Lukas Schwarz

Congenital infection with atypical porcine pestivirus (APPV) is associated with disease and viral persistence

In 2013, several Austrian piglet-producing farms recorded outbreaks of action-related repetitive myoclonia in newborn piglets (“shaking piglets”). Malnutrition was seen in numerous piglets as a complication of this tremor syndrome. Overall piglet mortality was increased and the number of weaned piglets per sow decreased by more than 10% due to this outbreak. Histological examination of the CNS of affected piglets revealed moderate hypomyelination of the white substance in cerebellum and spinal cord. We detected a recently discovered pestivirus, termed atypical porcine pestivirus (APPV) in all these cases by RT-PCR. A genomic sequence and seven partial sequences were determined and revealed a 90% identity to the US APPV sequences and 92% identity to German sequences. In confirmation with previous reports, APPV genomes were identified in different body fluids and tissues including the CNS of diseased piglets. APPV could be isolated from a “shaking piglet”, which was incapable of consuming colostrum, and passaged on different porcine cells at very low titers. To assess the antibody response a blocking ELISA was developed targeting NS3. APPV specific antibodies were identified in sows and in PCR positive piglets affected by congenital tremor (CT). APPV genomes were detected continuously in piglets that gradually recovered from CT, while the antibody titers decreased over a 12-week interval, pointing towards maternally transmitted antibodies. High viral loads were detectable by qRT-PCR in saliva and semen of infected young adults indicating a persistent infection.

Molecular investigations on the prevalence and viral load of enteric viruses in pigs from five European countries.

Abstract Enteric viral infections in pigs may cause diarrhea resulting in ill-thrift and substantial economic losses. This study reports the enteric infections with porcine astrovirus type 4 (PAstV4), porcine group A rotavirus (GARV), porcine group C rotavirus (GCRV), porcine circovirus type 2 (PCV2) and porcine kobuvirus (PKoV) in 419 pigs, comprising both healthy and diarrheic animals, from 49 farms in five European countries (Austria, Germany, Hungary, Spain and Sweden). Real-time RT-PCR assays were developed to test fecal samples and to compare the prevalence and viral load in relation to health status, farms of origin and age groups. The results showed that PAstV4 (70.4%) was the dominant virus species, followed by PKoV (56.7%), PCV2 (42.2%), GCRV (3%) and GARV (0.9%). Diarrheic pigs had a higher viral load of PAstV4 in the nursery and growing-finishing groups. Rotaviruses were mainly detected in diarrheic pigs, whereas PCV2 was more often detected in clinically healthy than in diarrheic pigs, suggesting that most PCV2 infections were subclinical. PAstV4, PCV2 and PKoV were considered ubiquitous in the European pig livestock and co-infections among them were frequent, independently of the disease status, in contrast to a low prevalence of classical rotavirus infections.

Isospora suis in an Epithelial Cell Culture System – An In Vitro Model for Sexual Development in Coccidia

Coccidian parasites are of major importance in animal production, public health and food safety. The most frequently used representative in basic research on this group is Toxoplasma gondii. Although this parasite is well investigated there is no adequate in vitro model for its sexual development available and knowledge on this important life cycle phase is therefore scarce. The use of Isospora suis , a sister taxon to T. gondii and the causative agent of piglet coccidiosis, could provide a solution for this. In the present study an in vitro model for neonatal porcine coccidiosis in cells representative for the in vivo situation in the piglet gut was developed and evaluated. The parasite development was investigated by light and transmission electron microscopy and optimum culture conditions were evaluated. Intestinal porcine epithelial cells (IPEC-J2) adequately representing the natural host cells supported the development of all endogenous life cycle stages of I . suis , including gametocytes and oocysts. A concentration of 5% fetal calf serum in the culture medium led to highest gametocyte densities on day 12 post infection. Low infection doses (≤1 sporozoite for 100 host cells) were best for oocyst and gametocyte development. The presented system can also be used for immunostaining with established antibodies developed against T. gondii (in our case, anti-TgIMC3 antibodies directed against the inner membrane complex 3). The complete life cycle of I . suis in a cell line representing the natural host cell type and species provides a unique model among coccidian parasites and can be used to address a wide range of topics, especially with regard to the sexual development of coccidia.

Novel Pestivirus Species in Pigs, Austria, 2015

A novel pestivirus species was discovered in a piglet-producing farm in Austria during virologic examinations of congenital tremor cases. The emergence of this novel pestivirus species, provisionally termed Linda virus, in domestic pigs may have implications for classical swine fever virus surveillance and porcine health management.

