Severe Neurologic Disease in a Horse Caused by Tick-Borne Encephalitis Virus, Austria, 2021.

As evidenced by sero-epidemiological studies, infections of horses with the tick-borne encephalitis virus (TBEV) occur frequently in TBEV-endemic areas. However, there are only very few reports of clinical cases. A possible underreporting may be due to a variety of diagnostic challenges. In this study, ELISA and neutralization tests were applied to serum samples. Brain tissue samples were investigated for the presence of nucleic acids of TBEV, Equid alphaherpesvirus 1, Borna disease virus 1, West Nile and Usutu viruses, rustrela virus, as well as Eastern, Western, and Venezuelan equine encephalitis viruses with RT-qPCR, RT-PCR, and qPCR, respectively. TBEV-specific amplification products were subjected to Sanger sequencing. In addition, a direct fluorescent antibody test for rabies was performed. Clinical and patho-histological findings are reported. Using specific RT-qPCR and RT-PCR assays, TBEV nucleic acids were demonstrated in brain tissue samples. Sequencing revealed the Western (formerly Central) European subtype of TBEV as the etiological agent. A high titer of TBEV-specific neutralizing antibodies was found in the serum. RNAscope in situ hybridization revealed TBEV RNA confined to neuronal cell bodies and processes. No other pathogens or nucleic acids thereof could be detected. Diagnostic procedures need to be carried out early after the onset of neurological signs to allow for a final etiological diagnosis of acute TBEV infections in horses.

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.

First Case of Autochthonous Equine Theileriosis in Austria.

A 23-year-old pregnant warmblood mare from Güssing, Eastern Austria, presented with apathy, anemia, fever, tachycardia and tachypnoea, and a severely elevated serum amyloid A concentration. The horse had a poor body condition and showed thoracic and pericardial effusions, and later dependent edema and icteric mucous membranes. Blood smear and molecular analyses revealed an infection with Theileria equi. Upon treatment with imidocarb diproprionate, the mare improved clinically, parasites were undetectable in blood smears, and 19 days after hospitalization the horse was discharged from hospital. However, 89 days after first hospitalization, the mare again presented to the hospital with an abortion, and the spleen of the aborted fetus was also PCR-positive for T. equi. On the pasture, where the horse had grazed, different developmental stages of Dermacentor reticulatus ticks were collected and subjected to PCR, and one engorged specimen was positive for T. equi. All three amplicon sequences were identical (T. equi genotype E). It is suspected that T. equi may repeatedly be transmitted in the area where the infected mare had grazed, and it could be shown that transmission to the fetus had occurred. Due to the chronic nature of equine theileriosis and the possible health implications of infection, it is advised to include this disease in the panel of differential diagnoses in horses with relevant clinical signs, including horses without travel disease, and to be aware of iatrogenic transmission from inapparent carrier animals.

Emergence of West Nile virus lineage 2 in Europe: Characteristics of the first seven cases of West Nile neuroinvasive disease in horses in Austria.

We report details of the first seven equine cases of confirmed West Nile neuroinvasive disease in Austria. The cases presented during summer and autumn of 2016 (n = 2), 2017 (n = 3) and 2018 (n = 2). All horses showed gait abnormalities and 6 of 7 horses exhibited fasciculations and/or tremors, and we provide video recordings of these. Three horses also showed cranial nerve involvement. Following rapid improvement, three horses were discharged. Four horses were euthanized due to the severity of clinical signs and subjected to neuropathological examination. West Nile virus (WNV) lineage 2 nucleic acid was detected in 5 of 7 horses, and WNV-specific neutralizing antibodies in all 7 horses. In addition, serologic evidence of WNV infection was found in two out of fourteen in-contact horses. Horses may be considered a sentinel species for human WNV infections, integrating human and veterinary medicine and thus contributing to the one health concept.

No Evidence of Mosquito Involvement in the Transmission of Equine Hepacivirus (Flaviviridae) in an Epidemiological Survey of Austrian Horses.

Prevalence studies have demonstrated a global distribution of equine hepacivirus (EqHV), a member of the family Flaviviridae. However, apart from a single case of vertical transmission, natural routes of EqHV transmission remain elusive. Many known flaviviruses are horizontally transmitted between hematophagous arthropods and vertebrate hosts. This study represents the first investigation of potential EqHV transmission by mosquitoes. More than 5000 mosquitoes were collected across Austria and analyzed for EqHV ribonucleic acid (RNA) by reverse transcription quantitative polymerase chain reaction (RT-qPCR). Concurrently, 386 serum samples from horses in eastern Austria were analyzed for EqHV-specific antibodies by luciferase immunoprecipitation system (LIPS) and for EqHV RNA by RT-qPCR. Additionally, liver-specific biochemistry parameters were compared between EqHV RNA-positive horses and EqHV RNA-negative horses. Phylogenetic analysis was conducted in comparison to previously published sequences from various origins. No EqHV RNA was detected in mosquito pools. Serum samples yielded an EqHV antibody prevalence of 45.9% (177/386) and RNA prevalence of 4.15% (16/386). EqHV RNA-positive horses had significantly higher glutamate dehydrogenase (GLDH) levels (p = 0.013) than control horses. Phylogenetic analysis showed high similarity between nucleotide sequences of EqHV in Austrian horses and EqHV circulating in other regions. Despite frequently detected evidence of EqHV infection in Austrian horses, no viral RNA was found in mosquitoes. It is therefore unlikely that mosquitoes are vectors of this flavivirus.

