Borna disease in an adult free-ranging Eurasian beaver (Castor fiber albicus).

Borna disease (BD) associated with a peracute bacterial septicaemia with Escherichia coli was diagnosed in an adult female, naturally infected, free-ranging Eurasian beaver of the subspecies Castor fiber albicus, clinically characterized by weight loss, depression, weakness and gurgled peristaltic sounds. The beaver was euthanized humanely. Necropsy and light microscopy revealed a non-purulent meningoencephalitis with typical mononuclear perivascular cuffs and parenchymal infiltrates. The diagnosis of BD was confirmed by detection of viral antigen and RNA by immunohistochemistry and reverse transcription-polymerase chain reaction (RT-PCR). The PCR product was sequenced and cluster analysis revealed a close relationship between endemic clusters in Saxony-Anhalt. This is the first report of naturally occurring BD in a free-ranging Eurasian beaver.

Pathogen Screening for Possible Causes of Meningitis/Encephalitis in Wild Carnivores From Saxony-Anhalt.

Inflammation in meninges and/or brain is regularly noticed in red foxes and other wild carnivores during rabies control programs. Despite negative rabies virus (RABV) results, the etiologies of these cases remain unknown. Thus, the aim of this study was to provide an overview of the occurrence of pathogens that may cause diseases in the brains of wild carnivores and pose a risk to humans and other animals. In addition to RABV and canine distemper virus (CDV), a variety of pathogens, including members of Flaviviridae, Bornaviridae, Herpesviridae, Circoviridae, as well as bacteria and parasites can also cause brain lesions. In 2016 and 2017, brain samples of 1,124 wild carnivores were examined by direct fluorescent antibody test for RABV as well as (reverse-transcriptase) quantitative polymerase chain reaction (PCR) for the presence of CDV as part of a monitoring program in Saxony-Anhalt, Germany. Here, we applied similar methods to specifically detect suid herpesvirus 1 (SuHV-1), West Nile virus (WNV), Borna disease virus 1 (BoDV-1), canid alphaherpesvirus 1 (CaHV-1), canine parvovirus type 2 (CPV-2), fox circovirus (FoxCV), and Neospora caninum (N. caninum). Further, bacteriogical examination for the existence of Listeria monocytogenes (L. monocytogenes) and immunohistochemistry of selected cases to detect Toxoplasma gondii (T. gondii) antigen were performed. Of all pathogens studied, CDV was found most frequently (31.05%), followed by FoxCV (6.80%), CPV-2 (6.41%), T. gondii (4/15; 26.67%), nematode larvae (1.51%), L. monocytogenes (0.3%), and various other bacterial pathogens (1.42%). In 68 of these cases (6.05%), multiple pathogen combinations were present simultaneously. However, RABV, WNV, BoDV-1, SuHV-1, CaHV-1, and N. caninum were not detected. The majority of the histopathological changes in 440 animals were inflammation (320/440; 72.73%), predominantly non-suppurative in character (280/320; 87.50%), and in many cases in combination with gliosis, satellitosis, neuronophagia, neuronal necrosis, and/or vacuolization/demyelination, or in single cases with malacia. Thus, it could be shown that wild carnivores in Saxony-Anhalt are carriers mainly for CDV and sometimes also for other, partly zoonotic pathogens. Therefore, the existing monitoring program should be expanded to assess the spill-over risk from wild carnivores to humans and other animals and to demonstrate the role of wild carnivores in the epidemiology of these zoonotic pathogens.

Two outbreaks of virulent systemic feline calicivirus infection in cats in Germany.

Over the last years, several outbreaks of virulent systemic feline calicivirus (VS-FCV) infection have been described in the USA and several European countries. The paper describes two outbreaks of VS-FCV infection in cats in Germany. Data concerning clinical, laboratory, and histopathological features ofVS-FCV infection were collected from two outbreaks affecting 55 and 4 cats, respectively. Presence of feline calicivirus was confirmed by PCR followed by sequencing of the PCR-products. Clinical signs were variable, including severe upper respiratory tract infection, dyspnoea, oral and footpad ulceration, facial oedema, enteritis, pneumonia, bleeding disorder, high fever, and icterus. Both outbreaks were characterized by a high mortality rate.The present report describes the first documented outbreaks of VS-FCV infection in cats in Germany. Clinical and histopathological features are comparable to outbreaks described in the USA and Europe. However, phylogenetic analysis of the virus genome suggests that virus strains involved in these outbreaks were different from each other and from virulent strains isolated before, confirming the known genetic variability of FCV.

Growth and differentiation of primary and passaged equine bronchial epithelial cells under conventional and air-liquid-interface culture conditions.

