Detection of a Novel Bovine Astrovirus in a Cow with Encephalitis.

Encephalitis can be caused by several infectious agents, including bacteria, fungi, parasites and viruses. In many cases, the causative agent cannot be identified, because the pathogens are unknown or detection methods are not routinely available. In our case, a 15-month-old cow developed central nervous disorders and died within 6 days after the onset of clinical signs. The histopathology revealed an acute encephalitis, predominantly in the brain stem, and a ganglionitis of the trigeminal ganglion with massive neuronal necroses in both the brain and the ganglion. However, a relevant panel of bacterial and viral infections of cattle could be routinely excluded. Therefore, a brain sample from the cow was analysed using a metagenomics approach with next-generation sequencing. A novel bovine astrovirus (BoAstV-BH89/14) could be identified using the analysis pipeline RIEMS, and the finding could be confirmed with a specific BoAstV RT-qPCR. The genome of the bovine astrovirus (BoAstV), belonging to the family Astroviridae in the genus Mamastrovirus, has a length of 6478 bp. Sequence identities between 71% to a sheep astrovirus and 69% to two recently described bovine astroviruses from the USA and Switzerland were ascertained. The latter were also connected to encephalitis cases in cattle. Like these, the new virus described here was detected in different brain sections using the specific BoAstV RT-qPCR and fluorescent in situ hybridization. In conclusion, while astroviruses so far were mainly found in relation to gastroenteritis in animals and humans, recently detected astrovirus infections were also related to encephalitis.

Organ distribution of Schmallenberg virus RNA in malformed newborns.

A novel orthobunyavirus was first detected in German dairy cows in autumn 2011 and was subsequently found in the brains of malformed lambs, kids and calves in the Netherlands, Germany, Belgium, France, Italy, Great Britain, Luxembourg and Spain. For rapid detection of this novel virus, named Schmallenberg virus, a real time quantitative reverse transcription PCR (RT-qPCR) was developed at the Friedrich-Loeffler-Institut and provided to the federal veterinary state laboratories in Germany. For diagnostic purposes, the organ distribution of this new virus was analyzed in several organs and body fluids of 15 lambs and two calves showing typical malformations. Spleen, cerebrum, meconium, spinal cord, rib cartilage, umbilical cord, placental fluid out of the stomach as well as external placental fluid scraped from the coat of the foetuses were collected during necropsy. All animals were tested RT-qPCR positive in the external placental fluid, and all but one were also RT-qPCR positive in the cerebrum, the umbilical and the spinal cord. Our results suggest that both the external placental fluid and the umbilical cord could be suitable sample materials for the confirmation of an infection with Schmallenberg virus in malformed newborns, at least in lambs. This is of special interest since those samples can be collected very easily on the farm without the need of a necropsy.

Bicarbonate exporting transporters in the ovine ruminal epithelium.

In order to stabilize the intraruminal pH, bicarbonate secretion by the ruminal epithelium seems to be an important prerequisite. The present study therefore focussed on the characterization of bicarbonate exporting systems in ruminal epithelial cells. Intracellular pH (pH(i)) was measured spectrofluorometrically in primary cultured ruminal epithelial cells loaded with the pH-sensitive fluorescent dye, 2,7-bis(carboxyethyl)-5(6')-carboxyfluorescein acetomethyl ester. Switching from CO2/HCO3- -buffered to HEPES-buffered solution caused a rapid intracellular alkalinization followed by a counter-regulation towards initial pH(i). The recovery of pH(i) was dependent upon extracellular chloride, but independent of extracellular sodium. Adding 500 microM H2DIDS significantly reduced the increase of pH(i). For further characterization of the bicarbonate exporting systems, we tested the ability to reverse the direction from HCO3- export to import in the absence of sodium and chloride. Under sodium and chloride-free conditions, counter-regulation after CO2-induced pH(i) decrease did not differ from pH(i) recovery in the presence of sodium and chloride. Existence of bicarbonate exporting systems in cultured ruminal epithelial cells and intact ruminal epithelium was verified by reverse transcription polymerase chain reaction (RT-PCR). Using RT-PCR and subsequent sequencing, expression of mRNA encoding for AE2, DRA and PAT1 could be found. Bicarbonate exporting systems could therefore be detected both on the functional and structural level.