Serological screening in wild ruminants in Germany, 2021/2022: No evidence of SARS-CoV-2, bluetongue virus or pestivirus spread but high seroprevalences against Schmallenberg virus.

Wildlife animals may be susceptible to multiple infectious agents of public health or veterinary relevance, thereby potentially forming a reservoir that bears the constant risk of re-introduction into the human or livestock population. Here, we serologically investigated 493 wild ruminant samples collected in the 2021/2022 hunting season in Germany for the presence of antibodies against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and four viruses pathogenic to domestic ruminants, namely, the orthobunyavirus Schmallenberg virus (SBV), the reovirus bluetongue virus (BTV) and ruminant pestiviruses like bovine viral diarrhoea virus or border disease virus. The animal species comprised fallow deer, red deer, roe deer, mouflon and wisent. For coronavirus serology, additional 307 fallow, roe and red deer samples collected between 2017 and 2020 at three military training areas were included. While antibodies against SBV could be detected in about 13.6% of the samples collected in 2021/2022, only one fallow deer of unknown age tested positive for anti-BTV antibodies, and all samples reacted negative for antibodies against ruminant pestiviruses. In an ELISA based on the receptor-binding domain (RBD) of SARS-CoV-2, 25 out of 493 (5.1%) samples collected in autumn and winter 2021/2022 scored positive. This sero-reactivity could not be confirmed by the highly specific virus neutralisation test, occurred also in 2017, 2018 and 2019, that is, prior to the human SARS-CoV-2 pandemic, and was likewise observed against the RBD of the related SARS-CoV-1. Therefore, the SARS-CoV-2 sero-reactivity was most likely induced by another hitherto unknown deer virus belonging to the subgenus Sarbecovirus of betacoronaviruses.

Prebiotic effect of galacto-oligosaccharides on the skin microbiota and determination of their diffusion properties.

OBJECTIVE: Recently, prebiotics are attracting plenty of attention in the field of skin care, since it is found that they are able to support the balance of beneficial and harmful microorganisms on the skin, and accordingly prevent several skin conditions associated with microbial imbalance. Topical application of prebiotics, although insufficiently investigated, holds great promise in improving skin health. The purpose of this research was to determine the prebiotic potential of galacto-oligosaccharides (GOS) for skin microbiota and suitability for incorporation in different topical formulations, and finally, provide insights into their diffusion properties. METHODS: The prebiotic effect of GOS was evaluated through the influence on the growth of Staphylococcus epidermidis and Staphylococcus aureus, the most common resident and pathogenic bacterium of the skin microbiota, respectively. Also, with the future use of GOS in cosmetic products in mind, the diffusion of GOS molecules from two different topical formulations, hydrogel and oil-in-water (O/W) gel emulsion, was monitored employing Franz diffusion cell and two systems-with cellulose acetate membrane and transdermal diffusion test model, Strat-M® membrane. Course of fermentation and the amount of diffused GOS molecules were monitored using high-performance liquid chromatography (HPLC). RESULTS: The in vitro results revealed that GOS at a concentration of 5% (w/v) has a pronounced stimulatory effect on S. epidermidis, while simultaneously showing an inhibitory effect on S. aureus, both in nutrient broth and cosmetic formulations. GOS trisaccharide and tetrasaccharide diffusion coefficients from O/W gel emulsion were calculated to be 5.61·10-6  cm2  s-1 and 1.41·10-8  cm2  s-1 , respectively. The diffusion coefficient of GOS trisaccharides from hydrogel was 3.22·10-6  cm2  s-1 , while it was not determined for tetrasaccharides due to low diffused concentration. Transdermal diffusion tests revealed that GOS incorporated in two formulations stays at the surface of the skin even after 24 h. CONCLUSION: When applied in adequate concentration, GOS has the potential to be used as a skin prebiotic. Novel GOS enriched formulations, Aristoflex® AVC-based hydrogel and Heliogel™-based O/W gel emulsion, provided efficient diffusion and delivery of prebiotic GOS molecules to the skin surface.

