Presence of Equine and Bovine Coronaviruses, Endoparasites, and Bacteria in Fecal Samples of Horses with Colic

Acute abdominal pain (colic) is one of the major equine health threats worldwide and often necessitates intensive veterinary medical care and surgical intervention. Equine coronavirus (ECoV) infections can cause colic in horses but are rarely considered as a differential diagnosis. To determine the frequency of otherwise undetected ECoV infections in horses with acute colic, fresh fecal samples of 105 horses with acute colic and 36 healthy control horses were screened for viruses belonging to the Betacoronavirus 1 species by RT-PCR as well as for gastrointestinal helminths and bacteria commonly associated with colic. Horses with colic excreted significantly fewer strongyle eggs than horses without colic. The prevalence of anaerobic, spore-forming, gram-positive bacteria (Clostridium perfringens and Clostridioides difficile) was significantly higher in the feces of horses with colic. Six horses with colic (5.7%) and one horse from the control group (2.8%) tested positive for Betacoronaviruses. Coronavirus-positive samples were sequenced to classify the virus by molecular phylogeny (N gene). Interestingly, in three out of six coronavirus-positive horses with colic, sequences closely related to bovine coronaviruses (BCoV) were found. The pathogenic potential of BCoV in horses remains unclear and warrants further investigation

Construction of an ORF34 deletion φCh1 strain and secretion of putative tail fibre proteins

