Third dose of COVID-19 mRNA vaccine closes the gap in immune response between naïve nursing home residents and healthy adults.

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.

Poor Antibody Response to BioNTech/Pfizer Coronavirus Disease 2019 Vaccination in Severe Acute Respiratory Syndrome Coronavirus 2-Naive Residents of Nursing Homes.

BACKGROUND: Residents of nursing homes (NHs) are at high risk of coronavirus disease 2019 (COVID-19)-related disease and death and may respond poorly to vaccination because of old age and frequent comorbid conditions. METHODS: Seventy-eight residents and 106 staff members, naive to infection or previously infected with severe acute respiratory syndrome coronavirus (SARS-CoV-2), were recruited in NHs in Belgium before immunization with 2 doses of 30 µg BNT162b2 messenger RNA (mRNA) vaccine at days 0 and 21. Binding antibodies (Abs) to SARS-CoV-2 receptor-binding domain (RBD), spike domains S1 and S2, RBD Ab avidity, and neutralizing Abs against SARS-CoV-2 wild type and B.1.351 were assessed at days 0, 21, 28, and 49. RESULTS: SARS-CoV-2-naive residents had lower Ab responses to BNT162b2 mRNA vaccination than naive staff. These poor responses involved lower levels of immunoglobulin (Ig) G to all spike domains, lower avidity of RBD IgG, and lower levels of Abs neutralizing the vaccine strain. No naive residents had detectable neutralizing Abs to the B.1.351 variant. In contrast, SARS-CoV-2-infected residents had high responses to mRNA vaccination, with Ab levels comparable to those in infected staff. Cluster analysis revealed that poor vaccine responders included not only naive residents but also naive staff, emphasizing the heterogeneity of responses to mRNA vaccination in the general population. CONCLUSIONS: The poor Ab responses to mRNA vaccination observed in infection-naive NH residents and in some naive staff members 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.

Safety and immunogenicity of a reduced dose of the BNT162b2 mRNA COVID-19 vaccine (REDU-VAC): A single blind, randomized, non-inferiority trial.

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. Trial Registration: The trial is registered at ClinicalTrials.gov (NCT04852861).

Performance of five rapid serological tests in mild-diseased subjects using finger prick blood for exposure assessment to SARS-CoV-2.

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.

Early Kinetics of Intestinal Infection and Immune Responses to Two Toxoplasma gondii Strains in Pigs.

Toxoplasma gondii is an obligate intracellular parasite, able to infect all homeothermic animals mostly through ingestion of (oo)cysts contaminated food or water. Recently, we observed a T. gondii strain-specific clearance from tissues upon infection in pigs: while the swine-adapted LR strain persisted in porcine tissues, a subsequent infection with the human-isolated Gangji strain cleared parasites from several tissues. We hypothesized that intestinal immune responses shortly after infection might play a role in this strain-specific clearance. To assess this possibility, the parasite load in small intestinal lymph node cells and blood immune cells as well as the IFNγ secretion by these cells were evaluated at 2, 4, 8, 14, and 28 days post oral inoculation of pigs with tissue cysts of both strains. Interestingly, at day 4 post inoculation with the LR strain the parasite was detected by qPCR only in the duodenal lymph node cells, while in the jejunal and ileal lymph node cells and PBMCs the parasite was detected from day 8 post inoculation onwards. Although we observed a similar profile upon inoculation with the Gangji strain, the parasite load in the examined cells was much lower. This was reflected in a significantly higher T. gondii-specific serum IgG response in LR compared to Gangji infected pigs at day 28 post inoculation. Unexpectedly, this was not reflected in the IFNγ secretion upon re-stimulation of the cells where almost equal IFNγ secretion was observed in both groups. In conclusion, our results show that T. gondii first enters the host at the duodenum and then probably disseminates from this site to the other tissues. How the early immune response influences the clearance of parasite from tissues needs further study.

Molecular Study of Toxoplasma gondii Isolates Originating from Humans and Organic Pigs in Belgium.

