Effect of Probiotic E. coli Nissle 1917 Supplementation on the Growth Performance, Immune Responses, Intestinal Morphology, and Gut Microbes of Campylobacter jejuni Infected Chickens.

Campylobacter jejuni is the most common cause of bacterial foodborne gastroenteritis and holds significant public health importance. The continuing increase of antibiotic-resistant Campylobacter necessitates the development of antibiotic-alternative approaches to control infections in poultry and in humans. Here, we assessed the ability of E. coli Nissle 1917 (EcN; free and chitosan-alginate microencapsulated) to reduce C. jejuni colonization in chickens and measured the effect of EcN on the immune responses, intestinal morphology, and gut microbes of chickens. Our results showed that the supplementation of 3-week-old chickens daily with free EcN in drinking water resulted in a 2.0 log reduction of C. jejuni colonization in the cecum, whereas supplementing EcN orally three times a week, either free or microencapsulated, resulted in 2.0 and 2.5 log reductions of C. jejuni colonization, respectively. Gavaged free and microencapsulated EcN did not have an impact on the evenness or the richness of the cecal microbiota, but it did increase the villous height (VH), crypt depth (CD), and VH:CD ratio in the jejunum and ileum of chickens. Further, the supplementation of EcN (all types) increased C. jejuni-specific and total IgA and IgY antibodies in chicken's serum. Microencapsulated EcN induced the expression of several cytokines and chemokines (1.6 to 4.3-fold), which activate the Th1, Th2, and Th17 pathways. Overall, microencapsulated EcN displayed promising effects as a potential nonantibiotic strategy to control C. jejuni colonization in chickens. Future studies on testing microencapsulated EcN in the feed and water of chickens raised on built-up floor litter would facilitate the development of EcN for industrial applications to control Campylobacter infections in poultry.

Naturally Occurring Animal Coronaviruses as Models for Studying Highly Pathogenic Human Coronaviral Disease.

Coronaviruses (CoVs) comprise a large group of positive stranded RNA viruses that infect a diverse host range including birds and mammals. Infection with CoVs typically presents as mild to severe respiratory or enteric disease, but CoVs have the potential to cause significant morbidity or mortality in highly susceptible age groups. CoVs have exhibited a penchant for jumping species barriers throughout history with devastating effects. The emergence of highly pathogenic or infectious CoVs in humans over the past 20 years, including severe acute respiratory syndrome CoV (SARS-CoV), Middle East respiratory syndrome CoV (MERS-CoV), and most recently severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), underscores the significant threat that CoV spillovers pose to humans. Similar to the emergence of SARS-CoV-2, CoVs have been devastating to commercial animal production over the past century, including infectious bronchitis virus in poultry and bovine CoV, as well as the emergence and reemergence of multiple CoVs in swine including transmissible gastroenteritis virus, porcine epidemic diarrhea virus, and porcine deltacoronavirus. These naturally occurring animal CoV infections provide important examples for understanding CoV disease as many animal CoVs have complex pathogenesis similar to SARS-CoV-2 and can shed light on the ongoing SARS-CoV-2 outbreak. We provide an overview and update regarding selected existing animal CoVs and their primary host species, diseases caused by CoVs, how CoVs jump species, whether these CoVs pose an outbreak risk or risk to humans, and how we can mitigate these risks.

Broad receptor engagement of an emerging global coronavirus may potentiate its diverse cross-species transmissibility.

Porcine deltacoronavirus (PDCoV), identified in 2012, is a common enteropathogen of swine with worldwide distribution. The source and evolutionary history of this virus is, however, unknown. PDCoV belongs to the Deltacoronavirus genus that comprises predominantly avian CoV. Phylogenetic analysis suggests that PDCoV originated relatively recently from a host-switching event between birds and mammals. Insight into receptor engagement by PDCoV may shed light into such an exceptional phenomenon. Here we report that PDCoV employs host aminopeptidase N (APN) as an entry receptor and interacts with APN via domain B of its spike (S) protein. Infection of porcine cells with PDCoV was drastically reduced by APN knockout and rescued after reconstitution of APN expression. In addition, we observed that PDCoV efficiently infects cells of unusual broad species range, including human and chicken. Accordingly, PDCoV S was found to target the phylogenetically conserved catalytic domain of APN. Moreover, transient expression of porcine, feline, human, and chicken APN renders cells susceptible to PDCoV infection. Binding of PDCoV to an interspecies conserved site on APN may facilitate direct transmission of PDCoV to nonreservoir species, including humans, potentially reflecting the mechanism that enabled a virus, ancestral to PDCoV, to breach the species barrier between birds and mammals. The APN cell surface protein is also used by several members of the Alphacoronavirus genus. Hence, our data constitute the second identification of CoVs from different genera that use the same receptor, implying that CoV receptor selection is subjected to specific restrictions that are still poorly understood.

