Pathogens in ticks collected from dogs in Berlin/Brandenburg, Germany

BackgroundTick-borne diseases are a major health risk for humans and dogs. In addition to collection and analysis of questing ticks, analysis of host- associated ticks for the presence of pathogens is a valuable method to gain insight into transmission patterns of tick-borne diseases.MethodsTicks were collected from dogs living in the Berlin/Brandenburg area. The three tick species Ixodes ricinus, Ixodes hexagonus and Dermacentor reticulatus were examined for the presence of Babesia spp., Borrelia spp., Rickettsia spp. and Anaplasmataceae. Conventional PCR followed by sequencing was used for pathogen detection and characterization.Results Babesia spp. were found in 2.5% and 3% of I. ricinus and I. hexagonus, respectively. Sequencing revealed the presence of Babesia microti, Babesia capreoli and Babesia venatorum. D. reticulatus were free of Babesia canis. Rickettsia spp. were detected in 61% of I. ricinus, 44% of I. hexagonus and 39% of D. reticulatus. Specifically detected were Rickettsia raoulti in D. reticulatus and I. hexagonus, Rickettsia helvetica in I. ricinus and I. hexagonus and Rickettsia monacensis in I. hexagonus. Anaplasma phagocytophilum and Candidatus Neoehrlichia mikurensis have been reported previously in I. ricinus (6.5% and 4.3%, respectively) and I. hexagonus (3.9% and 5.9%). Borrelia spp. were found in 11.6% of I. ricinus and 11.2% of I. hexagonus. Subsequent genospecies analysis revealed Borrelia afzelii, Borrelia garinii, Borrelia burgdorferi sensu stricto and Borrelia miyamotoi. Simultanous presence of more than one pathogen was found in 20% of I. ricinus and in 59% of I. hexagonus whereas the total frequency of any pathogen was 65% in I. ricinus, 59% in I. hexagonus and 64% in D. reticulatus. Ticks in which A. phagocytophilum was detected had a significantly increased risk of also containing Rickettsia. Ticks harbouring a pathogen had significantly higher scutal indices than ticks without presence of any pathogen.ConclusionsFrequencies of potential human or canine pathogens in ticks were considerable and DNA of all four groups of pathogens was detected. Differences in scutal indices might suggest that pathogens are frequently taken up by ticks when feeding on dogs in Berlin/Brandenburg

Cause and Effect Analysis between Influencing Factors Related to Environmental Conditions, Hunting and Handling Practices and the Initial Microbial Load of Game Carcasses

Environmental, hunting and handling factors affect the microbial load of hunted game and the resulting meat products. The aim of this study was to systematically investigate the influence of several factors on the initial microbial load (IML) of game carcasses during the early hunting chain. Eviscerated roe deer body cavities (n = 24) were investigated in terms of total viable count and the levels of Pseudomonas spp., Lactobacillus spp., Enterobacteriaceae and Escherichia coli (E. coli). Furthermore, a risk analysis based on the obtained original IML data, literature search and a Failure Mode and Effects Analysis (FMEA) was performed. The IML could be explained in a regression model by factors including the higher body weight (BW), damaged gastrointestinal tract by the shot, ambient temperature or rain. The levels of Lactobacillus spp. (p = 0.0472), Enterobacteriaceae (p = 0.0070) and E. coli (p = 0.0015) were lower on the belly flap surface when gloves were used during evisceration. The literature search revealed that studies examining influencing factors (IF) on the IML of game carcasses found contradictory effects of the comparable IF on IML. Potential handling failures may lead to a higher IML of game carcasses during the early hunting chain ranked by FMEA. Several handling practices for game carcasses are recommended, such as ensuring efficient cooling of heavier BW carcasses to limit bacterial growth or eviscerating heavier carcasses before lighter ones

Factors associated with diversity, quantity and zoonotic potential of ectoparasites on urban mice and voles

