Neurobehavioral Disorders and Pathological Changes in the Brain of Mice Are Caused by Chronic Toxocara canis Larval Invasion with Low to Moderate Inoculum.

Toxocara canis larvae are one of the most overlooked agents of nervous system infection in paratenic hosts. Previous studies in mouse models have shown that infection with various (mainly high) numbers of larvae leads to neurobehavioral disturbances and pathological changes. Our study investigated whether the infection with low and moderate numbers of larvae could affect the physical condition, motor skills, and pathogenesis in the brains of experimentally infected mice.Two groups of BALB/c mice were orally infected with 10 and 100 T. canis larvae per animal and examined regularly until the 97th week after infection. General appearance, specific antibody responses, and motor/balance skills were assessed. The number and viability of larvae in the liver, spleen, lungs, and brain were assessed by quantitative compressed biopsy technique, while the pathological changes of the brain infection were studied histologically.As a result, changes were observed in overall appearance, activity, as well as motor and balance ability. The infections were associated with an increased IgG antibody response to the specific anti-T. canis excretory/secretory antigen and tissue damage in the brain characterized by necrosis, cell infiltrations, including foamy cells, and hemorrhages.The study demonstrated the effects of low and moderate T. canis infection in a paratenic host during the chronic phase of infection, which lasted up to 97 weeks for the first time.

SARS-CoV-2 Specific Humoral Immune Responses after BNT162b2 Vaccination in Hospital Healthcare Workers.

BACKGROUND: COVID-19 pandemic has led to a loss of human life in millions and devastating socio-economic consequences worldwide. So far, vaccination is the most effective long-term strategy to control and prevent severe COVID-19 disease. The aim of the current study was to evaluate the humoral immune responses raised against the BNT162b2 vaccine in hospital healthcare workers. METHODS: Total number of 173 healthcare workers enrolled in the study. Their blood samples were collected in three different time intervals after the second SARS-CoV-2 vaccination and evaluated by the ELISA method to detect anti-spike protein IgM and IgG antibodies. The baseline characteristics of all participants were collected using questionnaires and were evaluated for finding any significant data. RESULTS: Our results demonstrated that the levels of antibodies were higher in the young group (21-30 years old) and also among male participants. Moreover, the highest levels of antibodies were detected from the group that received the third shot vaccination. CONCLUSIONS: Our results indicate that age, gender and third-dose vaccination can affect the levels of humoral immune responses against the BNT162b2 vaccine in healthcare workers.

Toxocara canis infection worsens the course of experimental autoimmune encephalomyelitis in mice.

Toxocara canis, a gastrointestinal parasite of canids, is also highly prevalent in many paratenic hosts, such as mice and humans. As with many other helminths, the infection is associated with immunomodulatory effects, which could affect other inflammatory conditions including autoimmune and allergic diseases. Here, we investigated the effect of T. canis infection on the course of experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. Mice infected with 2 doses of 100 T. canis L3 larvae 5 weeks prior to EAE induction (the Tc+EAE group) showed higher EAE clinical scores and greater weight loss compared to the non-infected group with induced EAE (the EAE group). Elevated concentrations of all measured serum cytokines (IL-1α, IL-2, IL-4, IL-6, IL-10, IL-17A, IFN-γ and TNF-α) were observed in the Tc+EAE group compared to the EAE group. In the CNS, the similar number of regulatory T cells (Tregs; CD4+FoxP3+Helios+) but their decreased proportion from total CD4+ cells was found in the Tc+EAE group compared to the EAE group. This could indicate that the group Tc+EAE harboured significantly more CD4+ T cells of non-Treg phenotype within the affected CNS. Altogether, our results demonstrate that infection of mice with T. canis worsens the course of subsequently induced EAE. Further studies are, therefore, urgently needed to reveal the underlying pathological mechanisms and to investigate possible risks for the human population, in which exposure to T. canis is frequent.

The Impact of Escherichia coli Probiotic Strain O83:K24:H31 on the Maturation of Dendritic Cells and Immunoregulatory Functions In Vitro and In Vivo.

