Comparison of B cells' immune response induced by PEDV virulent and attenuated strains.

Porcine epidemic diarrhea virus (PEDV) is an acute, highly contagious enterovirus that infects pigs of all ages. The B cells are important for antigen presentation, antibody production, and cytokine secretion to resist infection. However, the role of B cells in PEDV infection remains unclear. In this study, the effects of PEDV virulent (QY2016) and attenuated strains (CV777) on B cells sorted from neonatal piglets, nursery piglets, and gilts were investigated. The results showed that PEDV-QY2016 and PEDV-CV777 could significantly increase the expression of CD54 and CD27 in B cells from neonatal piglets. The percentages of CD80, MHC II, and IgM expressed on neonatal piglet B cells infected with PEDV-QY2016 were significantly lower than those expressed on the B cells infected with PEDV-CV777. Both PEDV-QY2016 and PEDV-CV777 could stimulate IFN-α and GM-CSF secretions in neonatal piglet B cells; IL-1, IFN-α, and IL-4 secretion in nursery piglet B cells; and IL-1, TGF-ß secretion, and GM-CSF in gilt B cells. Furthermore, both PEDV-QY2016 and PEDV-CV777 could induce the secretion of IgA, IgM, and IgG in nursery piglet B cells but could not induce the secretion of IgA, IgM, and IgG in neonatal piglet B cells. The secretion of IgA, IgM, and IgG was significantly higher by the PEDV-CV777 strains infected B cells than those by the PEDV-QY2016 strains infected gilt B cells. In conclusion, the surface molecule expression, cytokine secretion, and antibody production of B cells induced by PEDV are closely related to the ages of pigs and the virulence of the PEDV strain.

Effects of porcine epidemic diarrhea virus infection on CD21+ B cells activation.

Porcine epidemic diarrhea virus (PEDV) is a devastating pathogen of acute- gastrointestinal infectious diseases, which can cause vomiting, diarrhea, dehydration and high morbidity and mortality among neonatal piglets. Humoral immunity plays a vital role in the host anti-PEDV infection process, but the mechanism of PEDV-induced B-cell immune response remains unknown. In this study, the effects of PEDV infection on CD21+ B cell activation were systematically analyzed through animal experiments. Enzyme-linked immunosorbent assays (ELISA) revealed that low levels of serum-specific IgA, IgM, or IgG were detected in piglets after PEDV infection, respectively. Serum interleukin (IL)-6 levels increased significantly at 4 d after infection, and the levels of IL-4, B-cell activating factor (BAFF), interferon (IFN)-γ, transforming growth factor (TGF)-ß and IL-10 decreased at 7 d after infection. Fluorescence-activated cell sorting (FACS) showed that expression levels of CD21, MHC Ⅱ, CD40, and CD38 on B cell surfaces were significantly higher. In contrast, the proportions of CD21+IgM+ B cells were decreased in peripheral blood mononuclear cells (PBMCs) from the infected piglets. No differences were found in the percentage of CD21+CD80+ and CD21+CD27+ B cells in PBMCs from the infected piglets. In addition, the number of CD21+B cells in PBMCs stimulated with PEDV in vitro was significantly lower. No significant change in the mRNA expression of BCR molecules was found while the expression levels of paired immunoglobulin-like receptor B (PIR-B), B cell adaptor molecule of 32 kDa (Bam32) and BAFF were decreased. In conclusion, our research demonstrates that virulent strains of PEDV profoundly impact B cell activation, leading to alterations in phenotypic expression and BCR signaling molecules. Furthermore, this dysregulation results in compromised specific antibody secretion and perturbed cytokine production, highlighting the intricate immunological dysfunctions induced by PEDV infection.

Comparison of immune effects of porcine circovirus type 2d (PCV2d) capsid protein expressed by Escherichia coli and baculovirus-insect cells.

