Isolation and characterization of Leptospira licerasiae in Austrian swine - a first-time case report in Europe.

BACKGROUND: Leptospiraceae comprise a diverse family of spirochetal bacteria, of which many are involved in infectious diseases of animals and humans. Local leptospiral diversity in domestic animals is often poorly understood. Here we describe the incidental detection of Leptospira (L.) licerasiae in an Austrian pig. CASE PRESENTATION: During an experiment to characterize the pathogenesis of L. interrogans serovar Icterohaemorrhagiae in pigs, cultivation of a urine sample from a non-challenged contact pig resulted in growth of a spirochetal bacterium that tested negative for pathogenic Leptospira (LipL32 gene). PCR, Sanger sequencing and standard serotyping further confirmed that the recovered isolate was clearly different from the challenge strain L. interrogans serovar Icterohaemorrhagiae used in the animal experiment. Whole genome sequencing revealed that the isolate belongs to the species L. licerasiae, a tropical member of the Leptospiraceae, with no prior record of detection in Europe. CONCLUSIONS: This is the first report describing the occurrence of L. licerasiae in Europe. Since L. licerasiae is considered to have intermediate pathogenicity, it will be important to follow the geographical distribution of this species and its pathogenic and zoonotic potential in more detail.

Efficacy of live attenuated porcine reproductive and respiratory syndrome virus 2 strains to protect pigs from challenge with a heterologous Vietnamese PRRSV 2 field strain.

BACKGROUND: Effective vaccines against porcine reproductive and respiratory syndrome virus (PRRSV), especially against highly pathogenic (HP) PRRSV are still missing. The objective of this study was to evaluate the protective efficacy of an experimental live attenuated PRRSV 2 vaccine, composed of two strains, against heterologous challenge with a Vietnamese HP PRRSV 2 field strain. For this reason, 20 PRRSV negative piglets were divided into two groups. The pigs of group 1 were vaccinated with the experimental vaccine, group 2 remained unvaccinated. All study piglets received an intranasal challenge of the HP PRRSV 2 on day 0 of the study (42 days after vaccination). Blood samples were taken on days 7 and 21 after vaccination and on several days after challenge. On day 28 after challenge, all piglets were euthanized and pathologically examined. RESULTS: On days 7 and 21 after vaccination, a PRRSV 2 viraemia was seen in all piglets of group 1 which remained detectable in seven piglets up to 42 days after vaccination. On day 3 after challenge, all piglets from both groups were positive in PRRSV 2 RT-qPCR. From day 7 onwards, viral load and number of PRRSV 2 positive pigs were lower in group 1 than in group 2. All pigs of group 1 seroconverted after PRRSV 2 vaccination. PRRSV antibodies were detected in serum of all study pigs from both groups from day 14 after challenge onwards. In group 2, moderate respiratory symptoms with occasional coughing were seen following the challenge with HP PRRSV 2. Pigs of group 1 remained clinically unaffected. Interstitial pneumonia was found in four piglets of group 1 and in all ten piglets of group 2. Histopathological findings were more severe in group 2. CONCLUSIONS: It was thus concluded that the used PRRSV 2 live experimental vaccine provided protection from clinical disease and marked reduction of histopathological findings and viral load in pigs challenged with a Vietnamese HP PRRSV 2 field strain.

Bridging basic science and applied diagnostics: Comprehensive viral diagnostics enabled by graphene-based electronic biosensor technology advancements.

This study presents a graphene field-effect transistor (gFET) biosensor with dual detection capabilities for SARS-CoV-2: one RNA detection assay to confirm viral positivity and the other for nucleocapsid (N-)protein detection as a proxy for infectiousness of the patient. This technology can be rapidly adapted to emerging infectious diseases, making an essential tool to contain future pandemics. To detect viral RNA, the highly conserved E-gene of the virus was targeted, allowing for the determination of SARS-CoV-2 presence or absence using nasopharyngeal swab samples. For N-protein detection, specific antibodies were used. Tested on 213 clinical nasopharyngeal samples, the gFET biosensor showed good correlation with RT-PCR cycle threshold values, proving its high sensitivity in detecting SARS-CoV-2 RNA. Specificity was confirmed using 21 pre-pandemic samples positive for other respiratory viruses. The gFET biosensor had a limit of detection (LOD) for N-protein of 0.9 pM, establishing a foundation for the development of a sensitive tool for monitoring active viral infection. Results of gFET based N-protein detection corresponded to the results of virus culture in all 16 available clinical samples and thus it also proved its capability to serve as a proxy for infectivity. Overall, these findings support the potential of the gFET biosensor as a point-of-care device for rapid diagnosis of SARS-CoV-2 infection and indirect assessment of infectiousness in patients, providing additional information for clinical and public health decision-making.

Leptospira interrogans Serovar Icterohaemorrhagiae Failed to Establish Distinct Infection in Naïve Gilts: Lessons Learned from a Preliminary Experimental Challenge.

