jennes, M A1 - Dorny, P A1 - Goddeeris, B A1 - Cox, E KW - Animals KW - Antibodies, Protozoan KW - Antigens, Protozoan KW - brain KW - Cytokines KW - Heart KW - Interferon-gamma KW - Interleukin-10 KW - Muscle, Skeletal KW - Protozoan Proteins KW - Recombinant Proteins KW - Sheep KW - Sheep Diseases KW - Swine KW - Swine Diseases KW - Toxoplasma KW - Toxoplasmosis, Animal AB -

Livestock animals are a potential risk for transmission of toxoplasmosis to humans. Sheep and pigs still remain an important source because their meat is often eaten undercooked which has been regarded as a major route of infection in many countries. Moreover, porcine tissues are processed in many food products. In the current study, the IFN-gamma (T-helper 1 cells), IL-4 (Th2 cells) and IL-10 mRNA (Treg cells) expression by blood mononuclear cells, and the serum antibody response against Toxoplasma gondii total lysate antigen, recombinant T. gondii GRA1, rGRA7, rMIC3 and rEC2, a chimeric antigen composed of MIC2, MIC3 and SAG1, was studied in sheep the first two months after a T. gondii infection and compared with these responses in pigs. At the end of this period, the parasite distribution in heart, brain and two skeletal muscles in sheep was compared with this in pigs. Whereas the parasite distribution was similar in sheep and pigs, the antibody response differed considerably. In sheep, antibodies appeared against all tested T. gondii antigens, but mainly against rGRA7, rMIC3234307 and TLA whereas in pigs only rGRA7-specific antibodies could be demonstrated. Also, the cytokine response differed. Both in sheep and pigs an IFN-gamma response occurred which seemed to be a slightly more pronounced in sheep. In sheep, also IL-10 and IL-4 mRNA expression showed an increase, but later than IFN-gamma and with more variation. However, in pigs no such increase was seen. As concerning diagnosis, results indicate that serum antibodies against GRA7 in live sheep and pigs and heart tissue for bioassay and qPCR in slaughtered animals are the best targets to demonstrate presence of T. gondii infection.

VL - 207 CP - 1-2 U1 - http://www.ncbi.nlm.nih.gov/pubmed/25499128?dopt=Abstract M3 - 10.1016/j.vetpar.2014.11.014 ER - TY - JOUR T1 - Serologic screening for 13 infectious agents in roe deer (Capreolus capreolus) in Flanders. JF - Infect Ecol Epidemiol Y1 - 2015 A1 - Tavernier, Paul A1 - Sys, Stanislas U A1 - Kris De Clercq A1 - Ilse De Leeuw A1 - Ann Brigitte Cay A1 - Miet De Baere A1 - Nick De Regge A1 - David Fretin A1 - Virginie Roupie A1 - Govaerts, Marc A1 - Heyman, Paul A1 - Vanrompay, Daisy A1 - Yin, Lizi A1 - Kalmar, Isabelle A1 - Vanessa Suin A1 - Bernard Brochier A1 - Alexandre Dobly A1 - Stéphane De Craeye A1 - Sophie Roelandt A1 - Goossens, Els A1 - S. Roels AB -

INTRODUCTION: In order to investigate the role of roe deer in the maintenance and transmission of infectious animal and human diseases in Flanders, we conducted a serologic screening in 12 hunting areas.

MATERIALS AND METHODS: Roe deer sera collected between 2008 and 2013 (n=190) were examined for antibodies against 13 infectious agents, using indirect enzyme-linked immunosorbent assay, virus neutralisation, immunofluorescence, or microagglutination test, depending on the agent.

RESULTS AND DISCUSSION: High numbers of seropositives were found for Anaplasma phagocytophilum (45.8%), Toxoplasma gondii (43.2%) and Schmallenberg virus (27.9%), the latter with a distinct temporal distribution pattern following the outbreak in domestic ruminants. Lower antibody prevalence was found for Chlamydia abortus (6.7%), tick-borne encephalitis virus (5.1%), Neospora caninum (4.8%), and Mycobacterium avium subsp paratuberculosis (4.1%). The lowest prevalences were found for Leptospira (1.7%), bovine viral diarrhoea virus 1 (1.3%), and Coxiella burnetii (1.2%). No antibodies were found against Brucella sp., bovine herpesvirus 1, and bluetongue virus. A significant difference in seroprevalence between ages (higher in adults >1 year) was found for N. caninum. Four doubtful reacting sera accounted for a significant difference in seroprevalence between sexes for C. abortus (higher in females).

