Diarrhea In Neonatal Piglets Research Articles

Conditional expression of flagellar motility, curli fimbriae, and biofilms in Shiga toxin- producing Escherichia albertii.

Escherichia albertii is an emerging foodborne pathogen. We previously reported that some avian Shiga toxin-producing E. albertii strains exhibited higher or comparable cytotoxicity in Vero-d2EGFP cells with several enterohemorrhagic E. coli (EHEC) outbreak strains. To better understand the environmental persistence of this pathogen, comparative genomics and phenotypic assays were applied to assess adhesion capability, motility, and biofilm formation in E. albertii. Among the 108 adherence-related genes, those involved in biogenesis of curli fimbriae, hemorrhagic E. coli pilus, type 1 fimbriae, and Sfm fimbriae were conserved in E. albertii. All 20 E. albertii strains carried a complete set of primary flagellar genes that were organized into four gene clusters, while five strains possessed genes related to the secondary flagella, also known as lateral flagella. Compared to EHEC strain EDL933, the eight chemotaxis genes located within the primary flagellar gene clusters were deleted in E. albertii. Additional deletion of motility genes flhABCD and motBC was identified in several E. albertii strains. Swimming motility was detected in three strains when grown in LB medium, however, when grown in 5% TSB or in the pond water-supplemented with 10% pigeon droppings, an additional four strains became motile. Although all E. albertii strains carried curli genes, curli fimbriae were detected only in four, eight, and nine strains following 24, 48, and 120 h incubation, respectively. Type 1 fimbriae were undetectable in any of the strains grown at 37°C or 28°C. Strong biofilms were detected in strains that produced curli fimbriae and in a chicken isolate that was curli fimbriae negative but carried genes encoding adhesive fimbriae K88, a signature of enterotoxigenic E. coli strains causing neonatal diarrhea in piglets. In all phenotypic traits examined, no correlation was revealed between the strains isolated from different sources, or between the strains with and without Shiga toxin genes. The phenotypic variations could not be explained solely by the genetic diversity or the difference in adherence genes repertoire, implying complex regulation in expression of various adhesins. Strains that exhibited a high level of cytotoxicity and were also proficient in biofilm production, may have potential to emerge into high-risk pathogens.

Bacillus subtilis Feed Supplementation Combined with Oral E. coli Immunization in Sows as a Tool to Reduce Neonatal Diarrhea in Piglets.

To investigate the effects of B. subtilis on the specific immune response of lactating sows to E. coli and the diarrhea rate in suckling piglets, thirty large white sows with similar farrowing dates were randomly divided into two groups: a feedback feeding (i.e., feeding a homogenate of intestinal contents and tissues from E. coli-infected piglets to sows; FB) group and a feedback feeding with B. subtilis (FB + BS) group. Serum, colostrum, and intestinal tissues from sows and piglets were collected to assess the immune response and intestinal barrier function at weaning. T and B cells from Peyer's patches (PPs) and mesenteric lymph nodes (MLNs) in lactating mice (with treatments consistent with the sows') were isolated to explore the underlying mechanism. The results showed that, compared with the FB group, the reproductive performance of sows and the growth performance of their offspring were effectively improved in the FB + BS group. Moreover, the levels of IgG/IgA and those of IgG/IgA against E. coli in the serum and colostrum of sows in the FB+BS group were increased (p < 0.05). Meanwhile, the ratio of CD4+/CD8+, CD4+CXCR5+PD1+, and B220+IgA+ cells in MLNs and PPs, and the IgA levels in the mammary glands of mice, were also increased in the FB + BS group (p < 0.05). Notably, in suckling piglets in the FB + BS group, the diarrhea rate was decreased (p < 0.05), and the intestinal barrier function and intestinal flora composition at weaning were significantly improved. Overall, these results indicated that B. subtilis feed supplementation combined with feedback feeding in pregnant and lactating sows can reduce diarrhea in suckling piglets by enhancing the maternal immune response against E. coli and intestinal barrier function in their offspring, improving survival rates and pre-weaning growth.

