Porcine enteric coronaviruses include some of the most relevant viral pathogens to the swine industry such as porcine epidemic diarrhea virus (PEDV) or porcine transmissible gastroenteritis virus (TGEV) as well as several recently identified virus such as swine enteric coronavirus (SeCoV), porcine deltacoronavirus (PDCoV) or swine enteric alphacoronavirus (SeACoV). The aim of this study is the identification and characterization of enteric coronaviruses on Spanish pig farms between 2017 and 2019. The study was carried out on 106 swine farms with diarrhea outbreaks where a viral etiology was suspected by using two duplex RT-PCRs developed for the detection of porcine enteric coronaviruses. PEDV was the only coronavirus detected in our research (38.7% positive outbreaks, 41 out of 106) and neither TGEV, SeCoV, PDCoV nor SeACoV were detected in any of the samples. The complete S-gene of all the PEDV isolates recovered were obtained and compared to PEDV and SeCoV sequences available in GenBank. The phylogenetic tree showed that only PEDV of the INDEL 2 or G1b genogroup has circulated in Spain between 2017 and 2019. Three different variants were detected, the recombinant PEDV-SeCoV being the most widespread. These results show that PEDV is a relevant cause of enteric disorders in pigs in Spain while new emerging coronavirus have not been detected so far. However, the monitoring of these virus is advisable to curtail their emergence and spread.

Background: The aim of this work was to study the prevalence and distribution of Porcine astrovirus (PAstV), Porcine kobuvirus (PKoV), Porcine torovirus (PToV), Mammalian orthoreovirus (MRV) and Porcine mastadenovirus (PAdV) as well as their association with widely recognized virus that cause diarrhoea in swine such as coronavirus (CoVs) and rotavirus (RVs) in diarrhoea outbreaks from Spanish swine farms. Furthermore, a selection of the viral strains was genetically characterized. Results: PAstV, PKoV, PToV, MRV and PAdV were frequently detected. Particularly, PAstV and PKoV were detected in almost 50% and 30% of the investigated farms, respectively, with an age-dependent distribution; PAstV was mainly detected in postweaning and fattening pigs, while PKoV was more frequent in sucking piglets. Viral co-infections were detected in almost half of the outbreaks, combining CoVs, RVs and the viruses studied, with a maximum of 5 different viral species reported in three investigated farms. Using a next generation sequencing approach, we obtained a total of 24 ARN viral genomes (> 90% genome sequence), characterizing for first time the full genome of circulating strains of PAstV2, PAstV4, PAstV5 and PToV on Spanish farms. Phylogenetic analyses showed that PAstV, PKoV and PToV from Spanish swine farms clustered together with isolates of the same viral species from neighboring pig producing countries. Conclusions: Although further studies to evaluate the role of these enteric viruses in diarrhoea outbreaks are required, their wide distribution and frequent association in co-infections cannot be disregard. Hence, their inclusion into routine diagnostic panels for diarrhoea in swine should be considered.

Alternative antimicrobials require a deep understanding of their action mechanisms by in vitro assays which support science-based field use. This study focuses on the characterization of bactericidal mechanisms of potential antimicrobial compounds, two organic acids and three single essential oil (EO) compounds against swine enteropathogenic bacteria Escherichia coli, Salmonella enterica subsp. enterica serovar Typhimurium, and Clostridium perfringens. Target concentrations of the compounds were evaluated using the inhibitory potential of the vapor phase and bacterial viability after short-term exposure, while cell targets were disclosed using flow cytometry (FC), Fourier-transform infrared (FTIR) spectroscopy, and scanning electron microscopy (SEM). All tested compounds exhibited vapor phase activity against the three bacterial species, except sodium salt of coconut fatty acid distillates against C. perfringens. Survival test results evidenced that effects on bacterial viability were concentration dependent and higher in single EO compounds than in organic acids. In detail, thymol and its isomer carvacrol were the most effective compounds. Further characterization of thymol and cinnamaldehyde activity revealed that thymol main target was the cell membrane, since it caused striking damages in the membrane permeability, integrity and composition evidenced by FC and FTIR in the three enteric pathogens. In contrast, cinnamaldehyde was more effective against enterobacteria than against C. perfringens and only caused slightly damages at the highest concentration tested. Its target at the molecular level differed between enterobacteria and C. perfringens isolates. The SEM micrographs allowed us to confirm the results previously obtained for both EO compounds by other techniques. Altogether, the study showed the straight effect of these antimicrobials, which could constitute relevant information to optimize their feed inclusion rates in field studies or field use.

The aim of this study was to characterize the infection of weaned pigs with swine enteric coronavirus (SeCoV) - a chimeric virus most likely originated from a recombination event between porcine epidemic diarrhoea virus (PEDV) and transmissible gastroenteritis virus, or its mutant porcine respiratory coronavirus - and two PEDV G1b variants, including a recently described recombinant PEDV-SeCoV (rPEDV-SeCoV), as well as to determine the degree of cross-protection achieved against the rPEDV-SeCoV. For this purpose, forty-eight 4-week-old weaned pigs were randomly allocated into four groups of 12 animals. Piglets within each group were primary inoculated with one of the investigated viral strains (B: PEDV; C: SeCoV and D: rPEDV-SeCoV) or mock-inoculated (A), and exposed to rPEDV-SeCOV at day 20 post-infection; thus, group A was primary challenged (-/rPEDV-SeCoV), groups B and C were subjected to a heterologous re-challenge (PEDV/rPEDV-SeCoV and SeCoV/rPEDV-SeCoV, respectively), and group D to a homologous re-challenge (rPEDV-SeCoV/rPEDV-SeCoV), Clinical signs, viral shedding, microscopic lesions and specific humoral and cellular immune responses (IgG, IgA, neutralizing antibodies and IgA and IFN-gamma-secreting cells) were monitored. After primo-infection, all three viral strains induced an undistinguishable mild-to-moderate clinical disease with diarrhoea as the main sign and villus shortening lesions in the small intestine. In homologous re-challenged pigs, no clinical signs or lesions were observed, and viral shedding was only detected in a single animal. This fact may be explained by the significant high level of rPEDV-SeCoV-specific neutralizing antibodies found in these pigs before the challenge. In contrast, prior exposure to a different PEDV G1b variant or SeCoV only provided partial cross-protection, allowing rPEDV-SeCoV replication and shedding in faeces.