Vorkommenden Krankheitsfälle bei frei lebenden Waschbären (Procyon lotor) aus ruralen und urbanen Populationen in Nordostdeutschland

The North-American raccoon was first introduced to Germany in 1934. Currently, racoons occur throughout Germany with two major populations: one in the centre (Hessen), and the other in the northeast (Mecklenburg- Western Pomerania, Brandenburg). In their native North America raccoons are a well-known vector for infectious pathogens such as rabies, canine distemper virus or the zoonotic nematode Baylisascaris procyonis. Despite more than 70 years of successful introduction, there is a lack of knowledge regarding infectious diseases present in German raccoons. In order to provide information, two subpopulations of raccoons in north-eastern Germany were selected: one from a rural/sylvatic area (Müritz National Park (MNP) in Mecklenburg-Western Pomerania) and one from an urban settlement (Berlin metropolitan area). In total, 240 road-killed, hunted or euthanized animals were retrieved: 100 raccoons from MNP (2007-2011) and 140 animals from Berlin (2011-2013). Post- mortem examinations, histological, microbiological and molecular examinations of selected pathogens were performed on these animals. The results were published in four scientific articles: Article 1: Alaria alata, a previously detected parasite encysted in raccoon tongue tissue was investigated and identified. Mesocercariae were isolated from eleven selected raccoons and PCR assays successfully identified A. alata in nine animals from MNP and one raccoon from Berlin, the parasite was successfully identified as A. alata. The mesocercariae were only found in tissue of the tongue and it was absent from other organs during histological examinations suggesting that raccoons are acting as paratenic host for this trematode. The higher number of MNP positive cases in comparison with Berlin indicates that differences in food resources allow raccoons in MNP to more likely ingest A. alata intermediate hosts such as amphibians than their urban counterparts. Moreover, since raccoons are an introduced species they can extend the host range of some endemic European parasites. Article II: The second article of this project investigates sarcoptic mange in urban raccoons with three cases from Berlin and two cases from Kassel. Macroscopic skin lesions, histopathological findings and the morphology of the mites were described. In order to elucidate the infections possible origin, nine microsatellite markers were used to genotype isolated mites from these raccoons to compare the mites with S. scabiei derived from foxes, wild boar and chamois. The mites from the raccoons clustered together with fox-derived S. scabiei, suggesting a vulpine origin for the infection. These results indicate a cross-species transmission of S. scabiei between urban foxes and raccoons. Article III: The first major canine distemper outbreak in German raccoons is described in this article. It occurred in Berlin metropolitan area from winter 2012/2013. During this time, 97 raccoon carcasses suspicious for CDV infection were collected as part of this thesis. Histology, immunohistochemistry and RT-PCR assays were performed on organ tissue and 74 animals were confirmed positive for CDV. Additionally, phylogenetic analyses of the haemagglutinin gene of CDV strains isolated from 4 of these raccoons were conducted. These CDV strains clustered within the Europe lineage and were closely related to those of German foxes and Hungarian dogs. The results suggest interspecies transmission amongst raccoons and other carnivores, in this case most likely foxes or an unvaccinated dog. Article IV: This article is the summary of all investigations on the 240 raccoons examined in total. Besides post-mortem and histo-pathology investigations of all animals, screening for selected pathogens including the zoonotic nematodes B. procyonis were performed. Macrospcopic findings were mostly related to traumatic injuries as the causes of death. Histological examinations showed that 65.6% of all raccoons had inflammatory lesions, with gastro-intestinal- tract, liver, spleen and lymph nodes as the most commonly effected organs. Berlin animals more frequently carried viral or bacterial infections (CDV, PV, Leptospira