Integrated analysis of human-animal-vector surveillance: West Nile virus infections in Austria, 2015-2016.

The results of integrated human and veterinary surveillance for West Nile virus (WNV) infections in Austria during the transmission seasons 2015 and 2016 are shown. Altogether WNV nucleic acid was detected in 21 humans, horses, wild birds and mosquito pools. In detail: in four human clinical cases [two cases of West Nile fever (WNF) and two cases of West Nile neuroinvasive disease (WNND)]; eight blood donors [among 145,541 tested donations], of which three remained asymptomatic and five subsequently developed mild WNF; two horses with WNND, of which one recovered and one had to be euthanized; two wild birds [one goshawk and one falcon, both succumbed to WNND]; and five Culex pipiens mosquito pools. Compared to previous years the number of infections increased remarkably. All infections were recorded in the city of Vienna and neighboring regions of Lower Austria. Sixteen coding-complete WNV sequences were established which were closely related to each other and to other Austrian, Czech and Italian viruses, all belonging to the Central/Southern European cluster of WNV sublineage 2d. However, several genetically slightly different WNV strains seem to co-circulate in the same area, as demonstrated by phylogenetic analysis. Based on detailed sequence analysis, all newly discovered Austrian WNV strains had the potential to cause neurological disease, but no correlation was found between severity of disease and the analyzed genetic virulence/neuroinvasiveness markers. Results of integrated human-animal-vector surveillance presented in this paper provide a comprehensive description of WNV activity in the region and will facilitate proactive public health measures to prevent or mitigate potential outbreaks.

Changes in the Population of Methicillin-Resistant Staphylococcus pseudintermedius and Dissemination of Antimicrobial-Resistant Phenotypes in the Netherlands.

Methicillin-resistant Staphylococcus pseudintermedius (MRSP), which is often multidrug resistant (MDR), has recently emerged as a threat to canine health worldwide. Knowledge of the temporal distribution of specific MRSP lineages, their antimicrobial resistance phenotypes, and their association with clinical conditions may help us to understand the emergence and spread of MRSP in dogs. The aim of this study was to determine the yearly proportions of MRSP lineages and their antimicrobial-resistant phenotypes in the Netherlands and to examine possible associations with clinical conditions. MRSP was first isolated from a canine specimen submitted for diagnostics to the Faculty of Veterinary Medicine of Utrecht University in 2004. The annual cumulative incidence of MRSP among S. pseudintermedius increased from 0.9% in 2004 to 7% in 2013. MRSP was significantly associated with pyoderma and, to a lesser extent, with wound infections and otitis externa. Multilocus sequence typing (MLST) of 478 MRSP isolates yielded 39 sequence types (ST) belonging to 4 clonal complexes (CC) and 15 singletons. CC71 was the dominant lineage that emerged since 2004, and CC258, CC45, and several unlinked isolates became more frequent during the following years. All but two strains conferred an MDR phenotype, but strains belonging to CC258 or singletons were less resistant. In conclusion, our study showed that MDR CC71 emerged as the dominant lineage from 2004 and onward and that less-resistant lineages were partly replacing CC71.

Longitudinal study on methicillin-resistant Staphylococcus pseudintermedius in households.

Methicillin-resistant Staphylococcus pseudintermedius (MRSP) is an emerging pathogen in dogs and has been found in Europe, Asia and North America. To date most studies are one-point prevalence studies and therefore little is known about the dynamics of MRSP in dogs and their surrounding. In this longitudinal study MRSP colonization in dogs and the transmission of MRSP to humans, contact animals and the environment was investigated. Sixteen dogs with a recent clinical MRSP infection were included. The index dogs, contact animals, owners and environments were sampled once a month for six months. Samples taken from the nose, perineum and infection site (if present) of the index cases and contact animals, and the nares of the owners were cultured using pre-enrichment. Index cases were found positive for prolonged periods of time, in two cases during all six samplings. In five of the 12 households that were sampled during six months, the index case was intermittently found MRSP-positive. Contact animals and the environment were also found MRSP-positive, most often in combination with a MRSP-positive index dog. In four households positive environmental samples were found while no animals or humans were MRSP-positive, indicating survival of MRSP in the environment for prolonged periods of time. Genotyping revealed that generally similar or indistinguishable MRSP isolates were found in patients, contact animals and environmental samples within the same household. Within two households, however, genetically distinct MRSP isolates were found. These results show that veterinarians should stay alert with (former) MRSP patients, even after repeated MRSP-negative cultures or after the disappearance of the clinical infection. There is a considerable risk of transmission of MRSP to animals in close contact with MRSP patients. Humans were rarely MRSP-positive and never tested MRSP-positive more than once suggesting occasional contamination or rapid elimination of colonization of the owners.