Reduced neural progenitor cell count and cortical neurogenesis in guinea pigs congenitally infected with Toxoplasma gondii.

Toxoplasma (T.) gondii is an obligate intracellular parasite with a worldwide distribution. Congenital infection can lead to severe pathological alterations in the brain. To examine the effects of toxoplasmosis in the fetal brain, pregnant guinea pigs are infected with T. gondii oocysts on gestation day 23 and dissected 10, 17 and 25 days afterwards. We show the neocortex to represent a target region of T. gondii and the parasite to infect neural progenitor cells (NPCs), neurons and astrocytes in the fetal brain. Importantly, we observe a significant reduction in neuron number at end-neurogenesis and find a marked reduction in NPC count, indicating that impaired neurogenesis underlies the neuronal decrease in infected fetuses. Moreover, we observe focal microglioses to be associated with T. gondii in the fetal brain. Our findings expand the understanding of the pathophysiology of congenital toxoplasmosis, especially contributing to the development of cortical malformations.

Phytohormones regulate asexual Toxoplasma gondii replication.

The protozoan Toxoplasma gondii (T. gondii) is a zoonotic disease agent causing systemic infection in warm-blooded intermediate hosts including humans. During the acute infection, the parasite infects host cells and multiplies intracellularly in the asexual tachyzoite stage. In this stage of the life cycle, invasion, multiplication, and egress are the most critical events in parasite replication. T. gondii features diverse cell organelles to support these processes, including the apicoplast, an endosymbiont-derived vestigial plastid originating from an alga ancestor. Previous studies have highlighted that phytohormones can modify the calcium-mediated secretion, e.g., of adhesins involved in parasite movement and cell invasion processes. The present study aimed to elucidate the influence of different plant hormones on the replication of asexual tachyzoites in a human foreskin fibroblast (HFF) host cell culture. T. gondii replication was measured by the determination of T. gondii DNA copies via qPCR. Three selected phytohormones, namely abscisic acid (ABA), gibberellic acid (GIBB), and kinetin (KIN) as representatives of different plant hormone groups were tested. Moreover, the influence of typical cell culture media components on the phytohormone effects was assessed. Our results indicate that ABA is able to induce a significant increase of T. gondii DNA copies in a typical supplemented cell culture medium when applied in concentrations of 20 ng/µl or 2 ng/µl, respectively. In contrast, depending on the culture medium composition, GIBB may potentially serve as T. gondii growth inhibitor and may be further investigated as a potential treatment for toxoplasmosis.

Monocyte-Derived Chicken Macrophages Exposed to Eimeria tenella Sporozoites Display Reduced Susceptibility to Invasion by Toxoplasma gondii Tachyzoite.

Both Eimeria tenella and Toxoplasma gondii are common apicomplexan parasites in chickens. Host cell invasion by both protozoans includes gliding motility, host cell attachment and active penetration. Chicken macrophages as phagocytic cells participate in the innate host immune response against these two parasites. In this study, primary chicken monocyte-derived macrophages (MM) were infected with both pathogens to investigate mutual and host-parasite interactions. MM cultures were assigned to groups that were infected with E. tenella, T. gondii or both. In co-infected cultures, MM were first exposed to E. tenella sporozoites for 2 h. Afterwards, T. gondii tachyzoite infection was performed. Live-cell imaging was carried out to observe cell invasion and survival of T. gondii by single parasite tracking over a period of 20 h post infection (hpi). Quantitative analysis for parasite replication was performed by real-time quantitative PCR (qPCR) at 2, 6, 12 and 24 hpi. Overall, the ability of T. gondii to penetrate the cell membrane of the potential host cell was reduced, although high motility was displayed. We found that T. gondii tachyzoites adhered for more than 4 h to macrophages during early co-infection. qPCR results confirmed that significantly less T. gondii entered in E. tenella-activated MM at 2 hpi, and a reduced proportion of intracellular T. gondii survived and replicated in these cells at 24 hpi. We conclude that E. tenella modulates host cell responses to another apicomplexan agent, T. gondii, reducing active invasion and multiplication in chicken primary macrophages.

Interplay between Eimeria acervulina and Cryptosporidium parvum during In Vitro Infection of a Chicken Macrophage Cell Line (HD11).