Comparative Pathogenesis of Bovine and Porcine Respiratory Coronaviruses in the Animal Host Species and SARS-CoV-2 in Humans.

Discovery of bats with severe acute respiratory syndrome (SARS)-related coronaviruses (CoVs) raised the specter of potential future outbreaks of zoonotic SARS-CoV-like disease in humans, which largely went unheeded. Nevertheless, the novel SARS-CoV-2 of bat ancestral origin emerged to infect humans in Wuhan, China, in late 2019 and then became a global pandemic. Less than 5 months after its emergence, millions of people worldwide have been infected asymptomatically or symptomatically and at least 360,000 have died. Coronavirus disease 2019 (COVID-19) in severely affected patients includes atypical pneumonia characterized by a dry cough, persistent fever, and progressive dyspnea and hypoxia, sometimes accompanied by diarrhea and often followed by multiple organ failure, especially of the respiratory and cardiovascular systems. In this minireview, we focus on two endemic respiratory CoV infections of livestock: bovine coronavirus (BCoV) and porcine respiratory coronavirus (PRCV). Both animal respiratory CoVs share some common features with SARS-CoV and SARS-CoV-2. BCoV has a broad host range including wild ruminants and a zoonotic potential. BCoV also has a dual tropism for the respiratory and gastrointestinal tracts. These aspects, their interspecies transmission, and certain factors that impact disease severity in cattle parallel related facets of SARS-CoV or SARS-CoV-2 in humans. PRCV has a tissue tropism for the upper and lower respiratory tracts and a cellular tropism for type 1 and 2 pneumocytes in lung but is generally a mild infection unless complicated by other exacerbating factors, such as bacterial or viral coinfections and immunosuppression (corticosteroids).

Mechanism of Cell Culture Adaptation of an Enteric Calicivirus, the Porcine Sapovirus Cowden Strain.

UNLABELLED: The porcine sapovirus (SaV) (PoSaV) Cowden strain is one of only a few culturable enteric caliciviruses. Compared to the wild-type (WT) PoSaV Cowden strain, tissue culture-adapted (TC) PoSaV has two conserved amino acid substitutions in the RNA-dependent RNA polymerase (RdRp) and six in the capsid protein (VP1). By using the reverse-genetics system, we identified that 4 amino acid substitutions in VP1 (residues 178, 289, 324, and 328), but not the substitutions in the RdRp region, were critical for the cell culture adaptation of the PoSaV Cowden strain. The other two substitutions in VP1 (residues 291 and 295) reduced virus replication in vitro. Three-dimensional (3D) structural analysis of VP1 showed that residue 178 was located near the dimer-dimer interface, which may affect VP1 assembly and oligomerization; residues 289, 291, 324, and 328 were located at protruding subdomain 2 (P2) of VP1, which may influence virus binding to cellular receptors; and residue 295 was located at the interface of two monomeric VP1 proteins, which may influence VP1 dimerization. Although reversion of the mutation at residue 291 or 295 from that of the TC strain to that of the WT reduced virus replication in vitro, it enhanced virus replication in vivo, and the revertants induced higher-level serum and mucosal antibody responses than those induced by the TC PoSaV Cowden strain. Our findings reveal the molecular basis for PoSaV adaptation to cell culture. These findings may provide new, critical information for the cell culture adaptation of other PoSaV strains and human SaVs or noroviruses. IMPORTANCE: The tissue culture-adapted porcine sapovirus Cowden strain is one of only a few culturable enteric caliciviruses. We discovered that 4 amino acid substitutions in VP1 (residues 178, 289, 324, and 328) were critical for its adaptation to LLC-PK cells. Two substitutions in VP1 (residues 291 and 295) reduced virus replication in vitro but enhanced virus replication and induced higher-level serum and mucosal antibody responses in gnotobiotic pigs than those induced by the tissue culture-adapted strain. Structural modeling analysis of VP1 suggested that residue 178 may affect VP1 assembly and oligomerization; residues 289, 291, 324, and 328 may influence virus binding to cellular receptors; and residue 295 may influence VP1 dimerization. Our findings will provide new information for the cell culture adaptation of other sapoviruses and possibly noroviruses.

