Serological differentiation of antibodies against Rickettsia helvetica, R. raoultii, R. slovaca, R. monacensis and R. felis in dogs from Germany by a micro-immunofluorescent antibody test

Background Spotted Fever Group (SFG) Rickettsiae can cause febrile diseases with or without rash in humans worldwide. In Germany only limited data are available about their medical significance. Serological screening tests for antibodies against rickettsiae usually only distinguish between SFG and Typhus Group (TG) Rickettsiae due to the strong cross reactivities within the groups. Seroprevalence rates in dogs, as possible sentinels for tick-borne diseases, could serve as an indicator for the distribution of different Rickettsia species. Methods In this study, a micro-immunofluorescence assay (micro-IFA) was established for detection and differentiation of antibodies against five Rickettsia species in dogs (R. helvetica, R. raoultii, R. slovaca, R. monacensis and R. felis). Dogs that never left Germany (n = 605) previously investigated with an SFG-ELISA were included in this study and screened at a 1:128 dilution. Endpoint titres of fifty randomly selected seropositive samples of each of the five investigated regions in Germany were determined in order to allow a differentiation of the causative Rickettsia species. Sensitivity and specificity of the micro-IFA were compared with ELISA results of the previous study. Results A total of 93.9% of the dogs were positive for antibodies of the SFG Rickettsiae at the screening titer of 1:128. Differentiation of SFG Rickettsiae with the micro-IFA was possible in 70.4%, but in 29.6% of the cases the detected antibodies were not differentiable. Considering a clear differentiation by a twofold titre difference between observed reactions, the seroprevalence rates were 66.0% for R. helvetica, 2.8% for R. raoultii, 1.6% for R. slovaca, but no serological reaction could be clearly attributed to R. monacensis or R. felis. No statistically significant regional differences were found for R. helvetica, R. slovaca and R. raoultii comparing the five regions of Germany. Comparison of micro-IFA with ELISA revealed a sensitivity of 82.0% and a specificity of 83.8% for the Rickettsia SFG ELISA. Conclusions The micro-IFA is a useful serological tool to differentiate antibodies against different Rickettsia species in dogs. Seroprevalence rates in dogs correspond to the prevalence rates and distribution of Rickettsia-carrying tick species

Tick-borne lymphadenopathy (TIBOLA) acquired in Southwestern Germany

<p>Abstract</p> <p>Background</p> <p>Tick-borne lymphadenopathy (TIBOLA) was first described in 1997 in a patient in France. The causative agent, <it>Rickettsia slovaca</it>, is transmitted by <it>Dermacentor </it>ticks.</p> <p>Case presentation</p> <p>In southwestern Germany we encountered a patient with a tick bite at the dorsal scalp that resulted in an eschar and nuchal lymphadenopathy. Additionally, fever, malaise as well as elevated inflammatory markers and transaminases occurred. The characteristic clinical picture along with positive antibody testing for rickettsiae of the tick-borne spotted fever group strongly suggest the diagnosis TIBOLA.</p> <p>Conclusion</p> <p>Human rickettsioses are emerging infections. Clinicians should be aware of TIBOLA as a newly described rickettsial disease. As in our case, TIBOLA may be encountered in regions/countries where <it>R. slovaca </it>and <it>Dermacentor </it>ticks are prevalent but autochthonous acquisition was not described before.</p

Vergleichende Untersuchung zur Validität neuer Verfahren der LSD- und Ethanolbestimmung in Körperflüssigkeiten