Porcine circovirus type 2 (PCV2) is an important pathogen harmful to global pig production, which causes immunosuppression and serious economic losses. PCV2 capsid (Cap) protein expressed by E. coli or baculovirus-insect cells are often used in preparation of PCV2 subunit vaccines, but the latter is expensive to produce. It is therefore crucial to comparison of the immune effects of Cap protein expressed by the above two expression systems for reducing the production cost and guaranteeing PCV2 vaccine quality. In this study, the PCV2d-Cap protein lacking nuclear localization signal (NLS), designated as E. coli-Cap and Bac-Cap, was expressed by E. coli and baculovirus-Spodoptera frugiperda Sf9 (Bac-Sf9) cells, respectively. The expressed Cap proteins could self-assemble into virus-like particles (VLPs), but the Bac-Cap-assembled VLPs were more regular. The two system-expressed Cap proteins induced similar specific IgG responses in mice, but the neutralizing antibody levels of Bac-Cap-immunized mice was higher than those of E. coli-Cap. After PCV2 challenge, IL-10 in Bac-Cap immunized mice decreased significantly than that in E. coli-Cap. The lesions and PCV2 antigen positive cells in tissues of mice immunized with E. coli-Cap and Bac-Cap were significantly reduced, and Bac-Cap appeared mild lesions and fewer PCV2 antigen-positive cells compared with E. coli-Cap immunized mice. The study indicated that Cap proteins expressed by E. coli and Bac-Sf9 cells could induce specific protective immunity, but the latter induced more effective immunity, which provides valuable information for the research and development of PCV2 vaccine.

Comparative Analysis of Intestinal Inflammation and Microbiota Dysbiosis of LPS-Challenged Piglets between Different Breeds.

Post-weaning diarrhea is common in piglets, causing huge economic losses worldwide. Associations between LPS challenge, intestinal inflammation, and microbiota have been reported in Duroc × Landrace × Yorkshire (DLY) crossbred pigs. However, the effects of LPS challenge in other breeds remain unclear. In the current study, we performed a comprehensive comparative analysis of the effects of LPS challenge on jejunal mucosal morphology, jejunal microbial composition, and serum indexes in two pig breeds: DLY and Heigai, an indigenous Chinese breed. The results showed that LPS caused considerable damage to the mucosal morphology, enhanced serum levels of inflammatory cytokines and the intestinal permeability index, and lowered the antioxidant capacity index. LPS challenge also changed the microbial composition and structure of the jejunum, significantly increased the abundances of Escherichia-Shigella in DLY pigs, and decreased those of Gemella and Saccharimonadales in Heigai pigs. Furthermore, LPS challenge triggered functional changes in energy metabolism and activities related to the stress response in the jejunal bacterial community, alleviating the inflammatory response in Heigai pigs. This study also revealed that Heigai pigs had a weaker immune response to LPS challenge than DLY pigs, and identified several genera related to the breed-specific phenotypes of Heigai pigs, including Gemella, Saccharimonadales, Clostridia_UCG_014, Terrisporobacter, and Dielma. Our collective findings uncovered differences between Heigai and DLY pigs in intestinal inflammation and microbiota dysbiosis induced by LPS challenge, providing a theoretical basis for unraveling the mechanism of intestinal inflammation in swine and proposing microbial candidates involved in the resistance to diarrhea in piglets.

Intranasally inoculated bacterium-like particles displaying porcine epidemic diarrhea virus S1 protein induced intestinal mucosal immune response in mice.

Porcine epidemic diarrhea virus (PEDV) causes acute watery diarrhea and high mortality in newborn piglets. Activation of intestinal mucosal immunity is crucial to anti-PEDV infection. To develop a vaccine capable of stimulating intestinal mucosal immunity, we prepared a bacterium (Lactococcus lactis)-like particle (BLP) vaccine (S1-BLPs) displaying the S1 protein, a domain of PEDV spike protein (S), based on gram-positive enhancer matrix (GEM) particle display technology. We further compared the effects of different vaccination routes on mucosal immune responses in mice induced by S1-BLPs. The specific IgG titer in serum of intramuscularly immunized mice with S1-BLPs was significantly higher than that of the intranasally administered. The specific IgA antibody was found in the serum and intestinal lavage fluid of mice vaccinated intranasally, but not intramuscularly. Moreover, the intranasally inoculated S1-BLPs induced higher levels of IFN-γ and IL-4 in serum than the intramuscularly inoculated. In addition, the ratio of serum IgG2a/IgG1 of mice inoculated intramuscularly was significantly higher with S1-BLPs compared to that of with S1 protein, suggesting that the immune responses induced by S1-BLPs was characterized by helper T (Th) cell type 1 immunity. The results indicated that S1-BLPs induced systemic and local immunity, and the immunization routes significantly affected the specific antibody classes and Th immune response types. The intranasally administered S1-BLPs could effectively stimulate intestinal mucosal specific secretory IgA response. S1-BLPs have the potential to be developed as PEDV mucosal vaccine.