Early postnatal events are important for the development of the neonatal immune system. Harboring the pioneering microorganisms forming the microbiota of the neonatal gastrointestinal tract is important for priming the immune system, as well as inducing appropriate tolerance to the relatively innocuous environmental antigens and compounds of normal healthy microbiota. Early postnatal supplementation of suitable, safe probiotics could accelerate this process. In the current study, the immunomodulatory capacity of the probiotic strain of Escherichia coli O83:K24:H31 (EcO83) was characterized in vitro and in vivo. We compared the capacity of EcO83 with and without hemolytic activity on selected immune characteristics in vitro as determined by flow cytometry and quantitative real-time PCR. Both strains with and without hemolytic activity exerted comparable capacity on the maturation of dendritic cells while preserving the induction of interleukin 10 (Il10) expression in dendritic cells and T cells cocultured with EcO83 primed dendritic cells. Early postnatal supplementation with EcO83 led to massive but transient colonization of the neonatal gastrointestinal tract, as detected by in vivo bioimaging. Early postnatal EcO83 administration promoted gut barrier function by increasing the expression of claudin and occludin and the expression of Il10. Early postnatal EcO83 application promotes maturation of the neonatal immune system and promotes immunoregulatory and gut barrier functions.

Antigenic Proteins from the Excretory-Secretory Products of Toxocara canis Larvae and Evaluation of Their Potential for Immunodiagnostics of Larval Toxocarosis.

BACKGROUND: Larval toxocarosis is a zoonosis caused by larvae of Toxocara canis and T. cati, a gastrointestinal nematode of canids and felids, respectively. Diagnosis is usually performed by ELISA IgG using Toxocara excretory-secretory products as an antigen. Due to laboriousness of isolation of the products and subsequent process of standardization of antigenic compounds, routine use of this method is limited and can produce inaccurate diagnostical results. The purpose of this study was to discover new specific antigenic proteins that could be used in routine serological methods of larval toxocarosis. MATERIALS AND METHODS: Toxocara excretory-secretory products were collected and separated by SDS-PAGE. Proteins from the gel were electro-transferred to a membrane and incubated with mouse sera. Antigenic proteins were analyzed using the liquid chromatography-tandem mass spectrometry approach. Selected proteins were prepared in recombinant form and tested with mice and human sera by ELISA and Western blot. RESULTS: A total of four recombinant protein antigens were prepared (rTc-TES-26, rTc-ASA, rTc-PDP, and rTc-ASP). They were analyzed by ELISA and Western blot using mice and human sera. For all sera, three of the four recombinant antigens correlated with Toxocara excretory-secretory products in ELISA analysis. By Western blot, the infection was confirmed in all experimentally infected mice and two out of seven human patients. CONCLUSION: Combination of the presented methods and analyses represents a possible method of effective identification of Toxocara protein antigens for the purpose of routine serodiagnosis.

Effect of early postnatal supplementation of newborns with probiotic strain E. coli O83:K24:H31 on allergy incidence, dendritic cells, and microbiota.

Introduction: Probiotic administration seems to be a rational approach to promote maturation of the neonatal immune system. Mutual interaction of the microbiota with the host immune system is critical for the setting of appropriate immune responses including a tolerogenic one and thevmaintenance of homeostasis. On the other hand, our knowledge on the modes of actions of probiotics is still scarce. Methods: In our study, probiotic strain Escherichia coli O83:K24:H31 (EcO83) was administered to neonates of allergic mothers (AMs; neonates with increased risk for allergy development) within 48 h after the delivery, and the impact of this early postnatal supplementation on allergy incidence and selected immune markers has been analyzed 10 years after the primary EcO83 administration. Results: We have observed decreased allergy incidence in 10-year-old children supplemented with EcO83 (13 of 52 children were allergic) in comparison with non-supplemented children of AMs (16 of 42 children were allergic). The early postnatal EcO83 supplementation appeared to limit the allergy in the high-risk group (children of AMs) compared to that in the low-risk group (children of healthy mothers). Dendritic cells (DCs) in the peripheral blood of EcO83-supplemented children do not differ significantly in cell surface presence of CD83. The immunomodulatory capacity of EcO83 on DCs was tested in vitro as well. Both directly isolated myeloid and in vitro monocyte-derived DCs from cord blood increased CD83 expression together with interleukin (IL)-10 secretion after EcO83 stimulation. The effect of early postnatal EcO83 supplementation on the microbiota composition of 10-year-old children was characterized by next-generation sequencing, and we have not observed significant changes in the microbiota composition of EcO83-supplemented and non-supplemented children at the age of 10 years. Conclusions: Early postnatal EcO83 supplementation appears to lower allergy incidence in children of AMs. It seems that the beneficial effect of EcO83 is mediated via modulation of DC functional capacities without impacting the microbiota composition. Larger-scale studies will be necessary to confirm these preliminary findings.