CONCLUSIONS: Despite the more intensive landscape use in Flanders, the results are consistent with other European studies. Apart from maintaining C. abortus and MAP, roe deer do not seem to play an important role in the epidemiology of the examined zoonotic and domestic animal pathogens. Nevertheless, their meaning as sentinels should not be neglected in the absence of other wild cervid species.

VL - 5 U1 - http://www.ncbi.nlm.nih.gov/pubmed/26609692?dopt=Abstract M3 - 10.3402/iee.v5.29862 ER - TY - JOUR T1 - Parasite distribution and associated immune response during the acute phase of Toxoplasma gondii infection in sheep. JF - BMC Vet Res Y1 - 2014 A1 - Verhelst, Delfien A1 - Stéphane De Craeye A1 - Entrican, Gary A1 - Dorny, Pierre A1 - Cox, Eric KW - Acute-Phase Reaction KW - Animals KW - Antibodies, Protozoan KW - Antigens, Protozoan KW - Cytokines KW - Enterocytes KW - Enzyme-Linked Immunosorbent Assay KW - Fluorescent Antibody Technique, Indirect KW - Intestines KW - Lymph Nodes KW - Real-Time Polymerase Chain Reaction KW - Sheep KW - Sheep Diseases KW - Spleen KW - Toxoplasma KW - Toxoplasmosis, Animal AB -

BACKGROUND: In many countries, Toxoplasma gondii (T. gondii) is a major cause of reproductive disorders and abortions in the sheep industry, and therefore responsible for important financial and economic losses. In addition, undercooked infected lamb is an important risk factor for human toxoplasmosis. In the present study, the initial phase of the infection was investigated: the parasite's entry site, the subsequent distribution of the parasite and the host-immune response.

RESULTS: Parasite DNA was already detected in the cranial small intestinal mucosa the first days after oral infection with T. gondii tissue cysts. Simultaneously, high IFN-gamma and IL-12 responses were induced mainly in the mesenteric lymph nodes. The emergence of IgG1 (at 8dpi), and IgG2 (at 11 dpi) was accompanied by a decrease or even disappearance of the IFN-gamma and IL-12 response in the Peyers' patches (PP), PBMC's and popliteal LN's. Meanwhile the parasite DNA could be recovered from most mucosal and systemic tissues to become undetectable in the small intestine, popliteal LN, PBMC and spleen 3 weeks pi.

CONCLUSIONS: Our results indicate that parasites enter the cranial small intestine the first days after infection and that after an increase the first two weeks after infection, the parasite DNA levels in the intestine drop below the detection limit three weeks after infection. This coincides with an increase in parastic-specific serum IgG1 and IgG2 and a decrease of the antigen-specific IFN-gamma response in PP, PBMC and popliteal LN. We suggest a role for IFN-gamma and IL-12 in controlling the infection.

VL - 10 U1 - http://www.ncbi.nlm.nih.gov/pubmed/25511864?dopt=Abstract M3 - 10.1186/s12917-014-0293-5 ER - TY - JOUR T1 - Seroprevalence of Toxoplasma gondii in domestic sheep in Belgium. JF - Vet Parasitol Y1 - 2014 A1 - Verhelst, D A1 - Stéphane De Craeye A1 - Vanrobaeys, M A1 - Czaplicki, G A1 - Dorny, P A1 - Cox, E KW - Animals KW - Antibodies, Protozoan KW - Belgium KW - Immunoglobulin G KW - Seroepidemiologic Studies KW - Sheep KW - Sheep Diseases KW - Toxoplasma KW - Toxoplasmosis, Animal AB -

Even though infected sheep are a potential source of Toxoplasma gondii infection in humans, information is lacking concerning the seroprevalence of T. gondii infection in sheep in Belgium. We examined 3170 serum samples for anti-Toxoplasma IgG in sheep by total lysate antigen (TLA) enzyme-linked immunosorbent assay (ELISA). IgG to T. gondii was demonstrated in 87.4% of the tested sheep and in 96.2% of the 209 tested flocks. The seroprevalences in Antwerp (65.2%) and Wallonia (68.6%) are statistically lower than in the other regions in Belgium (96.7-97.8%) (P<0.05). The present study is the first report that analyzed the prevalence of T. gondii infection in sheep in Belgium and confirms the high prevalence of Toxoplasma-specific IgG antibodies in the sheep population.