RBM14 inhibits the replication of porcine epidemic diarrhea virus by recruiting p62 to degrade nucleocapsid protein through the activation of autophagy and interferon pathway.

Porcine epidemic diarrhea virus (PEDV) results in PED, which is an infectious intestinal disease with the representative features of diarrhea, vomiting, and dehydration. PEDV infects neonatal piglets, causing high mortality rates. Therefore, elucidating the interaction between the virus and host in preventing and controlling PEDV infection is of immense significance. We found a new antiviral function of the host protein, RNA-binding motif protein 14 (RBM14), which can inhibit PEDV replication via the activation of autophagy and interferon (IFN) signal pathways. We found that RBM14 can recruit cargo receptor p62 to degrade PEDV nucleocapsid (N) protein through the RBM14-p62-autophagosome pathway. Furthermore, RBM14 can also improve the antiviral ability of the hosts through interacting with mitochondrial antiviral signaling protein to induce IFN expression. These results highlight the novel mechanism underlying RBM14-induced viral restriction. This mechanism leads to the degradation of viral N protein via the autophagy pathway and upregulates IFN for inhibiting PEDV replication; thus, offering new ways for preventing and controlling PED.IMPORTANCEPorcine epidemic diarrhea virus (PEDV) is a vital reason for diarrhea in neonatal piglets, which causes high morbidity and mortality rates. There is currently no effective vaccine or drug to treat and prevent infection with the PEDV. During virus infection, the host inhibits virus replication through various antiviral factors, and at the same time, the virus antagonizes the host's antiviral reaction through its own encoded protein, thus completing the process of virus replication. Our study has revealed that the expression of RNA-binding motif protein 14 (RBM14) was downregulated in PEDV infection. We found that RBM14 can recruit cargo receptor p62 to degrade PEDV N protein via the RBM14-p62-autophagosome pathway and interacted with mitochondrial antiviral signaling protein and TRAF3 to activate the interferon signal pathway, resulting in the inhibition of PEDV replication.

The vaccination changed the profile of rotavirus infection with the increase of non-rotavirus A species diagnosis in one-week-old diarrheic piglets.

Porcine rotavirus (RV) is a major viral agent associated with severe diarrhea in newborn piglets. RVA, RVB, RVC, and RVH are RV species that have already been identified in pigs. RVA is considered the most prevalent and relevant virus in pig production worldwide. This study aimed to evaluate the frequency of RV infection associated with diarrhea in suckling piglets from regular RVA-vaccinated Brazilian pig herds between 2015 and 2021. Therefore, 511 diarrheic fecal samples were collected from suckling piglets aged up to 3weeks from 112 pig farms located in three main Brazilian pork production regions. All piglets were born to RVA-vaccinated sows. The nucleic acids of RVA, RVC, and RVH were investigated by RT-PCR assays and RVB by semi-nested RT-PCR assay. Of the diarrheic fecal samples analyzed, 221/511 (43.3%) were positive for at least one of the RV species. Regarding the distribution of RV species among the positive fecal samples that presented with only one RV species, 99 (44.8%), 63 (28.5%), and 45 (20.4%) were identified as RVB, RVC, and RVA, respectively. RVH was not identified in diarrheic piglets with a single infection. More than one RV species was identified in 14/221 (6.3%) of the diarrheic fecal samples evaluated. Co-detection of RVB + RVH (11/221; 5.0%), RVA + RVB (1/221; 0.4%), RVA + RVC (1/221; 0.4%), and RVB + RVC (1/221; 0.4%) was identified in fecal samples. The results demonstrated a significant increase in the RVC and, mainly, RVB detection rates in single infections. This study allowed us to characterize the importance of other RV species, in addition to RVA, in the etiology of neonatal diarrhea in piglets from pig herds with a regular vaccination program for RVA diarrhea control and prophylaxis.

Reverse Genetics Systems for Emerging and Re-Emerging Swine Coronaviruses and Applications.