Unravelling the genetic diversity and relatedness of Echinococcus multilocularis isolates in Eurasia using the EmsB microsatellite nuclear marker.

The cestode Echinococcus multilocularis is the causative agent of alveolar echinococcosis, a severe helminthic zoonotic disease distributed in the Northern Hemisphere. The lifecycle of the parasite is mainly sylvatic, involving canid and rodent hosts. The absence of genetic data from most eastern European countries is a major knowledge gap, affecting the study of associations with parasite populations in Western Europe. In this study, EmsB microsatellite genotyping of E. multilocularis was performed to describe the genetic diversity and relatedness of 785 E. multilocularis isolates from four western and nine eastern European countries, as well as from Armenia and the Asian parts of Russia and Turkey. The presence of the same E. multilocularis populations in the Benelux resulting from expansion from the historical Alpine focus can be deduced from the main profiles shared between these countries. All 33 EmsB profiles obtained from 528 samples from the nine eastern European countries belonged to the European clade, except one Asian profile form Ryazan Oblast, Russia. The expansion of E. multilocularis seems to have progressed from the historical Alpine focus through Hungary, Slovakia, the Czech Republic and southern Poland towards Latvia and Estonia. Most of the samples from Asia belong to the Asian clade, with one EmsB profile shared between Armenia and Turkey, and two between Turkey and Russia. However, two European profiles were described from two foxes in Turkey, including one harboring worms from both European and Asian clades. Three EmsB profiles from three Russian samples were associated with the Arctic clade. Two E. multilocularis profiles from rodents from Lake Baikal belonged to the Mongolian clade, described for the first time here using EmsB. Further worldwide studies on the genetic diversity of E. multilocularis using both mitochondrial sequencing and EmsB genotyping are needed to understand the distribution and expansion of the various clades.

Lower Functional and Proportional Characteristics of Cord Blood Treg of Male Newborns Compared with Female Newborns.

Understanding the early events involved in the induction of immune tolerance to harmless environmental antigens and microbiota compounds could reveal potential targets for allergic disease therapy or prevention. Regulatory T cells (Treg), particularly induced Treg (iTreg), are crucial for the induction and maintenance of tolerance against environmental antigens including allergens. A decrease in the number and/or function of Treg or iTreg could represent an early predictor of allergy development. We analyzed proportional and functional properties of Treg in the cord blood of children of allergic mothers (neonates at high risk of allergy development) and healthy mothers (neonates with relatively low risk of allergy development). We observed a higher number of induced Treg in the cord blood of females compared to males, suggesting an impaired capacity of male immunity to set up tolerance to allergens, which could contribute to the higher incidence of allergy observed in male infants. The decreased proportion of iTreg in cord blood compared with maternal peripheral blood documents the general immaturity of the neonatal immune system. We observed a positive correlation in the demethylation of the Treg-specific demethylated region (TSDR) and the proportion of Treg in cord blood. Our data suggest that immaturity of the neonatal immune system is more severe in males, predisposing them to increased risk of allergy development.

Seroprevalence of Larval Toxocarosis in the Czech Republic.

BACKGROUND: Larval toxocarosis (LT), a zoonotic disease transmitted by dogs, cats, and other carnivores, is caused by roundworms of the genus Toxocara. Humans become infected by ingesting embryonated eggs of this parasite. In this study, we present data on the seroprevalence of LT in the Czech Republic collected by the National Reference Laboratory for Tissue Helminthoses in 2012-2016. METHODS: Using enzyme-linked immunosorbent assay, a total of 4428 adults and children with or without clinical symptoms were examined for the presence of IgG antibodies against Toxocara canis excretory-secretory antigens. RESULTS: Of all the persons examined, specific Toxocara antibodies were detected in 160 (3.6%) individuals. There were, however, significant differences between various regions, with seropositivity rates ranging from 1.4 to 7.5%. CONCLUSION: In comparison to studies from 1998 and 2004, our results suggest a decrease in overall Toxocara seroprevalence in the Czech population, whereby the rates are similar to or even lower than rates in some other Central European countries.