Alkohol- und Drogenkonsum gehören aufgrund der Konsequenzen für das soziale Umfeld und der Gefährdungsmöglichkeiten am Arbeitsplatz und im Verkehr zu den größten sozialmedizinischen Problemen. Bei den Verfahren zur Bestimmung von Ethanol in Serum gibt es bezüglich der Präzision keine großen Unterschiede zwischen den meisten Assays. Lediglich der TDx REA® Ethanol Assay liegt teilweise außerhalb des festgelegten Grenzen für forensische Fragestellungen und scheint daher im Rahmen der forensischen Toxikologie weniger gut einsetzbar zu sein als die anderen Verfahren. Die Spezifität der meisten Tests ist gut, nur der ALC Ethyl Alcohol Assay weist eine erhebliche Störanfälligkeit gegenüber 1-Propanol und 1-Butanol auf. Ebenfalls von Interesse bei der Auswahl eines Verfahrens sind Praktikabilität, Zeitaufwand, Kosten und benötigtes Probenvolumen. Am einfachsten in der Handhabung sowie am günstigsten bezüglich des Zeitaufwandes sind der EMIT® ETS® Plus Ethyl Alcohol Assay von Syva Co./Behring und der Ethyl Alcohol Assay von Boehringer Mannheim. Sie weisen einen ausreichenden Linearitätsbereich (0,1-4 g/l) auf, so daß lediglich selten vorkommende, in der Blutalkoholkonzentration noch darüberliegende Proben verdünnt und erneut bestimmt werden müssen. Die mit diesen Assays gemessenen Werte liegen innerhalb des für forensische Zwecke geforderten Bereichs. Gemeinsam mit dem TDx REA® Ethanol Assay von Abbott sind sie auch was das benötigte Probenvolumen angeht am günstigsten. Der Vorteil der Headspace-Gaschromatographie liegt einerseits in der hohen Präzision der Methode, andererseits in der hohen Spezifität sowie der Möglichkeit der Begleitstoffanalyse. Gerade in der forensischen Toxikologie wird die Headspace-Gaschromatographie ungeachtet des relativ hohen Zeitaufwandes durch keine andere Methode zu ersetzen sein. Aufgrund der verbesserten Spezifität finden Immunoassays inzwischen auch beim LSD-Screening häufige Anwendung. Unsere Untersuchungen zum CEDIA® DAU- Test für LSD (Boehringer Mannheim) ergaben insgesamt eine geringe Störanfälligkeit dieser Methode. Bei den Untersuchungen zur Spezifität führte von den chemisch nicht verwandten Substanzen lediglich Ambroxol zu falsch positiven Meßwerten. Der Vergleich des CEDIA® DAU LSD mit dem EMIT® II LSD (Syva Co./Behring) zeigte die geringe Störanfälligkeit des CEDIA®- Tests. Von 21 falsch- positiven Ergebnissen des EMIT®- Tests fanden wir mit dem CEDIA®- Test nur noch 3 Proben positiv; diese enthielten alle Ambroxol. Bezüglich der Präzision, Spezifität und Praktikabilität ist der CEDIA® DAU LSD dem Abuscreen® OnLine LSD (Roche) in etwa gleichwertig. Der Microplate EIA von Cozard Bioscience hingegen schneidet, was die Spezifität angeht, schlechter ab. Die radioimmunchemischen Tests sind Interferenzen gegenüber am wenigsten störanfällig. Sie bergen aber den Nachteil, daß Abfälle gesondert entsorgt werden müssen, besondere Sicherheitsvorkehrungen getroffen werden müssen und die Reagenzien für die Tests schneller verfallen und somit auch höhere Gesamtkosten. Nur die chromatographischen Bestätigungsanalysen für den Nachweis von LSD sind so spezifisch, daß aufgrund des Drogennachweises strafrechtliche Konsequenzen möglich sind. Auf diesem Gebiet konnten in den letzten Jahren verschiedene Methoden mit ausreichender Empfindlichkeit etabliert werden. Es darf jedoch nicht vergessen werden, wie störanfällig und schwer der Einsatz solch komplizierter Verfahren im Laboralltag ist. Diese Schwierigkeiten werden vor allem durch die geringe Konzentration des Analyten und die störende Probenmatrix verursacht. Daher hat sich die Forschung der letzten Jahre auch darauf konzentriert, immunchemische Extraktionsverfahren zu entwickeln, die die gesuchte Substanz mit möglichst hoher Ausbeute aus der störenden Probenmatrix separieren. Unsere Erfahrungen mit dem immunchemischen Drogennachweis haben gezeigt, daß die Systeme teilweise erheblichen Qualitätsschwankungen unterliegen. Da ein positives Screeningergebnis allein keinen Beweiswert hat, ist es besonders wichtig, die Untersuchungsergebnisse kritisch zu hinterfragen, schließlich ist der Nachweis des Drogenkonsums für den Betroffenen oftmals folgenschwer. Ziel sollte es daher sein, vertrauenswürdige, störungsarme Bestätigungsanalysen zu entwickeln und positive Screeningergebnisse in jedem Fall einer solchen Analyse zu unterziehen