OBJECTIF: Récemment, les prébiotiques attirent beaucoup d'attention dans le domaine des soins de la peau, car il a été constaté qu'ils sont capables de soutenir l'équilibre des micro-organismes bénéfiques et nocifs sur la peau et, par conséquent, de prévenir plusieurs affections cutanées associées à un déséquilibre microbien. L'application topique de prébiotiques, bien qu'insuffisamment étudiée, est très prometteuse pour améliorer la santé de la peau. Le but de cette recherche était de déterminer le potentiel prébiotique des galacto-oligosaccharides (GOS) pour le microbiote cutané et leur aptitude à être incorporés dans différentes formulations topiques, et enfin, de fournir des informations sur leurs propriétés de diffusion. MÉTHODES: L'effet prébiotique du GOS a été évalué à travers l'influence sur la croissance de Staphylococcus epidermidis et de Staphylococcus aureus, les bactéries résidentes et pathogènes les plus courantes du microbiote cutané, respectivement. De plus, en gardant à l'esprit l'utilisation future du GOS dans les produits cosmétiques, la diffusion des molécules de GOS à partir de deux formulations topiques différentes, l'hydrogel et l'émulsion de gel huile-dans-eau (H/E), a été surveillée à l'aide d'une cellule de diffusion de Franz et de deux systèmes - avec de la cellulose membrane en acétate et modèle de test de diffusion transdermique, membrane Strat-M®. Le cours de la fermentation et la quantité de molécules de GOS diffusées ont été surveillés en utilisant la chromatographie liquide à haute performance (HPLC). RESULTATS: Les résultats in vitro ont révélé que le GOS à une concentration de 5% (p/v) a un effet stimulant prononcé sur S. epidermidis, tout en montrant simultanément un effet inhibiteur sur S. aureus, à la fois dans les bouillons nutritifs et les formulations cosmétiques. Les coefficients de diffusion GOS trisaccharide et tétrasaccharide de l'émulsion de gel H/E ont été calculés comme étant de 5,61·10−6 cm2 s−1 et 1,41·10-8 cm2 s−1 , respectivement. Le coefficient de diffusion des trisaccharides GOS à partir de l'hydrogel était de 3,22·10−6 cm2 s−1 , alors qu'il n'a pas été déterminé pour les tétrasaccharides en raison de la faible concentration diffusée. Des tests de diffusion transdermique ont révélé que le GOS incorporé dans deux formulations reste à la surface de la peau même après 24 h. CONCLUSION: Lorsqu'il est appliqué à une concentration adéquate, le GOS a le potentiel d'être utilisé comme prébiotique cutané. De nouvelles formulations enrichies en GOS, l'hydrogel à base d'Aristoflex® AVC et l'émulsion de gel H/E à base d'Heliogel™, ont permis une diffusion et une délivrance efficaces des molécules GOS prébiotiques à la surface de la peau.

Scent dog identification of SARS-CoV-2 infections in different body fluids.

BACKGROUND: The main strategy to contain the current SARS-CoV-2 pandemic remains to implement a comprehensive testing, tracing and quarantining strategy until vaccination of the population is adequate. Scent dogs could support current testing strategies. METHODS: Ten dogs were trained for 8 days to detect SARS-CoV-2 infections in beta-propiolactone inactivated saliva samples. The subsequent cognitive transfer performance for the recognition of non-inactivated samples were tested on three different body fluids (saliva, urine, and sweat) in a randomised, double-blind controlled study. RESULTS: Dogs were tested on a total of 5242 randomised sample presentations. Dogs detected non-inactivated saliva samples with a diagnostic sensitivity of 84% (95% CI: 62.5-94.44%) and specificity of 95% (95% CI: 93.4-96%). In a subsequent experiment to compare the scent recognition between the three non-inactivated body fluids, diagnostic sensitivity and specificity were 95% (95% CI: 66.67-100%) and 98% (95% CI: 94.87-100%) for urine, 91% (95% CI: 71.43-100%) and 94% (95% CI: 90.91-97.78%) for sweat, 82% (95% CI: 64.29-95.24%), and 96% (95% CI: 94.95-98.9%) for saliva respectively. CONCLUSIONS: The scent cognitive transfer performance between inactivated and non-inactivated samples as well as between different sample materials indicates that global, specific SARS-CoV-2-associated volatile compounds are released across different body secretions, independently from the patient's symptoms. All tested body fluids appear to be similarly suited for reliable detection of SARS-CoV-2 infected individuals.

