Julia Stadler

Comparison of Porcine Epidemic Diarrhea Viruses from Germany and the United States, 2014

Since 2013, highly virulent porcine epidemic diarrhea virus has caused considerable economic losses in the United States. To determine the relation of US strains to those recently causing disease in Germany, we compared genomes and found that the strain from Germany is closely related to variants in the United States.

Emergence of porcine epidemic diarrhea virus in southern Germany

BackgroundOver the last years, porcine epidemic diarrhea virus (PEDV) has caused devastating enteric diseases in the US and several countries in Asia, while outbreaks in Europe have only been reported sporadically since the 1980s. At present, only insufficient information is available on currently circulating PEDV strains in Europe and their impact on the European swine industry. In this case report, we present epidemic outbreaks of porcine epidemic diarrhea in three farms in South-Western Germany.Case presentationEpidemic outbreaks of diarrhea affecting pigs of all age groups were reported in three farms, one fattening farm and two piglet producing farms, in South-Western Germany between May and November 2014. In the fattening farm yellowish, watery diarrhea without evidence of mucus or blood was associated with a massive reduction of feed consumption. Severity of clinical signs and mortality in young suckling pigs varied significantly between the two affected sow farms. While mortality in suckling piglets reached almost 70 % in one sow herd, no increase in suckling piglet mortality was observed in the second sow farm. In all three cases, PEDV was confirmed in feces and small intestines by RT-qPCR. Phylogenetic analyses based on full-length PEDV genomes revealed high identity among strains from all three herds. Moreover, the German strains showed very high nucleotide identity (99.4 %) with a variant of PEDV (OH851) that was isolated in the United States in January 2014. This strain with insertions and deletions in the S-gene (so called INDEL strains) was reported to show lower virulence. Slightly lower identities were found with other strains from the US and Asia.ConclusionPhylogenetic information on the distribution of PEDV strains in Europe is severely lacking. In this case report we demonstrate that acute outbreaks of PEDV occurred in southern Germany in 2014. Current strains were clearly different from isolates found in the 1980s and were closely related to a PEDV variant found in the US in 2014. Moreover, the present case report indicates that variant strains of PEDV, containing insertions and deletions in the S gene, which were reported to be of lower virulence, might be able to cause high mortality in suckling piglets.

Porcine Epidemic Diarrhea in Europe: In-Detail Analyses of Disease Dynamics and Molecular Epidemiology

Porcine epidemic diarrhea (PED) is an acute and highly contagious enteric disease of swine caused by the eponymous virus (PEDV) which belongs to the genus Alphacoronavirus within the Coronaviridae virus family. Following the disastrous outbreaks in Asia and the United States, PEDV has been detected also in Europe. In order to better understand the overall situation, the molecular epidemiology, and factors that might influence the most variable disease impact; 40 samples from swine feces were collected from different PED outbreaks in Germany and other European countries and sequenced by shot-gun next-generation sequencing. A total of 38 new PEDV complete coding sequences were generated. When compared on a global scale, all investigated sequences from Central and South-Eastern Europe formed a rather homogeneous PEDV S INDEL cluster, suggesting a recent re-introduction. However, in-detail analyses revealed two new clusters and putative ancestor strains. Based on the available background data, correlations between clusters and location, farm type or clinical presentation could not be established. Additionally, the impact of secondary infections was explored using the metagenomic data sets. While several coinfections were observed, no correlation was found with disease courses. However, in addition to the PEDV genomes, ten complete viral coding sequences from nine different data sets were reconstructed each representing new virus strains. In detail, three pasivirus A strains, two astroviruses, a porcine sapelovirus, a kobuvirus, a porcine torovirus, a posavirus, and an enterobacteria phage were almost fully sequenced.

Clinical and haematological characterisation of Mycoplasma suis infections in splenectomised and non-splenectomised pigs.

Mycoplasma suis causes infectious anaemia in pigs (IAP), which can manifest in various degrees of severity depending on the virulence and the hosts susceptibility. As M. suis cannot be cultured in vitro experimental infections of splenectomised animals play an essential role for pathogenesis research. The aim of the present study was to characterise the course of experimental infection using the highly virulent and red blood cell (RBC-) invasive M. suis strain KI3806, to compare the experimental course in splenectomised and non-splenectomised pigs and to correlate clinical and haematological parameters with M. suis blood loads. All infected splenectomised pigs (n=7) were PCR-positive 2 days post infection (DPI) with maximum mean bacterial loads of 1.61 × 10(10)M. suis/mL on 8 DPI. They developed severe anaemia and massive hypoglycaemia by 8 DPI and had to be euthanised preterm (until 8 DPI) without seroconversion. The non-splenectomised pigs (n=7) became PCR-positive within 23 DPI and reached a maximum mean M. suis load of 1.64 × 10(5)M. suis/mL on 8 DPI. They developed mild anaemia, massive skin alterations with petechiae and haemorrhagic diathesis and seroconverted within 35 DPI. The study demonstrated that experimental infection of splenectomised pigs with the highly virulent M. suis strain KI3806 induces a fulminant course of infection. In contrast, M. suis strain KI3806 induces a mild course of disease in non-splenectomised pigs, which resembles the situation in naturally infected pigs. Therefore, these infection models are valuable for future pathogenesis studies on acute and chronic M. suis infections.