First detection of Hyalomma rufipes in Germany

Hyalomma rufipes, a two-host tick, is the most widespread Hyalomma species in Africa. In December 2015, an ixodid tick male with an unusual morphology was detected on a horse in a stable near Mainz in the Federal State Rhineland-Palatine. For identification purposes, the tick was preserved in alcohol and sent to our laboratory. The morphology of the tick showed specific characteristics of H. rufipes. The 16S rDNA sequence of H. rufipes from Germany was identical to the corresponding 16S rDNA sequence of H. rufipes from Tanzania, and they both were closely related to Hyalomma marginatum. The tick was tested with a real-time PCR for rickettsiae and Crimean-Congo hemorrhagic fever (CCHF) virus with negative results.EEA RafaelaFil: Chitimia-Dobler, Lidia. German Center of Infection Research (DZIF). Bundeswehr Institute of Microbiology; AlemaniaFil: Nava, Santiago. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Rafaela; Argentina.Fil: Bestehorn, Malena. German Center of Infection Research (DZIF). Bundeswehr Institute of Microbiology; AlemaniaFil: Dobler, Gerhard. German Center of Infection Research (DZIF). Bundeswehr Institute of Microbiology; AlemaniaFil: Wölfel, Silke. German Center of Infection Research (DZIF). Bundeswehr Institute of Microbiology; Alemani

HLA class I loss in metachronous metastases prevents continuous T cell recognition of mutated neoantigens in a human melanoma model

T lymphocytes against tumor-specific mutated neoantigens can induce tumor regression. Also, the size of the immunogenic cancer mutanome is supposed to correlate with the clinical efficacy of checkpoint inhibition. Herein, we studied the susceptibility of tumor cell lines from lymph node metastases occurring in a melanoma patient over several years towards blood-derived, neoantigen-specific CD8+ T cells. In contrast to a cell line established during early stage III disease, all cell lines generated at later time points from stage IV metastases exhibited partial or complete loss of HLA class I expression. Whole exome and transcriptome sequencing of the four tumor lines and a germline control were applied to identify expressed somatic single nucleotide substitutions (SNS), insertions and deletions (indels). Candidate peptides encoded by these variants and predicted to bind to the patient’s HLA class I alleles were synthesized and tested for recognition by autologous mixed lymphocyte-tumor cell cultures (MLTCs). Peptides from four mutated proteins, HERPUD1G161S, INSIG1S238F, MMS22LS437F and PRDM10S1050F, were recognized by MLTC responders and MLTC-derived T cell clones restricted by HLA-A*24:02 or HLA-B*15:01. Intracellular peptide processing was verified with transfectants. All four neoantigens could only be targeted on the cell line generated during early stage III disease. HLA loss variants of any kind were uniformly resistant. These findings corroborate that, although neoantigens represent attractive therapeutic targets, they also contribute to the process of cancer immunoediting as a serious limitation to specific T cell immunotherapy

Ixodes inopinatus − Occurring also outside the Mediterranean region

We report the presence of Ixodes inopinatus and its sympatric occurrence with Ixodes ricinus in southeastern Germany, western Austria, and Romania. The identification of I. inopinatus was based on morphological and molecular 16S rRNA and 12S rRNA gene features. We also report the finding of Rickettsia monacensis and Rickettsia helvetica in I. inopinatus collected from a fox and a sheep in Romania. Although the vector competence of I. inopinatus for these pathogens remains to be proven, there is evidence of transstadial persistence, an important prerequisite for acting as a vector.EEA RafaelaFil: Chitimia-Dobler, Lidia. German Center of Infection Research. Bundeswehr Institute of Microbiology; AlemaniaFil: Rieß, Ramona. German Center of Infection Research. Bundeswehr Institute of Microbiology; AlemaniaFil: Kahl, Olaf. Tick-radar GmbH; AlemaniaFil: Wölfel, Silke. German Center of Infection Research. Bundeswehr Institute of Microbiology; AlemaniaFil: Dobler, Gerhard. German Center of Infection Research. Bundeswehr Institute of Microbiology; Alemania. University of Hohenheim. Parasitology Unit; AlemaniaFil: Nava, Santiago. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Rafaela; ArgentinaFil: Estrada-Peña, Agustin. University of Zaragoza. Faculty of Veterinary Medicine. Department of Animal Health; Españ

Detection of Rickettsia monacensis and Rickettsia amblyommatis in ticks collected from dogs in Costa Rica and Nicaragua

