Dual topology of equine arteritis virus GP3 protein and the role of arginine motif RXR in GP3 ER retention.

Glycoprotein 3 (GP3) serves as a structural protein in equine arteritis virus (EAV), forming a heterotrimeric complex that plays a pivotal role in virus tropism. In this study, we tested the membrane topology of GP3, both when expressed separately and during infection with recombinant tagged EAV GP3-HA. In our antibody accessibility experiment, we made a noteworthy discovery: GP3, when expressed separately, exhibits a dual topology. We introduced an additional N-glycosylation site, which was only partially used, providing further evidence for the dual topology of GP3. Intriguingly, this mutated GP3 was secreted into the medium, a result of the disruption of the ER retention motif RXR. The additional glycosylation site was not used when we examined the recombinant EAV virus with the same mutation. Despite the fact of higher expression levels of mutant GP3-HA, the protein was not secreted, and the recombinant mutant virus did not have growth delay compared to the EAV wild-type virus. This finding suggests that GP3 has a single type one membrane topology in virus infected cells, whereas the expression of GP3 in trans results in the dual topology of this protein. The RXR motif in the C-terminus is a co-factor of ER retention of the protein, but the main retention signal remains elusive.

Occurrence and molecular characterization of Escherichia coli strains isolated from black grouse (Lyrurus tetrix) from the Karkonosze National Park in Poland.

The purpose of this study was to characterize Escherichia coli (E. coli) strains isolated from wild black grouse (Lyrurus tetrix), carried out due to the crossing of hiking trails with wild bird habitats from the Karkonosze National Park. Twenty-seven E. coli isolates were obtained from fecal samples collected during the winter months of 2017 and 2018. The strains were assigned to their relevant phylo-groups and the prevalence of virulence genes characteristic of APEC strains (irp2, astA, iss, iucD, papC, tsh, vat, cva/cvi, stx2f) was checked using PCR analysis. In addition, the phenotypic and genotypic resistance to antibiotics was determined. The entire study provided a better understanding of the potential bacteriological threat to wild birds of the Karkonosze National Park. The results showed that 55.6% of the strains belonged to phylo-group B1 (15/27), 33.3% to group B2 (9/27) and 11.1% to group D (3/27). Among the virulence genes tested, irp2 was detected in 25.9% of isolates (7/27), vat in 22.2% (6/27) and iucD in 3.7% (1/27). The tested E. coli strains showed susceptibility to most antimicrobials, only 14 (51.9%) of them were intermediate resistant or resistant to sulfamethoxazole. The presence of none of the tested genes responsible for resistance to selected antibiotics was identified. Our research indicates a low level of transfer of antimicrobial substances to the natural environment and confirms the effectiveness of the Karkonosze National Park's activities to protect and restore black grouse habitats.

Genetic Evaluation of Bovine Papillomavirus Types Associated with Teat Papillomatosis in Polish Dairy Cattle with the Report of a New Putative Type.

Teat papillomatosis is reported to be one of the factors causing mastitis and milk losses in dairy cattle. Little is known about bovine papillomavirus (BPV) circulation in the European cattle population, and no reports can yet be found about its prevalence in Polish herds. In this study, 177 BPV-like lesions were collected from teats of 109 slaughtered cows. BPV was identified in 39 of the examined animals, using PCR amplification and Sanger dideoxy sequencing. In total, 10 BPV types were isolated, among which the most common were infections caused by types 8 and 7. Macroscopically, "rice-grain" type lesions dominated (76%) and were mainly found on one teat (57.4%). The diversity of BPV types causing teat papillomatosis in Polish cows seems to be large, with nine already known types isolated and a new putative type found. The spread of new types among the worldwide cattle population can be seen for the first time, as type 25 and so called isolates BPV42 and BPV43 were found in the European cattle population.

The Prevalence, Coexistence, and Correlations between Seven Pathogens Detected by a PCR Method from South-Western Poland Dairy Cattle Suffering from Bovine Respiratory Disease.