Emerging and re-emerging swine coronaviruses (CoVs), including porcine epidemic diarrhea virus (PEDV), porcine deltacoronavirus (PDCoV), and swine acute diarrhea syndrome-CoV (SADS-CoV), cause severe diarrhea in neonatal piglets, and CoV infection is associated with significant economic losses for the swine industry worldwide. Reverse genetics systems realize the manipulation of RNA virus genome and facilitate the development of new vaccines. Thus far, five reverse genetics approaches have been successfully applied to engineer the swine CoV genome: targeted RNA recombination, in vitro ligation, bacterial artificial chromosome-based ligation, vaccinia virus -based recombination, and yeast-based method. This review summarizes the advantages and limitations of these approaches; it also discusses the latest research progress in terms of their use for virus-related pathogenesis elucidation, vaccine candidate development, antiviral drug screening, and virus replication mechanism determination.

Association between the MUC4 g.243A&gt;G Polymorphism and Production Performance of Landrace and Yorkshire Pigs in Vietnam

Porcine mucin 4 (MUC4) is a candidate gene for controlling the adhesion of the enterotoxigenic Escherichia coli (ETEC) F4 receptor. Polymorphisms of the MUC4 gene have been used as markers to identify the susceptibility of neonatal diarrhea in piglets for breeding selection. The objective of this study was to evaluate the effects of MUC4 g.243A&gt;G polymorphisms on the production traits of Landrace and Yorkshire pigs in Vietnam. A total of 1,057 Landrace and 1,361 Yorkshire piglets were used to estimate the allelic and genotypic frequencies of the polymorphisms. Body weights at birth, at weaning, at initial fattening (IBW), and at the end of fattening period (FBW), backfat thickness (BFT), and depth of the longissimus dorsi muscle (DLD) were measured and lean meat percentage was estimated. Frequency of the susceptibility allele A to ETEC was higher than the resistance allele G for both breeds based on genotyping piglet tails collected at birth. The AA, AG, and GG genotypes were present in Yorkshire while GG was not found in Landrace. The production traits were not affected (P &gt;0.05) by MUC4 polymorphisms except BFT and DLD (P &lt;0.05). There were interactions between gender and MUC4 genotype (P &lt;0.05) for IBW, FBW, average daily gain, and DLD. These traits of GG males were significantly higher than those of GG females (P &lt;0.05). The results suggest that selecting pigs carrying the GG genotype of MUC4, known as providing resistance to ETEC, do not negatively affect productive performance in Landrace and Yorkshire pigs.

Impact of glyphosate residues in sow diets on neonatal piglets: tail kinks, stillborn and diarrhoea

Among the applications, glyphosate-based herbicides are used for pre-harvest crop desiccation and weed management in glyphosate resistant crops. Due to these applications glyphosate residues have been found in feedstuff for conventional livestock. Besides its herbicidal property, glyphosate has documented mineral chelating and antimicrobial properties. Previous studies have found glyphosate residuals in animal organs, and glyphosate may cause adverse effects in animals during the embryonic or neonatal period. The present study has two endpoints; (1) whether glyphosate concentration in the diets fed to sows during early gestation, was associated with the frequency of congenital tail kinks and stillborn piglets, and (2) whether the glyphosate concentration in diets fed to lactating sows, is associated with the frequency of diarrhoea in neonatal piglets. The concentration of glyphosate in 78 gestation diets obtained from seven Danish conventional sow herds ranged from 0.08 to 1.52 mg/kg dry matter (DM) and in 34 lactation diets from 0.06 to 0.87 mg/kg DM. When correcting for an average DM content of 88.3% in gestation diets and 89.0% in lactation diets the concentration of glyphosate ranged from 0.07 to 1.34 and 0.05 to 0.77 mg/kg feed, respectively. In the first study, 28,336 docked tails from 2 to 4-day-old piglets were assessed for congenital tail kinks and the number of kinky tails per herd per week was linked to the glyphosate concentration in sow diets during week 1 to 6 of gestation. Percentage of kinky tails and stillborn piglets ranged from 0 to 2.60% and 0.7 to 3.2% per week, respectively. No associations between the glyphosate concentration in sow gestation diets and the frequency of kinky tails or stillborn piglets were found. In the second study, faeces consistency of 5,526 piglets from 386 litters was linked to the glyphosate concentration in sow lactation diets. A proportion of 0.15 of the 0- to 5-day-old piglets had diarrhoea. No association was found between the concentration of glyphosate in sow lactation diets and the frequency of diarrhoea in neonatal piglets.