Inactivation of Classical Swine Fever Virus in Porcine Serum Samples Intended for Antibody Detection.

Shipping of serum samples that were taken from pigs infected with classical swine fever (CSF) virus is frequently requested with the objective of serological analyses, not only for diagnostic purposes but also for exchange of reference materials that are used as control material of diagnostic assays. On the basis of the fact that an outbreak with CSF is associated with enormous economic losses, biological safety during the exchange of reference material is of great importance. The present study aimed to establish a pragmatic approach for reliable CSF virus (CSFV) inactivation in serum without impairing antibody detection. Considering the fact that complement inactivation through heating is routinely applied, the basic idea was to combine heat treatment with the dilution of serum in a detergent containing buffer in order to facilitate the inactivation process. The results show that treatment of serum samples with phosphate buffered saline-Tween20 (final concentration = 0.15%) along with incubation at 56 °C for 30 min inactivated CSFV and such treatment with ≤ 0.25% PBS-Tween20 does not impair subsequent antibody detection by ELISA or virus neutralization test. This minimizes the risk of virus contamination and represents a valuable contribution to a safer CSF diagnosis on a national and international level.

Epidemiology, diagnosis and control of classical swine fever: Recent developments and future challenges.

Classical swine fever (CSF) represents a major health and trade problem for the pig industry. In endemic countries or those with a wild boar reservoir, CSF remains a priority for Veterinary Services. Surveillance as well as stamping out and/or vaccination are the principle tools of prevention and control, depending on the context. In the past decades, marker vaccines and accompanying diagnostic tests allowing the discrimination of infected from vaccinated animals have been developed. In the European Union, an E2 subunit and a chimeric live vaccine have been licensed and are available for the use in future disease outbreak scenarios. The implementation of commonly accepted and globally harmonized concepts could pave the way to replace the ethically questionable stamping out policy by a vaccination-to-live strategy and thereby avoid culling of a large number of healthy animals and save food resources. Although a number of vaccines and diagnostic tests are available worldwide, technological advancement in both domains is desirable. This work provides a summary of an analysis undertaken by the DISCONTOOLS group of experts on CSF. Details of the analysis can be downloaded from the web site at http://www.discontools.eu/.

Classical swine fever virus marker vaccine strain CP7_E2alf: Shedding and dissemination studies in boars.

Over the last decade, pestivirus chimaera CP7_E2alf has proven to be a most promising marker vaccine candidate against classical swine fever (CSF). To provide further background data for the risk assessment towards licensing and release, especially on presence of the vaccine chimaera in faeces, urine, and organs of the male reproductive tract, supplementary studies were carried out under controlled laboratory conditions. In detail, the shedding and dissemination pattern of Suvaxyn(®) CSF Marker ("CP7_E2alf") was assessed in 12 adult boars after single intramuscular vaccination with a tenfold vaccine dose. Four and seven days post vaccination, six animals were subjected to necropsy and triplicate samples were obtained from reproductive and lymphatic organs as well as urine, faeces, blood, and several additional organs and matrices. The sampling days were chosen based on pre-existing data that indicated the highest probability of virus detection. Upon vaccination, neither local nor systemic adverse effects were observed in the experimental animals. It was confirmed that primary replication is restricted to the lymphatic tissues and especially the tonsil. While viral genome was detectable in several samples from lymphatic tissues at four and seven days post vaccination, infectious virus was only demonstrated at four days post vaccination in one tonsil sample and one parotid lymphnode. Sporadic detection at a very low level occurred in some replicates of liver, lung, bone marrow, and salivary gland samples. In contrast, viral genome was not detected in any sample from reproductive organs and accessory sex glands, in faeces, urine, or bile. The presented data on the dissemination of the vaccine virus CP7_E2alf in adult boars are supplementing existing safety and efficacy studies and indicate that the use of the vaccine is also safe in reproductive boars.