Low prevalence of porcine circovirus type 2 infections in farrowing sows and corresponding pre-suckling piglets in southern German pig farms

Porcine circovirus type 2 (PCV2) is the assumed causative agent of a number of different diseases summarized as porcine circovirus diseases (PCVD). The virus is shed via different se- and excretions of PCV2 infected pigs. Transmission of the virus occurs horizontally and vertically either by oronasal or diaplacental infection. Recent research emphasizes the importance of diaplacental PCV2 infection or the infection in early stages of the piglets life attributable to excretion of PCV2 by the dams within the suckling period. To estimate the prevalence of intrauterine PCV2 infections under field conditions in Bavaria the PCV2 status of farrowing sows (n=198) and corresponding pre-suckling piglets (n=590) of 20 piglet producing farms was examined. PCV2 viral load and anti-PCV2 antibodies in the serum of the sows and piglets were examined at time of farrowing or before colostrum intake, respectively. PCV2 excretion of the sows via saliva, feces and urine was examined additionally. PCV2 specific antibodies in the serum of the sows were detectable on 11 farms with a mean in herd seroprevalence of 35.5% in these farms. Only 0.65% of all samples collected from 198 sows were positive for PCV2 DNA (serum: 1%; feces: 0.5%; saliva: 0.5%; urine: 0.6%). PCV2 DNA was detectable in sample material from seronegative sows as well as from seropositive sows. In none of the pre-suckling serum samples of the piglets IgG antibodies against PCV2 or PCV2 DNA were present. No correlation between the antibody- and viremia status of the sows and the PCV2 excretion was detectable. In contrast to reports about a high prevalence of viremic pre-suckling piglets in the suckling period in North America, the results of the present study reveal that diaplacental infection with PCV2 is comparatively rare in Southern Germany and infection of piglets within the suckling period seems to be more likely.

Quantitative PCR analysis of Mycoplasma suis shedding patterns during experimental infection

The uncultivable hemotrophic bacterium Mycoplasma suis causes infectious anemia in pigs worldwide. The mechanisms by which M. suis is transmitted from pig to pig are largely unknown. Thus, the present study aimed at investigating urine, feces, saliva, nasal and vaginal secrets as well as environmental samples for the presence of M. suis DNA to get insights into potential transmission routes. Seven pigs were experimentally infected with M. suis KI3806. Samples were taken for 8 days post infection (p.i.). A quantitative LightCycler msg1 PCR was used to detect and quantify M. suis. Shedding was found in saliva as well as nasal and vaginal secrets from day 6 p.i. on with a quantity of 3.4 × 10(2) to 2.7 × 10(5)M. suis/swab. In urine M. suis DNA could be detected in 100.0% of the samples from day 6 p.i. on with a quantity of 4.7 × 10(2) to 6.3 × 10(5)M. suis per mL. When shedding patterns were correlated to the median bacterial blood loads shedding was observed at loads of 2.0 × 10(9)-7.0 × 10(10)M. suis per mL blood. No M. suis DNA could be amplified from feces. Dust and water samples of the pig drinking troughs were positive for M. suis on days 2 and 6 post infection, air samples were M. suis-negative throughout the experiment. Our results indicate that blood independent direct transmission as well as indirect transmission via environmental contamination could play a role in the epidemiology of M. suis infections.

Assessment of safety and reproductive performance after vaccination with a modified live-virus PRRS genotype 1 vaccine in pregnant sows at various stages of gestation

The objective of the present study was to assess safety and efficacy of a new modified live-virus porcine reproductive and respiratory syndrome (PRRS) genotype 1 vaccine in pregnant sows at various stages of gestation under field conditions. A total of 505 sows and gilts were allocated to two treatment groups and maintained in separate facilities. Animals of group 1 were vaccinated with a commercial modified live genotype 1 PRRSV vaccine (control product, CP), while animals of group 2 were immunized with a new modified live genotype 1 PRRSV vaccine (investigational veterinary product, IVP) (ReproCyc® PRRS EU, Boehringer Ingelheim Vetmedica GmbH). Injection site reactions were noted to be significantly less frequent in the IVP group compared to the CP group for pain (p=0.039), redness (p=0.030), heat (p=0.016) and swelling (p=0.002). The mean total number of piglets alive at weaning did not differ significantly between both study groups (10.6 vs. 11.0, p=0.375). However, pre-weaning mortality was significantly higher (p=0.005) in piglets from the CP group (14.1% vs. 10.9%). Analyses of reproductive performance data for both groups did not result in statistically significant differences between CP group and IVP group for number of piglets alive (12.7 and 12.6, respectively), healthy live (11.9 and 11.8), weak (0.7 and 0.5), stillborn (1.0 and 0.8) and mummified piglets (0.3 and 0.2) per litter. No differences were detected between both groups for piglet birth weights, while body weights at weaning (7.2kg vs. 6.6kg, p=0.026) and average daily gain (0.2445kg vs. 0.2211kg, p=0.037) were significantly higher in piglets from the IVP group. In conclusion, the administration of a single dose of ReproCyc® PRRS EU to sows and gilts at various stages of gestation confirmed non-inferiority to a commercial PRRS vaccine regarding safety and efficacy parameters under field conditions.