BACKGROUND: Eimeria acervulina is a frequent intestinal pathogen of chickens, causing economic impact on the poultry industry. Cryptosporidium parvum is a neglected parasite in chickens. However, because of its zoonotic potential, poultry cryptosporidiosis may pose a risk to public health. Little is known about the parasite-host interactions during coinfection with both parasites. In this study, we investigated the possible interactions during in vitro coinfection of E. acervulina and C. parvum in a chicken macrophage cell line (HD11). METHODS: HD11 cells were inoculated with E. acervulina and C. parvum sporozoites and incubated 2, 6, 12, 24, and 48 h post infection (hpi). Mono-infections for each parasite were also investigated. Real-time PCR was used to quantify parasite replication. Additionally, macrophage mRNA expression levels of IFN-γ, TNF-α, iNOS, and IL-10 were measured. RESULTS: For both parasites, multiplication was, in most groups, lower in the coinfection group (COIG) compared with mono-infections. However, at 6 hpi, the number of C. parvum copies was higher in co-infections. Intracellular replication started to decrease from 12 hpi onward, and it was almost undetectable by 48 hpi in all groups. Infections resulted in low expression of all cytokines, except at 48 hpi. CONCLUSIONS: Infection of avian macrophages with both E. acervulina and C. parvum seemed to hinder intracellular replication for both parasites in comparison to mono-infection. A clear reduction in intracellular parasites from 12 hpi onward details the important role potentially played by macrophages in host control of these parasites.

Apicomplexan parasites are attenuated by low-energy electron irradiation in an automated microfluidic system and protect against infection with Toxoplasma gondii.

Radiation-attenuated intracellular parasites are promising immunization strategies. The irradiated parasites are able to invade host cells but fail to fully replicate, which allows for the generation of an efficient immune response. Available radiation technologies such as gamma rays require complex shielding constructions and are difficult to be integrated into pharmaceutical production processes. In this study, we evaluated for the first time low-energy electron irradiation (LEEI) as a method to generate replication-deficient Toxoplasma gondii and Cryptosporidium parvum. Similar to other radiation technologies, LEEI mainly damages nucleic acids; however, it is applicable in standard laboratories. By using a novel, continuous, and microfluidic-based LEEI process, tachyzoites of T. gondii and oocysts of C. parvum were irradiated and subsequently analyzed in vitro. The LEEI-treated parasites invaded host cells but were arrested in intracellular replication. Antibody-based analysis of surface proteins revealed no significant structural damage due to LEEI. Similarly, excystation rates of sporozoites from irradiated C. parvum oocysts were similar to those from untreated controls. Upon immunization of mice, LEEI-attenuated T. gondii tachyzoites induced high levels of antibodies and protected the animals from acute infection. These results suggest that LEEI is a useful technology for the generation of attenuated Apicomplexan parasites and has potential for the development of anti-parasitic vaccines.

Rapid Reverse Purification DNA Extraction Approaches to Identify Microbial Pathogens in Wastewater.

Wastewater monitoring became a promising solution in the early detection of outbreaks. Despite the achievements in the identification of pathogens in wastewater using real-time PCR, there is still a lack of reliable rapid nucleic acid extraction protocols. Therefore, in this study, samples were subjected to alkali, proteinase K and/or bead-beating followed by reverse purification magnetic beads-based separation. Wastewater samples spiked with S. aureus, E. coli and C. parvum were used as examples for Gram-positive and -negative bacteria and protozoa, respectively. All results were compared with a spin column technology as a reference method. Proteinase K with bead beating (vortexing with 0.1 mm glass beads for three minutes) was particularly successful for bacterial DNA extraction (three- to five-fold increase). The most useful extraction protocol for protozoa was pre-treatment with proteinase K (eight-fold increase). The selected methods were sensitive as far as detecting one bacterial cell per reaction for S. aureus, ten bacterial cells for E. coli and two oocysts for C. parvum. The extraction reagents are cold chain independent and no centrifuge or other large laboratory equipment is required to perform DNA extraction. A controlled validation trial is needed to test the effectiveness at field levels.

Fenbendazole treatment against Dentostomella translucida in Syrian golden hamsters.