Calves are susceptible to infection with the newly emerged porcine deltacoronavirus, but not with the swine enteric alphacoronavirus, porcine epidemic diarrhea virus.

Fecal virus shedding, seroconversion and histopathology were evaluated in 3-7-year-old gnotobiotic calves orally inoculated with porcine deltacoronavirus (PDCoV) (9.0-9.6 log10 genomic equivalents [GE] of OH-FD22-P5; n=4) or porcine epidemic diarrhea virus (PEDV) (10.2-12.5 log10 GE of PC21A; n=3). In PDCoV-inoculated calves, an acute but persisting fecal viral RNA shedding and PDCoV-specific serum IgG antibody responses were observed, but without lesions or clinical disease. However, no fecal shedding, seroconversion, histological lesions, and clinical disease were detected in PEDV-inoculated calves. Our data indicate that calves are susceptible to infection by the newly emerged PDCoV, but not by the swine coronavirus, PEDV.

Experimental infection of gnotobiotic pigs with the cell-culture-adapted porcine deltacoronavirus strain OH-FD22.

Porcine deltacoronavirus (PDCoV) is a novel enteropathogenic coronavirus in pigs. We have isolated and passaged the PDCoV strain OH-FD22 in an LLC porcine kidney (LLC-PK) cell line. Our study investigated the pathogenicity of the tissue-culture-grown PDCoV (TC-PDCoV) OH-FD22 at cell passages 5, 20 and 40 in LLC-PK cells, in eight 14-day-old gnotobiotic (Gn) pigs. Pigs (n = 3) were euthanized for pathologic examination at post-inoculation day (PID) 3, and the remainder were monitored for clinical signs, virus shedding, and serum antibody responses until PID 28. All inoculated pigs developed watery diarrhea and/or vomiting at PID 1-2 and shed the highest amount of viral RNA in feces at PID 3-5, accompanied by severe atrophic enteritis. They developed high titers of PDCoV-specific IgG/IgA and virus-neutralizing antibodies in serum at PID 23-24. Histologic lesions were limited to the villous epithelium of the jejunum and ileum at PID 3. Two inoculated pigs tested at PID 23-24 had small to moderate numbers of PDCoV antigen-positive cells in the intestinal lamina propria and mesenteric lymph nodes, but not in enterocytes. An analysis of full-length S and N genes of TC- and Gn-pig-passaged OH-FD22 revealed a high genetic stability in cell culture and pigs. TC-PDCoV OH-FD22 (cell passages 5, 20 and 40) was enteropathogenic, and the pathogenicity was similar to that of the original field virus. The TC-PDCoV OH-FD22 will be useful for further pathogenesis studies and for evaluating if higher-level cell-culture passaged virus becomes attenuated for vaccine development.

Pathogenicity of 2 porcine deltacoronavirus strains in gnotobiotic pigs.

To verify whether porcine deltacoronavirus infection induces disease, we inoculated gnotobiotic pigs with 2 virus strains (OH-FD22 and OH-FD100) identified by 2 specific reverse transcription PCRs. At 21-120 h postinoculation, pigs exhibited severe diarrhea, vomiting, fecal shedding of virus, and severe atrophic enteritis. These findings confirm that these 2 strains are enteropathogenic in pigs.

Isolation and characterization of porcine deltacoronavirus from pigs with diarrhea in the United States.

