Reconstruction of the molecular evolution of Usutu virus in Germany: Insights into virus emersion and circulation.

Usutu virus (USUV) is a mosquito-borne flavivirus that is widely distributed in southern and central Europe. The zoonotic virus circulates primarily between birds and mosquitoes, can, however, in rare cases infect other mammals including humans. In the past, USUV has been repeatedly associated with mass mortalities in birds, primarily blackbirds and owls. Birds commonly succumb either due to the peracute nature of the infection or due to severe encephalitis. In Germany, USUV has spread rapidly since its first detection in 2010 in mosquitoes under the presence of susceptible host and vector species. Nonetheless, there is to date limited access to whole genome sequences resulting in the absence of in-depth phylogenetic and phylodynamic analyses. In this study, 118 wild and captive birds were sequenced using a nanopore sequencing platform with prior target enrichment via amplicons. Due to the high abundancy of Europe 3 and Africa 3 in Germany an ample quantity of associated whole genome sequences was generated and the most recent common ancestor could be determined for each lineage. The corresponding clock phylogeny revealed an introduction of USUV Europe 3 and Africa 3 into Germany three years prior to their first isolation in the avifauna in 2011 and 2014, respectively. Based on the clustering and temporal history of the lineages, evidence exists for the genetic evolution of USUV within Germany as well as new introductions thereof into the country.

Genotyping of Streptococcus uberis isolates in healing process of bovine clinical mastitis.

In the present work, macrorestriction analysis was applied to characterize 44 S. uberis field strains isolated from lactating cows suffering from mastitis in three dairy herds in Hesse State, Germany. Analysis of the obtained data by Pulse-Field Gel Electrophoresis (PFGE) showed that most of the isolates originating from different herds and cows were not related to each other. However, identical macrorestriction patterns were noted in 12 of 13 mastitic quarters in healing process, in three quarters even over the whole sampling period indicating persistent infection. In the present work, PFGE could detect variable levels of similarity ranging from 76 to 100%. The macrorestriction analyses revealed the presence of 10 S. uberis PFGE pattern with more than four bands difference. PFGE profiles with minor differences (only one to three bands) were considered to be subtypes. The use of sensitive genotyping methods like macrorestriction analyses by PFGE enables the differentiation among new and persistent infections. Nevertheless minor changes in macrorestriction profiles could occur which are clearly distinguishable from totally unrelated strains.

[Pathologic-anatomical changes in newborn goats caused by an intrauterine Schmallenberg virus infection]. / Pathologisch-anatomische Veränderungen bei Ziegenlämmern, verursacht durch eine intrauterine Schmallenberg-Virus- Infektion.

A complex of various malformations in newborns was observed to an increased extent in sheep farms in the 2011/2012 lambing season. An intrauterine Schmallenberg virus (SBV) infection was identified as the cause of these malformations. To date, a detailed pathological description of the deformity complex has only been given for bovine and ovine newborns.The aim of this study was therefore to provide a description of pathologic-anatomical congenital malformations in goat kids caused by intrauterine SBV infection. To this end, pathologic-anatomical and molecular biological investigations by PCR were carried out on 37 goat kids and 457 lambs from 238 sheep and goat farms in order to carry out an interspecies comparison. Of the 37 goat kids dissected, it was possible to identify a SBV infection in twelve animals (32.4%) by RT-PCR. In nine animals (24.3%) displaying pathological-anatomical malformations SBV could not be detected by PCR. The following malformations were observed: athrogryposis, deformation of spinal column, torticollis, asymmetry of the skull, brachygnathia inferior, cerebellar hypoplasia, cerebellar aplasia and internal hydrocephalus. Arthogryposis was the most common malformation, both in animals with positive PCR results and those with negative PCR results. This study documents congenital malformations caused by an intrauterine SBV infection for the first time on a large number of newborn goats.

Use of competition ELISA for monitoring of West Nile virus infections in horses in Germany.

West Nile virus (WNV) is a mosquito-borne viral pathogen of global importance and is considered to be the most widespread flavivirus in the World. Horses, as dead-end hosts, can be infected by bridge mosquito vectors and undergo either subclinical infections or develop severe neurological diseases. The aim of this study was to detect WNV specific antibodies in horses in Germany as an indicator for an endemic circulation of WNV. Sera from more than 5,000 horses (primarily fallen stock animals) were collected in eight different federal states of Germany from 2010 to 2012. Sera were screened by a competitive ELISA and positive reactions were verified by an indirect IgM ELISA and/or by virus neutralization tests (VNT) for WNV and Tick-borne encephalitis virus (TBEV) in order to exclude cross-reacting antibody reactions. In essence WNV specific antibodies could not be detected in any of the horse sera. Not surprisingly, a small number of sera contained antibodies against TBEV. It is noteworthy that equine sera were often collected from horse carcasses and therefore were of poor quality. Nonetheless, these sera were still suitable for WNV ELISA testing, i.e., they did not produce a high background reaction which is a frequently observed phenomenon. According to these data there is no evidence for indigenous WNV infections in horses in Germany at present.

Detection and characterization of Histoplasma capsulatum in a German badger (Meles meles) by ITS sequencing and multilocus sequencing analysis.

A wild badger (Meles meles) with a severe nodular dermatitis was presented for post mortem examination. Numerous cutaneous granulomas with superficial ulceration were present especially on head, dorsum, and forearms were found at necropsy. Histopathological examination of the skin revealed a severe granulomatous dermatitis with abundant intralesional round to spherical yeast-like cells, 2-5 µm in diameter, altogether consistent with the clinical appearance of histoplasmosis farciminosi. The structures stained positively with Grocott's methenamine silver and Periodic acid-Schiff stains, but attempts to isolate the etiologic agent at 25 and 37°C failed. DNA was directly extracted from tissue samples and the ribosomal genes ITS1-5.8S-ITS2 were partially sequenced. This revealed 99% identity to sequences from Ajellomyces capsulatus, the teleomorph of Histoplasma capsulatum, which was derived from a human case in Japan, as well as from horses from Egypt and Poland. Phylogenetic multi-locus sequence analysis demonstrated that the fungus in our case belonged to the Eurasian clade which contains members of former varieties H. capsulatum var. capsulatum, H. capsulatum var. farciminosum. This is the first study of molecular and phylogenetic aspects of H. capsulatum, as well as evidence for histoplasmosis farciminosi in a badger, further illuminating the role of this rare pathogen in Central Europe.