Wild rodents are important hosts for tick larvae but co-infestations with other mites and insects are largely neglected. Small rodents were trapped at four study sites in Berlin, Germany, to quantify their ectoparasite diversity. Host-specific, spatial and temporal occurrence of ectoparasites was determined to assess their influence on direct and indirect zoonotic risk due to mice and voles in an urban agglomeration. Rodent-associated arthropods were diverse, including 63 species observed on six host species with an overall prevalence of 99%. The tick Ixodes ricinus was the most prevalent species, found on 56% of the rodents. The trapping location clearly affected the presence of different rodent species and, therefore, the occurrence of particular host-specific parasites. In Berlin, fewer temporary and periodic parasite species as well as non-parasitic species (fleas, chiggers and nidicolous Gamasina) were detected than reported from rural areas. In addition, abundance of parasites with low host-specificity (ticks, fleas and chiggers) apparently decreased with increasing landscape fragmentation associated with a gradient of urbanisation. In contrast, stationary ectoparasites, closely adapted to the rodent host, such as the fur mites Myobiidae and Listrophoridae, were most abundant at the two urban sites. A direct zoonotic risk of infection for people may only be posed by Nosopsyllus fasciatus fleas, which were prevalent even in the city centre. More importantly, peridomestic rodents clearly supported the life cycle of ticks in the city as hosts for their subadult stages. In addition to trapping location, season, host species, body condition and host sex, infestation with fleas, gamasid Laelapidae mites and prostigmatic Myobiidae mites were associated with significantly altered abundance of I. ricinus larvae on mice and voles. Whether this is caused by predation, grooming behaviour or interaction with the host immune system is unclear. The present study constitutes a basis to identify interactions and vector function of rodent-associated arthropods and their potential impact on zoonotic diseases

Der Einfluss von Koinfektionen auf die Reservoirkompetenz von peridomestischen Nagetieren für Zecken-übertragene Krankheitserreger