Value of cord blood Treg population properties and function-associated characteristics for predicting allergy development in childhood.

Allergic diseases represent some of the most common immunological disorders with high clinical and economic impact. Despite intensive research, there are still few universally accepted and reliable biomarkers capable of predicting their development at an early age. There is therefore a pressing need for identification of potential predictive factors and validation of their prognostic value by correlating them with allergy development. Dysbalance of the branches of immune response, most often excessive Th2 polarization, is the principal cause of allergic diseases. Regulatory T cells (Treg) are a crucial population for the timely establishment of physiological immune polarization and induction and maintenance of tolerance against environmental antigens. This makes them a potentially promising candidate for an early marker predicting allergy development. In our study, we analysed samples of cord blood of children of allergic mothers and children of healthy mothers by flow cytometry and retrospectively correlated the data with clinical allergy status of the children at the age of 6 to 10 years. Studied parameters included cord blood Treg population proportions and functional properties - intracellular presence of IL-10 and TGF-b, MFI of FoxP3. We observed higher percentage of Tregs in cord blood of children who did not develop allergy compared with allergic children. Further, we found higher numbers of IL-10+ Tregs in cord blood of healthy children of healthy mothers than in cord blood of children of allergic mothers and decreased TGF-b+ cord blood Tregs in the group of allergic children of allergic mothers compared to all other groups.

Distinct characteristics of Tregs of newborns of healthy and allergic mothers.

Allergic diseases represent a major issue in clinical and experimental immunology due to their high and increasing incidence worldwide. Allergy status of the mother remains the best predictor of an individual's increased risk of allergy development. Dysregulation of the balance between different branches of immune response, chiefly excessive polarization towards Th2, is the underlying cause of allergic diseases. Regulatory T cells (Tregs) play a pivotal role in the timely establishment of physiological immune polarization and are crucial for control of allergy. In our study we used flow cytometry to assess Tregs in cord blood of newborns of healthy (n = 121) and allergic (n = 108) mothers. We observed a higher percentage of Tregs (CD4+CD25+CD127lowFoxP3+) in cord blood of children of allergic mothers. However, the percentage of cells expressing extracellular (PD-1, CTLA-4, GITR) and intracellular (IL-10, TGF-ß) markers of function was lower (significantly for PD-1 and IL-10) within Tregs of these children. Furthermore, Helios- induced Tregs in the cord blood of children of allergic mothers were decreased. These results were supported by a decrease in plasma levels of IL-10 and TGF-ß in cord blood of newborns of allergic mothers, implying lower tolerogenic capacity on the systemic level. Taken together, these findings reflect deficient function of Tregs in the group with higher risk of allergy development. This may be caused by a lower maturation status of the immune system, specifically Tregs, at birth. Such immaturity may represent an important mechanism involved in the increased risk of allergy in children of allergic mothers.

Decreased allergy incidence in children supplemented with E. coli O83:K24:H31 and its possible modes of action.

The growing knowledge of the key role of microbiota in the maturation of neonatal immune system suggests that manipulation of microbiota could be exploited in hampering allergy development. In this study, Escherichia coli O83:K24:H31 (EcO83) was administered to newborns that were followed prospectively. Several immunological characteristics (cytokines, specific IgE, total T regulatory cells (Treg) and subpopulation of natural Treg (nTreg) and induced Treg (iTreg)) were tested in peripheral blood of 8-year-old children. Incidence of allergic disease was decreased in EcO83 supplemented children and significantly elevated levels of IL-10 and IFN-É£ were detected in serum of EcO83 supplemented children. Probiotic supplementation did not influence the numbers of the total Treg population but their functional capacity (intracellular expression of IL-10) was significantly increased in children supplemented with EcO83 in comparison to non-supplemented children. Morover, decreased proportion of iTreg was present in peripheral blood of non-supplemented in comparison to EcO83 supplemented children. Finally, stimulation of cord blood cells with EcO83 promoted both gene expression and secretion of IL-10 and IFN-É£ suggesting that beneficial effect of EcO83 in prevention of allergy development could be mediated by promotion of regulatory responses (by IL-10) and Th1 immune response (by IFN-É£).