Porcine deltacoronavirus (PDCoV) is a novel coronavirus that causes diarrhea in nursing piglets. Following its first detection in the United States in February 2014, additional PDCoV strains have been identified in the United States and Canada. Currently, no treatments or vaccines for PDCoV are available. In this study, U.S. PDCoV strain OH-FD22 from intestinal contents of a diarrheic pig from Ohio was isolated in swine testicular (ST) and LLC porcine kidney (LLC-PK) cell cultures by using various medium additives. We also isolated PDCoV [OH-FD22(DC44) strain] in LLC-PK cells from intestinal contents of PDCoV OH-FD22 strain-inoculated gnotobiotic (Gn) pigs. Cell culture isolation and propagation were optimized, and the isolates were serially propagated in cell culture for >20 passages. The full-length S and N genes were sequenced to study PDCoV genetic changes after passage in Gn pigs and cell culture (passage 11 [P11] and P20). Genetically, the S and N genes of the PDCoV isolates were relatively stable during the first 20 passages in cell culture, with only 5 nucleotide changes, each corresponding to an amino acid change. The S and N genes of our sequenced strains were genetically closely related to each other and to other U.S. PDCoV strains, with the highest sequence similarity to South Korean strain KNU14-04. This is the first report describing cell culture isolation, serial propagation, and biological and genetic characterization of cell-adapted PDCoV strains. The information presented in this study is important for the development of diagnostic reagents, assays, and potential vaccines against emergent PDCoV strains.

Determination of the infectious titer and virulence of an original US porcine epidemic diarrhea virus PC22A strain.

The infectious dose of a virus pool of original US PEDV strain PC22A was determined in 4-day-old, cesarean-derived, colostrum-deprived (CDCD) piglets. The median pig diarrhea dose (PDD50) of the virus pool was determined as 7.35 log10 PDD50/mL, similar to the cell culture infectious titer, 7.75 log10 plaque-forming units (PFU)/mL. 100 PDD50 caused watery diarrhea in all conventional suckling piglets (n = 12) derived from a PEDV-naive sow, whereas 1000 and 10 000 PDD50 did not cause diarrhea in piglets derived from two PEDV-field exposed-recovered sows. This information is important for future PEDV challenge studies and validation of PEDV vaccines.

Pathology of US porcine epidemic diarrhea virus strain PC21A in gnotobiotic pigs.

To understand the progression of porcine epidemic diarrhea virus infection, we inoculated gnotobiotic pigs with a newly emerged US strain, PC21A, of the virus. At 24­48 hours postinoculation, the pigs exhibited severe diarrhea and vomiting, fecal shedding, viremia, and severe atrophic enteritis. These findings confirm that strain PC21A is highly enteropathogenic.

Comparative pathology of pigs infected with Korean H1N1, H1N2, or H3N2 swine influenza A viruses.

BACKGROUND: The predominant subtypes of swine influenza A virus (SIV) in Korea swine population are H1N1, H1N2, and H3N2. The viruses are genetically close to the classical U.S. H1N1 and triple-reassortant H1N2 and H3N2 viruses, respectively. Comparative pathogenesis caused by Korean H1N1, H1N2, and H3N2 SIV was evaluated in this study. FINDINGS: The H3N2 infected pigs had severe scores of gross and histopathological lesions at post-inoculation days (PID) 2, and this then progressively decreased. Both the H1N1 and H1N2 infected pigs lacked gross lesions at PID 2, but they showed moderate to severe pneumonia on PID 4, 7 and 14. The pigs infected with H1N1 had significant scores of gross and histopathological lesions when compared with the other pigs infected with H1N2, H3N2, and mock at PID 14. Mean SIV antigen-positive scores were rarely detected for pigs infected with H1N2 and H3N2 from PID 7, whereas a significantly increased amount of viral antigens were found in the bronchioles and alveolar epithelium of the H1N1infected pigs at PID 14. CONCLUSIONS: We demonstrated that Korean SIV subtypes had different pulmonary pathologic patterns. The Korean H3N2 rapidly induced acute lung lesions such as broncho-interstitial pneumonia, while the Korean H1N1 showed longer course of infection as compared to other strains.

Failure of propagation of human norovirus in intestinal epithelial cells with microvilli grown in three-dimensional cultures.

Human noroviruses (HuNoVs) are a leading cause of acute gastroenteritis. Establishment of a cell culture system for in vitro HuNoV growth remains challenging. Replication of HuNoVs in human intestinal cell lines (INT-407 and Caco-2) that differentiate to produce microvilli in rotation wall vessel (RWV) three-dimensional cultures has been reported (Straub et al. in Emerg Infect Dis 13:396-403, 2007; J Water Health 9:225-240, 2011, and Water Sci Technol 67:863-868, 2013). We used a similar RWV system, intestinal cell lines, and the same (Genogroup [G] I.1) plus additional (GII.4 and GII.12) HuNoV strains to test the system's reproducibility and to expand the earlier findings. Apical microvilli were observed on the surface of both cell lines by light and electron microscopy. However, none of the cell types tested resulted in productive viral replication of any of the HuNoV strains, as confirmed by plateau or declining viral RNA titers in the supernatants and cell lysates of HuNoV-infected cells, determined by real-time reverse transcription PCR. These trends were the same when culture supplements were added that have been reported to be effective for replication of other fastidious enteric viruses in vitro. Additionally, by confocal microscopy and orthoslice analysis, viral capsid proteins were mainly observed above the actin filament signals, which suggested that the majority of viral antigens were on the cell surface. We conclude that even intestinal cells displaying microvilli were not sufficient to support HuNoV replication under the conditions tested.