Der Virus φCh1, welcher das haloalkaliphile Archaeon Natrialba magadii infiziert, besitzt eine spezialisierte Gensequenz mit einem invertierbaren Bereich. Dieser umfasst die mutmaßliche Invertase int1, flankiert von den beiden offenen Leserahmen 34 und 36 welche nach einer Inversionsreaktion zu unterschiedlichen Genprodukten führt, die als putative „tail fibre“ Proteine dienen sollen und durch die entstehende Vielfalt ein erweitertes Wirtspektrum sicherstellen sollen. Dieses Ereignis führt unter anderem zu einem Austausch der 3´Enden von ORF34 und ORF36 wobei das Genprodukt 3452 entsteht und frühere Studien konnten die Interaktion mit der Zelloberfläche von Nab. magadii nachweisen (Klein et al., 2010). In diesem Zusammenhang wurde im Zuge dieser Arbeit eine Deletionsmutante dieser fraglichen Region ORF34 hergestellt, wobei die Abwesenheit des vermeintlichen „Infektionswerkzeuges“ des Viruses φCh1 einen Verlust der Infektion zur Folge haben sollte. Dieser Nab. magadii L11-ΔORF34 Stamm wurde mittels homologer Rekombination mit einer Novobiocin Kassette hergestellt. Die Abwesenheit dieses Produktes wurde anhand Western Blot nachgewiesen und die Bestätigung eines homozygoten Stammes wurde mit einem Southern Blot durchgeführt. Nachdem festgestellt wurde, dass keine intakten Virus Partikel freigesetzt werden konnten, wurde auf das Vorhandensein des Hauptcapsidprotein E getestet. Dieses Protein ist unter anderem für das Entlassen von reifen Viren verantwortlich. Es konnte keine Expression nachgewiesen werden und lässt daraus schließen, dass die eingefügte Mutation den Leserahmen der Transkription beeinflusst und somit keine gänzlich funktionierende Virus Partikel produziert. Im Anschluss wurde versucht die ursprüngliche Funktion dieser „Knock out“ Mutante L11-ΔORF34 durch das Einbringen einer funktionellen Sequenz, welche für das Genprodukt 3452 kodiert, widerherzustellen. Für diese Komplementationsanalyse wurden die Transformanten unter Durchführung eines Phagen Titers untersucht wobei resultierende Plaques auf wieder intakte wild typ Viren schließen lässt. Es konnte keine Plaque Formation nachgewiesen werden und somit keine funktionstüchtigen Viren. Um diesen Einfluss der Deletion auf nachfolgende Genexpression unter Kontrolle zu halten, soll ein zusätzliches Stopp-codon, spezifisch für Archaea, eingefügt werden und weiter Erfolge versprechen. Der zweite Teil dieser Arbeit umfasst ein weiteres Experiment welches die Funktion des Genproduktes 3452 als „Infektionswerkzeug“ nachweisen soll. Dafür wurde ein weiteres Produkt dieser bereits erwähnten Inversionsreaktion ausgewählt (Genprodukt 341) von dem bekannt ist, dass es nicht an die Zelloberfläche von Nab. magadii bindet. Das Ziel war es, durch Sekretion beider Genprodukte 341 und 3452 aus Nab. magadii die Infektion des wildtyp Virus φCh1 zu verhindern oder einzuschränken. Dies wurde mithilfe der Natrialba Extrazellulären Protease (Nep) durchgeführt. Es ist bekannt, dass dieses proteolytische Enzym zu Beginn der Stationären Phase autokatalytisch expremiert und prozessiert wird, wobei der N-Terminus während des Exportes abgespalten wird um die Protease in den Extrazellulären Raum zu entlassen (De Castro et al., 2008). Diese Eigenschaften wurden als Hilfsmittel zum Export der vermeintlichen „tail fibre“ Proteinen genutzt und Konstrukte hergestellt, welche den N-Terminus der Protease fusioniert mit den jeweiligen Varianten der Inversionsprodukte von ORF34/36 tragen. Diese Konstrukte wurden in den Stamm Nab. magadii P3 transformiert welcher defizient für diese Protease ist und mittels Western Blot konnte die erfolgreiche Sekretion dieser Proteine nachgewiesen werden. Die anschließende Durchführung eines Phagen Titers, wobei die exportierenden Stämme Nab. magadii P3 (pNB102 pNPro-341), Nab. magadii P3 (pNB102 pNPro-3452) und Nab. magadii P3 (pNB102, leerer Vektor) mit dem wild typ Virus infiziert und mit der Infektionsrate des Kontrollstammes Nab. magadii P3 (pNB102) verglichen wurden. Die Ergebnisse zeigten eine Reduktion der Infektionsrate des Stammes Nab. magadii P3 (pNB102 pNPro-3452). Dieser Stamm exportiert das vermutete „tail fibre“ Protein welches an die Zelloberfläche binden kann und lässt die Infektionsrate im Vergleich zum Kontrollstamm Nab. magadii P3 (pNB102) mindestens um ein Achtfaches sinken und etwa eine Fünffache Abnahme der Infektion konnte bei einer Gegenüberstellung mit dem Stamm Nab. magadii P3 (pNB102 pNPro-341) festgestellt werden. Diese Ergebnisse unterstreichen deutlich die bereits angestellte Vermutung, dass das Genprodukt 3452 eine essentielle Rolle im Infektionsprozess des Viruses φCh1 mit Natrialba magdii spielt.The virus φCh1, which infects the haloalkaliphilic archaeon Natrialba magadii contains an invertible genomic region where a recombination event leads to an exchange of the 3´ends of the convergent open reading frames (ORFs) 34 and 36. During the lysogenic life cycle, this inversion reaction yields in various gene products coding for putative tail fibre proteins. Increased emphasis has been put on two selected products of ORF34, the non-inverted variant gp341 and the variant comprising the C-terminus of gp36 gp3452. Previous studies identified that just gp3452 is able to bind to the cell surface of Nab.magadii, the only known host of φCh1, yet (Klein et al., 2012). The aim of this work was another confirmation of the protein gp3452 as a putative tail fibre protein. Therefore an ORF34 deletion mutant was created by homologous recombination, using a selection cassette (NovR) for gene replacement. Per Western blot and Southern blot the absence of the respective gene product and homozygation of the strain L11ΔORF34 respectively, were demonstrated. However, the elimination of ORF34, which is supposed to represent the sequence of encoded putative tail fibre proteins, yielded no mature phage particles. Furthermore, the presence of the major capsid protein E, which is thought to be essential for release of progeny precursors from the host membrane, could not be detected by Western blot analysis. This result may explain why the deletion mutant Nab. magadii L11ΔORF34 is not able to enter the lytic life cycle. By introducing a sequence expressing ORF3452 it was tested if the deletion mutant is able to recover wild type behavior. The complementation application was performed with investigation of potential plaque formation, which would be due to regained infectivity of the mutant virus. However, complementation of the mutant strain resulted in no plaque formation. It is presumably that the introduced deletion mutation affects transcription. In order to achieve a proper transcription, an archaeal termination sequence integrated in the deletion construction sequence might be an approach. In the second part of this work, an experiment was performed in order to prevent infection of the virus ФCh1 with the purpose of occupying the receptors on the Nab. magadii cell surface, by exporting the protein variants of gp34 (gp341 and gp3452). Therefore, Nep (Natrialba extracellular protease) was used as facility for secretion of the putative tail fibre protein by fusion of the respective proteins with the N-terminus of the protease. Those in frame fusion constructions were transformed by a shuttle vector into Nab. magadii P3, a protease deficient strain. The autocatalytically features of Nep enables to export the putative tail fibre proteins without residues from the N-terminus of the protease because of the cleavage, before export occurs. The confirmation of a successful secretion was performed by western blot analysis which was followed with an infectivity analyses with the wild type φCh1. The results showed that the strain Nab. magadii P3 (pNB102 pNPro-3452) exporting the putative tail fibre protein gp3452 yielded a reduction of infectivity of at least 8 orders of magnitude compared to the control strain Nab. magadii P3 (pNB102) and 5 orders of magnitude compared to Nab. magadii P3 (pNB102 pNPro-341), indicating that infectivity of φCh1 can be hindered by occupying the receptor with gp3452.These results gave another hind, that gp34 acts as a tail fibre protein

