First detection of autochthonous Lamanema chavezi infections in llamas (Lama glama) in Europe.

Lamanema chavezi is one of the most pathogenic nematode species of South American camelids (SAC), with a homoxenous life cycle involving enterohepatic migration of its larvae in the host. So far, it has been found in the Americas and New Zealand. The first autochthonous L. chavezi infections in SAC in Europe are reported here. On a SAC farm in Germany, a 15-month-old male llama with a short history of diarrhoea died in September 2017, followed nine months later by a three-year-old female llama with a history of emaciation, apathy, anorexia, anaemia and tetraparesis with retained sensorium. Both animals were born and raised on the farm, which had imported three llamas directly from Chile 4-14 years earlier. At necropsy, the main lesions in both cases were numerous white-yellow to dark red foci, up to 3 mm in size, close to the Glisson's capsule and deep in the parenchyma of the liver. Histologically, the livers showed haemorrhagic tracks by and with nematode larvae and a necro-haemorrhagic to fibrinous inflammation with a predominantly lymphohistiocytic infiltration. The larvae were 30-50 µm in diameter and had external longitudinal cuticular ridges. Larvae extracted from unfixed liver tissue were 1800-2000 µm long and about 80 µm in diameter, with a terminal spine at the posterior end, which is characteristic of female L. chavezi stages. The ribosomal DNA including the almost complete 18S rRNA gene, the first internal transcribed spacer, the 5.8S RNA gene, the second internal transcribed spacer (ITS) and the partial 28S rRNA gene from isolated larvae were amplified using nematode-specific oligonucleotide primers and then sequenced. The assembled nematode sequence of 3448 bp showed an identity of 99.4% to previously published L. chavezi sequences in the BLASTN search. Low numbers of L. chavezi-like eggs were found in the faeces of seven (29%) of 24 llamas and alpacas in the herd, including some farm-born crias, tested two years after the last fatal case. The results show for the first time that L. chavezi has not only been imported into Europe from South America, but has also completed its life cycle locally, resulting in autochthonous infections of SAC. This was also suspected to be the cause of the fatal disease in two llamas.

Tissue Distribution of Parrot Bornavirus 4 (PaBV-4) in Experimentally Infected Young and Adult Cockatiels (Nymphicus hollandicus).

Proventricular dilatation disease (PDD) caused by parrot bornavirus (PaBV) infection is an often-fatal disease known to infect Psittaciformes. The impact of age at the time of PaBV infection on organ lesions and tissue distribution of virus antigen and RNA remains largely unclear. For this purpose, tissue sections of 11 cockatiels intravenously infected with PaBV-4 as adults or juveniles, respectively, were examined via histology, immunohistochemistry applying a phosphoprotein (P) antibody directed against the bornaviral phosphoprotein and in situ hybridisation to detect viral RNA in tissues. In both groups of adult- and juvenile-infected cockatiels, widespread tissue distribution of bornaviral antigen and RNA as well as histologic inflammatory lesions were demonstrated. The latter appeared more severe in the central nervous system in adults and in the proventriculus of juveniles, respectively. During the study, central nervous symptoms and signs of gastrointestinal affection were only demonstrated in adult birds. Our findings indicate a great role of the age at the time of infection in the development of histopathological lesions and clinical signs, and thus provide a better understanding of the pathogenesis, possible virus transmission routes, and the development of carrier birds posing a risk to psittacine collections.

Experimental Infection of Embryonic Cells and Embryonated Eggs of Cockatiels (Nymphicus hollandicus) with Two Parrot Bornavirus Isolates (PaBV-4 and PaBV-2).