Comparative Analysis of Structural Composition and Function of Intestinal Microbiota between Chinese Indigenous Laiwu Pigs and Commercial DLY Pigs.

Intestinal microbiota has an important impact on pig phenotypes. Previous studies mainly focused on the microbiota of feces and worldwide farmed commercial pigs, while research on the microbiota of various intestinal sections and indigenous pig breeds is very limited. This study aimed to characterize and compare the biogeography of intestinal microbiota in pigs of one Chinese indigenous breed and one commercial crossbred. In this study, we sequenced the microbiota of six intestinal segments in the grown-up pigs of a Chinese indigenous breed, Laiwu pigs, and the worldwide farmed crossbred Duroc × Landrace × Yorkshire (DLY) pigs by 16S rRNA sequencing, characterized the biogeography of intestinal microbiota, and compared the compositional and functional differences between the two breeds. The results showed that there were obvious differences in microbial structure and abundance between the small and large intestines. Laiwu pigs had higher large intestinal diversity than DLY pigs, while DLY pigs had higher small intestinal diversity than Laiwu pigs. Moreover, some specific bacterial taxa and Kyoto Encyclopedia of Genes and Genomes pathways were found to be related to the high fat deposition and good meat quality of Laiwu pigs and the high growth speed and lean meat rate of DLY pigs. This study provides an insight into the shifts in taxonomic composition, microbial diversity, and functional profile of intestinal microbiota in six intestinal segments of Laiwu and DLY pigs, which would be essential for exploring the potential influence of the host's genetic background on variation in microbiota composition and diversity.

Porcine Circovirus 2 Increases the Frequency of Transforming Growth Factor-ß via the C35, S36 and V39 Amino Acids of the ORF4.

Porcine circovirus 2 (PCV2) is one of the most important endemic swine pathogens, inducing immunosuppression in pigs and predisposing them to secondary bacterial or viral infections. Our previous studies show that PCV2 infection stimulated pig intestinal epithelial cells (IPEC-J2) to produce the secretory transforming growth factor-ß (TGF-ß), which, in turn, caused CD4+ T cells to differentiate into regulatory T cells (Tregs). This may be one of the key mechanisms by which PCV2 induces immunosuppression. Here, we attempt to identify the viral proteins that affect the TGF-ß secretion, as well as the key amino acids that are primarily responsible for this occurrence. The three amino acids C35, S36 and V39 of the ORF4 protein are the key sites at which PCV2 induces a large amount of TGF-ß production in IPEC-J2 and influences the frequency of Tregs. This may elucidate the regulatory effect of PCV2 on the Tregs differentiation from the perspective of virus structure and intestinal epithelial cell interaction, laying a theoretical foundation for improving the molecular mechanism of PCV2-induced intestinal mucosal immunosuppression in piglets.

Chemokines induced by PEDV infection and chemotactic effects on monocyte, T and B cells.

Porcine epidemic diarrhea virus (PEDV) is a re-emerging pathogen that causes severe economic loss in the pig industry. The host's innate immune system is the first line of defense on virus invasion of the small intestinal epithelial cells. Chemokines, as a part of the innate immune system, play an important role in host immunity against infection, however, and their expression and chemotactic effect on key immune cells in PEDV infection remains unclear. In this study, cDNA microarray was firstly performed to analyzed ileum tissue of piglets on the third day after PEDV infection. The differentially expressed genes mainly involved in multiple biological processes, chemokine signaling pathway and cytokine receptor interaction signaling pathway had the highest enrichment according to GO and KEGG enrichment analysis. The expression levels of chemokines MCP-1, MIP-1ß, IL-8, CXCL9, CXCL10 and CXCL13 in ileum of PEDV- infected piglets were significantly higher than those in the control group. The expression of chemokines in vivo experiment was further verified by RT-qPCR and ELISA using PEDV-infected IPEC-J2 cells. The results showed that the PEDV-infected IPEC-J2 cells had significantly induced protein expression of MCP-1, MIP-1ß, IL-8, CXCL9, CXCL-10 and CXCL13. These results indicated that the changes of chemokines expressed in the ileum of piglets (in vivo) were consistent with those in IPEC-J2 cells (in vitro) after PEDV infection. Finally, the role of chemokines in immune cell migration during PEDV infection was illustrated by siRNA-mediated knock down method and the co-culture model of IPEC-J2 cells with peripheral blood leukocyte cells (PBLCs). The FACS analysis showed that MCP-1 induced by PEDV infection played a chemotactic effect on CD14+ cells, CXCL9 on CD3+CD4-CD8-γδ T, CD3+CD4-CD8+ Tc, CD3+CD4+CD8- Th and CD3+CD4+CD8+ Tm subsets, and CXCL13 on CD19+ B cells. Collectively, our findings first indicate that PEDV-induced chemokines MCP-1, CXCL-9 and CXCL-13 attracted CD14+ cells, T cells and B cells, respectively. These results provide a theoretical basis for studying the mechanism of anti-PEDV infection in piglets.