Small rodents and their nests are fascinating and existential biotopes for numerous arthropod, helminth and protozoan animals. On the other side, some of the parasite species can cause disease in humans and companion animals and wild mice and voles are reservoir hosts for many tick-borne pathogens. Despite that, the biodiversity and quantitative occurrence of many wild rodent parasites and their zoonotic potential is largely unknown. Six rodent species from the genera Apodemus, Myodes and Microtus are presumably the most abundant non-commensal rodent species in Europe and also among the most important hosts for immature life stages of ticks in urban areas like Berlin. In the first instance, the present thesis therefore reviewed for the first time all the 460 invertebrate and 69 protozoan species which were reported from these six rodent species in Europe and gave a brief overview about the taxonomy, major morphological characteristics, life cycles and zoonotic potential of the macroparasites and intestinal Coccidia associated with these rodents. In a longitudinal field study, 257 rodents of these six species were trapped at four study sites in Berlin and examined for intestinal Coccidia, macroparasites and other invertebrates. The ectoparasites, as well as phoretic and non-parasitic arthropods detected in the fur and on the skin of the rodents were covered in manuscript 1, while helminths and intestinal Coccidia were addressed in manuscript 2. A high taxon richness of at least 84 species, comprising 63 arthropod, 20 helminth and one higher coccidian taxon was observed on/in the rodents in Berlin and no rodent individual was free of parasite infections. The tick Ixodes ricinus was the most frequent species with 56% prevalence and a mean intensity of 9.4 ticks per rodent host. Eight mite and one nematode species represent new records for the fauna of Germany. Rodent species, trapping location and season were clearly associated with the quantitative occurrence of most parasite groups. Mice were more often parasitised by digenean flukes, hymenlepidid tapeworms, the nematode Heterakis spumosa, lice and listrophorid mites but less often by anoplocephalid tapeworms, trombiculid and myocoptid mites than voles. In non-metric multidimensional scaling, host species even appeared to be more important than trapping location, since rodent individuals could mostly be allocated to one of three host taxon groups, only according to the parasite taxa they harboured. However, the different degrees of urbanisation between the trapping locations affected not only the presence and abundance of the rodent species, but also the species richness of rodent parasites. Compared to rural areas, mice and voles in Berlin carried less arthropod species with low host dependence and host specifity, such as fleas, trombiculid and non-parasitic gamasid mites. Furthermore, ecto- and endoparasites with lower host adaptation and high dependence on intermediate hosts or other external factors were decreasingly diverse, prevalent and/or numerous with increasing degree of urbanisation from periurban to urban sites. Endoparasites without host change (monoxenous) and stationary ectoparasites, in contrast, were more prevalent and/or numerous at urban sites. Apart from ticks, the parasites detected on the six peridomestic rodent species in Berlin only pose a minor zoonotic risk of infection for people, since species causing severe diseases, such as Echinococcus multilocularis were absent. In contrast, the role of these hosts in the urban life cycle of ticks is important and they most certainly participate in the maintenance of well-known and emerging tick-borne pathogens in Berlin. Hence, regression analyses were performed to assess the influence of co-infections on tick abundance on the rodent host. Considering several confounding factors, the abundance of I. ricinus larvae was