Effects of oral probiotic and lactoferrin interventions on iron-zinc homeostasis, oxidant/antioxidant equilibrium and diarrhoea incidence of neonatal piglets.

The objective of this study was to examine the effects of early-life host specific probiotic and lactoferrin (LF) supplementations on diarrhoea incidence, iron (Fe)-zinc (Zn) balance and antioxidant capabilities in serum of neonatal piglets. A total of eight sow litters obtained from parity matched sows were randomly divided into four groups and assigned to one of the four interventions: control (2.0 ml normal saline), bovine lactoferrin (bLF) (100 mg bLF in normal saline), probiotic (Pb) (1×109 cfu of swine origin Pediococcus acidilactici FT28 strain) and bLF+Pb (both 100 mg bLF and 1×109 cfu of P. acidilactici FT28). All the piglets received supplementations once daily orally for first 7 days of life. The incidence of diarrhoea markedly decreased in bLF group compared to control group. Notably, no incidences of diarrhoea were recorded in Pb and bLF+Pb groups. The Zn and Fe concentrations were significantly increased from day 7 to 21 in bLF and on day 21 in bLF+Pb group. No such changes were noted in Pb group. Total antioxidant capacity (TAC) in serum was significantly increased on days 7 and 15 in bLF group and on days 7 and 21 in bLF+Pb group. Malonaldehyde concentration was markedly reduced from day 7 to 21 in bLF and bLF+Pb groups. The concentrations of nitrate on days 15 and 21 and malonaldehyde on day 7 were significantly higher in Pb group, but mean TAC was unaltered from day 0 to 21. Although no correlation between the incidence of diarrhoea and Zn/Fe and oxidant/antioxidant homeostasis was noted in the Pb group, the supplementation of P. acidilactici FT28 alone was sufficient to prevent the incidence of diarrhoea in neonatal piglets. Taken together, it is concluded that strategic supplementation of P. acidilactici FT28 in early life could help in preventing diarrhoea until weaning of piglets.

Pathogenic and metagenomic evaluations reveal the correlations of porcine epidemic diarrhea virus, porcine kobuvirus and porcine astroviruses with neonatal piglet diarrhea.

Porcine epidemic diarrhea virus (PEDV) frequently causes diarrhea outbreaks. However, whether newly discovered enteric viruses such as porcine kobuvirus (PKV) and porcine astroviruses (PAstVs) are also correlated with diarrhea is still unclear. Diarrhea outbreaks were reported in a PEDV-vaccinated pig farm in Xinjiang Uygur Autonomous Region of China from 2019 to 2020. PEDV was a common pathogen detected in fecal samples by routine RT-PCR assays. The PEDV positive fecal sample was used for pathogenic analysis due to the failure isolation of PEDV. The challenged neonatal piglets appeared watery diarrhea within one day post infection (dpi) and all died within 6 dpi. Histopathological and immunohistochemical examinations supported that PEDV is a major pathogen causing intestinal lesions. To further explore enteric viruses associated with neonatal piglet diarrhea, metagenomics sequencing was performed for the diarrheic piglets. Remarkably, PKV was the most abundant virus (58.33%) followed by PEDV (34.45%) and PAstVs (7.22%), which were also confirmed by real-time RT-PCR assays. Significant in vivo replications of PEDV and PKV could only be observed in challenged piglets whilst PAstVs maintained similar virus loads in both challenged and mock infected piglets. Overall, this study provides first pathogenic and metagenomic evidence that significant proliferations of PEDV and PKV are closely associated with severe diarrhea in neonatal piglets, while PAstVs likely play limited roles in neonatal piglet diarrhea.

Emodin from Aloe inhibits Swine acute diarrhea syndrome coronavirus in cell culture.

