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.

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 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.

A SYBR green I real-time polymerase chain reaction (PCR) assay for detection and quantification of Trichomonas gallinae.

Trichomonas gallinae are parasitic flagellates of importance in wild and domestic birds. The parasite is worldwide distributed, and Columbine birds are its main host. Current research focuses mostly on epidemiological and phylogenetic studies. However, there is still a lack of knowledge regarding parasite-host interaction or therapy development. Real-time PCR is a useful tool for diagnostic and quantification of gene copies in a determined sample. By amplification of a 113-bp region of the 18S small subunit ribosomal RNA gene, a SYBR green-based real-time PCR assay was developed. A standard curve was prepared for quantification analysis. Assay efficiency, linearity, and dissociation analysis were successfully performed. Specificity, sensibility, and reproducibility analysis were tested. This assay could be a useful tool not only for diagnostic purposes but also for future in vivo and in vitro T. gallinae studies.

A modified method for purification of Eimeria tenella sporozoites.

Coccidiosis is an economically important gastrointestinal disease in domestic fowl. Eimeria species are the causative agents of avian coccidiosis. Current challenges in management and prevention of eimeriosis enhance the need for research in this field. Sporozoite purification is a necessary step for Eimeria spp. in vitro infection models. Current alternatives such as DE-52 anion exchange chromatography and Percoll gradient require time and resources. We present a modified protocol consisting on vacuum filtration of sporozoites using a disposable 5-µL filter. Yield percentages were similar to those reported for Percoll gradient purification. By reducing time and efforts during sporozoite purification, it could be possible to increase resources in other areas of Eimeria studies.

Alaria spp. mesocercariae in Eurasian badger (Meles meles) and wild boar (Sus scrofa) from the Bialowieza Forest, north-eastern Poland.

Alaria spp. mesocercariae are commonly found in wild boar and other omnivorous mammals. In Europe, the number of cases presenting Alaria mesocercariae infections has been on the rise in the last years. From October to December 2016, samples of muscle from tongue, neck, and mandibular regions were collected from 1 Eurasian badger (Meles meles) and 14 wild boars (Sus scrofa) hunted in the Bialowieza Forest, north-eastern Poland. Using the Alaria migration technique (AMT), Alaria mesocercariae were isolated and morphologically identified in one badger and one wild boar. To the authors' knowledge, this is the first report of Alaria mesocercariae in paratenic hosts from the Bialowieza Forest.

First detection of Baylisascaris procyonis in wild raccoons (Procyon lotor) from Leipzig, Saxony, Eastern Germany.

Baylisascaris procyonis is a zoonotic nematode mainly harbored by the North American raccoon. It can cause severe neurological problems in paratenic hosts and humans. In Germany, raccoons are spread throughout the country. However, the presence of B. procyonis in the German raccoon population has not been thoroughly studied. For this study, 32 wild raccoons were collected in the urban area Leipzig, Saxony, Eastern Germany. Adult ascaroid nematodes were isolated from the intestines and morphologically identified as B. procyonis. Species confirmation was conducted through PCR. In total, adult B. procyonis worms were found in 24 raccoons. The results of the present study add new information about the presence of the parasite in Saxony, Germany. Similarly, the results highlight the importance of the raccoon as a reservoir of zoonotic parasites.

Alaria alata mesocercariae in raccoons (Procyon lotor) in Germany.

Alaria alata is a trematode of carnivores from Europe. The mesocercarial stage was recently identified in wild boar meat from Europe. Previous histopathologic studies showed the presence of unidentified parasitic cysts within the tongues of raccoons from northern Germany. For identification of the parasite species, tissue samples of 105 raccoons originating from a National Park in northern Germany and from Berlin metropolitan area were collected. Histological examination of cryotome sections of frozen as well as paraffin-embedded tongues were used to identify parasite cysts. These were located in the connective and adipose tissue and in close proximity to small arterioles, suggesting a hematogenous spread of the parasite. Often, cysts were surrounded with mild infiltration by inflammatory cells. Additionally, mesocercariae were isolated from defrosted tongue samples of 11 raccoons. Molecular-biology assays confirmed the parasite species as A. alata. Except for one positive raccoon from Berlin City, all other positive raccoons originated from the sylvan Müritz National park, indicating an abundance of intermediate hosts in this area. Our results show that raccoons can act as paratenic hosts for A. alata and extend the broad host range of this parasite to a species introduced into Germany.