negatively associated with the abundance of nematodes from the superfamily Heligmosomoidea and Syphacia sp., as well as of gamasid mite parasites from the family Laelapidae, while that of hymenolepidid tapeworms was positively associated. To verify a relationship of cause and effect, laboratory co-infection experiments were conducted and published in manuscript 3. Mice simultaneously infected with Heligmosomoides polygyrus nematodes as well as with larval or nymphal I. ricinus ticks exhibited substantially higher systemic type 2 T helper cell (Th2) responses, based on interleukin 13 and GATA 3 expression compared to single-infected mice. However, the development of partial immunity and the Th2 reactivity towards ticks were unaffected by the nematode infection during repeated larval tick infestations. Co-infections with H. polygyrus were also unable to affect the susceptibility for tick-transmitted Borrelia afzelii Lyme disease spirochetes and their replication, dissemination and induction of signs of pathology in the rodent host. The negative association between heligmosomoid nematodes and ticks in wild rodents could not be confirmed in laboratory mice. However, the observed negative effect of laelapid mites on tick abundance, which may be caused by predation, should be further investigated. These mites have the potential to affect feeding success of ticks and therefore the transmission of many tick-borne pathogens. The present field study constitutes a current basis for studies on parasite diversity, arthropod vector competence and natural co-infections.Kleine Nagetiere und ihre Nester sind faszinierende und existentielle Biotope für zahlreiche Arthropoden-, Helminthen-, und intestinale Protozoenarten. Auf der anderen Seite können einige der parasitischen Arten Krankheiten bei Menschen und Haustieren verursachen. Zudem sind wilde Wühlmäuse und Mäuse Reservoirwirte für viele Zecken übertragene Pathogene. Trotz allem sind die Biodiversität und das quantitative Vorkommen vieler Wildnagerparasiten, sowie ihr zoonotisches Potential noch immer weitgehend unbekannt. Sechs Nagetierarten aus den Gattungen Apodemus, Myodes und Microtus umfassen wahrscheinlich die individuenreichsten, nicht kommensalen Nagetierarten in Europa und gehören zugleich zu den wichtigsten Wirten für juvenile Zeckenstadien in urbanen Gebieten wie Berlin. Daher fasst diese Thesis zu Beginn erstmalig alle 460 Invertebraten- und 69 Protozoenarten zusammen, welche diese sechs Nagetierarten in Europa parasitieren oder anderweitig mit ihnen assoziiert sind und gibt zudem einen kurzen Überblick über die Taxonomie, die wichtigsten morphologischen Charakteristiken, die Lebenszyklen und das zoonotische Potential der Makroparasiten und intestinalen Kokzidien. In einer longitudinalen Freilandstudie wurden 257 Nagetiere dieser sechs Arten an vier Fangorten in Berlin gefangen und auf intestinale Kokzidien, Makroparasiten und andere Invertebraten untersucht. Die Ektoparasiten, sowie phoretische und nicht parasitische Arthropoden, welche im Fell und auf der Haut der Nagetiere gefunden wurden, werden im Manuskript 1 behandelt, während Helminthen und intestinale Kokzidien in Manuskript 2 präsentiert werden. Eine hohe Taxonvielfalt von mindestens 84 Arten, welche sich aus 63 Arthropoden , 20 Helminthenarten und einem höheren Kokzidientaxon zusammensetzt, wurde auf/in den Nagetieren in Berlin festgestellt und kein einziges Tier war frei von parasitischen Infektionen. Die Zecke Ixodes ricinus war die häufigste Art mit einer Prävalenz von 56% und einer mittleren Intensität von 9,4 Zecken pro Nagetierwirt. Acht Milbenarten und eine Nematodenart stellten Erstnachweise für die deutsche Fauna dar. Nagetierart, Fangort und Jahreszeit hatten deutlichen Einfluss auf das quantitative Vorkommen der meisten Parasitengruppen. Mäuse waren häufiger mit Trematoden, hymenolepididen Bandwürmern, dem Nematoden