Dentostomella translucida is an oxyurid nematode that was first discovered in the Mongolian gerbil but has also been detected in other wild and housed rodents. In conventional laboratory animals, oxyurid nematode parasites are widespread infections. A proven treatment strategy for pinworm eradication is the oral application of benzimidazoles, such as fenbendazole. In general, this drug is regarded as safe with minimal side effects. Nevertheless, in Sprague Dawley rats, a significantly reduced litter size could be seen after longer treatment with fenbendazole. Even though Dentostomella translucida was already described in Syrian golden hamsters (Mesocricetus auratus), data on treatment with fenbendazole and its effects on reproduction is lacking. Therefore, the main purposes of the study were (1) the verification of the effectiveness of fenbendazole as medicated feed (150 ppm) against this parasite in naturally infected Syrian golden hamsters in conventional husbandry and (2) monitoring of possible effects on reproduction during the treatment. Results show that fenbendazole treatment was highly effective against Dentostomella translucida, as numbers of pinworm eggs in the faeces were significantly reduced already after the first week of treatment in all animals. After four weeks of treatment, eggs were eradicated entirely. Interestingly, the average weaning weight was significantly reduced during treatment, but the litters were in good health.

Gastrointestinal parasites in young dogs and risk factors associated with infection.

Young dogs are particularly susceptible to infections with endoparasites. The occurrence of endoparasites was investigated in young dogs from Central Germany between July 2020 and July 2022. In total, 386 fecal samples originating from 171 dogs were examined for the prevalence of endoparasites using a combined flotation- and sedimentation technique and conventional PCR. Overall, in 41.2% (159/386) of the examined samples, endoparasites were detected. The most frequently occurring endoparasites were Giardia duodenalis (29%), Cryptosporidium spp. (9.1%), Cystoisospora spp. (7.3%), and Toxocara canis (6%). Sequencing of G. duodenalis positive samples showed that most infections belonged to the host-specific assemblages C (38.4% (43/112)) and D (35.7% (40/112)). The zoonotic assemblage A was identified in 8% (9/112) of the samples. Moreover, mixed infections were observed as follows: C/D in 5 (4.5%), D/A in 4 (3.6%), and C/A in 3 (2.7%) samples. All assemblage A infections were assigned to the potentially zoonotic subassemblage AI. Co-infections of G. duodenalis and Cryptosporidium spp. were observed in 3.1% (12/386) of the samples. Analyzing several host factors for their potential association with endoparasitic infection, the origin of dogs, as well as the living environment were identified as the main risk factors for infection with endoparasites. Overall, this study shows a high infection rate with endoparasites, especially G. duodenalis, in young dogs from Germany. The results of this study contribute to further insight into the distribution and potential risk factors associated with endoparasitic infections, as well as the zoonotic potential these parasites may present.

Prevalence and molecular characterization of Toxoplasma gondii in different types of poultry in Greece, associated risk factors and co-existence with Eimeria spp.

Toxoplasma gondii is a protozoan parasite of public health importance, infecting all warm-blooded animals, including chickens. Undercooked chicken meat or relevant products such as sausages could lead to human infections. In free-range, organic and slow-growth farming systems where the susceptibility period for chickens is extended, more knowledge about potential risk factors is essential. This study is the first seroepidemiological survey in different regions and types of chicken farms in Greece, using a major tachyzoite surface antigen-based ELISA (TgSAG1), combined with magnetic-capture PCR (mc-PCR) and bioassay for the isolation of strains from the chickens' tissues. Potential risk factors for T. gondii infection in these hosts were also investigated. Additionally, the co-existence of T. gondii and Eimeria spp. infections was assessed to elucidate epidemiological links between these two protozoan infections. Overall T. gondii seroprevalence was 9.5%. Of the backyard chickens sampled, 41.2% were seropositive and 70% of the organic and free-range layer farms had at least one T. gondii seropositive hen. No serologically positive broilers were found, although mc-PCR revealed a positive sample, highlighting the importance of accurate early-infection direct detection of T. gondii infections to ensure public health. T. gondii isolates obtained by mouse bioassay were genotyped. All belonged to type II (ToxoDB#3) as confirmed also by microsatellite typing. Production system, type of nutrition, and feeding system automation were identified as the most significant risk factors, while no association was found between the presence of cats and T. gondii seropositivity as calculated on both a farm level and per individual bird sampled.

Distribution of Cryptosporidium parvum subgenotypes in pre-weaned calves in Germany.