The neotropical climate of Central America provides ideal conditions for ticks, which may transmit several human pathogens, including spotted-fever group Rickettsia. Dogs may act as sentinels or reservoirs for human tick-borne diseases due to shared tick species. Here, ticks were collected from 680 client-owned dogs in Nicaragua and Costa Rica, and a total of 316 tick pools were investigated for Rickettsia infection by quantitative real-time PCR (qPCR) targeting the gltA gene. Subsequently, up to six further genomic targets (16S rDNA, gltA, sca4, ompA, ompB and the 23S-5S intergenic spacer) were investigated for Rickettsia species determination. The predominant tick species was Rhipicephalus sanguineus sensu lato (s.l.) (19.9% of dogs infested in Costa Rica, 48.0% in Nicaragua), followed by Ixodes boliviensis (3.1% in Costa Rica / none in Nicaragua) and Amblyomma ovale (4.8% in Costa Rica, 0.9% in Nicaragua). In total, 22 of 316 tick pools containing 60 of 1023 individual ticks were Rickettsia-positive as determined by qPCR, resulting in a minimum infection rate (MIR) of 2.2%. In detail, MIR in Rh. sanguineus s.l. was 0.7% (7/281 pools), in I. boliviensis 33.3% (12/13 pools) and in A. ovale 9.7% (3/22 pools). For 11 of 12 positive I. boliviensis pools and one of six positive Rh. sanguineus s.l. pools, the species could be determined as R. monacensis. R. amblyommatis was identified in one Rh. sanguineus s.l. pool from Costa Rica and one A. ovale pool from Nicaragua. Nine of 12 R. monacensis-positive tick pools were collected in San Rafael de Heredia, Costa Rica, indicating a high local occurrence in this area. This study supports recent evidence that R. monacensis is present on the American continent. Its high local occurrence among dog-asso ciated I. boliviensis, which may also parasitize humans, in Costa Rica gives cause for concern, as R. monacensis is also pathogenic to humansEl clima neotropical de Centroamérica ofrece condiciones ideales para las garrapatas, que pueden transmitir varios patógenos humanos, entre ellos la Rickettsia del grupo de la fiebre manchada. Los perros pueden actuar como centinelas o reservorios de enfermedades humanas transmitidas por garrapatas debido a las especies de garrapatas que comparten. En este caso, se recogieron garrapatas de 680 perros propiedad de clientes en Nicaragua y Costa Rica, y se investigaron 316 grupos de garrapatas para detectar la infección por Rickettsia mediante PCR cuantitativa en tiempo real (qPCR) dirigida al gen gltA. Posteriormente, se investigaron hasta seis objetivos genómicos más (16S rDNA, gltA, sca4, ompA, ompB y el espaciador intergénico 23S-5S) para determinar la especie de Rickettsia. La especie de garrapata predominante fue Rhipicephalus sanguineus sensu lato (s.l.) (19,9% de los perros infestados en Costa Rica, 48,0% en Nicaragua), seguida de Ixodes boliviensis (3,1% en Costa Rica / ninguna en Nicaragua) y Amblyomma ovale (4,8% en Costa Rica, 0,9% en Nicaragua). En total, 22 de 316 grupos de garrapatas que contenían 60 de 1023 garrapatas individuales fueron positivas a Rickettsia según la qPCR, lo que dio lugar a una tasa de infección mínima (MIR) del 2,2%. En detalle, la TMI en Rh. sanguineus s.l. fue del 0,7% (7/281 pools), en I. boliviensis del 33,3% (12/13 pools) y en A. ovale del 9,7% (3/22 pools). En 11 de los 12 pozos positivos de I. boliviensis y en uno de los seis pozos positivos de Rh. sanguineus s.l., la especie pudo determinarse como R. monacensis. R. amblyommatis se identificó en un grupo de Rh. sanguineus s.l. de Costa Rica y en un grupo de A. ovale de Nicaragua. Nueve de los 12 charcos de garrapatas positivos para R. monacensis se recogieron en San Rafael de Heredia, Costa Rica, lo que indica una elevada presencia local en esta zona. Este estudio respalda las pruebas recientes de que R. monacensis está presente en el continente americano. Su elevada presencia local entre las garrapatas I. boliviensis asociadas a los perros, que también pueden parasitar a los humanos, en Costa Rica es motivo de preocupación, ya que R. monacensis también es patógena para los humanos.Universidad Nacional, Costa Rica.Escuela de Medicina Veterinari