Humoral response of mice infected with Toxocara canis following different infection schemes.

The study was focused on the dynamics of humoral response to Toxocara canis excretory-secretory antigens (TES antigens) in mice experimentally infected by T. canis L3 larvae in different ways. In particular, we compared the effect of infection with two doses of 1000 larvae vs. repeated infections with a low number of larvae (daily infection with 10 larvae and weekly infection with 100 larvae in the course of 22 weeks). In ELISA, all infections, including both schemes with lower larval doses, elicited significant antibody response. Elevated levels of total IgE and TES-antigen-specific IgM were detected on day 12 after the first infection, followed by IgG and IgG1, and later by IgG3, IgG2a and IgG2b; specific IgE response was not detected. It seems that the high levels of IgM and IgG1 represent the best markers of infection. In addition, gradual increase of IgG2a and IgG2b could help in determination of the infection course. As a byproduct of our work, a new method of infection by repeated drinking of larvae was introduced; it minimizes the pain and discomfort for the experimental mice.

Different capacity of in vitro generated myeloid dendritic cells of newborns of healthy and allergic mothers to respond to probiotic strain E. coli O83:K24:H31.

Allergic diseases belong to one of the most common diseases with steadily increasing incidence even among young children. There is an urgent need to identify a prognostic marker pointing to increased risk of allergy development enabling early preventive measures introduction. It has been shown that administration of selected probiotic strains or mixtures could prevent allergy development. In our study, we have tested the capacity of probiotic strain Escherichia coli O83:K24:H31 (E. coli O83) to promote dendritic cell (DC) maturation and polarisation of immune responses. Increased presence of activation marker CD83 was observed on DC stimulated by E. coli O83 and DC of newborns of allergic mothers have significantly more increased cell surface presence of CD83 in comparison to children of healthy mothers. Increased gene expression and secretion of IL-10 was detected in DC stimulated with E. coli O83 being higher in DC of newborns of healthy mothers in comparison to allergic ones. Generally, increased presence of intracellular cytokines (IL-4, IL-13, IFN-gamma, IL-17A, IL-22, IL-10) was detected in CD4+ T cells cocultured with DC of children of allergic mothers in comparison to healthy ones. E. coli O83 primed DC significantly increased IL-10 and IL-17A in CD4 T cells of newborns of healthy mothers in comparison to the levels detected in CD4 T cells cocultured with control non-stimulated DC. We can conclude E. coli O83 induces dendritic cell maturation and IL-10 production in DC. Newborns of allergic mothers have generally increased reactivity of both DC and CD4 T cells which together with decreased capacity of DC of newborns of allergic mothers to produce IL-10 could support inappropriate immune responses development after allergen encounter.

Dirofilaria repens: emergence of autochthonous human infections in the Czech Republic (case reports).

BACKGROUND: Human dirofilariasis is a zoonotic infection that continues to spread to previously unaffected areas of Europe. In the South Moravian Region of the Czech Republic (CR), imported as well as autochthonous canine infections were recorded in the last decade, and parasite DNA was detected in mosquitoes of Aedes vexans. In the present paper, human Dirofilaria infections are reported from the country for the first time. CASE PRESENTATION: The samples from five patients with suspected tissue helminthiases were investigated. In particular cases, nematodes were isolated from various tissues including skin of lower leg, soft tissues of finger, subcutaneous tissue of hypogastrium, lymph node and peritoneum. The diagnosis was based on light microscopic morphology and/or DNA analysis of the worms. In addition, ELISA examination of patients' sera for anti-filaria IgG antibodies was performed. CONCLUSIONS: In the CR, five cases of human dirofilariasis caused by Dirofilaria repens were recorded during 2010-2014 (species determination for three of them was confirmed besides morphological also by DNA analysis). At least, three of the cases were of autochthonous origin (the patients are Czech citizens residing in South Moravian Region who have never travelled abroad). The findings confirm the natural setting of D. repens in South Moravian Region of the CR. Dirofilariasis should be therefore considered as endemic in this area where it may represent a significant risk factor for public health.