Cryptosporidium parvum is a main cause of severe diarrhea in young calves leading to economic loss and animal suffering. Little is known about the epidemiology of the genetic subtypes that may differ in their infectiousness and zoonotic potential. The present study analyzes the distribution of Glycoprotein 60 (GP60) variants in C. parvum positive samples obtained from young calves in Germany where individual fecal samples of 441 pre-weaned calves collected from 98 German dairy herds were evaluated for cryptosporidiosis (Göhring et al., 2014). DNA was extracted for Restriction Fragment Length Polymorphism (RFLP) analysis from 268 samples that were positive for Cryptosporidium by coproantigen EIA originating from 91 farms confirming C. parvum as the only species occurring. GP60 subtype analysis revealed 12C. parvum subtypes, all belonging to the zoonotic allele family IIa. The most widespread subtype was IIaA15G2R1 (71.4%). Other subtypes occurred sporadically: IIaA16G2R1, IIaA16G3R1 and IIaA17G2R1 (6.2%), IIaA13G1R2 (3.5%), IIaA14G1R1 (3.1%), IIaA14G2R1 (1.3%), IIaA17G1R1 (0.9%) and IIaA13G2R1, IIaA16G1R2, IIaA17G1R2 and IIaA24G1R1 (0.4%). Altogether a high diversity of subgenotypes was found with IIaA15G2R1 clearly dominating irrespective of geographic region. All subgenotypes are considered zoonotic underscoring that pre-weaned calves are likely to be a reservoir for zoonotic C. parvum and thus may be a risk for animal-to-human transmission.

Cryptosporidium infection in young dogs from Germany.

Cryptosporidium is an enteric protozoan parasite which is able to cause severe gastrointestinal disease and is distributed all over the world. Since information about the prevalence of cryptosporidiosis in German dogs is rare, the aim of this study was to examine the occurrence of Cryptosporidium spp. in dogs and the potential zoonotic risk emanating from these infected animals. In total, 349 fecal samples of 171 dogs were collected during the dogs' first year of life. The samples were examined for Cryptosporidium spp. using PCR, targeting the small subunit ribosomal RNA gene (SSU rRNA). Further analysis of Cryptosporidium parvum and Cryptosporidium canis positive samples was accomplished using the 60 kDa glycoproteine gene (GP60). Overall, 10.0% (35/349) of the specimens were tested positive for Cryptosporidium. Cryptosporidium canis was found in 94.3% (33/35) of these samples and the zoonotic type C. pavum in 5.7% (2/35). Both C. parvum infections were subtyped as IIaA15G2R1. Sixteen of the C. canis positive samples were successfully amplified at the GP60 gene locus. These isolates were identified to belong to the subtype families XXd, XXe, or XXb; however, 2 samples could not be assigned to any of the described subtype families. Considering the close contact between pets and their owners, dogs may act as a potential source of infection for human cryptosporidiosis. The results of this study, in context with other studies from different countries, provide important further insights into the distribution of Cryptosporidium species in dogs and their zoonotic potential.

Octaarginine Improves the Efficacy of Nitazoxanide against Cryptosporidium parvum.

Cryptosporidiosis is an intestinal disease that affects a variety of hosts including animals and humans. Since no vaccines exist against the disease till date, drug treatment is the mainstay of disease control. Nitazoxanide (NTZ) is the only FDA-approved drug for the treatment of human cryptosporidiosis. However, its efficacy in immunocompromised people such as those with AIDS, in malnourished children, or those with concomitant cryptosporidiosis is limited. In the absence of effective drugs against cryptosporidiosis, improving the efficacy of existing drugs may offer an attractive alternative. In the present work, we have assessed the potential of the cell-penetrating peptide (CPP) octaarginine (R8) to increase the uptake of NTZ. Octaarginine (R8) was synthetically attached to NTZ in an enzymatically releasable manner and used to inhibit growth of Cryptosporidium parvum in an in vitro culture system using human ileocecal adenocarcinoma (HCT-8) cell line. We observed a significant concentration-dependent increase in drug efficacy. We conclude that coupling of octaarginine to NTZ is beneficial for drug activity and it represents an attractive strategy to widen the repertoire of anti-cryptosporidial therapeutics. Further investigations such as in vivo studies with the conjugate drug will help to further characterize this strategy for the treatment of cryptosporidiosis.

Literature Review: Coinfection in Young Ruminant Livestock-Cryptosporidium spp. and Its Companions.