BACKGROUND: Horses develop recurrent airway obstruction (RAO) that resembles human bronchial asthma. Differentiated primary equine bronchial epithelial cells (EBEC) in culture that closely mimic the airway cells in vivo would be useful to investigate the contribution of bronchial epithelium in inflammation of airway diseases. However, because isolation and characterization of EBEC cultures has been limited, we modified and optimized techniques of generating and culturing EBECs from healthy horses to mimic in vivo conditions. RESULTS: Large numbers of EBEC were obtained by trypsin digestion and successfully grown for up to 2 passages with or without serum. However, serum or ultroser G proved to be essential for EBEC differentiation on membrane inserts at ALI. A pseudo-stratified muco-ciliary epithelium with basal cells was observed at differentiation. Further, transepithelial resistance (TEER) was more consistent and higher in P1 cultures compared to P0 cultures while ciliation was delayed in P1 cultures. CONCLUSIONS: This study provides an efficient method for obtaining a high-yield of EBECs and for generating highly differentiated cultures. These EBEC cultures can be used to study the formation of tight junction or to identify epithelial-derived inflammatory factors that contribute to lung diseases such as asthma.

The equine endometrosis: new insights into the pathogenesis.

This paper describes the histomorphological and immunohistochemical characterisation of phenotypic variations of endometrosis as well as potential etiological factors which may influence disease progression. In total, 779 endometrial biopsies were examined. These biopsies were taken in the breeding and non-breeding season (n=509), on defined days during the estrous cycle (n=70) and before and after experimentally induced bacterial endometritis (n=200). In addition to conventional histopathology, selected biopsies were investigated using alcianblue staining as well as immunohistochemical methods for the detection of steroid hormone receptors, Ki-67-antigen, vimentin, desmin, fibronectin, smooth-muscle-alpha-actin and laminin. The equine endometrosis can be divided into a destructive and a non-destructive form. Based on the morphology of the stromal cells involved, an active or inactive state can be distinguished in fibrotic foci. In all types of endometrosis, fibrotic stromal cells show a distinctly reduced expression of steroid hormone receptors in comparison to the intact stroma, indicating their dedifferentiation. However, the steroid hormone receptor expression of involved glandular epithelia seems to depend on the activity of the fibrosis. These results suggest an independency of all fibrotic foci from the hormonal control mechanism of the uterus. The characteristical features of destructive endometrosis are a large number of smooth-muscle-alpha-actin containing myofibroblasts, a pronounced epithelial vimentin expression, excessive extracellular matrix accumulation and a progressive alteration of the basal lamina. Furthermore, the frequently seen cystic glandular dilatation and mechanical destruction of the uterine glands may occur due to the contractibility of the myofibroblasts involved. As shown in this study, a simultaneous endometritis can cause a temporary activation of fibrotic stromal cells. However, cyclic and seasonal endocrine changes seem to have no effects on progression of the disease. It can be concluded that the various types of endometrosis represent different stages in the fibrotic process, possibly leading to the destruction of the glands and subsequently resulting in the development of a stromal fibrosis.

Isolation and culture of primary equine tracheal epithelial cells.

Culture of airway epithelial cells is a useful model to investigate physiology of airway epithelia and airway disease mechanisms. In vitro models of airway epithelial cells are established for various species. However, earlier published method for isolation and culture of equine tracheal epithelial cells requires significant improvements. In this report, the development of a procedure for efficient isolation, characterization, culture, and passage of primary equine tracheal epithelial cells are described. Epithelial cells were isolated from adult equine trachea by exposing and stripping the mucosal epithelium from the adjacent connective tissue and smooth muscle. The tissue was minced and dissociated enzymatically using 0.25% trypsin-ethylenediaminetetraacetic acid (EDTA) solution for 2 h at 37 degrees C. Cells were collected by sieving and centrifugation, and contaminating fibroblasts were removed by differential adhesion. This procedure resulted in a typical yield of 1 x 10(7) cytokeratin-positive epithelial cells per gram tracheal lining tissue. Viability was 95% by trypan blue exclusion and isolates contained approximately 94% cytokeratin-positive cells of epithelial origin. Cells seeded at a density of 6.9 x 10(4) cells/cm2 in serum-free airway epithelial cell growth medium formed monolayers near confluency within a week. Confluent cells were dissociated using dispase II and first passages (P1) and second passages (P2) were successfully established in serum-free medium. Collagen coating of tissue culture flask was not required for cell adhesion, and cultures could be maintained at the level of P2 over 30 d. In the present study, we could establish a high-yield protocol for isolation and culture of equine tracheal epithelial cells that can serve for in vitro/ex vivo studies on the (patho-)physiology of equine airway disease as well as pharmacological and toxicological targets relevant to airway diseases.