Toxoplasma gondii is a worldwide prevalent, zoonotic parasite of major importance for public health, which can infect any warm-blooded animal species, including humans. Humans can get infected by consumption of meat from a chronically infected animal, by ingestion of sporulated oocysts (resulting from the sexual replication in felids), via contaminated water, soil, or vegetables, and by vertical transmission via the placenta. Infection through meat consumption is estimated to be one of the main sources of human toxoplasmosis cases in developed countries, and more specifically pork is considered to be responsible for 41% of foodborne human toxoplasmosis cases in the United States. To better assess the role of pork as a source of T. gondii infection in humans in Belgium, parasites were isolated from pigs to compare with human clinical isolates in a molecular epidemiological study. A positive result was obtained by magnetic capture-quantitative polymerase chain reaction for T. gondii in 14 out of the 92 hearts sampled during 2016 and 2017 from pigs raised in organic farms. From 9 of these 14 samples, parasites were isolated by mouse bioassay, demonstrating the presence of viable T. gondii in animals intended for human consumption. When genotyped and compared with 15 human isolates obtained during 2015 and 2016, a highly related structured population was demonstrated. Overall, these findings demonstrate the presence of infectious T. gondii in pigs intended for human consumption. Therefore, a potential transmission of T. gondii strains from pigs to humans could occur. However, both species could also be infected via a common source of infection such as oocysts. Furthermore, Belgium does not have an official surveillance program for T. gondii in human cases or food-producing animals; as a consequence, the detection of the infection source of a patient is very rare. Overall, this study reinforces the identification of pork as a potential risk for the consumers.

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.

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.

A more sensitive, efficient and ISO 17025 validated Magnetic Capture real time PCR method for the detection of archetypal Toxoplasma gondii strains in meat.

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.

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.

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-CD8αbright 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.

Toxoplasma gondii in stranded marine mammals from the North Sea and Eastern Atlantic Ocean: Findings and diagnostic difficulties.

The occurrence of the zoonotic protozoan parasite Toxoplasma gondii in marine mammals remains a poorly understood phenomenon. In this study, samples from 589 marine mammal species and 34 European otters (Lutra lutra), stranded on the coasts of Scotland, Belgium, France, The Netherlands and Germany, were tested for the presence of T. gondii. Brain samples were analysed by polymerase chain reaction (PCR) for detection of parasite DNA. Blood and muscle fluid samples were tested for specific antibodies using a modified agglutination test (MAT), a commercial multi-species enzyme-linked immunosorbent assay (ELISA) and an immunofluorescence assay (IFA). Out of 193 animals tested by PCR, only two harbour porpoise (Phocoena phocoena) cerebrum samples, obtained from animals stranded on the Dutch coast, tested positive. The serological results showed a wide variation depending on the test used. Using a cut-off value of 1/40 dilution in MAT, 141 out of 292 animals (41%) were positive. Using IFA, 30 out of 244 tested samples (12%) were positive at a 1/50 dilution. The commercial ELISA yielded 7% positives with a cut-off of the sample-to-positive (S/P) ratio≥50; and 12% when the cut-off was set at S/P ratio≥20. The high number of positives in MAT may be an overestimation due to the high degree of haemolysis of the samples and/or the presence of lipids. The ELISA results could be an underestimation due to the use of a multispecies conjugate. Our results confirm the presence of T. gondii in marine mammals in The Netherlands and show exposure to the parasite in both the North Sea and the Eastern Atlantic Ocean. We also highlight the limitations of the tests used to diagnose T. gondii in stranded marine mammals.

Molecular Analysis of Rising Fluoroquinolone Resistance in Belgian Non-Invasive Streptococcus pneumoniae Isolates (1995-2014).

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.

Serologic screening for 13 infectious agents in roe deer (Capreolus capreolus) in Flanders.

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.

Parasite distribution and associated immune response during the acute phase of Toxoplasma gondii infection in sheep.

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.

Isolation and genotyping of viable Toxoplasma gondii from sheep and goats in Ethiopia destined for human consumption.