Retrospective serosurveillance of bovine norovirus (GIII.2) and nebovirus in cattle from selected feedlots and a veal calf farm in 1999 to 2001 in the United States.

There is a dearth of information on the seroprevalence of bovine norovirus (BoNoV) and nebovirus in cattle of the US. In this retrospective study, serum IgG antibodies to two bovine enteric caliciviruses, GIII.2 BoNoV (Bo/CV186-OH/00/US) and genetically and antigenically distinct nebovirus (Bo/NB/80/US), were evaluated in feedlot and veal calves from different regions of the US during 1999-2001. Three groups of 6- to 7-month-old feedlot calves from New Mexico (NM) (n=103), Arkansas (AR) (n=100) and Ohio (OH) (n=140) and a group of 7- to 10-day-old Ohio veal calves (n=47) were studied. Serum samples were collected pre-arrival or at arrival to the farms for the NM, AR and OH calves and 35 days after arrival for all groups for monitoring seroconversion rates during the period. Virus-like particles of Bo/CV186-OH/00/US and Bo/NB/80/US were expressed using the baculovirus expression system and were used in ELISA to measure antibodies. A high seroprevalence of 94-100 % and 78-100 % was observed for antibodies to GIII.2 BoNoV and nebovirus, respectively, in the feedlot calves tested. In the Ohio veal farm, an antibody seroprevalence of 94-100 % and 40-66 % was found for GIII.2 BoNoV and nebovirus, respectively. Increased seropositive rates of 38-85 % for GIII.2 BoNoV and 26-83 % for nebovirus were observed at 35 days after arrival and commingling on farms for all groups. Infection of calves with either GIII.2 BoNoV or nebovirus, or both viruses, appeared to be common in the regions studied in the US during 1999-2001. These two viruses likely remain endemic because no commercial vaccines are available.

Insights into potential pathogenesis mechanisms associated with Campylobacter jejuni-induced abortion in ewes.

BACKGROUND: Campylobacter jejuni is commonly found in the gastrointestinal tract of many food-animals including sheep without causing visible clinical symptoms of disease. However, C. jejuni has been implicated in ovine abortion cases worldwide. Specifically, in the USA, the C. jejuni sheep abortion (SA) clone has been increasingly associated with sheep abortion. In vivo studies in sheep (the natural host) are needed to better characterize the virulence potential and pathogenesis of this clone. RESULTS: Pregnant ewes intravenously (IV) or orally inoculated with ovine or bovine abortion-associated C. jejuni SA clones exhibited partial or complete uterine prolapse with retained placenta, and abortion or stillbirth, whereas delivery of healthy lambs occurred in pregnant ewes inoculated with C. jejuni 81-176 or in the uninfected group. In sheep inoculated with the SA clone, histopathological lesions including suppurative necrotizing placentitis and/or endometritis coincided with: 1) increased apoptotic death of trophoblasts, 2) increased expression of the host genes (e.g. genes encoding interleukin IL-6 and IL-15) related to cellular necrosis and pro-inflammatory responses in uterus, and 3) decreased expression of the genes encoding GATA binding protein 6, chordin, and insulin-like 3 (INSL3) that account for embryonic development in uterus. Immunohistochemistry revealed localization of bacterial antigens in trophoblasts lining the chorioallantoic membrane of ewes inoculated with the C. jejuni SA clone. CONCLUSIONS: The results showed that C. jejuni SA clones are capable of causing abortion or stillbirth in experimentally infected sheep. Furthermore, down- or up-regulation of specific genes in the uterus of infected pregnant ewes might implicate host genes in facilitating the disease progression. Since the C. jejuni SA strains share genotypic similarities with clones that have been isolated from human clinical cases of gastroenteritis, these strains might represent a potential public health risk.