Taenia martis in a white-headed lemur (Eulemur albifrons) from a zoological park in North Rhine-Westphalia, Germany.

We present the case of Taenia martis metacestode infection in a white-headed lemur (Eulemur albifrons) from a zoological park. A post-mortem examination was conducted on the unexpectedly perished animal and focal granulomatous pneumonia with metacestodic tissue was discovered. Identification of T. martis was conducted through amplification and sequencing of a 12S rRNA gene fragment. We discuss the possible sources of infection and underline the importance of this infection in public health and conservation.

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.

Spotted Fever Group Rickettsiae in Ticks and Small Mammals from Grassland and Forest Habitats in Central Germany.

Rickettsiae of the spotted fever group (SFG) are zoonotic tick-borne pathogens. Small mammals are important hosts for the immature life stages of two of the most common tick species in Europe, Ixodes ricinus and Dermacentor reticulatus. These hosts and vectors can be found in diverse habitats with different vegetation types like grasslands and forests. To investigate the influence of environmental and individual factors on Rickettsia prevalence, this study aimed to analyse the prevalence of SFG rickettsiae in ticks and small mammals in different small-scale habitats in central Germany for the first time. Small mammals of ten species and ticks of two species were collected from grasslands and forests in the Hainich-Dün region, central Germany. After species identification, DNA samples from 1098 ticks and ear snips of 1167 small mammals were screened for Rickettsia DNA by qPCR targeting the gltA gene. Positive samples were retested by conventional PCR targeting the ompB gene and sequencing. Rickettsia DNA was detected in eight out of ten small mammal species. Small mammal hosts from forests (14.0%) were significantly more often infected than those from grasslands (4.4%) (p < 0.001). The highest prevalence was found in the mostly forest-inhabiting genus Apodemus (14.8%) and the lowest in Microtus (6.6%), which inhabits grasslands. The prevalence was higher in D. reticulatus (46.3%) than in the I. ricinus complex (8.6%). Adult ticks were more often infected than nymphs (p = 0.0199). All sequenced rickettsiae in I. ricinus complex ticks were R. helvetica, and the ones in D. reticulatus were R. raoultii. Unlike adults, questing nymphs have had only one blood meal, which explains the higher prevalence in I. ricinus adults. Interestingly, habitat type did influence infection probability in small mammals, but did not in ticks. A possible explanation may be the high prevalence in Apodemus flavicollis and A. sylvaticus which were more abundant in the forest.

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.

Morphological and Molecular Identification of Physaloptera alata (Nematoda: Spirurida) in a Booted Eagle (Aquila pennata) from Portugal.

Physaloptera spp. are parasitic nematodes that infect the gastrointestinal tracts of many carnivores and omnivores. Although they are distributed worldwide, Physaloptera spp. have not been studied in raptors in Portugal. In this study, we report Physaloptera alata in a booted eagle (Aquila pennata) in Portugal. Adult nematodes were discovered in the gizzard of a young booted eagle, and morphological features were consistent with those of the genus Physaloptera. DNA was extracted and a PCR assay performed to amplify a region of the 18S small subunit of the ribosomal RNA gene and the cytochrome c oxidase subunit I gene. The resulting PCR products were Sanger-sequenced, and comparison with the available sequences in the GenBank database confirmed the initial morphological classification as Physaloptera sp. Phylogenetic analysis clustered the sequence within the Physaloptera group. The presence of this parasite in raptors from Portugal is of particular importance to wildlife rehabilitation centers, disease ecologists, and wildlife professionals. Furthermore, we produced a new genetic sequence and have added it to the GenBank database of parasites in birds of prey.