spp.). Meanwhile, lesions caused by nematodes or A. alata were most often found in raccoons from MNP. B. procyonis, Trichinella spp., Lyssavirus, CAV-1, and SuHv-1 were not detected in any animal. The differences in pathogen occurrence are most likely driven by the respective environment. For urban raccoons habitat defragmentation, reduced home ranges, abundance of anthropogenic food and shelter as well as higher interspecies contact rate can result in easier pathogen transmission between raccoons and other urban wildlife or domestic animals. While in MNP ideal habitat, wide home ranges, diverse food resources with abundant intermediate hosts on the one side and rare interspecies contact on the other reduce the spectrum of infectious pathogens. Overall, the low number of infectious diseases in raccoons suggests that this animal species does not play an important role in disease transmission in North-eastern Germany. However, because raccoons explore human settlements closer than other wild species, their potential in pathogen transmission highlights the need for continuous investigation of urban raccoons with regard to public health.Seit seiner ersten, 1934 erfolgten, Einbürgerung ist der Nordamerikanische Waschbär (Proyon lotor) eine invasive Tierart in Deutschland. Waschbären sind in Deutschland weit verbreitet, können aber in zwei Hauptpopulationen differenziert werden: Eine im Zentrum (Hessen), eine andere im nordöstlichen Landesteil (Mecklenburg-Vorpommern, Brandenburg). In Nordamerika gilt der Waschbär als bekannter Überträger von Infektionserregern wie Tollwut, Staupe oder dem zoonotischen Nematoden Baylisascaris procyonis. Aber trotz ihrer 70 Jahre währenden, erfolgreichen Einbürgerung gibt es wenig Kenntnis zu Infektionskrankheiten bei Waschbären in Deutschland. Um zu untersuchen, welche Krankheiten oder Krankheitserreger bei diesen Tieren vorkommen, wurden zwei Teilpopulationen in Nordostdeutschland ausgewählt: eine in einem ländlichen Waldgebiet (Müritz Nationalpark (MNP), Mecklenburg-Vorpommern), ein urbane im Großraum Berlin. Insgesamt wurden 240 Verkehrsopfer, jagdlich erlegte oder eingeschläferte Waschbären untersucht: 100 aus dem MNP (2007 bis 2011) und 140 aus Berlin (2011-2013). Tierkörpersektionen, histologische, mikrobiologische und molekularbiologische Untersuchungen von ausgewählten Erregern wurden mit diesen Tieren durchgeführt. Die Ergebnisse sind in vier wissenschaftlichen Artikeln veröffentlicht: Artikel I: In vorangegangenen Studien histologisch entdeckte Parasitenzysten im Zungengewebe von Waschbären wurden untersucht und ihre Artzugehörigkeit identifiziert. Mesozerkarien konnten aus neun Tieren vom MNP und einem Tier aus Berlin isoliert und mittels PCR als Alaria alata in identifiziert werden. In histologischen Untersuchungen wurden A. alata Mesozerkarien nur in Zungengewebe detektiert, jedoch nicht in anderen Organen. Das deutet darauf hin, dass Waschbären für diesen Trematoden als paratenische Wirte auftreten. Die höhere Anzahl positiver A. alata Fälle im MNP im Vergleich zu Berlin läßt sich durch Unterschiede in der Nahrungszusammensetzung erklären, da den Waschbären im MNP häufiger Zwischenwirte von A. alata, wie Amphibien, zur Verfügung stehen als den urbanen Waschbären. Es konnte hier gezeigt werden, dass eine neueingebürgerte Art wie der Waschbär das Wirtsspektrum endemischer Parasiten erweitern kann. Artikel II: Der zweite Artikel aus diesem Projekt beschreibt Sarcoptesräude in urbanen Waschbären mit drei Fällen aus Berlin und zwei Fällen aus Kassel. Makroskopische Hautläsionen, histo-pathologische Befunde und die Morphologie der Milben werden beschrieben. Um den möglichen Ursprung der Infektionen zu finden, wurden neun Mikrosatellitenmarker für die Genotypisierung der von Waschbären isolierten Milben verwendet, um sie mit S. scabiei von Füchsen, Wildschweinen und Gämsen zu vergleichen. Die Milben der Waschbären lagen in einem Cluster mit S. scabiei von Füchsen, was für einem Infektionsursprung aus Füchsen spricht. Diese Ergebnisse deuten auf eine zwischenartliche Übertragung von S. scabiei zwischen urbanen Füchsen und Waschbären hin. Artikel III: Der erste große Staupeausbruch von Waschbären in Deutschland wird in diesem Artikel beschrieben, der im Winter 2012/2013 im Großraum Berlin stattfand. Während dieser