VL - 205 CP - 1-2 U1 - http://www.ncbi.nlm.nih.gov/pubmed/25065982?dopt=Abstract M3 - 10.1016/j.vetpar.2014.07.001 ER - TY - JOUR T1 - A two-step lyssavirus real-time polymerase chain reaction using degenerate primers with superior sensitivity to the fluorescent antigen test. JF - Biomed Res Int Y1 - 2014 A1 - Vanessa Suin A1 - Nazé, Florence A1 - Aurélie Francart A1 - Lamoral, Sophie A1 - Stéphane De Craeye A1 - Kalai, Michael A1 - Steven Van Gucht KW - Animals KW - Base Sequence KW - brain KW - Cats KW - Chiroptera KW - Computer Simulation KW - Dogs KW - Fluorescent Antibody Technique KW - Humans KW - Limit of Detection KW - Lyssavirus KW - mice KW - Molecular Sequence Data KW - Real-Time Polymerase Chain Reaction KW - Reproducibility of Results KW - Reverse Transcriptase Polymerase Chain Reaction KW - Rhabdoviridae Infections KW - RNA, Viral KW - Sequence Alignment AB -

A generic two-step lyssavirus real-time reverse transcriptase polymerase chain reaction (qRT-PCR), based on a nested PCR strategy, was validated for the detection of different lyssavirus species. Primers with 17 to 30% of degenerate bases were used in both consecutive steps. The assay could accurately detect RABV, LBV, MOKV, DUVV, EBLV-1, EBLV-2, and ABLV. In silico sequence alignment showed a functional match with the remaining lyssavirus species. The diagnostic specificity was 100% and the sensitivity proved to be superior to that of the fluorescent antigen test. The limit of detection was ≤ 1 50% tissue culture infectious dose. The related vesicular stomatitis virus was not recognized, confirming the selectivity for lyssaviruses. The assay was applied to follow the evolution of rabies virus infection in the brain of mice from 0 to 10 days after intranasal inoculation. The obtained RNA curve corresponded well with the curves obtained by a one-step monospecific RABV-qRT-PCR, the fluorescent antigen test, and virus titration. Despite the presence of degenerate bases, the assay proved to be highly sensitive, specific, and reproducible.

VL - 2014 U1 - http://www.ncbi.nlm.nih.gov/pubmed/24822188?dopt=Abstract M3 - 10.1155/2014/256175 ER - TY - JOUR T1 - A non-invasive intranasal inoculation technique using isoflurane anesthesia to infect the brain of mice with rabies virus. JF - J Virol Methods Y1 - 2011 A1 - Rosseels, Valérie A1 - Nazé, Florence A1 - Stéphane De Craeye A1 - Aurélie Francart A1 - Kalai, Michael A1 - Steven Van Gucht KW - Anesthetics, Inhalation KW - Animals KW - brain KW - Disease Models, Animal KW - Isoflurane KW - mice KW - Rabies KW - Survival Analysis AB -

Methods for intranasal inoculation of viruses are often described poorly and the effects of variations in the technique on the outcome are unknown. Standardization of protocols is key to compare studies and minimize animal use. The clinical and virological outcome of infection with rabies virus (genotypes 1 and 5) upon administration of different inoculum volumes (25, 50 and 100μl) and different anesthetic regimens were examined. Administration of 25μl of virus as a drop on both nostrils under brief superficial isoflurane anesthesia (92μl/dm(3), recovery after 85 ± 1 0s) was the most effective to infect the brain and induced 100% lethal infection 9 days later. Increasing the inoculum volume reduced infectivity significantly, with decreased viral loads in the brain and only 40% mortality. Increasing the depth of isoflurane anesthesia (230μl/dm(3)) improved the infectivity of the large-volume inoculum (90% mortality), probably because of suppression of swallow and sneeze reflexes. Compared to isoflurane anesthesia, xylazine-ketamine anesthesia reduced the infectivity of the inoculum significantly. Thus, administration of a small volume of virus on the nostrils under brief gas anesthesia is a safe and reproducible technique to induce infection of the brain. Since needles are not required, this helps to preserve the integrity of the physical barriers, animal welfare and the manipulator's safety.