Swine acute diarrhea syndrome coronavirus (SADS-CoV) is an emerging swine enteropathogenic coronavirus that causes severe diarrhea in neonatal piglets, leading to serious economic losses to the pig industries. At present, there are no effective control measures for SADS, making an urgent need to exploit effective antiviral therapies. Here, we confirmed that Aloe extract (Ae) can strongly inhibit SADS-CoV in Vero and IPI-FX cells in vitro. Furthermore, we detected that Emodin from Ae had anti-SADS-CoV activity in cells but did not impair SADS-CoV infectivity directly. The time-of-addition assay showed that Emodin inhibits SADS-CoV infection at the whole stages of the viral replication cycle. Notably, we found that Emodin can significantly reduce virus particles attaching to the cell surface and induce TLR3 (p < 0.001), IFN-λ3 (p < 0.01), and ISG15 (p < 0.01) expressions in IPI-FX cells, indicating that the anti-SADS-CoV activity of Emodin might be due to blocking viral attachment and the activation of TLR3-IFN-λ3-ISG15 signaling axis. These results suggest that Emodin has the potential value for the development of anti-SADS-CoV drugs.

Porcine intraepithelial lymphocytes undergo migration and produce an antiviral response following intestinal virus infection

The location of intraepithelial lymphocytes (IELs) between epithelial cells provide a first line of immune defense against enteric infection. It is assumed that IELs migrate only along the basement membrane or into the lateral intercellular space (LIS) between epithelial cells. Here, we identify a unique transepithelial migration of porcine IELs as they move to the free surface of the intestinal epithelia. The major causative agent of neonatal diarrhea in piglets, porcine epidemic diarrhea virus (PEDV), increases the number of IELs entering the LIS and free surface of the intestinal epithelia, driven by chemokine CCL2 secreted from virus-infected intestinal epithelial cells. Remarkably, only virus pre-activated IELs inhibits PEDV infection and their antiviral activity depends on the further activation by virus-infected cells. Although high levels of perforin is detected in the co-culture system, the antiviral function of activated IELs is mainly mediated by IFN-γ secretion inducing robust antiviral response in virus-infected cells. Our results uncover a unique migratory behavior of porcine IELs as well as their protective role in the defense against intestinal infection.

CASE REPORT: ENTEROCOCCUS HIRAE INFECTION IN SUCKLING PIGS

This case report describes New Neonatal Porcine Diarrhea Syndrome (NNPDS) caused by Enterococcus hirae. Four piglets at 3–5 days old with diarrhea were submitted for diagnostic investigation. The histopathological analysis revealed numerous large Gram-positive cocci adherent to the luminal surface from the tip to the base of the villi. The organisms were tightly adherent to enterocytes on the villous tips and exfoliated enterocytes within the luminal detritus. In situ hybridization revealed positive signals of E. hirae, the enteroadherent Gram-positive cocci, in the intestinal tissue. The swab of distal jejunum and gut content was cultured positive for E. hirae. Taken all results together, it was concluded that neonatal diarrhea in piglets was caused by E. hirae.

The Characteristics of Spike Glycoprotein Gene of Swine Acute Diarrhea Syndrome Coronavirus Strain CH/FJWT/2018 Isolated in China.

Swine acute diarrhea syndrome (SADS) is a highly contagious infectious disease characterized by acute vomiting and watery diarrhea in neonatal piglets. The causative agent for SADS is the swine acute diarrhea syndrome coronavirus (SADS-CoV), an alphacoronavirus in the family Coronaviridae. Currently, SADS-CoV was identified only in Guangdong and Fujian provinces of China, not in any other regions or countries in the world. To explore the genetic diversity of SADS-CoV isolates, herein we comparatively analyzed 44 full-length genomes of viruses isolated in Guangdong and Fujian provinces during 2017–2019. The spike glycoprotein gene of SADS-CoV strain CH/FJWT/2018 isolated in Fujian province is distinct from that of other viral isolates in either spike glycoprotein gene-based phylogenetic analysis or whole genome-based gene similarity analysis. Moreover, at least 7 predicted linear B cell epitopes in the spike glycoprotein of CH/FJWT/2018 would be affected by amino acid variations when compared with a representative virus isolated in Guangdong province. The spike glycoprotein of coronaviruses determines viral host range and tissue tropism during virus infection via specific interactions with the cellular receptor and also plays critical roles in eliciting the production of neutralizing antibodies. Since SADS-CoVs have a broad cell tropism, the results in this report further emphasize that the spike glycoprotein gene is a pivotal target in the surveillance of SADS-CoV.