Linguatula serrata in an imported dog in Germany: Single-case or emerging disease?

Linguatula serrata is a worm-like parasite with zoonotic potential that inhabits the nasal cavities of canids. Although most cases of linguatulosis are associated with unspecific and rather mild respiratory symptoms, cases of unusual infestations and severe courses in both animals and humans have been reported. In central and northern Europe, the pathogen used to appear only sporadically, however, within the last few years the number of detections has increased noticeably. In July 2020 an approximately nine-month-old dog, imported from Romania, was presented in a veterinary practice in Gotha, central Germany, due to persistent worsening cough. Despite antibiotic treatment the tussis became more severe until the dog expectorated multiple worm-like structures. Three of these specimens were sent to the Institute of Parasitology (Faculty of Veterinary Medicine, University of Leipzig, Leipzig) for morphological and genetic species identification. The latter was based on a 1000-bp fragment of the mitochondrial cytochrome c oxidase subunit I gene (cox1) and the complete nuclear 18S rRNA gene. The dog presented in this study suffered from a severe respiratory impairment caused by worm-like parasites inhabiting its upper respiratory tract. The detected parasites were morphologically identified as female specimens of the so-called tongue-worm L. serrata, which was confirmed by pairwise alignment and phylogenetic analysis of the produced sequences. We report an unusually severe case of L. serrata infection in an imported dog and discuss the spread of this potentially dangerous parasite in central and northern Europe.

First report about a cerebrospinal nematode infection in an alpaca (Vicugna pacos).

The number of New World camelids in European farms is rising and thus, the need for veterinary care towards these animals arises. However, veterinary care requires sophisticated knowledge on disease and pathogen occurrence within New World camelids. Here, an alpaca cria with neurological signs was admitted to the veterinary clinic. Although the animal was treated with antibiotics, vitamins and dexamethason, it refused to drink milk and the clinical status worsened. After euthanasia, necropsy and histopathological examination were carried out and revealed intracerebral nematode larvae. The morphology of these larvae strongly suggests them to be Baylisascaris procyonis, a parasite of raccoons. The extended history revealed that a fully grown raccoon was living within farm enclosures, suggesting an infection of the alpaca and the development of a cerebrospinal larva migrans. This zoonotic disease is characterized by aberrant larval migration that typically shows extraintestinal migration in dead-end hosts. The aim of this report is to sensitize practical colleagues towards this rare, but occasionally fatal infection in New World camelids while familiarizing diagnostic pathologists with the morphological characteristics of this disease.

Diversity of Borrelia burgdorferi sensu lato in ticks and small mammals from different habitats.

BACKGROUND: Ixodid ticks are important vectors for zoonotic pathogens, with Ixodes ricinus being the most important in Europe. Rodents are hosts of immature life stages of I. ricinus ticks and are considered main reservoirs for tick-borne pathogens, e.g. Borrelia burgdorferi. The aim of this study was to analyse the prevalence as well as genospecies and sequence type (ST) diversity of Borrelia burgdorferi sensu lato in ticks and small mammals from central Germany and to elaborate on the influence of environmental and/or individual host and vector factors on Borrelia prevalence. METHODS: After species identification, 1167 small mammal skin samples and 1094 ticks from vegetation were screened by B. burgdorferi sensu lato real-time polymerase chain reaction, and positive samples were characterized by multilocus sequence typing. Generalized linear (mixed) models were used to estimate how seasonality, small mammal species/tick life stage and habitat affect individual infection status. RESULTS: In total, 10 small mammal species and three tick species, Ixodes ricinus, Ixodes inopinatus (both considered members of the I. ricinus complex) and Dermacentor reticulatus, were investigated. Borrelia DNA was detected in eight host species, i.e. the striped field mouse (Apodemus agrarius), the yellow-necked field mouse (Apodemus flavicollis), the wood mouse (Apodemus sylvaticus), the water vole (Arvicola amphibius), the bank vole (Clethrionomys glareolus), the field vole (Microtus agrestis), the common vole (Microtus arvalis), and the common shrew (Sorex araneus). Two species were Borrelia negative, the greater white-toothed shrew (Crocidura russula) and the pygmy shrew (Sorex minutus). The average prevalence was 6.2%, with two genospecies detected, Borrelia afzelii and Borrelia garinii, and at least three STs that had not been previously reported in small mammals. Borrelia prevalence in small mammals did not differ between seasons. Six genospecies of Borrelia-Borrelia afzelii, Borrelia valaisiana, Borrelia garinii, Borrelia lusitaniae, Borrelia spielmanii, and Borrelia burgdorferi sensu stricto-and 25 STs of Borrelia, of which 12 have not been previously described at all and five have not been previously reported in Germany, were detected in 13% of I. ricinus complex ticks. Prevalence was highest in adult females (25.3%) and lowest in nymphs (11.4%). Prevalence was significantly higher in ticks from grassland (16.8%) compared to forests (11.4%). CONCLUSIONS: The high level of small mammal diversity in this region of Germany seems to be reflected in a wide variety of genospecies and STs of B. burgdorferi.