The protozoan Cryptosporidium parvum is one of the major causative pathogens of diarrhoea in young ruminants; therefore, it causes economic losses and impairs animal welfare. Besides C. parvum, there are many other non-infectious and infectious factors, such as rotavirus, Escherichia coli, and Giardia duodenalis, which may lead to diarrhoeic disease in young livestock. Often, more than one infectious agent is detected in affected animals. Little is known about the interactions bet-ween simultaneously occurring pathogens and their potential effects on the course of disease. In this review, a brief overview about pathogens associated with diarrhoea in young ruminants is presented. Furthermore, information about coinfections involving Cryptosporidium is provided.

TIDE Analysis of Cryptosporidium Infections by gp60 Typing Reveals Obscured Mixed Infections.

BACKGROUND: Cryptosporidiosis is a parasitic disease associated with potentially fatal diarrhea. The most used method in Cryptosporidium subtyping is based on the glycoprotein gene gp60. Each infection can represent a parasite population, and it is important to investigate the influence on transmission and virulence, as well as any impact on public health investigations. However, an easy-to-use method for detection is lacking. METHODS: Here we report on the use of the bioinformatic program TIDE for deconvolution of gp60 chromatograms. A combination of single oocyst analysis and cloning successfully confirmed the within-sample parasite population diversity. Retrospective sample analysis was conducted on archived chromatograms. RESULTS: For Cryptosporidium parvum, 8.6% multistrain infections (13 of 152) obscured by currently used consensus base calling were detected. Importantly, we show that single oocysts can harbor a mixed population of sporozoites. We also identified a striking dominance of unappreciated polymerase stutter artefacts in all 218 chromatograms analyzed, challenging the uncritical use of gp60 typing. CONCLUSIONS: We demonstrate the value of a new, easy-to-use analytical procedure for critical characterization of C. parvum and Cryptosporidium hominis in epidemiological investigations, also applicable retrospectively. Our findings illuminate the hidden parasite diversity with important implications for tracing zoonotic and person-to-person transmissions.

In vitro activity of selected natural products against Eimeria tenella sporozoites using reproduction inhibition assay.

Eimeria tenella is the causative agent of cecal coccidiosis in poultry characterized by weight loss, hemorrhagic diarrhea, and high mortality rates. Research into herbal candidates with possible anticoccidial activity has increased lately. As an alternative to animal experiments, an in vitro reproduction inhibition assay (RIA) was previously designed to determine the sensitivity of E. tenella isolates against ionophores. In this study, the RIA was used to test the anticoccidial activity of nutmeg oil, cinnamon oil, and glabridin. The concentration of nutmeg oil used in this study ranged between 1.1 and 139.1 µg/ml. Nutmeg oil exhibited a moderate in vitro inhibitory activity ranging from 35.5 to 49.5%. In contrast, no inhibitory effect was detected when incubating E. tenella sporozoites for 24 h with cinnamon oil at concentrations of 0.3 to 80.5 µg/ml. Glabridin (0.08-41.7 µg/ml) prevented the replication of sporozoites at a rate of 14.1 to 81.7% of inhibition. The calculated minimum concentrations of glabridin needed to inhibit parasite replication by 75%, 50%, and 30% (MIC75, MIC50, and MIC30) were 21.43 µg/ml, 5.28 µg/ml, and 0.96 µg/ml, respectively. Further studies to assess the in vitro efficacy of glabridin were performed by studying mRNA gene expression of stress-induced protein genes (HSP-70, NADPH, and EtPP5) after exposure of E. tenella sporozoites to glabridin at MIC75 for 0.5 h, 1 h, 2 h, and 4 h (a time-dependent experiment). Moreover, a dose-dependent experiment was performed using glabridin at a concentration matching MIC75, MIC50, and MIC30 for 24 h. In the time-dependent experiment, a significant (p < 0.05) increase of expression in NADPH and EtPP5 were detected after 4 h of incubation with glabridin at a concentration of 21.43 µg/ml. The dose-dependent experiment exhibited a gradual increase of expression in all studied genes, which indicates stress imposed on E. tenella sporozoites by glabridin. In our hands, RIA was suitable to assess the anticoccidial activity exhibited by the tested natural products as a precursor to in vivo studies which will help in the identification of novel anticoccidial candidates.

First Metabolic Insights into Ex Vivo Cryptosporidium parvum-Infected Bovine Small Intestinal Explants Studied under Physioxic Conditions.