Development of an indirect ELISA for detecting swine acute diarrhoea syndrome coronavirus IgG antibodies based on a recombinant spike protein.

Swine acute diarrhoea syndrome coronavirus (SADS-CoV) is a newly identified swine enteropathogenic coronavirus that causes watery diarrhoea in neonatal piglets, leading to significant economic losses to the swine industry. Currently, there are no suitable serological methods to assess the infection of SADS-CoV and effectiveness of vaccines, making an urgent need to exploit effective enzyme-linked immunosorbent assay (ELISA) to compensate for this deficiency. Here, a recombinant plasmid that expresses the spike (S) protein of SADS-CoV fused to the Fc domain of human IgG was constructed to generate recombinant baculovirus and expressed in HEK 293F cells. The S-Fc protein was purified with protein G Resin, which retained reactivity with anti-human Fc and anti-SADS-CoV antibodies. The S-Fc protein was then used to develop an indirect ELISA (S-iELISA) and the reaction conditions of S-iELISA were optimized. As a result, the cut-off value was determined as 0.3711 by analyzing OD450nm values of 40 SADS-CoV-negative sera confirmed by immunofluorescence assay (IFA) and western blot. The coefficient of variation (CV) of 6 SADS-CoV-positive sera within and between runs of S-iELISA were both less than 10%. The cross-reactivity assays demonstrated that S-iELISA was non-cross-reactive with other swine viruses' sera. Furthermore, the overall coincidence rate between IFA and S-iELISA was 97.3% based on testing 111 clinical serum samples. Virus neutralization test with seven different OD450nm values of the sera showed that the OD450nm values tested by S-iELISA are positively correlated with the virus neutralization assay. Finally, a total of 300 pig field serum samples were tested by S-iELISA and commercial kits of other swine enteroviruses showed that the IgG-positive for SADS-CoV, TGEV, PDCoV and PEDV was 81.7, 54, 65.3 and 6%, respectively. The results suggest that this S-iELISA is specific, sensitive, repeatable and can be applied for the detection of the SADS-CoV infection in the swine industry.

Red blood cells serve as a vehicle for PEDV transmission

As the most abundant cell type in the blood, red blood cells (RBCs) are serving for transporting oxygen. However, the mechanism by which RBCs binding virus remains largely unknown. Here, we demonstrated that porcine epidemic diarrhea virus (PEDV), a kind of coronavirus, could hijack RBCs and cause typical diarrhea in neonatal piglets. In an epidemiology investigation of PEDV, the RBCs samples from diarrheic pigs in several pig farms were found to be PEDV-positive. PEDV could bind to neonatal RBCs through CD71 and clathrin-mediated endocytosis, and its viability was maintained for 12 h. PEDV-loaded RBCs could transfer the virus to CD3+ T cells by conjugation and reach the intestine mucosa, where it caused infection. Finally, a further animal challenge revealed that transfusing with PEDV-loaded RBCs could cause intestinal epithelial cells (IECs) infection and typical diarrhea symptom. Therefore, our studies illustrated the mechanism by which PEDV could cause intestinal infection through hijacking RBCs, further providing a novel insight into the role of RBCs as potential cells for viral transmission in coronavirus pathogenesis.

Increased viral read counts and metagenomic full genome characterization of porcine astrovirus4 and Posavirus 1 in sows in a swine farm with unexplained neonatal piglet diarrhea.