Zeit, wurden im Rahmen dieser Doktorarbeit 97 Waschbärkadaver gesammelt. Histologische, immunhistochemische und RT-PCR Untersuchungen wurden mit Organproben durchgeführt und 74 Tiere als Staupe-positiv bestätigt. Zusätzlich erfolgten phylogenetische Analysen des Hämagglutiningens von Staupevirusisolaten aus vier dieser Waschbären. Alle diese Virusisolate lagen im phylogenetischen Cluster der „Europe“-Linie und sind eng verwandt zu Isolaten von Füchsen aus Deutschland und Hunden aus Ungarn. Diese Ergebnisse lassen eine Übertragung zwischen Waschbären und anderen Fleischfressern vermuten, in diesem Fall Füchsen oder möglicherweise auch einem ungeimpften Hund. Artikel IV: Dieser Artikel beinhaltet die Untersuchungen aller 240 Waschbären der gesamten Studie. Neben Sektionen und histo-pathologischen Untersuchungen wurden Organproben auf ausgewählte Krankheitserreger, die beim Waschbären beschrieben wurden, untersucht. Diese umfassen die zoonotischen Nematoden Baylisascaris procyonis, Trichinella spp., Tollwutvirus, Canines Adenovirus 1 (CAV-1), Suis herpesvirus 1 (SuHV 1, Aujeszkysche Krankheit), Parvovirus (PV), Canines Staupevirus (CDV) sowie Leptospira spp.. Makroskopische Befunde waren meist traumatischen Verletzungen, die in direktem Zusammenhang mit der Todesursache standen. Histologische Untersuchungen wiesen bei 65,6% der Waschbären entzündliche Läsionen auf, wobei Magen-Darm-Trakt, Leber, Milz und Lymphknoten am stärksten betroffen waren. Bei Berliner Waschbären fanden sich häufiger virale oder bakterielle Pathogene: CDV, PV, oder Leptospira spp., während Tiere aus dem MNP am häufigsten parasitäre Infektionen aufwiesen. In keinem der untersuchten Waschbären fanden sich weder B. procyonis oder Trichinella spp. noch Tollwutvirus, CAV-1, oder SuHv-1. Die Unterschiede im Pathogenvorkommen könnten durch die unterschiedlichen Habitate erklärt werden. Für urbane Waschbären kann es durch zersiedelte Habitat, reduzierte Streifgebietsgrößen, reiches Angebot sowohl anthropogener Nahrungsquellen als auch Zufluchtsorte zu erhöhten inner- als auch zwischenartlichen Kontaktraten kommen, die eine Pathogenübertragung erleichtern – nicht nur zwischen Waschbären als auch zwischen Waschbären und anderen urbanen Wildtieren sowie Haustieren oder auch Menschen. Im Unterschied dazu scheinen im MNP ideale Habitate und große Streifgebiete, verschiedenartige Futterressourcen, inklusive zahlreicher Zwischenwirte auf der einen Seite und seltene zwischenartliche Kontakte auf der anderen Seite zu einem geringerem Pathogenspektrum aber mit Schwerpunkt auf Parasiten zu führen. Generell deuten die geringen Zahlen von Infektionskrankheiten bei Waschbären im Nordosten Deutschlands daraufhin, dass die Waschbären gegenwärtig keine herausragende Rolle in der Verbreitung oder Übertragung von Pathogenen zu spielen scheinen. Dennoch sollte, gerade aufgrund der Tatsache, dass Waschbären menschliche Siedlungen intensiver nutzen können als andere Wildtiere, die potenzielle Übertragung von Krankheitserregern nicht außer Acht gelassen werden und im Interesse des Gesundheitswesens urbane Waschbären kontinuierlich untersucht werden

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

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

Interplay between <i>Eimeria acervulina</i> and <i>Cryptosporidium parvum</i> 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

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

Abstract 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

Presence of enterobacteriaceae, listeria spp., vibrio spp and staphylococcus spp in house fly (musca domestica l.), collected and macerated from different sites in contact with a few animals species

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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

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 C. parvum, 8.6% multi-strain infections (13 out 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. DISCUSSION We demonstrate the value of a new easy-to-use analytical procedure for critical characterization of C. parvum and C. hominis in epidemiological investigations, also applicable in retrospect. Our findings illuminate the hidden parasite diversity with important implications for tracing zoonotic and person-to-person transmissions