Course and transmission characteristics of oral low-dose infection of domestic pigs and European wild boar with a Caucasian African swine fever virus isolate.

In 2007, African swine fever virus (ASFV) was introduced into the Transcaucasian countries and Russia. Since then, it has spread alarmingly and reached the European Union. ASFV strains are highly virulent and lead to almost 100% mortality under experimental conditions. However, the possibility of dose-dependent disease courses has been discussed. For this reason, a study was undertaken to assess the risk of chronic disease and the establishment of carriers upon low-dose oronasal infection of domestic pigs and European wild boar. It was demonstrated that very low doses of ASFV are sufficient to infect especially weak or runted animals by the oronasal route. Some of these animals did not show clinical signs indicative of ASF, and they developed almost no fever. However, no changes were observed in individual animal regarding the onset, course and outcome of infection as assessed by diagnostic tests. After amplification of ASFV by these animals, pen- and stablemates became infected and developed acute lethal disease with similar characteristics in all animals. Thus, we found no indication of prolonged or chronic individual courses upon low-dose infection in either species. The scattered onset of clinical signs and pathogen detection within and among groups confirms moderate contagiosity that is strongly linked with blood contact. In conclusion, the prolonged course at the "herd level" together with the exceptionally low dose that proved to be sufficient to infect a runted wild boar could be important for disease dynamics in wild-boar populations and in backyard settings.

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.

Development and validation of a harmonized TaqMan-based triplex real-time RT-PCR protocol for the quantitative detection of normalized gene expression profiles of seven porcine cytokines.

Dysregulation of cytokine responses plays a major role in the pathogenesis of severe and life-threatening infectious diseases like septicemia or viral hemorrhagic fevers. In pigs, diseases like African and classical swine fever are known to show exaggerated cytokine releases. To study these responses and their impact on disease severity and outcome in detail, reliable, highly specific and sensitive methods are needed. For cytokine research on the molecular level, real-time RT-PCRs have been proven to be suitable. Yet, the currently available and most commonly used SYBR Green I assays or heterogeneous gel-based RT-PCRs for swine show a significant lack of specificity and sensitivity. The latter is however absolutely essential for an accurate quantification of rare cytokine transcripts as well as for detection of small changes in gene expressions. For this reason, a harmonized TaqMan-based triplex real-time RT-PCR protocol for the quantitative detection of normalized gene expression profiles of seven porcine cytokines was designed and validated within the presented study. Cytokines were chosen to represent different immunological pathways and targets known to be involved in the pathogenesis of the above mentioned porcine diseases, namely interleukin (IL)-1ß, IL-2, IL-4, IL-6, IL-8, tumor necrosis factor (TNF)-α and interferon (IFN)-α. Beta-Actin and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) served as reference genes for normalization. For absolute quantification a synthetic standard plasmid was constructed comprising all target cytokines and reference genes within a single molecule allowing the generation of positive control RNA. The standard as well as positive RNAs from samples, and additionally more than 400 clinical samples, which were collected from animal trials, were included in the validation process to assess analytical sensitivity and applicability under routine conditions. The resulting assay allows the reliable assessment of gene expression profiles and provides a broad applicability to any kind of immunological research in swine.

Alternative sampling strategies for passive classical and African swine fever surveillance in wild boar.