Neonatal diarrhea in piglets may cause major losses in affected pig herds. The present study used random high-throughput RNA sequencing (metagenomic next generation sequencing, mNGS) to investigate the virome of sows from a farm with persistent neonatal piglet diarrhea in comparison to two control farms without diarrhea problems. A variety of known swine gastrointestinal viruses was detected in the control farms as well as in the problem farm (Mamastrovirus, Enterovirus, Picobirnavirus, Posavirus 1, Kobuvirus, Proprismacovirus). A substantial increase in normalized viral read counts was observed in the affected farm compared to the control farms. The increase was attributable to a single viral species in each of the sampled sows (porcine astrovirus 4 and Posavirus 1). The complete genomes of a porcine astrovirus4 and two co-infecting Posavirus 1 were de novo assembled and characterized. The 6734 nt single-stranded RNA genome of porcine astrovirus 4 (PoAstV-4) strain Belgium/2019 contains three overlapping open reading frames (nonstructural protein 1ab, nonstructural protein 1a, capsid protein). Posavirus 1 strains Belgium/01/2019 and Belgium/02/2019 have a 9814 nt single-stranded positive-sense RNA genome encoding a single open reading frame (polyprotein precursor) containing the five expected Picornavirales-conserved protein domains. The study highlights the potential of mNGS workflows to study unexplained neonatal diarrhea in piglets and contributes to the scarce availability of both PoAstV-4 and Posavirus-1 whole genome sequences from Western Europe.

Porcine enteric alphacoronavirus Inhibits IFN-α, IFN-β, OAS, Mx1, and PKR mRNA Expression in Infected Peyer's Patches in vivo.

Porcine enteric alphacoronavirus (PEAV) is a newly identified swine enteropathogenic coronavirus that causes watery diarrhea in neonatal piglets. The pathogenesis and host immune responses of PEAV infection are not fully characterized. The reason lies in the stomach environment, which would degrade cell-cultured live viruses via oral infection, making it difficult to establish an effective infection model to study the pathogenesis and host immune responses in pigs with a mature immune system. To solve this problem, in this study, coated PEAV-loaded microspheres were developed by centrifugal granulation-fluidized bed coating and demonstrated as an effective oral delivery system/animal infection model to protect PEAV virion against the complex gastrointestinal environment in vitro and to cause infection in weaned piglets in vivo. Weaned piglets orally inoculated with coated PEAV-loaded microspheres developed diarrhea and virus RNA was detected in rectal swabs from one to seven days post inoculation. In addition, microscopic lesions in the small intestine were observed, and viral antigens were also detected in the small intestines with PEAV immunohistochemical staining. Importantly, PEAV significantly inhibited mRNA expression of IFN-α, IFN-β, OAS, Mx1, and PKR, the genes involved in modulation of the host immune responses, in infected Peyer's patches, indicating that PEAV can overcome the antiviral response to cause damage when infection occurs. Collectively, our research successfully established a PEAV animal infection model in weaned piglets and suggested that the observed gene expression profile might help explain immunological changes associated with PEAV infection.

Humoral immune responses in piglets experimentally infected with a field strain of porcine epidemic diarrhea virus

Porcine epidemic diarrhea virus (PEDV) causes severe clinical diarrhea in neonatal piglets, with reported mortality rates between 70–100%. The humoral immunity, especially the local intestinal IgA responses, plays an important role in the immune protection against PEDV infection. In this study, we evaluated the isotype antibody responses against the PEDV nucleocapsid (N) protein and the spike (S) protein subunits 1 (S1) and 2 (S2) in the serum and intestine of piglets. We also determined its serum neutralizing activity against the PEDV field strain HBMC2012 in 21-day-old piglets. Enzyme-linked immunosorbent assays (ELISA) revealed that the production of IgM against the N protein and S1 subunit was higher compared to the S2 subunit. The anti-S2 IgA antibodies were higher than the anti-N protein and anti-S1 IgA at 3 days post-infection (dpi). The specific IgA responses to the S2 subunit were higher than the responses observed in S1. The specific IgG responses against S1 and S2 subunits exceeded those of N protein. The serum neutralizing activities against PEDV were relatively low with a tendency to decline over time. No isotype-specific antibodies were found in the intestinal contents from infected pigs, except the one with weak IgA responses against N protein at 28 dpi. Immunohistochemical staining showed that a few IgM, IgA, and IgG antibody-secreting cells were mainly located in the mucosa of the duodenum and ileum of PEDV-infected pigs at 3 dpi. This study suggests poor systemic and intestinal isotype-specific antibody responses, especially those of IgA, and weak serum neutralizing activities against the field PEDV strain in piglets.