Maternal immunization and vitamin A sufficiency impact sow primary adaptive immunity and passive protection to nursing piglets against porcine epidemic diarrhea virus infection.

Porcine epidemic diarrhea virus (PEDV) causes a highly contagious enteric disease with major economic losses to swine production worldwide. Due to the immaturity of the neonatal piglet immune system and given the high virulence of PEDV, improving passive lactogenic immunity is the best approach to protect suckling piglets against the lethal infection. We tested whether oral vitamin A (VA) supplementation and PEDV exposure of gestating and lactating VA-deficient (VAD) sows would enhance their primary immune responses and boost passive lactogenic protection against the PEDV challenge of their piglets. We demonstrated that PEDV inoculation of pregnant VAD sows in the third trimester provided higher levels of lactogenic protection of piglets as demonstrated by >87% survival rates of their litters compared with <10% in mock litters and that VA supplementation to VAD sows further improved the piglets' survival rates to >98%. We observed significantly elevated PEDV IgA and IgG antibody (Ab) titers and Ab-secreting cells (ASCs) in VA-sufficient (VAS)+PEDV and VAD+VA+PEDV sows, with the latter maintaining higher Ab titers in blood prior to parturition and in blood and milk throughout lactation. The litters of VAD+VA+PEDV sows also had the highest serum PEDV-neutralizing Ab titers at piglet post-challenge days (PCD) 0 and 7, coinciding with higher PEDV IgA ASCs and Ab titers in the blood and milk of their sows, suggesting an immunomodulatory role of VA in sows. Thus, sows that delivered sufficient lactogenic immunity to their piglets provided the highest passive protection against the PEDV challenge. Maternal immunization during pregnancy (± VA) and VA sufficiency enhanced the sow primary immune responses, expression of gut-mammary gland trafficking molecules, and passive protection of their offspring. Our findings are relevant to understanding the role of VA in the Ab responses to oral attenuated vaccines that are critical for successful maternal vaccination programs against enteric infections in infants and young animals.

Detection of Porcine Deltacoronavirus RNA in the Upper and Lower Respiratory Tract and Biliary Fluid and the Effect of Infection on Serum Cholesterol Levels and Blood T Cell Population Frequencies in Gnotobiotic Piglets.

Porcine deltacoronavirus (PDCoV) was first identified approximately a decade ago, but much is still obscure in terms of its pathogenesis. We aimed to further characterize PDCoV infection by investigating the presence of virus in respiratory and biliary tissues or fluids; T cell population frequencies in blood; and altered serum cholesterol levels. Twelve, 6-day-old, gnotobiotic piglets were inoculated oronasally with PDCoV OH-FD22 (2.6 × 107 FFU/pig). Six control piglets were not inoculated. Rectal swab (RS), nasal swab (NS), nasal wash (NW), bronchoalveolar lavage (BAL), and biliary fluid (BF) samples were collected at 2, 4, and 7 days post-inoculation (DPI) and tested for PDCoV RNA by RT-qPCR. Blood T cell populations and serum cholesterol levels were determined by flow cytometry and a colorimetric assay, respectively. Moderate to high, and low to moderate titers of PDCoV RNA were detected in RS and in NS, NW, BAL, and BF samples, respectively, of inoculated piglets. There were trends toward decreased CD4+CD8-, CD4-CD8+, and CD4+CD8+ blood T cell frequencies in inoculated piglets. Furthermore, serum cholesterol levels were increased in inoculated piglets. Overall, we found that PDCoV infection does not exclusively involve the intestine, since the respiratory and biliary systems and cholesterol metabolism also can be affected.

Intestinal colonization with Escherichia fergusonii enhances infectivity of GII.12 human norovirus in gnotobiotic pigs.

