Diagnosis of Schmallenberg virus infection in malformed lambs and calves and first indications for virus clearance in the fetus

Abstract

Since mid-December 2011, samples from malformed lambs and calves are sent to CODA-CERVA in Belgium for diagnosis of Schmallenberg virus (SBV), a novel Orthobunyavirus that was first detected by researchers of the Friedrich-Loeffler-Institut (FLI, Germany) in German cattle in autumn 2011 and was later shown to be involved in congenital malformations in lambs, goat kids and calves. Surprisingly, by making use of real time RT-PCR (rRT-PCR) assays developed by the FLI, presence of SBV RNA could only be confirmed in part of the SBV suspected newborns examined. To investigate possible causes for non-confirmation by rRT-PCR, a comparative analysis between different organs and tissues (cerebrum, cerebellum, brain stem, spinal cord, thymus, spleen, lymph nodes, meconium) originating from respectively 90 and 81 malformed lambs and calves was undertaken. Furthermore, thoracic fluids of respectively 55 malformed lambs and calves were examined by a virus neutralization test (VNT) to evaluate the presence of neutralizing anti-SBV antibodies in these animals. Our results show that among the different organs tested by rRT-PCR, brain stem material is the most appropriate tissue for SBV detection while it could also be detected in all other tissues but to a more variable degree. The VNT test showed that 95% of the malformed lambs were positive for anti-SBV neutralizing antibodies while this was only the case for 44% of malformed calves. These immunological data suggest that a humoral immune response could assist in the clearance of SBV from the fetus during gestation and that SBV specific antibody testing should be considered together with rRT-PCR analysis for confirmation of SBV infection.

Introduction

In November 2011, a new virus was detected by metagenomic analysis at the Friedrich-Loeffler-Institut (FLI, Germany) that had caused milk drop, diarrhea and fever in adult cattle during the summer of 2011 in Germany and the Netherlands (Hoffmann et al., 2012) and later was shown to be involved in congenital malformations in lambs, calves and goat kids (Herder et al., 2012, Van den Brom et al., 2012). The virus was named Schmallenberg virus (SBV) and belongs to the Simbu serogroup of Orthobunyaviruses (Goller et al., 2012, Hoffmann et al., 2012). Malformations that are observed in aborted, stillborn or neonatal lambs and calves tend from arthrogryposis to torticollis, scoliosis, kyphosis and brachygnathia inferior. At necropsy, deformities of the central nervous system like hydranencephaly and hypoplasia of the cerebrum, cerebellum and/or spinal cord are commonly observed (Garigliany et al., 2012a, Garigliany et al., 2012b, Herder et al., 2012, Van den Brom et al., 2012).

Since the emergence of SBV, real time RT-PCR (rRT-PCR) assays suitable to detect the L and S segment of the SBV genome have been developed and distributed by the FLI (Germany) and it has been described that using these PCRs, SBV can be found in multiple tissues and organs (spleen, cerebrum, spinal cord, rib cartilage, placental fluid, umbilical cord and meconium) of aborted lambs and calves (Bilk et al., 2012). CODA-CERVA, the Belgian reference laboratory responsible for SBV diagnosis since the first suspected cases appeared from mid-December 2011 in Belgium, has used these PCRs for the diagnosis of SBV in samples of suspected aborted lambs and calves. Because of practical and budgetary constraints, only one sample of each SBV suspected animal was tested. The overall results of this rRT-PCR diagnosis have been published by the Belgian government (www.afsca.be/diergezondheid/schmallenberg) and show that despite viral detection in a part of the SBV suspected lambs and calves (report of July 3, 2012: respectively 66% and 35% of SBV suspected lambs and calves were confirmed by rRT-PCR since the emergence of SBV in Belgium), a considerable number of suspected cases could not be confirmed by rRT-PCR. A similar discrepancy between SBV suspected and rRT-PCR confirmed lambs and calves has been reported by the Dutch authorities (www.vwa.nl/onderwerpen/dierziekten/dossier/schmallenbergvirus). The reason for these unconfirmed cases is still unclear. It has been hypothesized that maybe insufficient amounts of virus were present in these organs at the time of rRT-PCR analysis or that the virus had been cleared from the fetus during the considerable time gap between the moment of SBV infection of the fetus and the moment the symptoms are observed at parturition. The longer gestation period in cattle in comparison to sheep might explain the lower percentage of SBV confirmed calves than lambs (ProMED-Mail, 2012, Garigliany et al., 2012a).

In this manuscript we describe our research efforts to identify the most appropriate tissue or organ(part) for SBV detection in aborted lambs and calves by rRT-PCR. This study was performed to maximize the likelihood of SBV detection if only one sample for each SBV suspected animal can be tested. We also present the results of serological testing carried out in aborted lambs and calves to find indications of an SBV infection in animals that were SBV negative by rRT-PCR at birth or abortion.