The apicomplexan Cryptosporidium parvum causes thousands of human deaths yearly. Since bovines represent the most important reservoir of C. parvum, the analysis of infected bovine small intestinal (BSI) explants cultured under physioxia offers a realistic model to study C. parvum-host cell-microbiome interactions. Here, C. parvum-infected BSI explants and primary bovine small intestinal epithelial cells were analysed for parasite development and metabolic reactions. Metabolic conversion rates in supernatants of BSI explants were measured after infection, documenting an immediate parasite-driven metabolic interference. Given that oxygen concentrations affect cellular metabolism, measurements were performed at both 5% O2 (physiological intestinal conditions) and 21% O2 (commonly used, hyperoxic lab conditions). Overall, analyses of C. parvum-infected BSI explants revealed a downregulation of conversion rates of key metabolites-such as glucose, lactate, pyruvate, alanine, and aspartate-at 3 hpi, followed by a rapid increase in the same conversion rates at 6 hpi. Moreover, PCA revealed physioxia as a driving factor of metabolic responses in C. parvum-infected BSI explants. Overall, the ex vivo model described here may allow scientists to address pending questions as to how host cell-microbiome alliances influence intestinal epithelial integrity and support the development of protective intestinal immune reactions against C. parvum infections in a realistic scenario under physioxic conditions.

Establishment and validation of a guinea pig model for human congenital toxoplasmosis.

BACKGROUND: Toxoplasma gondii is an obligate intracellular parasite with a worldwide distribution. Congenital infection in humans and animals may lead to severe symptoms in the offspring, especially in the brain. A suitable animal model for human congenital toxoplasmosis is currently lacking. The aim of this study is to establish and validate the guinea pig as a model for human congenital toxoplasmosis by investigating the impact of the T. gondii infection dose, the duration of infection and the gestational stage at infection on the seroconversion, survival rate of dams, fate of the offspring, T. gondii DNA loads in various offspring tissues and organs and the integrity of the offspring brain. METHODS: Pregnant guinea pigs were infected with three different doses (10, 100, 500 oocysts) of T. gondii strain ME49 at three different time points during gestation (15, 30, 48 days post-conception). Serum of dams was tested for the presence of T. gondii antibodies using immunoblotting. T. gondii DNA levels in the dam and offspring were determined by qPCR. Offspring brains were examined histologically. RESULTS: We found the survival rate of dams and fate of the offspring to be highly dependent on the T. gondii infection dose with an inoculation of 500 oocysts ending lethally for all respective offspring. Moreover, both parameters differ depending on the gestational stage at infection with infection in the first and third trimester of gestation resulting in a high offspring mortality rate. The duration of infection was found to substantially impact the seroconversion rate of dams with the probability of seroconversion exceeding 50% after day 20 post-infection. Furthermore, the infection duration of dams influenced the T. gondii DNA loads in the offspring and the integrity of offspring brain. Highest DNA levels were found in the offspring brain of dams infected for ≥ 34 days. CONCLUSION: This study contributes to establishing the guinea pig as a suitable model for human congenital toxoplasmosis and thus lays the foundation for using the guinea pig as a suitable animal model to study scientific questions of high topicality and clinical significance, which address the pathogenesis, diagnosis, therapy and prognosis of congenital toxoplasmosis.

Burden and regional distribution of Toxoplasma gondii cysts in the brain of COBB 500 broiler chickens following chronic infection with 76K strain.

Toxoplasmosis is a worldwide zoonosis caused by the obligate intracellular apicomplexan parasite Toxoplasma gondii (T. gondii). Chickens are ground-feeders and represent, especially if free-range, important intermediate hosts in the epidemiology of toxoplasmosis and are used as sentinels of environmental contamination with T. gondii oocysts. Until now, little is known about the burden and regional distribution of T. gondii cysts in the chicken brain. It was therefore the aim of this study to investigate the abundance and specific distribution of T. gondii cysts within the chicken brain following chronic infection with a type II strain (76 K) of T. gondii. A total of 29 chickens were included in the study and divided into control group (n = 9) and two different infection groups, a low dose (n = 10) and a high dose (n = 10) group, which were orally inoculated with 1500 or 150,000 T. gondii oocysts per animal, respectively. Seroconversion was detected in the majority of chickens of the high dose group, but not in the animals of the low dose and the control group. Moreover, T. gondii DNA was detected most frequently in the brain and more frequently in the heart than in liver, spleen, thigh and pectoral muscle using qPCR analysis. The number of T. gondii cysts, quantified in the chicken brain using histological analysis, seems to be considerably lower as compared to studies in rodents, which might explain why T. gondii infected chickens very rarely, if at all, develop neurological deficits. Similar to observations in mice, in which no lateralisation for T. gondii cysts was reported, T. gondii cysts were distributed nearly equally between the left and right chicken brain hemispheres. When different brain regions (fore-, mid- and hindbrain) were compared, all T. gondii cysts were located in the forebrain with the overwhelming majority of these cysts being present in the telencephalic pallium and subpallium. More studies including different strains and higher doses of T. gondii are needed in order to precisely evaluate its cyst burden and regional distribution in the chicken brain. Together, our findings provide insights into the course of T. gondii infection in chickens and are important to understand the differences of chronic T. gondii infection in the chicken and mammalian brain.

