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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.

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.

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. Graphical abstract

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 beDie Zahl landwirtschaftlich gehaltener Neweultkameliden nimmt in Europa stetig zu und daher wächst der Bedarf an veterinärmedizinischer Betreuung dieser Tiere. Eine kompetente Betreuung benötigt jedoch umfassendes Wissen zum Vorkommen von Erkrankungen und Krankheitserregern bei Neuweltkameliden. Im vorliegenden Fall wurde ein Alpaka-Cria mit neurologischen Ausfallerscheinungen in die Tierklinik eingewiesen. Trotz der Gabe von Antibiotika, Vitaminen und Dexamethason verschlechterte sich der Zustand des Tieres, und es verweigerte die Milchaufnahme. Nach der Euthanasie wurden eine Sektion und eine histopathologische Untersuchung durchgeführt, bei der intrazerebrale Nematodenlarven festgestellt wurden. Die Morphologie der Larven spricht für

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 E. acervulina gene copies were considerably reduced while E. tenella continued to multiply (p 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.

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.

Objective To investigate the presence of neutrophil extracellular traps (NETs) in vivo by analysing intestinal sections from experimentally Eimeria bovis - and naturally Eimeria arloingi -infected animals. Methods Intestinal samples of Eimeria arloingi - and Eimeria bovis -infected animals were analysed by using immunohistochemical and fluorescence approach by using monoclonal antibodies. Results Classical NET components were confirmed by co-localization of extracellular DNA being decorated with neutrophil elastase and histones in Eimeria -infected tissue samples. Here, extrusion of NETs was exclusively detected in intestinal polymorphonuclear neutrophils infiltrating Eimeria -infected sites. In vivo NETs were either found in close proximity or in direct contact to different Eimeria stages suggesting a stage-independent process. NETs were also found within the gut lumen driven by polymorphonuclear neutrophils that were contacting released oocysts. Conclusions We postulate that NETs might play an important role in innate defence reactions in coccidiosis therefore significantly altering the outcome of infection.

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.

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.

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 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.

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.

Raccoons are an invasive species continuously spreading throughout Europe with Germany as the core area. Here, raccoons mostly live in rural regions, but some urban populations are already established (city of Kassel) or starting to build up (Berlin). The objective of this study was to investigate Sarcoptes infections in racoons in these two urban areas and try to identify the putative origin of the parasite. Parasite morphology, gross and histopathology examinations of diseased skin tissue were consistent with Sarcoptes scabiei infection. Moreover, using 9 microsatellite markers we genotyped individual mites from five raccoons and compared them with fox-, wild boar- and Northern chamois-derived Sarcoptes mites, originating from Italy and Switzerland. The raccoon-derived mites clustered together with the fox samples and were clearly differentiated from those of the wild boar and chamois suggesting a fox origin for the raccoon mange infection. These results are evidence of cross-transmission of S. scabiei among wild carnivores. Although our results cannot elucidate whether raccoons became infected by frequent interaction with endemically or epidemically infected foxes or if these cases were the result of occasional contacts among these animal species. Nevertheless, they show that pathogens can be shared among urban populations of native and invasive carnivores

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 Muritz 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.