Presence of different genotypes of Helicobacter pylori in patients with chronic tonsillitis and sleep apnoea syndrome.

Helicobacter pylori, a well-known gastric pathogen, has been detected in the oral cavity and oropharynx in tonsillar tissue. In our study, the presence of H. pylori in the tonsillar tissue of patients with chronic tonsillitis and sleep apnoea syndrome (SAS) was investigated. The aim was to detect and genotype H. pylori for a collection of data supporting the possible role of H. pylori in the aetiology of chronic tonsillitis and SAS. Helicobacter pylori was detected by real-time polymerase chain reaction (rt-PCR). 89 patients, 60 with a diagnosis of chronic tonsillitis and 29 with SAS, were tested. In the chronic tonsillitis group, Helicobacter was detected in 48 (80 %) specimens, cagA gene was detected in 12 samples (25 %) and 12 samples were negative. In SAS group, Helicobacter was found in 24 samples (82.76 %), cagA gene was detected in 5 (20.83 %) and 5 samples (17.24 %) were negative. Helicobacter pylori-specific immunoglobulins were tested by ELISA in the serum of 57 patients only with 41 (71.93 %) showing positive. Our results on H. pylori DNA detection and H. pylori seropositivity show 26.32 % discrepancy, slightly in favour of rt-PCR (15.79 % compared to 10.53 %). The H. pylori presence in tonsillar tissue does not depend on the type of oropharyngeal disease (p = 0.756). This study shows that oropharynx constitutes an extragastric reservoir of H. pylori infection which could serve as an aetiopathogenetic factor for chronic tonsillitis and tonsillar hyperplasia by SAS. No conclusion has yet been drawn about the mechanism of the process.

Trichobilharzia mergi sp. nov. (Trematoda: Digenea: Schistosomatidae), a visceral schistosome of Mergus serrator (L.) (Aves: Anatidae).

Parasitological investigations on red-breasted mergansers (Mergus serrator L.) in Iceland revealed digenean flukes of the family Schistosomatidae. Adult worms were detected in blood vessels of the large intestine and eggs were deposited in the mucosa and surrounded by granulomatous reactions. Traditional morphological methods showed that the flukes have very slender filiform bodies, males are equipped with a short gynaecophoric canal and both suckers and spatulate ends are present on each sex. Among characteristics of the flukes which render them morphologically distinct from other Trichobilharzia species are: i) males-well developed vesicula seminalis (v.s.) consisting of a short v.s. externa and a significantly longer (approx. 3 times) v.s. interna, unusually well developed genital papilla and localization of the first testis a relatively long distance posterior to the gynaecophoric canal; ii) eggs-small and elongated with slightly rounded poles and a short terminal spine. DNA taxonomic techniques confirmed that a new species had been identified, Trichobilharzia mergi sp. n. The sequence data were deposited in GenBank under the accession numbers JX456151 to JX456172. Comparison of the results with our previously published data on characterization of DNA of cercariae isolated from freshwater lymnaeid snails showed that larval development of T. mergi is associated with Radix balthica L. (=Radix peregra Müller, 1774;=Radix ovata Draparnaud, 1805).

Cercarial dermatitis, a neglected allergic disease.

Cercarial dermatitis (swimmer's itch) is a common non-communicable water-borne disease. It is caused by penetration of the skin by larvae (cercariae) of schistosomatid flukes and develops as a maculopapular skin eruption after repeated contacts with the parasites. The number of outbreaks of the disease is increasing, and cercarial dermatitis can therefore be considered as an emerging problem. Swimmer's itch is mostly associated with larvae of the bird schistosomes of Trichobilharzia spp. Recent results have shown that mammalian infections (including man) manifest themselves as an allergic reaction which is able to trap and eliminate parasites in the skin. Studies on mammals experimentally infected by bird schistosome cercariae revealed, however, that during primary infection, parasites are able to escape from the skin to the lungs or central nervous system. This review covers basic information on detection of the infectious agents in the field and the clinical course of the disease, including other pathologies which may develop after infection by cercariae, and diagnosis of the disease.