Occurrence of dysentery-like diarrhoea associated with Brachyspira suanatina infection on a German fattening pig farm

The anaerobic intestinal spirochaete Brachyspira (B.) suanatina was first described in 2007 but since then no further isolates have been reported from pigs. Accordingly, when the species was validly published in 2016, the overall occurrence and clinical relevance in pigs were unknown. In a fattening farm in southern Germany, mucohaemorrhagic diarrhoea was observed in 60 per cent (750 animals) of the finisher pigs. A diagnostic workup including Brachyspira culture, Salmonella culture, Lawsonia intracellularis-specific, B. hyodysenteriae-specific and B. pilosicoli-specific multiplex PCR and postmortem examination of severely affected pigs was performed. Tests for Salmonella species, Lawsonia intracellularis and B. hyodysenteriae were all negative. Gross and microscopic lesions were in agreement with dysentery and spirochaetes could be demonstrated by silver staining in tissue samples of the caecum at the ileal papilla. B. suanatina was cultured from faeces or colon of all (five) animals sampled and identified using nox-RFLP, partial nox-gene-sequencing and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). According to the initial report from Scandinavia, B. suanatina can be isolated from birds and cross-species infection could be demonstrated infecting pigs with an avian isolate. Thus outdoor production as in the case presented here and international trade may pose a risk for infection of naive herds.

Updating the proteome of the uncultivable hemotrophic Mycoplasma suis in experimentally infected pigs

Mycoplasma suis belongs to the hemotrophic mycoplasmas that are associated with acute and chronic anemia in a wide range of livestock and wild animals. The inability to culture M. suis in vitro has hindered its characterization at the molecular level. Since the publication of M. suis genome sequences in 2011 only one proteome study has been published. Aim of the presented study was to significantly extend the proteome coverage of M. suis strain KI_3806 during acute infection by applying three different protein extraction methods followed by 1D SDS‐PAGE and LC‐MS/MS. A total of 404 of 795 M. suis KI_3806 proteins (50.8%) were identified. Data analysis revealed the expression of 83.7% of the predicted ORFs with assigned functions but also highlights the expression of 179 of 523 (34.2%) hypothetical proteins with unknown functions. Computational analyses identified expressed membrane‐associated hypothetical proteins that might be involved in adhesion or host–pathogen interaction. Furthermore, analyses of the expressed proteins indicated the existence of a hexose‐6‐phosphate‐transporter and an ECF transporter. In conclusion, our proteome study provides a further step toward the elucidation of the unique life cycle of M. suis and the establishment of an in vitro culture. All MS data have been deposited in the ProteomeXchange with identifier PXD002294 (http://proteomecentral.proteomexchange.org/dataset/PXD002294).

Investigation of three outbreaks of Porcine Epidemic Diarrhea in Germany in 2016 demonstrates age dependent differences in the development of humoral immune response

Porcine epidemic diarrhea (PED) has reemerged in Europe since 2014. Characterized by a rapid onset of diarrhea in pigs of all ages, morbidity can reach up to 100% whereas mortality is variable. The virus strains involved in the recent European outbreaks all cluster together with US strains (S INDEL) that lead to less severe clinical signs. In this study, fattening pigs and suckling piglets (n = 105) on farms with no prior PED history were monitored after an acute outbreak of the disease, caused by an S INDEL strain of PED virus (PEDV). For diagnostic investigations in the affected farms, real time RT-PCR was performed to detect PEDV RNA in individually taken fecal samples, and two commercial ELISA kits, both based on the N protein of PEDV, were used to detect IgG in serum samples of pigs experiencing acute signs of the disease. PEDV RNA could be detected in fecal samples up to 14 days after initial sampling. Comparing both ELISAs by Cohens Kappa showed substantial agreement (κ = 0,771). Antibodies were detectable in all fattening pigs (100%) within 10 days after the occurrence of first clinical signs and remained detectable for about two months at least in 20.6% (farm 1) and 45.7% (farm 2) of the animals, respectively. In contrast, only 18 of 34 (52.9%) suckling piglets seroconverted. Although, PEDV RNA was found in fecal samples of all piglets, 13 piglets did not demonstrate antibodies at any sampling day. PCR to detect PEDV RNA in fecal samples seems to be a reliable diagnostic tool during and after the acute outbreak. In the present study, IgG ELISA kits proved to be a feasible diagnostic tool, but age dependent differences in detection rate and persistence of antibodies need to be considered.