Bovine respiratory disease (BRD) is a very important disease that contributes to economic losses in dairy and beef cattle breeding worldwide. The molecular testing of material from 296 calves showing BRD symptoms from 74 dairy herds located in south-western Poland was performed in 2019-2021. Molecular tests were performed using a commercial kit "VetMAXTM Ruminant Respiratory Screening Kit" (Thermo Fisher Scientific) for the simultaneous detection of genetic material of seven pathogens responsible for BRD. At least one pathogen was detected in 95.95% of herds. The overall prevalence was: Pasteurella multocida 87.84%, Mannheimia haemolytica 44.59%, bovine coronavirus (BcoV) 32.43%, Mycoplasma bovis 29.73%, Histophilus somni 28.38%, bovine parainfluenza virus type 3 (BPIV-3) 13.51%, and bovine respiratory syncytial virus (BRSV) 10.81%. Twenty-nine configurations of pathogen occurrences were found. Bacterial infections were the most frequently recorded as 56.7% of all results. Coinfections mainly consisted of two pathogens. Not a single purely viral coinfection was detected. The most frequent result was a single P. multocida infection accounting for 18.31% of all results. The statistically significant correlation (p = 0.001) with the highest strength of effect (ϕ 0.38) was between M. bovis and H. somni.

Molecular detection of avian hepatitis E virus (Orthohepevirus B) in chickens, ducks, geese, and western capercaillies in Poland.

Orthohepevirus B, commonly known as avian hepatitis E virus (aHEV), causes big liver and spleen disease (BLS) or hepatitis-splenomegaly syndrome (HSS) in chickens. BLS is an emerging disease among chicken flocks in several countries around the world. In our previous studies, serology and molecular biology screening revealed that chicken flocks are widely affected by aHEV in Poland. The present study, which was conducted between 2019 and 2020, aimed to investigate the prevalence of aHEV in chicken flocks and other poultry, including ducks, geese, and turkeys. A total of 307 flocks were examined. In addition, 29 samples from captive wild birds (western capercaillies, Tetrao urogallus) were analyzed. In all the investigated poultry species, except turkeys, the nucleic acid sequence covering part of the ORF1 gene of the aHEV genome was detected (34/336 samples, 10.1%). The infection rate was found to be the highest in broiler breeder chicken flocks (14/40 samples; 35%). Phylogenetic analysis of partial ORF1 gene, which encodes helicase, revealed that the obtained sequences belonged to genotypes 2 and 4, while one belonged to genotype 3. Genotype 2 was detected for the first time in domestic geese and ducks, and genotype 4 was detected for the first time in Poland. The study demonstrated the presence of aHEV among the investigated western capercaillies, suggesting that this species is susceptible to aHEV infections and biosecurity is therefore required in western capercaillie breeding facilities.

Phenotypic and genotypic characterization of mcr-1-positive multidrug-resistant Escherichia coli ST93, ST117, ST156, ST10, and ST744 isolated from poultry in Poland.

BACKGROUND: A plasmid-mediated mechanism of bacterial resistance to polymyxin is a serious threat to public health worldwide. The present study aimed to determine the occurrence of plasmid-mediated colistin resistance genes and to conduct the molecular characterization of mcr-positive Escherichia coli strains isolated from Polish poultry. METHODS: In this study, 318 E. coli strains were characterized by the prevalence of mcr1-mcr5 genes, antimicrobial susceptibility testing by minimal inhibitory concentration method, the presence of antimicrobial resistance genes was screened by PCR, and the biofilm formation ability was tested using the crystal violet staining method. Genetic relatedness of mcr-1-positive E. coli strains was evaluated by multilocus sequence typing method. RESULTS: Among the 318 E. coli isolates, 17 (5.35%) harbored the mcr-1 gene. High antimicrobial resistance rates were observed for ampicillin (100%), tetracycline (88.24%), and chloramphenicol (82.35%). All mcr-1-positive E. coli strains were multidrug-resistant, and as many as 88.24% of the isolates contained the blaTEM gene, tetracycline (tetA and tetB), and sulfonamide (sul1, sul2, and sul3) resistance genes. Additionally, 41.18% of multidrug-resistant, mcr-1-positive E. coli isolates were moderate biofilm producers, while the rest of the strains showed weak biofilm production. Nine different sequence types were identified, and the dominant ST was ST93 (29.41%), followed by ST117 (17.65%), ST156 (11.76%), ST 8979 (11.76%), ST744 (5.88%), and ST10 (5.88%). Moreover, the new ST was identified in this study. CONCLUSIONS: Our results showed a low occurrence of mcr-1-positive E. coli strains isolated from Polish poultry; however, all the isolated strains were resistant to multiple antimicrobial agents and were able to form biofilms at low or medium level.