Leptospira is a pathogen involved in fertility problems in pigs. Nevertheless, little information is available on pathogenicity, transmission, tissue tropism, and immune response. The objective of this preliminary study was to induce a diagnostically detectable infection in naïve gilts using Leptospira interrogans serovar Icterohaemorrhagiae to gain the knowledge required for designing a large-scale trial. Eight seronegative fertile gilts were divided into three groups: control (n = 2), challenge (n = 3; 10 mL of 108 leptospires/mL intravenously), and contact (n = 3). A daily clinical examination and periodic sampling of blood, urine, and vaginal swabs were performed until four weeks after infection when necropsy was undertaken. Seroconversion of infected animals was detected first by a microscopic agglutination test (MAT) between four and seven days after inoculation. No clinical signs were observed except pyrexia. Laboratory data primarily remained within reference intervals. Leptospira were undetectable in all groups by real-time PCR (sera, urine, vaginal swabs, and tissue samples) and bacterial culture (urine and tissue samples). However, histologic evidence for tubulo-interstitial nephritis could be found. Based on the study results and limitations, questions to be solved and approaches to be reconsidered are raised for the conduction of further experimental studies to understand the pathogenesis and the role of Icterohaemorrhagiae in pig health.

Influence of Selective Agents (EMJH-STAFF), Sample Filtration and pH on Leptospira interrogans Serovar Icterohaemorrhagiae Cultivation and Isolation from Swine Urine.

Leptospira spp. cause the zoonotic disease leptospirosis, which occurs in numerous mammalians worldwide. Isolation is still important for serotyping and genotyping of Leptospira, which in turn is essential for epidemiological surveillance of leptospirosis and the development of diagnostic tests and vaccines. However, isolation of Leptospira from clinical specimens is inherently insensitive. This study was conducted to examine the influence of selective agents, sample filtration, sample pH and the use of phosphate buffered saline (PBS) buffer for sample storage to improve the success of cultivation and isolation of Leptospira interrogans serovar Icterohaemorrhagiae from swine urine. EMJH (Ellinghausen McCullough, Johnson and Harris) medium including the selective agents sulfamethoxazole, trimethoprim, amphotericin, fosfomycin and 5-fluorouracil (STAFF) increased the success of Leptospira isolation from spiked swine urine samples. Sample filtration yielded only negative results. Isolation in EMJH-STAFF was successful from swine urine with a density as low as 104 Leptospira/mL, and urine with pH ≤ 7 impaired the cultivation rate. Cultivation and isolation were not improved by the addition of PBS to spiked urine samples prior to storage for 24 h at 4 °C. The results of the study demonstrate that cultivation and isolation of leptospires from swine urine can be improved by enhanced methods.

Comparison of eleven in vitro diagnostic assays for the detection of SARS-CoV-2 RNA.

Transmission mitigation of SARS-CoV-2 requires the availability of accurate and sensitive detection methods. There are several commercial ad hoc molecular diagnostic kits currently on the market, many of which have been evaluated by different groups. However, in low resource settings the availability and cost of these commercial kits can be a limiting factor for many diagnostic laboratories. In such cases alternatives need to be identified. With this in mind, eight commercial reverse transcription quantitative real-time PCR (RT-qPCR) master mixes from Applied Biosystems (Thermo Fisher Scientific), Bio-Rad, Biotech Rabbit, Promega, Qiagen, QuantaBio, Invitrogen (Thermo Fisher Scientific) and Takara using the same commercial primer and probe mix [LightMix® Modular SARS and Wuhan CoV E-gene mix (TIB MolBiol, Germany)] were evaluated. Three ad hoc molecular diagnostic kits [GeneFinder™ COVID-19 Plus RealAmp kit (Osang Healthcare); genesig® Real-Time PCR Coronavirus COVID-19 (Primerdesign); and ViroReal® Kit SARS-CoV-2 & SARS-CoV (Ingenetix)] were also included in the study. The limit of detection was calculated for each assay using serial dilutions of a defined clinical sample. The performances of the assays were compared using a panel of 178 clinical samples and their analytical specificity assessed against a panel of human betacoronaviruses. Inter assay agreement was assessed using statistical tests (Bland-Altman, Fleiss-Kappa and Cohen's Kappa) and was shown to be excellent to good in all cases. We conclude that all of the assays evaluated in this study can be used for the routine detection of SARS-CoV-2 and that the RT-qPCR master mixes are a valid alternative to ad hoc molecular diagnostic kits.

Can We Protect Those We Care for in A Pandemic? - Prevalence of Neutralizing Antibodies against SARS-CoV-2 in Nursing Homes.

In December 2019, the People's Republic of China and the World Health Organization first reported on a cluster of pneumonia with an unknown cause. Nine months later more than 1.4 million people have died from COVID 19. In this work, the effects of the COVID 19 pandemic on five nursing homes in Austria, which cared for 889 residents in the first half of 2020, were examined. The research question was whether the measures taken were appropriate to prevent an outbreak within the individual facilities. To detect previously unrecognized infections, the present study evaluated the prevalence of neutralizing antibodies against the SARS-CoV-2 virus in residents and employees of the nursing homes. Following the analysis of blood samples, the prospectively collected data was connected to data from screening examinations and data from contact tracing. The present study demonstrated an overall prevalence of neutralizing antibodies against the SARS-CoV-2 virus in nursing homes of 3.7%. Whereas the prevalence in those facilities that have never been hit by an outbreak is 0%, the prevalence in those facilities with an outbreak is up to 4.9%. Neutralizing antibodies against SARS-CoV-2 were detected in 35 persons. A retrospective analysis of all 5 included nursing homes demonstrated that upon regular clinical screening in combination with PCRs an infection with SARS-COV-2 was detected in 66 residents and 24 employees from different professional groups. In only 25 of the 35 persons with neutralizing antibodies against SARS-CoV-2 an infection was proven in advance. This study suggests that specific measures can prevent transmission within a health care facility. Nevertheless, the results also show that a risk reduction to 0% cannot be achieved. In preparation for further pandemic waves there is still the need to reduce the probability of a transmission in nursing homes with specific test strategies.

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.