VL - 173 CP - 1 U1 - http://www.ncbi.nlm.nih.gov/pubmed/21295615?dopt=Abstract M3 - 10.1016/j.jviromet.2011.01.019 ER - TY - JOUR T1 - Partial depletion of CD4(+)CD25(+)Foxp3(+) T regulatory cells significantly increases morbidity during acute phase Toxoplasma gondii infection in resistant BALB/c mice. JF - Microbes Infect Y1 - 2011 A1 - Morampudi, Vijay A1 - Stéphane De Craeye A1 - Le Moine, Alain A1 - Detienne, Sophie A1 - Braun, M Y A1 - D'Souza, Sushila KW - Acute Disease KW - Animals KW - Antibodies, Monoclonal KW - Antigens, CD KW - Cells, Cultured KW - Cytokines KW - Disease Progression KW - Female KW - Ileum KW - Lymphocyte Depletion KW - mice KW - Mice, Inbred BALB C KW - Mice, Inbred C57BL KW - T-Lymphocytes, Regulatory KW - Toxoplasma KW - Toxoplasmosis AB -

CD4(+)CD25(+)Foxp3(+) T regulatory (Treg) cells, are known to regulate responses to infectious agents. Here we compared disease progression in BALB/c and C57BL/6(B6) mice infected perorally with Toxoplasma gondii for 7 days and examined the affect of partial depletion of Treg cells in these mice. BALB/c mice were seen to be resistant to peroral infection whereas B6 mice were susceptible in terms of mortality. Although the depletion of Treg cells before infection had no effect on the survival of B6 or BALB/c mice, it resulted in increased parasite burdens in BALB/c mice, especially in the lamina propria, but not in B6 mice. Pro-inflammatory cytokines were also increased in Treg cells depleted BALB/c mice as compared to B6 mice. In addition Treg cell depleted BALB/c mice displayed increased ileal histopathology compared to their non-treated counterparts. These findings provide evidence for the contribution of Treg cells, in the resistance of BALB/c mice against peroral T. gondii infection.

VL - 13 CP - 4 U1 - http://www.ncbi.nlm.nih.gov/pubmed/21262371?dopt=Abstract M3 - 10.1016/j.micinf.2011.01.006 ER - TY - JOUR T1 - Toxoplasma gondii and Neospora caninum in wildlife: common parasites in Belgian foxes and Cervidae? JF - Vet Parasitol Y1 - 2011 A1 - Stéphane De Craeye A1 - Speybroeck, N A1 - Ajzenberg, D A1 - Dardé, M L A1 - Collinet, F A1 - Tavernier, P A1 - Steven Van Gucht A1 - Dorny, P A1 - Katelijne Dierick KW - Animals KW - Belgium KW - brain KW - Coccidiosis KW - Deer KW - DNA, Protozoan KW - Foxes KW - Genotype KW - Neospora KW - Toxoplasma KW - Toxoplasmosis, Animal AB -

Sera from Cervidae were tested for the presence of antibodies against Neospora caninum using ELISA; and against Toxoplasma gondii using SAG1-ELISA and a commercially available agglutination test. The T. gondii seroprevalence was 52% (38/73) in roe deer (Capreolus capreolus), 0% in bred fallow deer (0/4) (Dama dama) and red deer (0/7) (Cervus elaphus). We found 2.7% of the roe deer samples and none of the bred deer samples positive for N. caninum. Brain samples from wild roe deer, red deer and red foxes (Vulpes vulpes) were tested for the presence of T. gondii and N. caninum DNA using multiplex real-time PCR. We detected T. gondii in 18.8% (57/304) of the red foxes and in 1 of the 33 deer samples. N. caninum was found in 6.6% of the red foxes and in 2 roe deer samples. Twenty-six of the T. gondii positive DNA extracts from the red fox samples were genotyped. Twenty-five were type II and only one was found to be type III.