In vitro infection of Madin-Darby bovine kidney (MDBK) cells with Eimeria acervulina sporozoites: quantitative analysis of parasite cellular invasion and replication using real-time polymerase chain reaction (PCR).

Poultry coccidiosis causes considerable economical losses to the livestock industry. Eimeria parasites are responsible for this disease. On a global scale, E. acervulina and E. tenella are amongst the most common Eimeria spp. infecting broilers. E. tenella is commonly used as infection model in in vivo and in vitro studies. On the other hand, E. acervulina has barely been studied under in vitro conditions. A well established and widely used in vitro model for E. tenella infection is the Madin-Darby bovine kidney cell line (MDBK); however, little is known regarding suitability of MDBK cells as host cells for E. acervulina. We infected MDBK monolayers with two different doses, 5 × 104 and 2 × 105, of E. acervulina sporozoites and evaluated cultures at 24 and 96 h post infection (hpi). For comparison, we ran an identical infection assay using E. tenella sporozoites. To assess parasite reproduction, the number of DNA copies of E. acervulina SCAR marker and E. tenella ITS-1 gene was quantified using real-time quantitative PCR. We found that the number of E. acervulina copies increased significantly at 24 hpi in comparison to E. tenella (p < 0.05). After 96 hpi, E. acervulina gene copies were considerably reduced while E. tenella continued to multiply (p < 0.05). Our results show that MDBK monolayers could be used for in vitro research aimed to study E. acervulina sporozoite cell invasion. Nevertheless, modifications of in vitro cultivation appear necessary to allow qualitative and quantitative studies over longer periods of parasite reproduction.

Metabolic Signatures of Cryptosporidium parvum-Infected HCT-8 Cells and Impact of Selected Metabolic Inhibitors on C. parvum Infection under Physioxia and Hyperoxia.

Cryptosporidium parvum is an apicomplexan zoonotic parasite recognized as the second leading-cause of diarrhoea-induced mortality in children. In contrast to other apicomplexans, C. parvum has minimalistic metabolic capacities which are almost exclusively based on glycolysis. Consequently, C. parvum is highly dependent on its host cell metabolism. In vivo (within the intestine) infected epithelial host cells are typically exposed to low oxygen pressure (1-11% O2, termed physioxia). Here, we comparatively analyzed the metabolic signatures of C. parvum-infected HCT-8 cells cultured under both, hyperoxia (21% O2), representing the standard oxygen condition used in most experimental settings, and physioxia (5% O2), to be closer to the in vivo situation. The most pronounced effect of C. parvum infection on host cell metabolism was, on one side, an increase in glucose and glutamine uptake, and on the other side, an increase in lactate release. When cultured in a glutamine-deficient medium, C. parvum infection led to a massive increase in glucose consumption and lactate production. Together, these results point to the important role of both glycolysis and glutaminolysis during C. parvum intracellular replication. Referring to obtained metabolic signatures, we targeted glycolysis as well as glutaminolysis in C. parvum-infected host cells by using the inhibitors lonidamine [inhibitor of hexokinase, mitochondrial carrier protein (MCP) and monocarboxylate transporters (MCT) 1, 2, 4], galloflavin (lactate dehydrogenase inhibitor), syrosingopine (MCT1- and MCT4 inhibitor) and compound 968 (glutaminase inhibitor) under hyperoxic and physioxic conditions. In line with metabolic signatures, all inhibitors significantly reduced parasite replication under both oxygen conditions, thereby proving both energy-related metabolic pathways, glycolysis and glutaminolysis, but also lactate export mechanisms via MCTs as pivotal for C. parvum under in vivo physioxic conditions of mammals.