BACKGROUND: Toxoplasma gondii is an obligate intracellular protozoan parasite that infects humans and a broad spectrum of warm-blooded vertebrates. The present study was undertaken with the objectives of isolation and determining the genotypes of T. gondii strains from sheep and goats slaughtered in East and West Shewa Zones of Oromia Regional State, Central Ethiopia. METHODS: Hearts of 47 sheep and 44 goats that were seropositive in the Direct Agglutination Test (DAT) were bioassayed in mice. A multiplex PCR assay with 15 microsatellite markers was employed for genotyping of T. gondii isolates from sheep and goats. RESULTS: Viable T. gondii were isolated from 47 (51.65%) animals, 27 sheep and 20 goats. Most isolates caused sub-clinical infections in mice, however, 2 sheep and 1 goat isolates were mouse-virulent, killing mice between 19-27 days post-inoculation. The success of T. gondii isolation in mice increased significantly (P = 0.0001) with higher DAT antibody titers in sheep and goats. Genotyping revealed that 29 (87.88%) of the 33 isolates were Type II, 3 (9.09%) were Type III and 1 (3.03%) was atypical. Three strains (one type II, one type III, and the atypical genotype) were virulent for mice. CONCLUSIONS: T. gondii tissue cysts in sheep and goats slaughtered for human consumption are widespread. This is the first report on isolation and genotyping of T. gondii from sheep and goats of Ethiopia.

A two-step lyssavirus real-time polymerase chain reaction using degenerate primers with superior sensitivity to the fluorescent antigen test.

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.

Seroprevalence of zoonotic parasites in pigs slaughtered in the Kathmandu Valley of Nepal.

For several years, the demand for pork has been on the rise in Nepal. To assess the importance of pork as a carrier of zoonotic agents, we performed a cross-sectional study in the Kathmandu Valley of Nepal, in which we serologically determined the infection status of slaughtered pigs with regard to three of the most important parasites transmitted through pork consumption: Trichinella spp., Taenia solium cysticerci, and Toxoplasma gondii. From 2007 to 2010, 742 pigs were sampled at slaughter, of which 0.1% (95% confidence interval [CI] 0.0-0.7%) were found positive for Trichinella infection, 13.8% (95% credibility interval [CrI] 0.8-28.5%) for T. solium cysticercosis, and 11.7% (95% CI 5.2-17.5%) for Toxoplasma infection. Further monitoring of the related animal and human disease burden and strengthening of food safety protocols throughout the pork production chain are strongly recommended.

A non-invasive intranasal inoculation technique using isoflurane anesthesia to infect the brain of mice with rabies virus.

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.

Partial depletion of CD4(+)CD25(+)Foxp3(+) T regulatory cells significantly increases morbidity during acute phase Toxoplasma gondii infection in resistant BALB/c mice.

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.

Direct detection and genotyping of Toxoplasma gondii in meat samples using magnetic capture and PCR.

Different transmission routes, including the ingestion of undercooked meat, can result in Toxoplasma gondii infection in humans. The development of effective prevention strategies is hampered by a lack of quantitative information on the contamination level of different types of meat. Therefore, we developed a method for detection and quantification of T. gondii. The method involved preparation of crude DNA extract from hundred gram samples of meat, magnetic capture of T. gondii DNA and, quantitative real-time PCR targeting the T. gondii 529-bp repeat element. The detection limit of this assay was approximately 230 tachyzoites per 100 g of meat sample. There was a linear relation between the number of parasites added to the samples and Cp-values. Results obtained with the PCR method were comparable to bioassay results for experimentally infected pigs, and to serological findings for sheep. In addition, the T. gondii in 50% of the positive sheep samples could be genotyped by sequencing of the GRA6 gene, after isolation of the gene by magnetic capture. Two subtypes of GRA6 type II were identified in the 16 samples from sheep. For seven samples, the identification of T. gondii as type II was confirmed by microsatellite typing. The PCR method can be used as an alternative to bioassay for detection and genotyping of T. gondii, and to quantify the organism in meat samples of various sources.

Functional characterization of in vivo effector CD4(+) and CD8(+) T cell responses in acute Toxoplasmosis: an interplay of IFN-gamma and cytolytic T cells.

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.