Antimicrobial Resistance Profiles and Characterization of Escherichia coli Strains from Cases of Neonatal Diarrhea in Spanish Pig Farms.

Escherichia coli is considered one of the most common agents associated with neonatal diarrhea in piglets. The aim of this work was to characterize the pathogenic and antimicrobial resistance (AMR) profiles of 122 E. coli strains isolated from pigs suffering diarrhea (n = 94) and pigs without diarrhea (n = 28) of 24 farms in Spain. Virulence factors, toxins and AMR (ESBL and colistin) genes and AMR phenotypes of E. coli isolates were analyzed. Low prevalence of pathogenic E. coli strains (26%) was found in both groups. However, ETEC and VTEC strains were more frequently isolated from diarrheic piglets. Irrespectively of diarrhea occurrence, 97.5% of the strains showed a multidrug-resistance (MDR) profile to aminopenicillins, sulfonamides and tetracyclines. It was found that 22% of E. coli was CTX-M+, with CTX-M-14 being the principal allelic variant. Remarkably, 81.5% of CTX-M+ strains were isolated from diarrheic animals and presented an extended MDR profile to aminopenicillins, quinolones and aminoglycosides. Finally, low frequencies of colistin resistance genes mcr-1 (4/122) and mcr-4 (1/122) were found. MDR E. coli strains are circulating in pig farms of Spain, representing a serious threat to animal and public health. More appropriate diagnostic approaches (genetic and AMR phenotypic analysis) should be implemented in animal health to optimize antibiotic treatments.

ITGB5 Plays a Key Role in Escherichia coli F4ac-Induced Diarrhea in Piglets.

Enterotoxigenic Escherichia coli (ETEC) that expresses F4ac fimbriae is the major pathogenic microorganism responsible for bacterial diarrhea in neonatal piglets. The susceptibility of piglets to ETEC F4ac is determined by a specific receptor on the small intestinal epithelium surface. We performed an iTRAQ-labeled quantitative proteome analysis using a case-control design in which susceptible and resistant full-sib piglets were compared for the protein expression levels. Two thousand two hundred forty-nine proteins were identified, of which 245 were differentially expressed (fold change > 1.5, FDR-adjusted P < 0.05). The differentially expressed proteins fell into four functional classes: (I) cellular adhesion and binding, (II) metabolic process, (III) apoptosis and proliferation, and (IV) immune response. The integrin signaling pathway merited particular interest based on a pathway analysis using statistical overexpression and enrichment tests. Genomic locations of the integrin family genes were determined based on the most recent porcine genome sequence assembly (Sscrofa11.1). Only one gene, ITGB5, which encodes the integrin β5 subunit that assorts with the αv subunit to generate integrin αvβ5, was located within the SSC13q41 region between 13:133161078 and 13:139609422, where strong associations of markers with the ETEC F4ac susceptibility were found in our previous GWAS results. To identify whether integrin αvβ5 is the ETEC F4acR, we established an experimental model for bacterial adhesion using IPEC-J2 cells. Then, the ITGB5 gene was knocked out in IPEC-J2 cell lines using CRISPR/Cas9, resulting in a biallelic deletion cell line (ITGB5−/−). Disruption of ITGB5 significantly reduced ETEC F4ac adhesion to porcine intestinal epithelial cells. In contrast, overexpression of ITGB5 significantly enhanced the adhesion. A GST pull-down assay with purified FaeG and ITGB5 also showed that FaeG binds directly to ITGB5. Together, the results suggested that ITGB5 is a key factor affecting the susceptibility of piglets to ETEC F4ac.