Short Beak and Dwarfism Syndrome in Ducks in Poland Caused by Novel Goose Parvovirus.

Short beak and dwarfism syndrome (SBDS), which was previously identified only in mule ducks, is now an emerging disease of Pekin ducks in China and Egypt. The disease is caused by the infection of ducks with a genetic variant of goose parvovirus-novel goose parvovirus (nGPV). In 2019, SBDS was observed for the first time in Poland in eight farms of Pekin ducks. Birds in the affected flock were found to show growth retardation and beak atrophy with tongue protrusions. Morbidity ranged between 15% and 40% (in one flock), while the mortality rate was 4-6%. Co-infection with duck circovirus, a known immunosuppressive agent, was observed in 85.7% of ducks. The complete coding regions of four isolates were sequenced and submitted to GenBank. The phylogenetic analysis revealed a close relationship of Polish viral sequences with the Chinese nGPV. Genomic sequence alignments showed 98.57-99.28% identity with the nGPV sequences obtained in China, and 96.42% identity with the classical GPV (cGPV; Derzsy's disease). The rate of amino acid mutations in comparison to cGPV and Chinese nGPV was higher in the Rep protein than in the Vp1 protein. To our knowledge, this is the first report of nGPV infection in Pekin ducks in Poland and Europe. It should be emphasized that monitoring and sequencing of waterfowl parvoviruses is important for tracking the viral genetic changes that enable adaptation to new species of waterbirds.

Electrochemical Detection of Dopamine and Serotonin in the Presence of Interferences in a Rotating Droplet System.

In this Article, a rotating droplet system is used for simultaneous detection of dopamine and serotonin. Carbon nanoparticles functionalized with sulfonic groups on the electrode surface enables potential discrimination between the neurotransmitters and the most common interferences, whereas the efficient and low-volume hydrodynamic system helps to lower the detection limit toward physiologically relevant concentrations. Here, we present results with a 10 nM limit of detection for serotonin and a 100 nM to 2 µM linear response range from the system in a sample containing an equimolar concentrations of dopamine and serotonin and 0.5 mM concentration of both uric and ascorbic acids. Demonstrating the practical applicability of this method, we measure the concentration of serotonin in 70 µL of mice blood serum samples without additional pretreatment.

Pencil Lead as a Material for Microfluidic 3D-Electrode Assemblies.

We present an electrochemical, microfluidic system with a working electrode based on an ordered 3D array of pencil leads. The electrode array was integrated into a plexiglass/PDMS channel. We tested the setup using a simple redox probe and compared the results with computer simulations. As a proof of concept application of the device we showed that the setup can be used for determination of dopamine concentration in physiological pH and ultrasensitive, although only qualitative, detection of p-nitrophenol with a limit of detection below 1 nmol L-1. The observed limit of detection for p-nitrophenol is not only much lower than achieved with similar methods but also sufficient for evaluation of exposure to pesticides such as methyl parathion through urinalysis. This low cost setup can be fabricated without the need for clean room facilities and in the future, due to the ordered structure of the electrode could be used to better understand the process of electroanalysis and electrode functionalization. To the best of our knowledge it is the first application of pencil leads as 3D electrochemical sensor in a microfluidic channel.