VL - 178 CP - 1-2 U1 - http://www.ncbi.nlm.nih.gov/pubmed/21236577?dopt=Abstract M3 - 10.1016/j.vetpar.2010.12.016 ER - TY - JOUR T1 - Functional characterization of in vivo effector CD4(+) and CD8(+) T cell responses in acute Toxoplasmosis: an interplay of IFN-gamma and cytolytic T cells. JF - Vaccine Y1 - 2010 A1 - Jongert, Erik A1 - Lemiere, Arnaud A1 - Van Ginderachter, Jo A1 - Stéphane De Craeye A1 - Huygen, Kris A1 - D'Souza, Sushila KW - Animals KW - Antigens, Protozoan KW - CD4-Positive T-Lymphocytes KW - CD8-Positive T-Lymphocytes KW - Cytotoxicity, Immunologic KW - Female KW - Interferon-gamma KW - Macrophages KW - Membrane Proteins KW - mice KW - Mice, Inbred C3H KW - Protozoan Proteins KW - Protozoan Vaccines KW - Th1 Cells KW - Toxoplasmosis KW - Vaccines, DNA AB -

Development of prophylactic vaccines against Toxoplasma gondii is based on the observation that latently infected subjects are protected against secondary infection during pregnancy. Cocktail DNA vaccines have been shown to provide high resistance to parasite challenge, and latently infected mice are protected against acute disease. In order to characterize the associated Th1 cellular immune responses in vivo, we used H2-K(k) bone marrow macrophage cell lines constitutively expressing T. gondii GRA1, GRA7 or ROP2 antigens, for the in vivo characterization of antigen-specific T cells in an antigenic challenge model, and as target cells in an in vivo CTL assay. In latently infected C3H/HeN mice, CD4(+) and CD8(+) T cells were recruited to the peritoneal cavity after i.p. challenge with these syngeneic cell lines. GRA1 and GRA7-specific T cells from infected mice were IFN-gamma(+) FasL(-) CD107(-). No IFN-gamma or lytic markers were observed against ROP2. In cocktail DNA vaccinated C3H/HeN mice, the response was restricted to GRA1-specific CD8(+) IFN-gamma(-) FasL(-) CD107(+) T cells. Target cells expressing GRA1 and GRA7, but not ROP2, were efficiently killed in an in vivo CTL assay in latently infected mice, while in DNA vaccinated mice only lysis of GRA1 expressing target cells was observed. Both forms of immunization, DNA vaccination and latent infection, completely protected mice against acute Toxoplasmosis. The results obtained in this work suggest that distinct in vivo cytolytic effector mechanisms are at work in DNA vaccinated and latently infected mice, but both converge to protect against acute toxoplasmosis.

VL - 28 CP - 13 U1 - http://www.ncbi.nlm.nih.gov/pubmed/20117266?dopt=Abstract M3 - 10.1016/j.vaccine.2010.01.031 ER - TY - JOUR T1 - An enhanced GRA1-GRA7 cocktail DNA vaccine primes anti-Toxoplasma immune responses in pigs. JF - Vaccine Y1 - 2008 A1 - Jongert, E A1 - Melkebeek, V A1 - Stéphane De Craeye A1 - Dewit, J A1 - Verhelst, D A1 - Cox, E KW - Adjuvants, Immunologic KW - Animals KW - Antibodies, Protozoan KW - Antigens, Protozoan KW - Bacterial Toxins KW - Cell Proliferation KW - Enterotoxins KW - Escherichia coli Proteins KW - Immunoglobulin G KW - Injections, Intradermal KW - Interferon-gamma KW - Protozoan Proteins KW - Protozoan Vaccines KW - Swine KW - T-Lymphocytes KW - Toxoplasma KW - Toxoplasmosis KW - Vaccines, DNA AB -

The protozoan parasite Toxoplasma gondii is the causative agent of a worldwide zoonosis and high prevalencies can be found both in animals and humans. An important source of human contamination with T. gondii is the consumption of raw or undercooked meat products. In this study, we evaluated whether DNA vaccination against T. gondii in pigs is able to generate immune responses known to be protective against tissue cyst formation. A GRA1-GRA7 DNA vaccine cocktail was enhanced by codon optimization of the encoding antigens and addition of heat labile enterotoxin expressing vectors as genetic adjuvant. Pigs vaccinated intradermally with this enhanced GRA1-GRA7 DNA vaccine cocktail developed high antibody levels against GRA1, GRA7 and a T. gondii lysate, and lymphocyte proliferation and production of IFN-gamma could be detected in these animals after challenge with the parasite. These results indicate that pigs can be efficiently primed against T. gondii infection by means of a DNA vaccine.