Active equine parvovirus‐hepatitis infection is most frequently detected in Austrian horses of advanced age

Background: Equine parvovirus-hepatitis (EqPV-H) research is in its infancy. Information regarding prevalence, geographical distribution, genetic diversity, pathogenesis and risk factors enhances understanding of this potentially fatal infection. Objectives: Determining the prevalence of EqPV-H in Austrian equids. Investigating factors increasing probability of infection, liver-associated biochemistry parameters, concurrent equine hepacivirus (EqHV) infection and phylogenetic analysis of Austrian EqPV-H variants. Study design: Cross-sectional study. Methods: Sera from 259 horses and 13 donkeys in Austria were analysed for anti-EqPV-H VP1-specific antibodies by luciferase immunoprecipitation system (LIPS) and EqPV-H DNA by nested polymerase chain reaction (PCR). Associations between infection status, sex and age were described. Glutamate dehydrogenase (GLDH), gamma-glutamyl transferase (GGT), bile acids and albumin concentrations were compared between horses with active infection and PCR-negative horses. PCR targeting partial EqPV-H NS1 was performed and phylogenetic analysis of Austrian EqPV-H variants was conducted. Complete coding sequences (CDS) of four Austrian variants were determined by next-generation sequencing (NGS) and compared with published sequences. Results: Horses' EqPV-H seroprevalence was 30.1% and DNA prevalence was 8.9%. One horse was co-infected with EqHV. Significantly, higher probability of active EqPV-H infection was identified in 16- to 31-year-old horses, compared with 1- to 8-year-old horses (P = 0.002; OR = 8.19; 95% CI = 1.79 to 37.50) and 9- to 15-year-old horses (P = 0.03; OR = 2.96; 95% CI = 1.08 to 8.17). Liver-associated plasma parameters were not significantly different between horses with active infection and controls. Austrian EqPV-H variants revealed high similarity to sequences worldwide. No evidence of EqPV-H was detected in donkeys. Main limitations: Equids' inclusion depended upon owner consent. There was only one sampling point per animal and the sample of donkeys was small. Conclusions: EqPV-H antibodies and DNA are frequently detected in Austrian horses, without associated hepatitis in horses with active infection. The risk of active EqPV-H infection increases with increasing age. Phylogenetic evidence supports close relation of EqPV-H variants globally, including Austrian variants

West Nile Virus and Tick-Borne Encephalitis Virus Are Endemic in Equids in Eastern Austria

The emergence of West Nile virus (WNV) and Usutu virus (USUV) in addition to the autochthonous tick-borne encephalitis virus (TBEV) in Europe causes rising concern for public and animal health. The first equine case of West Nile neuroinvasive disease in Austria was diagnosed in 2016. As a consequence, a cross-sectional seroprevalence study was conducted in 2017, including 348 equids from eastern Austria. Serum samples reactive by ELISA for either flavivirus immunoglobulin G or M were further analyzed with the plaque reduction neutralization test (PRNT-80) to identify the specific etiologic agent. Neutralizing antibody prevalences excluding vaccinated equids were found to be 5.3% for WNV, 15.5% for TBEV, 0% for USUV, and 1.2% for WNV from autochthonous origin. Additionally, reverse transcription quantitative polymerase chain reaction (RT-qPCR) was performed to detect WNV nucleic acid in horse sera and was found to be negative in all cases. Risk factor analysis did not identify any factors significantly associated with seropositivity

Porcine Circoviruses and Herpesviruses Are Prevalent in an Austrian Game Population