Heterakis spumosa, Tierläusen und listrophoriden Milben, aber seltener mit anoplocephaliden Bandwürmern, trombiculiden und myocoptiden Milben befallen als Wühlmäuse. Unter Verwendung einer Nicht-metrischen multidimensionalen Skalierung erschien die Wirtsart von größerer Bedeutung zu sein als der Fangort, da die Nagetierindividuen allein aufgrund ihrer Parasitentaxa einer von drei Wirtstaxongruppen zugeordnet werden konnten. Der unterschiedliche Grad an Urbanisierung zwischen den Fangorten hingegen beeinflusste nicht nur die Präsenz und Abundanz der Nagetierarten, sondern auch den Artenreichtum der Nagetierparasiten. Im Vergleich zu ländlichen Gebieten waren die Mäuse und Wühlmäuse in Berlin mit weniger Arthropodenarten mit geringer Wirtsabhängigkeit und spezifität, wie Flöhen, trombiculiden und nicht parasitischen gamasiden Milben, infestiert. Zudem waren Ekto- und Endoparasiten mit geringerer Wirtsadaptation und starker Abhängigkeit von Zwischenwirten oder anderen externen Faktoren weniger divers, prävalent und/oder individuenreich mit steigendem Grad der Urbanisierung von periurbanen zu urbanen Fangorten. Endoparasiten ohne Wirtswechsel (monoxen) und stationäre Ektoparasiten waren dagegen prävalenter und/oder zahlreicher an den urbanen Standorten. Mit Ausnahme der Zecken stellen die in dieser Studie gefundenen Parasiten der sechs peridomestischen Nagetierarten in Berlin nur ein geringes zoonotisches Infektionsrisiko für den Menschen dar. Arten wie der Fuchsbandwurm Echinococcus multilocularis, welche schwere Krankheiten verursachen, fehlten. Im Gegensatz dazu ist die Rolle dieser Wirte bedeutend für die urbanen Lebenszyklen von Zecken und sie nehmen unzweifelhaft auch an der Zirkulation von bekannten und neuartigen Zecken-übertragenen Krankheitserregern in Berlin teil. Aus diesem Grund wurden Regressionsanalysen zum Einfluss von Koinfektionen auf die Abundanz von Zecken auf ihren Nagetierwirten durchgeführt. Unter Berücksichtigung von wichtigen Störfaktoren war die Abundanz von I. ricinus Larven sowohl mit der Abundanz von Nematoden der Überfamilie Heligmosomoidea und von Syphacia sp., als auch mit der Abundanz von parasitischen, gamasiden Milben der Familie Laelapidae negativ assoziiert, während jene von hymenolepididen Bandwürmern positiv assoziiert war. Für den Nachweis einer kausalen Beziehung zwischen den Parasiten wurden experimentelle Koinfektionen in Labornagern durchgeführt, welche in Manuskript 3 publiziert wurden. Mäuse, welche gleichzeitig sowohl mit Heligmosomoides polygyrus Nematoden, als auch mit Zeckenlarven und nymphen von I. ricinus infiziert waren, zeigten beträchtlich erhöhte systemische Typ 2 T Helfer Zellantworten (Th2), auf der Basis der Interleukin 13- und GATA 3-Expression im Vergleich zu Mäusen mit Einzelinfektionen. Die Entwicklung einer teilweisen Immunität und die Th2 Reaktivität gegenüber Zecken waren hingegen unbeeinflusst von der Nematodeninfektion während wiederholter Infestationen mit Zeckenlarven. Koinfektionen mit H. polygyrus waren zudem nicht in der Lage, die Suszeptibilität gegenüber Zecken-übertragenen Borrelia afzelii Lyme-Spirochäten, sowie deren Replikation, Dissemination und Induktion von pathologischen Veränderungen im Nagetierwirt zu beeinflussen. Die in Wildnagern beobachtete negative Assoziation zwischen heligmosomoiden Nematoden und Zecken konnte somit in Labormäusen nicht bestätigt werden. Allerdings sollte ein womöglich negativer Effekt von laelapiden Milben auf die Zeckenabundanz, welcher eventuell auf Prädation beruht, in Zukunft näher untersucht werden. Diese Milben haben das Potential, den Erfolg der Blutmahlzeit von Zecken und somit die Transmission von Zecken-übertragenen Pathogenen zu beeinflussen. Die hier präsentierte Feldstudie bietet eine aktuelle Basis für weitere Studien zur Biodiversität von Parasiten, zur Vektorkompetenz von Arthropoden und zu natürlichen Koinfektionen