Parrot bornavirus (PaBV) might be transmitted vertically. Cockatiel embryonic brain cells and embryonated eggs of cockatiels (ECE) were infected with PaBV-2 and PaBV-4. In embryonic brain cells, PaBV-2 and PaBV-4 showed no differences in viral spread despite the slower growth of PaBV-2 compared with PaBV-4 in CEC-32 cells. ECE were inoculated with PaBV-4 and 13-14 dpi, organs were sampled for RT-PCR, immunohistochemistry/histology, and virus isolation. In 28.1% of the embryos PaBV-4-RNA and in 81.3% PaBV-4-antigen was detected in the brain. Virus isolation failed. Division of organ samples and uneven tissue distribution of the virus limited the results. Therefore, 25 ECE were inoculated with PaBV-4 (group 1) and 15 ECE with PaBV-2 (group 3) in the yolk sac, and 25 ECE were inoculated with PaBV-4 (group 2) and 15 eggs with PaBV-2 (group 4) in the chorioallantoic membrane to use the complete organs from each embryo for each examination method. PaBV-RNA was detected in the brain of 80% of the embryos in groups 1, 2, 3 and in 100% of the embryos in group 4. In 90% of the infected embryos of group 1, and 100% of group 2, 3 and 4, PaBV antigen was detected in the brain. PaBV antigen-positive brain cells were negative for anti-neuronal nuclear protein, anti-glial fibrillary acidic protein, and anti S-100 staining. Virus was not re-isolated. These results demonstrated a specific distribution pattern and spread of PaBV-4 and PaBV-2 in the brain when inoculated in ECE. These findings support a potential for vertical transmission.

Age-dependent development and clinical characteristics of an experimental parrot bornavirus-4 (PaBV-4) infection in cockatiels (Nymphicus hollandicus).

Parrot bornavirus (PaBV) is a pathogen often found in psittacine populations. Infected, clinically healthy carrier birds are of major importance for epidemiology, but the underlying pathomechanism of this carrier status is poorly understood. The age, implying the maturation status of the immune system, at the time of infection might be significant for the clinical outcome. Therefore, two groups of 11 cockatiels of different ages (adult and newly hatched) were inoculated with a PaBV-4 isolate intravenously. The trial lasted for 233 days and all birds were observed for clinical signs, PaBV-RNA shedding and anti-PaBV antibody production. At the end of the trial, histopathology, immunohistochemistry, PCR and virus re-isolation were performed. All 22 birds seroconverted and shed PaBV-RNA during the investigation period; the juvenile group earlier and more homogeneously. Nine of 11 birds of the adult group developed clinical signs; five birds died or had to be euthanized before the end of the study. In the juvenile group none of the birds developed clinical signs and only one bird died due to bacterial septicaemia. Eight birds of the adult group, but none of the juvenile group, showed a dilatation of the proventriculus. PaBV-RNA detection and virus re-isolation were successful in all birds. Immunohistochemically, PaBV antigen was found in all birds. Histopathology revealed mononuclear infiltrations in organs in birds of both groups, but the juveniles were less severely affected in the brain.Thus, PaBV infection at an age with a more naïve immune system makes the production of carrier birds more likely.RESEARCH HIGHLIGHTS PaBV infection at a young age might favour the development of carrier birds.Cockatiels infected at a very young age showed inflammation but no clinical signs.The juvenile group started seroconversion and PaBV-RNA shedding earlier.Seroconversion and PaBV-RNA shedding occurred more homogeneously in the juveniles.

AKAP6 orchestrates the nuclear envelope microtubule-organizing center by linking golgi and nucleus via AKAP9.

The switch from centrosomal microtubule-organizing centers (MTOCs) to non-centrosomal MTOCs during differentiation is poorly understood. Here, we identify AKAP6 as key component of the nuclear envelope MTOC. In rat cardiomyocytes, AKAP6 anchors centrosomal proteins to the nuclear envelope through its spectrin repeats, acting as an adaptor between nesprin-1α and Pcnt or AKAP9. In addition, AKAP6 and AKAP9 form a protein platform tethering the Golgi to the nucleus. Both Golgi and nuclear envelope exhibit MTOC activity utilizing either AKAP9, or Pcnt-AKAP9, respectively. AKAP6 is also required for formation and activity of the nuclear envelope MTOC in human osteoclasts. Moreover, ectopic expression of AKAP6 in epithelial cells is sufficient to recruit endogenous centrosomal proteins. Finally, AKAP6 is required for cardiomyocyte hypertrophy and osteoclast bone resorption activity. Collectively, we decipher the MTOC at the nuclear envelope as a bi-layered structure generating two pools of microtubules with AKAP6 as a key organizer.