Superinfection of sows with Cystoisospora suis ante partum leads to a milder course of cystoisosporosis in suckling piglets.

Cystoisospora (syn. Isospora) suis is a leading cause of diarrheal disease in neonatal piglets. To address the possibility of maternal immunization against C. suis infection six non-naïve pregnant sows were superinfected with 100,000 oocysts 2 weeks ante partum and compared to non-superinfected animals. Their piglets were infected with 1000 oocysts on the third day of life. Clinical and parasitological parameters as well as antibody titers in colostrum/milk and blood of sows and in the blood of piglets were evaluated by IFAT against sporozoites and merozoites from 2 weeks ante partum until the 35th day after birth. For IFAT two different invasive stages of C. suis were used to find possible differences between the immune response against the initially infectious stages (sporozoites) and later occurring asexual developmental stages (merozoites), which might be responsible for persisting/extraintestinal infections. IFN-γ production of PBMC and piglet splenocytes was determined by ELISPOT. Maternal superinfection resulted in increased titers of IgA, IgM and IgG in colostrum and milk as well as in the blood of sows and their piglets. Oocyst shedding and diarrhea were observed in the offspring of both groups, but piglets of superinfected sows showed significantly reduced oocyst shedding and less diarrhea. This protective effect was correlated with increased titers of antibodies, especially IgA, in colostrum, milk and blood serum of sows and piglets, and with the reactivity of splenocytes to parasite antigen. Superinfection of sows ante partum could partially protect piglets against the clinical outcome of experimental infection. Both colostrum and milk contain maternal protective substances as the effect of protection was highly correlated with antibody titers during the first 2 weeks of life. IgA in different substrates may serve as a marker for the level of protection against clinical cystoisosporosis.

Identification of a novel bufavirus in domestic pigs by a viral metagenomic approach.

Bufavirus is a single-stranded DNA virus belonging to the genus Protoparvovirus. This study reports the identification and characterization of a porcine bufavirus by a metagenomic approach, and a limited epidemiology investigation of bufavirus in six swine farms. A comparative genome analysis showed a similarity of 93 % to a Hungarian porcine bufavirus. Bayesian and maximum-likelihood analyses of genome sequences showed a close relationship of porcine bufaviruses to human and monkey bufaviruses. Molecular dating of the most recent common ancestors supported a recent introduction of bufaviruses into human and pig populations, respectively. A real-time PCR method was developed to screen 60 faecal samples for the porcine bufavirus DNA, and eight positive samples were found in two neighbouring farms, suggesting a relatively low prevalence (13.3 %). No direct transmission of porcine bufaviruses between two neighbouring farms was found, suggesting that bufaviruses may have spread widely in different geographical regions.

Development and application of a recombinant protein-based indirect ELISA for the detection of serum antibodies against Cystoisospora suis in swine

The apicomplexan parasite Cystoisospora suis which causes neonatal porcine coccidiosis is one of the predominant parasite in suckling piglets. Currently, the immunofluorescence antibody test (IFAT) is the only available serological tool for detecting serum antibodies against C. suis which has several limitations, including bias from interpretation and low throughput. In the present study, an indirect enzyme-linked immunosorbent assay (ELISA) was developed using a previously characterized recombinant merozoite protein for the detection of specific immunoglobulin G (IgG) and IgA against C. suis. The recombinant protein was expressed in Escherichia coli as a N-terminal histidine fusion protein, and its specificity was confirmed in an immunoblot probed with highly positive anti-C. suis sera from experimentally infected piglets. Optimal dilutions of recombinant protein, sera and conjugate were determined by checkerboard titrations, and the serum dilution that gave the greatest ratio between the positive and the negative sera was selected for subsequent analyses. Agreement between the IFAT and the newly developed ELISA was assessed with kappa statistics. The receiver operating characteristic (ROC) curve analysis based on 185 serum samples with known C. suis exposure previously tested in the reference IFAT was used to determine the cut-off value, sensitivity and specificity of the ELISA. For IgG, the ELISA had an estimated cut-off value of 0.82 and sensitivity and specificity values of 94.7% and 98%, respectively, whereas for IgA the estimated cut-off value was 0.41 and sensitivity and specificity values were both100%. According to kappa coefficient, an excellent correlation (κ > 0.8) was found between IFAT and ELISA for both isotypes. The diagnostic accuracy of the test measured as the area under the ROC curve index scaled between 0.98 and 1.0, indicating high discriminatory capacity and its possible application as a serological tool for detecting antibody response in the host following C. suis exposure/immunization and large-scale surveillance studies.