Standardised Sampling Approach for Investigating Pathogens or Environmental Chemicals in Wild Game at Community Hunts

Wildlife may host pathogens and chemicals of veterinary and public health relevance, as well as pathogens with significant economic relevance for domestic livestock. In conducting research on the occurrence and distribution of these agents in wildlife, a major challenge is the acquisition of a sufficient number of samples coupled with efficient use of manpower and time. The aim of this article is to present the methodology and output of a sampling approach for game animals, which was implemented from 2017/18 to 2020/21 at drive hunts in Brandenburg, Germany. The central element was a framework agreement with the BImA, whereby federal forest officials and other hunters collected most of the samples during field dressing. Further samples of game carcasses were obtained by scientists during subsequent gathering at a collection point. Altogether, 3185 samples from 938 wild ungulates of four species were obtained for various studies analysing—in this case—food-borne agents in game animals. Sampling was representative and reflected the proportional distribution of ungulate species hunted in Brandenburg. Hunting district and hunting season strongly influenced hunting bag and hence sampling success. This sampling approach was demonstrated to be a suitable basis for monitoring programs, that can be adapted to other regions

Small rodents as paratenic or intermediate hosts of carnivore parasites in Berlin, Germany.

Rodents are important intermediate and paratenic hosts for carnivore parasites, including the important zoonotic agents Toxoplasma, Echinococcus and Toxocara. Monitoring of such parasites in rodents can be used to detect increasing risks for human and veterinary public health. Rodents were trapped at four sites in Berlin, two near the city center, two at the periphery. PCRs were conducted to detect Coccidia (target ITS-1) and specifically Toxoplasma gondii (repetitive element) in brain and ascarids (ITS-2) in muscle or brain tissue. During necropsies, metacestodes were collected and identified using ITS-2 and 12S rRNA PCRs. An ELISA to detect antibodies against Toxocara canis ES antigens was performed. Within the 257 examined rodents, the most frequently observed parasite was Frenkelia glareoli predominantly found in Myodes glareolus. T. gondii was only detected in 12 rodents and Microtus spp. (although strongly underrepresented) had a significantly increased chance of being positive. Neither Echinococcus nor typical Taenia parasites of dogs and cats were found but Mesocestoides litteratus and Taenia martis metacestodes were identified which can cause severe peritoneal or ocular cysticercosis in dogs, primates and humans. Using PCR, the ascarids T. canis (n = 8), Toxocara cati (4) and Parascaris sp. (1) were detected predominantly in muscles. Seroprevalence of T. canis was 14.2% and ELISA was thus more sensitive than PCR to detect infection with this parasite. Non-parametric multidimensional scaling and cluster analysis revealed that parasite communities could be grouped into an urban and a peri-urban cluster with high frequency of ascarid-positive rodents in urban and high frequency of F. glareoli in peri-urban sites. Prevalence rates of parasites in rodents with potential impact for human or veterinary public health are considerable and the monitoring of transmission cycles of carnivore parasites in intermediate rodent hosts is recommended to estimate the health risks arising from wild and domesticated carnivores

Standardised Sampling Approach for Investigating Pathogens or Environmental Chemicals in Wild Game at Community Hunts

Wildlife may host pathogens and chemicals of veterinary and public health relevance, as well as pathogens with significant economic relevance for domestic livestock. In conducting research on the occurrence and distribution of these agents in wildlife, a major challenge is the acquisition of a sufficient number of samples coupled with efficient use of manpower and time. The aim of this article is to present the methodology and output of a sampling approach for game animals, which was implemented from 2017/18 to 2020/21 at drive hunts in Brandenburg, Germany. The central element was a framework agreement with the BImA, whereby federal forest officials and other hunters collected most of the samples during field dressing. Further samples of game carcasses were obtained by scientists during subsequent gathering at a collection point. Altogether, 3185 samples from 938 wild ungulates of four species were obtained for various studies analysing—in this case—food-borne agents in game animals. Sampling was representative and reflected the proportional distribution of ungulate species hunted in Brandenburg. Hunting district and hunting season strongly influenced hunting bag and hence sampling success. This sampling approach was demonstrated to be a suitable basis for monitoring programs, that can be adapted to other regions

Identification of patterns using non-metric multidimensional scaling (NMDS) and k-means cluster analysis.

<p>NMDS plots show similarity for trapped rodent species (A) or parasite-positive rodents (C) for the different trapping sites (G, Gatow, M, Moabit, S, Steglitz, T, Tegel) and trapping blocks (1–8). Rodents were considered to be positive for ascarids if the <i>T</i>. <i>canis</i> ELISA was positive or any ascarid species was detected by PCR. For every trapping week, the number of rodents of a particular species or the number positive for a particular parasite was used to calculate a Bray-Curtis dissimilarity matrix followed by NMDS. The matrix was used to identify the minimum number of k-means clusters which were indicated on the NMDS plots using ellipses of different colors. (B) Clusters were analyzed for differences in the proportions of particular rodent or parasite species (species relative to total number in that cluster) using mid-P exact tests. Numbers with different indices in the same column indicate significant differences (p<0.01) while identical indices indicate non-significant differences (p>0.05).</p

Odds ratios with 95% confidence intervals for variables determining the risk for detection of antibodies against <i>Toxocara canis</i>.

<p>Logistic regression was conducted using the variables study location (A), rodent genus (B) and a combination of both (C). Reference categories were <i>Apodemus</i> for genus and Gatow for study location. The odds ratios for the genus <i>Microtus</i> were 1.1×10⁻⁷ in both models in (B) and (C) but are not presented since antibodies against <i>T</i>. <i>canis</i> were not found in any sample from this genus and the very wide 95% confidence intervals. *, p < 0.05; **, p < 0.01.</p

Rodent species trapped at the different study sites and included in the study.

<p>Rodent species trapped at the different study sites and included in the study.</p