Diplotriaena obtusa infection in an Eurasian blue tit (Cyanistes caeruleus) in Germany. Pathology and phylogenetic analysis.

Diplotriaena obtusa is a nematode found in air sacs of a wide number of birds, including Passerine species. During a period of increased mortality of blue tits (Cyanistes caeruleus) in Germany, we collected adult nematode worms from the air sacs of a deceased male blue tit. The nematodes showed morphological features consistent with Diplotriaena ssp. A polymerase chain reaction (PCR) assay targeting the small subunit (SSU) 18S rRNA gene identified the parasite species as Diplotriaena obtusa. Phylogenetic analysis confirmed species identification. Further examinations against infectious pathogens like Suttonella ornithocola, Salmonella spp., Pasteurella spp., Chlamydia spp., Influenza A virus, Usutu virus and West Nile virus were negative. This is the first report of D. obtusa in a blue tit from Germany.

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.

Cryptosporidium sp. skunk genotype in wild raccoons (Procyon lotor) naturally infected with Baylisascaris procyonis from Central Germany.

Cryptosporidium spp. are apicomplexan parasites of public health concern. They are one of the main causes of intestinal diseases in humans and animals. Contaminated water is among the main sources of infection for humans and mammals. Raccoons are an introduced species in Germany. They are anthropogenic adapters with a natural affinity for water bodies. We collected samples from wild raccoons in the Federal States of Saxony and Thuringia, Central Germany. Through molecular genotyping, we found Cryptosporidium sp. skunk genotype in one raccoon from Saxony (1/24) and in one animal from Thuringia (1/27). Both raccoons were also infected with the zoonotic nematode Baylisascaris procyonis. This is the first report of co-infection with these two parasites in raccoons from Germany. Our study highlights the potential of these animals as carriers of zoonotic pathogens. Since raccoons can thrive in human settlements, this study provides data that can be used as a baseline for preventive programs.

Canine distemper outbreak in raccoons suggests pathogen interspecies transmission amongst alien and native carnivores in urban areas from Germany.

From December 2012 to May 2013, an outbreak occurred among urban wild carnivores from Berlin. We collected 97 free-ranging raccoons from the city area. PCR assays, histopathology and immunohistochemistry confirmed canine distemper virus (CDV) infection in 74 raccoons. Phylogenetic analysis of haemagglutinin gene fragments (1767 nucleotides) of CDV isolated from four raccoons showed close relation to CDV isolates from foxes from Germany and a domestic dog from Hungary; all belonging to the "Europe" lineage of CDV. These study results suggest an inter-species transmission of CDV as the origin for the outbreak among the raccoon population. Implications for domestic pets and suggested interspecies transmission between urban wildlife and raccoons are discussed. This is the first major outbreak of CDV amongst free-ranging raccoons in Europe.