VL - 26 CP - 8 U1 - http://www.ncbi.nlm.nih.gov/pubmed/18221825?dopt=Abstract M3 - 10.1016/j.vaccine.2007.11.058 ER - TY - JOUR T1 - Prevalence of Toxoplasma gondii infection in Belgian house cats. JF - Vet Parasitol Y1 - 2008 A1 - Stéphane De Craeye A1 - Aurélie Francart A1 - Chabauty, Julie A1 - De Vriendt, Veerle A1 - Steven Van Gucht A1 - Leroux, Ingrid A1 - Jongert, Erik KW - Animals KW - Antibodies, Protozoan KW - Belgium KW - Cat Diseases KW - Cats KW - Enzyme-Linked Immunosorbent Assay KW - Fluorescent Antibody Technique, Indirect KW - Immunoglobulin G KW - Immunoglobulin M KW - prevalence KW - Protozoan Proteins KW - Seroepidemiologic Studies KW - Toxoplasma KW - Toxoplasmosis, Animal AB -

Five hundred and sixty seven sera of healthy house cats aged 3 months to 7 years, were examined for the presence of anti-toxoplasma antibodies by indirect immunofluorescence assay and compared to SAG1 and TLA enzyme linked immunosorbent assays as alternative test. Twenty-five percent of cats tested positive for IgG and/or IgM. Seroprevalence increased with age from 2% below 12 months of age up to 44% at age 7. Sensitivities of SAG1 and TLA ELISA were 84.1% and 88.6%, respectively. Peak levels in seroprevalence were correlated to increased IgG titers in TLA ELISA. Our results suggest that T. gondii infections are common in house cats and that there is a high chance for a negative cat to seroconvert in its second life-year.

VL - 157 CP - 1-2 U1 - http://www.ncbi.nlm.nih.gov/pubmed/18707811?dopt=Abstract M3 - 10.1016/j.vetpar.2008.07.001 ER - TY - JOUR T1 - GRA7 provides protective immunity in cocktail DNA vaccines against Toxoplasma gondii. JF - Parasite Immunol Y1 - 2007 A1 - Jongert, E A1 - Stéphane De Craeye A1 - Dewit, J A1 - Huygen, K KW - Animals KW - Antibodies, Protozoan KW - Antigens, Protozoan KW - Female KW - Interferon-gamma KW - Membrane Proteins KW - mice KW - Mice, Inbred C3H KW - Protozoan Proteins KW - Protozoan Vaccines KW - Toxoplasma KW - Toxoplasmosis, Animal KW - Vaccines, DNA AB -

In a previous study, single-gene vaccination with GRA1, GRA7 or ROP2 was shown to elicit partial protection against Toxoplasma gondii. In this study, the contribution of each antigen in the evoked humoral and cellular immune responses was evaluated after vaccination with plasmid mixtures containing GRA1, GRA7 and ROP2. Cocktail DNA vaccinated mice developed high antibody titers against the antigens from two-gene DNA vaccine cocktails, but lower titres when immunized with the three-gene cocktail. High numbers of IFN-gamma secreting splenocytes were generated predominantly against GRA7. Brain cyst burden was reduced by 81% in mice vaccinated with the three-gene mixture and they were completely protected against acute toxoplasmosis. Similar high levels of brain cyst reductions were obtained after vaccination with cocktails composed of GRA1 and GRA7 (89% reduction), or GRA7 and ROP2 (79% reduction), but not with the cocktail composed of GRA1 and ROP2. In low dose single-gene vaccinations, IFN-gamma and strong protection could only be elicited by GRA7. Hence, the presence of GRA7 in the DNA vaccine formulation was important for optimal protection and this was correlated with GRA7-specific IFN-gamma production. We propose GRA7 as a main component in cocktail DNA vaccines for vaccination against T. gondii.

VL - 29 CP - 9 U1 - http://www.ncbi.nlm.nih.gov/pubmed/17727568?dopt=Abstract M3 - 10.1111/j.1365-3024.2007.00961.x ER -