During the annual hunt in a privately owned Austrian game population in fall 2019 and 2020, 64 red deer (Cervus elaphus), 5 fallow deer (Dama dama), 6 mouflon (Ovis gmelini musimon), and 95 wild boars (Sus scrofa) were shot and sampled for PCR testing. Pools of spleen, lung, and tonsillar swabs were screened for specific nucleic acids of porcine circoviruses. Wild ruminants were additionally tested for herpesviruses and pestiviruses, and wild boars were screened for pseudorabies virus (PrV) and porcine lymphotropic herpesviruses (PLHV-1-3). PCV2 was detectable in 5% (3 of 64) of red deer and 75% (71 of 95) of wild boar samples. In addition, 24 wild boar samples (25%) but none of the ruminants tested positive for PCV3 specific nucleic acids. Herpesviruses were detected in 15 (20%) ruminant samples. Sequence analyses showed the closest relationships to fallow deer herpesvirus and elk gammaherpesvirus. In wild boars, PLHV-1 was detectable in 10 (11%), PLHV-2 in 44 (46%), and PLHV-3 in 66 (69%) of animals, including 36 double and 3 triple infections. No pestiviruses were detectable in any ruminant samples, and all wild boar samples were negative in PrV-PCR. Our data demonstrate a high prevalence of PCV2 and PLHVs in an Austrian game population, confirm the presence of PCV3 in Austrian wild boars, and indicate a low risk of spillover of notifiable animal diseases into the domestic animal population

Active equine parvovirus-hepatitis infection is most frequently detected in Austrian horses of advanced age.

ckground: Equine parvovirus-hepatitis (EqPV-H) research is in its infancy. Information regarding prevalence, geographical distribution, genetic diversity, pathogenesis and risk factors enhances understanding of this potentially fatal infection. Objectives: Determining the prevalence of EqPV-H in Austrian equids. Investigating factors increasing probability of infection, liver-associated biochemistry parameters, concurrent equine hepacivirus (EqHV) infection and phylogenetic analysis of Austrian EqPV-H variants. Study design: Cross-sectional study. Methods: Sera from 259 horses and 13 donkeys in Austria were analysed for anti-EqPV-H VP1-specific antibodies by luciferase immunoprecipitation system (LIPS) and EqPV-H DNA by nested polymerase chain reaction (PCR). Associations between infection status, sex and age were described. Glutamate dehydrogenase (GLDH), gamma-glutamyl transferase (GGT), bile acids and albumin concentrations were compared between horses with active infection and PCR-negative horses. PCR targeting partial EqPV-H NS1 was performed and phylogenetic analysis of Austrian EqPV-H variants was conducted. Complete coding sequences (CDS) of four Austrian variants were determined by next-generation sequencing (NGS) and compared with published sequences. Results: Horses' EqPV-H seroprevalence was 30.1% and DNA prevalence was 8.9%. One horse was co-infected with EqHV. Significantly, higher probability of active EqPV-H infection was identified in 16- to 31-year-old horses, compared with 1- to 8-year-old horses (P = 0.002; OR = 8.19; 95% CI = 1.79 to 37.50) and 9- to 15-year-old horses (P = 0.03; OR = 2.96; 95% CI = 1.08 to 8.17). Liver-associated plasma parameters were not significantly different between horses with active infection and controls. Austrian EqPV-H variants revealed high similarity to sequences worldwide. No evidence of EqPV-H was detected in donkeys. Main limitations: Equids' inclusion depended upon owner consent. There was only one sampling point per animal and the sample of donkeys was small. Conclusions: EqPV-H antibodies and DNA are frequently detected in Austrian horses, without associated hepatitis in horses with active infection. The risk of active EqPV-H infection increases with increasing age. Phylogenetic evidence supports close relation of EqPV-H variants globally, including Austrian variants

New Emergence of the Novel Pestivirus Linda Virus in a Pig Farm in Carinthia, Austria

Linda virus (LindaV) was first identified in a pig farm in Styria, Austria in 2015 and associated with congenital tremor (CT) type A-II in newborn piglets. Since then, only one more LindaV affected farm was retrospectively discovered 10 km away from the initially affected farm. Here, we report the recent outbreak of a novel LindaV strain in a farrow-to-finish farm in the federal state Carinthia, Austria. No connection between this farm and the previously affected farms could be discovered. The outbreak was characterized by severe CT cases in several litters and high preweaning mortality. A herd visit two months after the onset of clinical symptoms followed by a diagnostic workup revealed the presence of several viremic six-week-old nursery pigs. These animals shed large amounts of virus via feces and saliva, implying an important epidemiological role for within- and between-herd virus transmission. The novel LindaV strain was isolated and genetically characterized. The findings underline a low prevalence of LindaV in the Austrian pig population and highlight the threat when introduced into a pig herd. Furthermore, the results urge the need to better understand the routes of persistence and transmission of this enigmatic pestivirus in the pig population