The role of gut microbiota [especially, histo-blood group antigen (HBGA)-expressing bacteria] in influencing human norovirus (HuNoV) infections is unclear. We investigated if infectivity of GII.12 HuNoV in gnotobiotic (Gn) pigs is altered by intestinal colonization with Escherichia fergusonii known to express HBGA A and H on their cell surface. Fifteen piglets were randomly grouped: (1) E. fergusonii + HuNoV (n = 6), (2) HuNoV alone (n = 6), and (3) Mock-inoculated (n = 3). Pigs (8-11-day-old) were inoculated orally with GII.12 HuNoV strain HS206 (9.5 log10 genomic equivalents/pig) or mock. For 2 days prior to viral inoculation, pigs were inoculated orally with E. fergusonii [8 log10 colony forming units/pig/day]. Daily fecal consistency, fecal viral RNA or E. fergusonii shedding, and histopathology (at euthanasia) were evaluated. Unlike the reduced infectivity of GII.4 HuNoV observed previously in Gn pigs colonized with Enterobacter cloacae known to express HBGA A, B, and H on the surface, E. fergusonii + HuNoV pigs exhibited significantly higher cumulative fecal HuNoV RNA shedding at PIDs 6-14 and 1-21 compared with HuNoV alone pigs. Mean days of fecal HuNoV RNA shedding were also significantly greater in E. fergusonii + HuNoV pigs (11.8 ± 1.6 days) compared with HuNoV alone pigs (7.0 ± 1.0 days). By immunofluorescent staining, HuNoV antigen-positive bacteria were detected on the surface of the intestinal epithelium, possibly enhancing attachment of HuNoV to enterocytes, suggesting a potential mechanism by which intestinal colonization with E. fergusonii promoted infectivity of GII.12 HuNoV in Gn pigs.

Vitamin A deficiency and vitamin A supplementation affect innate and T cell immune responses to rotavirus A infection in a conventional sow model.

Rotavirus A (RVA) causes ~200,000 diarrheal deaths annually in children <5yrs, mostly in low- and middle-income countries. Risk factors include nutritional status, social factors, breastfeeding status, and immunodeficiency. We evaluated the effects of vitamin A (VA) deficiency/VA supplementation and RVA exposure (anamnestic) on innate and T cell immune responses in RVA seropositive pregnant and lactating sows and passive protection of their piglets post-RVA challenge. Sows were fed VA deficient (VAD) or sufficient (VAS) diets starting at gestation day (GD)30. A subset of VAD sows received VA supplementation from GD|76 (30,000IU/day, VAD+VA). Sows (6 groups) were inoculated with porcine RVA G5P[7] (OSU strain) or Minimal Essential Medium (mock) at GD~90: VAD+RVA; VAS+RVA; VAD+VA+RVA; VAD-mock; VAS-mock; and VAD+VA-mock. Blood, milk, and gut-associated tissues were collected from sows at several time points to examine innate [natural killer (NK), dendritic (DC) cells], T cell responses and changes in genes involved in the gut-mammary gland (MG)-immunological axis trafficking. Clinical signs of RVA were evaluated post inoculation of sows and post-challenge of piglets. We observed decreased frequencies of NK cells, total and MHCII+ plasmacytoid DCs, conventional DCs, CD103+ DCs and CD4+/CD8+ and T regulatory cells (Tregs) and NK cell activity in VAD+RVA sows. Polymeric Ig receptor and retinoic acid receptor alpha (RARα) genes were downregulated in mesenteric lymph nodes and ileum of VAD+RVA sows. Interestingly, RVA-specific IFN-γ producing CD4+/CD8+ T cells were increased in VAD-Mock sows, coinciding with increased IL-22 suggesting inflammation in these sows. VA supplementation to VAD+RVA sows restored frequencies of NK cells and pDCs, and NK activity, but not tissue cDCs and blood Tregs. In conclusion, similar to our recent observations of decreased B cell responses in VAD sows that led to decreased passive immune protection of their piglets, VAD impaired innate and T cell responses in sows, while VA supplementation to VAD sows restored some, but not all responses. Our data reiterate the importance of maintaining adequate VA levels and RVA immunization in pregnant and lactating mothers to achieve optimal immune responses, efficient function of the gut-MG-immune cell-axis and to improve passive protection of their piglets.

Porcine Deltacoronaviruses: Origin, Evolution, Cross-Species Transmission and Zoonotic Potential.

Porcine deltacoronavirus (PDCoV) is an emerging enteropathogenic coronavirus of swine that causes acute diarrhoea, vomiting, dehydration and mortality in seronegative neonatal piglets. PDCoV was first reported in Hong Kong in 2012 and its etiological features were first characterized in the United States in 2014. Currently, PDCoV is a concern due to its broad host range, including humans. Chickens, turkey poults, and gnotobiotic calves can be experimentally infected by PDCoV. Therefore, as discussed in this review, a comprehensive understanding of the origin, evolution, cross-species transmission and zoonotic potential of epidemic PDCoV strains is urgently needed.