In view of the fact that African swine fever (ASF) was recently introduced into the wild boar population of the European Union and that classical swine fever (CSF) keeps reoccurring, targeted surveillance is of utmost importance for early detection. Introduction of both diseases is usually accompanied by an increased occurrence of animals found dead. Thus, fallen wild boar are the main target for passive surveillance. However, encouraging reporting by hunters and sampling of these animals is difficult. Partly, these problems could be solved by providing a pragmatic sampling approach. For this reason, we assessed the applicability of three different dry/semi-dry blood swabs, namely a cotton swab, a flocked swab, and a forensic livestock swab, for molecular swine fever diagnosis. After nucleic acid extraction using manual and automated systems, routine quantitative real-time polymerase chain reactions (qPCR) were carried out. Results obtained from swabs or their fragments were compared to results generated from EDTA blood. It was shown that reliable detection of both pathogens was possible by qPCR. Shifts in genome copy numbers were observed, but they did not change the qualitative results. In general, all swabs were suitable, but the forensic swab showed slight advantages, especially in terms of cutting and further storage. Robustness of the method was confirmed by the fact that different extraction methods and protocols as well as storage at room temperature did not have an influence on the final outcome. Taken together, swab samples could be recommended as a pragmatic approach to sample fallen wild boar.

Comparative analyses of host responses upon infection with moderately virulent classical swine fever virus in domestic pigs and wild boar.

BACKGROUND: Classical swine fever (CSF) is one of the most important viral diseases of pigs. Clinical signs may vary from almost inapparent infection to a hemorrhagic fever like illness. Among the host factors leading to different disease courses are age, breed, and immune status. The aim of this study was to compare host responses of different pig breeds upon infection with a recent moderately virulent CSF virus (CSFV) strain, and to assess their impact on the clinical outcome and the efficiency of immune responses. To this means, two domestic pig types (German Landrace and hybrids), were compared to European wild boar. Along with clinical and pathological assessments and routine virological and serological methods, kinetics of immune-cellular parameters were evaluated. FINDINGS: All animals were susceptible to infection and despite clinical differences, virus could be detected in all infected animals to similar amounts. All but one animal developed an acute disease course, two landrace animals recovered after a transient infection. One wild boar got chronically infected. Changes in the percentages of lymphocyte subsets in peripheral blood did not show a clear correlation with the clinical outcome. High and early titers of neutralizing antibodies were especially detected in wild boar and German Landrace pigs. CONCLUSIONS: While differences among breeds did not have the expected impact on course and outcome of CSFV infection, preload with facultative pathogens and even small differences in age seemed to be more relevant. Future studies will target the characterization of responses observed during different disease courses including cytokine reactions and further analyses of lymphocyte subsets.

Novel rope-based sampling of classical swine fever shedding in a group of wild boar showing low contagiosity upon experimental infection with a classical swine fever field strain of genotype 2.3.

Several classical swine fever (CSF) epidemics in wild boar and domestic pigs in Europe during the last decades have been caused by CSF virus (CSFV) strains of genotype 2.3. This genotype is known to be virulent leading to high morbidity and mortality. We experimentally infected two eight months old wild boar with 10(5,5) TCID50 of CSFV genotype 2.3 and kept the animals together with five noninoculated wild boar of the same age. Our original purpose was to evaluate a non-invasive sampling method based on saliva collection using "rope-in-a-bait" sampling baits. While expecting high morbidity, high level of virus shedding and some mortality, we actually observed a subclinical course of infection with an unexpected low contagiosity. The two inoculated animals infected only three contact animals while two contact animals remained uninfected. These findings substantially add to our epidemiological understanding of CSFV circulation in wild boar populations. CSFV infected animals older than six months and in good condition may not shed sufficient virus to transmit infection to all seronegative in-contact animals. The contagiosity in relation to the animal's age is discussed. This supports the hypothesis of silent perpetuation of CSFV in wild boar populations for several months if the wild boar density is sufficiently high. The feasibility of the "rope-in-a-bait" sampling method could be proven during the short viraemic phase of infected animals during the second week of infection.