Detection of PRRSV-1 in tongue fluids under experimental and field conditions and comparison of different sampling material for PRRSV sow herd monitoring

Abstract Background Infection with porcine reproductive and respiratory syndrome virus (PRRSV) leads to significant economic losses worldwide. One of the initial measures following an outbreak is to stabilise the herd and to prevent vertical transmission of PRRSV. The objective of this study was to detect PRRSV in different sampling material, both in an experimental model and on a commercial piglet producing farm, with a focus on evaluating the suitability of tongue fluid samples. Results In the experimental model, PRRSV negative pregnant gilts were infected with PRRSV-1 AUT15-33 on gestation day 85 and necropsy of gilts and foetuses was performed three weeks later. 38.3% of individual foetal serum and 39.4% of individual foetal thymus samples were considered PRRSV RT-qPCR positive. Tongue fluids from individual foetuses showed a 33.0% positivity rate. PRRSV RNA was detected in all but one sample of litter-wise pooled processing fluids and tongue fluids. In the field study, the investigated farm remained PRRSV positive and unstable for five consecutive farrowing groups after the start of the sampling process. Tongue fluid samples pooled by litter in the first investigated farrowing group had a 54.5% positivity rate, with the overall highest viral load obtained in the field study. In this farrowing group, 33.3% of investigated litter-wise pooled processing fluid samples and all investigated serum samples (pools of 4–6 individuals, two piglets per litter) were considered positive. Across all investigated farrowing groups, tongue fluid samples consistently showed the highest viral load. Moreover, tongue fluid samples contained the virus in moderate amounts for the longest time compared to the other investigated sampling material. Conclusion It can be concluded that the viral load in individual foetuses is higher in serum or thymus compared to tongue fluid samples. However, litter-wise pooled tongue fluid samples are well-suited for detecting vertical transmission within the herd, even when the suspected prevalence of vertical transmission events is low

Mosquitoes collected in the city of Vienna between 28 August and 10 September 2014.

<p>The two WNV-positive samples are marked with (+). Abbreviations used: BFH, Baumgarten Cemetery; FH Ottakring, Ottakring Cemetery; <i>An</i>., <i>Anopheles</i>; <i>Cs</i>., <i>Culiseta</i>; <i>Cx</i>., <i>Culex</i>; A, adults; E, egg rafts; L, larvae; P, pupae; MEM, minimal essential medium.</p><p>Mosquitoes collected in the city of Vienna between 28 August and 10 September 2014.</p

Estimates of evolutionary pairwise distances A. over the Austrian strains (including the SMB₁ plasma isolate) and their nine closest relatives, B. five major groups (clades), and C. six minor groups (clusters) among clade 2d, all defined according to the clustering in the phylogenetic tree (Fig 1).

<p>The average numbers of substitutions per site between nucleotide and amino acid sequences are indicated below and above the diagonal, respectively. Estimation of evolutionary distances was conducted in MEGA6 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0126381#pone.0126381.ref019" target="_blank">19</a>] using the MCL and p-distance algorithms for nucleotide and amino acid sequences, respectively. Group 2d-1 contains the Central/Southern European viruses including Austrian strains, and group 2d-5 consists of the Eastern European WNVs.</p><p>Abbreviations used: At-bd = strain Blood donor/Vienna/2014Austria (KP109691), SMB₁ = first passage of the suckling mouse brain isolate from the Austrian blood donor’s plasma, Cz 329 = strain Cz 13–329 (KM203861), Cz 479 = strain Cz 13–479 (KM203862), At-Cx = strain Cx pipiens/Vienna/2014Austria (KP109692), It AN2 = isolate Italy/2011/AN-2 (JN858070), It 32.1 = strain Italy/2013/Rovigo/32.1 (KF588365), It 33.2 = strain Italy/2013/Rovigo/33.2 (KF647249), It 34.1 = strain Italy/2013/Padova/34.1 (KF647251), Cz 104 = s train CZ 13–104 (KM203860), Cz 502 = strain Cz 13–502 (KM203863), At-gh = WNV strain Austria/2008_goshawk (KF179640).</p><p>Estimates of evolutionary pairwise distances A. over the Austrian strains (including the SMB₁ plasma isolate) and their nine closest relatives, B. five major groups (clades), and C. six minor groups (clusters) among clade 2d, all defined according to the clustering in the phylogenetic tree (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0126381#pone.0126381.g001" target="_blank">Fig 1</a>).</p