Recombinant spider silk protein eADF4(C16)-RGD coatings are suitable for cardiac tissue engineering.

Cardiac tissue engineering is a promising approach to treat cardiovascular diseases, which are a major socio-economic burden worldwide. An optimal material for cardiac tissue engineering, allowing cardiomyocyte attachment and exhibiting proper immunocompatibility, biocompatibility and mechanical characteristics, has not yet emerged. An additional challenge is to develop a fabrication method that enables the generation of proper hierarchical structures and constructs with a high density of cardiomyocytes for optimal contractility. Thus, there is a focus on identifying suitable materials for cardiac tissue engineering. Here, we investigated the interaction of neonatal rat heart cells with engineered spider silk protein (eADF4(C16)) tagged with the tripeptide arginyl-glycyl-aspartic acid cell adhesion motif RGD, which can be used as coating, but can also be 3D printed. Cardiomyocytes, fibroblasts, and endothelial cells attached well to eADF4(C16)-RGD coatings, which did not induce hypertrophy in cardiomyocytes, but allowed response to hypertrophic as well as proliferative stimuli. Furthermore, Kymograph and MUSCLEMOTION analyses showed proper cardiomyocyte beating characteristics on spider silk coatings, and cardiomyocytes formed compact cell aggregates, exhibiting markedly higher speed of contraction than cardiomyocyte mono-layers on fibronectin. The results suggest that eADF4(C16)-RGD is a promising material for cardiac tissue engineering.

miR-27a/b is a posttranscriptional regulator of Gpr126 (Adgrg6).

Gpr126 (Adgrg6), a member of the adhesion G protein-coupled receptor family, has been associated with a variety of human diseases. Yet, despite its clinical importance, the mechanisms regulating Gpr126 expression are poorly understood. Here, we aimed at identifying upstream regulatory mechanisms of Gpr126 expression utilizing the heart as model organ in which Gpr126 regulates trabeculation. Here, we focused on possible regulation of Gpr126 regulation by microRNAs, which have emerged as key players in regulating development, have a critical role in disease progression, and might serve as putative therapeutic targets. In silico analyses identified one conserved binding site in the 3' UTR of Gpr126 for microRNA 27a and 27b (miR-27a/b). In addition, miR-27a/b and Gpr126 expression were differentially expressed during rat heart development. A regulatory role of miR-27a/b in controlling Gpr126 expression was substantiated by reduced Gpr126 mRNA levels upon ectopic expression of miR-27a/b in HEK293T cells and miR-27b in zebrafish embryos. Regulation of Gpr126 expression by direct binding of miR-27a/b to the 3' UTR of Gpr126 was verified by luciferase reporter assays in HEK293T cells. Finally, the modulation of gpr126 expression in zebrafish by injection of either miR-27b or miR-27b inhibitor in single cell-stage embryos resulted in hypo- or hypertrabeculation, respectively. Collectively, the data indicate that Gpr126 expression is regulated by miR-27a/b.

Distribution of zoonotic variegated squirrel bornavirus 1 in naturally infected variegated and Prevost's squirrels.

Recently, the zoonotic capacity of the newly discovered variegated squirrel bornavirus 1 (VSBV-1) was confirmed in humans with a lethal encephalitis. Transmission to humans occurred by variegated and Prevost's squirrels as presumed reservoir hosts but possible ways of virus shedding and the route of infection still need to be elucidated. Thus, the tissue distribution of VSBV-1 antigen and RNA was investigated in detail via immunohistochemistry (IHC) in six variegated and eight Prevost's squirrels and by in situ hybridisation (ISH) in one Prevost's squirrel, respectively. VSBV-1 antigen and RNA positive cells were most numerous in the nervous system and were also found in nearly all tissues and different cell types indicating a broad organ and cell tropism of VSBV-1. Presence of VSBV-1 in several organs might indicate potential virus shedding via various routes and implies the risk of intra- and interspecies transmission, respectively.