TGF-ß from the Porcine Intestinal Cell Line IPEC-J2 Induced by Porcine Circovirus 2 Increases the Frequency of Treg Cells via the Activation of ERK (in CD4+ T Cells) and NF-κB (in IPEC-J2).

Porcine circovirus 2 (PCV2) causes immunosuppression. Piglets infected with PCV2 can develop enteritis. Given that the gut is the largest immune organ, however, the response of the gut's immune system to PCV2 is still unclear. Here, IPEC-J2 cells with different treatments were co-cultured with PBMC or CD4+ T cells (Transwell). Flow cytometry and Western blotting revealed that PCV2-infected IPEC-J2 increased the frequency of CD4+ T cells among piglets' peripheral blood mononuclear cells (PBMCs) and caused CD4+ T cells to undergo a transformation into Foxp3+ regulatory T cells (Treg cells) via activating CD4+ T ERK. Cytokines production and an inhibitor assay showed that the induction of Tregs by PCV2-infected IPEC-J2 was dependent on TGF-ß induced by PCV2 in IPEC-J2, which was associated with the activation of NF-κB. Taken together, PCV2-infected IPEC-J2 activated NF-κB to stimulate the synthesis of TGF-ß, which enhanced the differentiation of CD4+ T cells into Treg cells through the activation of ERK in CD4+ T cells. This information sheds light on PCV2's function in the intestinal immune system and suggests a potential immunosuppressive mechanism for PCV2 infection.

Differences in cytokines expression between Vero cells and IPEC-J2 cells infected with porcine epidemic diarrhea virus.

Porcine epidemic diarrhea virus (PEDV) primarily infects suckling piglets and causes severe economic losses to the swine industry. Cytokines, as part of the innate immune response, are important in PEDV infection. The cytokines secreted by cell infection models in vitro might reflect true response to viral infection of target cells in vivo. Vero cells and IPEC-J2 are commonly used as an in vitro model to investigate PEDV infection. However, it is not clear which type of cells is more beneficial to the study of PEDV. In our study, firstly, Vero cells and IPEC-J2 were successfully infected with PEDV virulent strains (HBQY2016) and attenuated vaccine strains (CV777) and were capable of supporting virus replication and progeny release. Moreover, cytokine differences expression by Vero cells and IPEC-J2 cells infected with two PEDV strains were analyzed. Compared with IPEC-J2 cells, only the mRNA levels of TGF-ß, MIP-1ß and MCP-1 were detected in Vero cells. ELISA assay indicated that compared to the control group, the PEDV-infected group had significantly induced expression levels of IL-1ß, MIP-1ß, MCP-1, IL-8, and CXCL10 in IPEC-J2 cells, while only secretion level of IL-1ß, MIP-1ß and IL-8 in Vero cells were higher in PEDV infected group. Finally, cytokines change of piglets infected PEDV-HBQY2016 strains were detected by cDNA microarray, and similar to those of IPEC-J2 cells infected PEDV. Collectively, these data determined that the IPEC-J2 could be more suitable used as a cell model for studying PEDV infection in vitro compared with Vero cells, based on the close approximation of cytokine expression profile to in vivo target cells.

Detection of Porcine Circovirus Type 2a and Pasteurella multocida Capsular Serotype D in Growing Pigs Suffering from Respiratory Disease.

In order to diagnose a respiratory disease in a pig farm, the lungs, spleen, and lymph nodes of three dead pigs were collected for pathogen detection by PCR and isolation on the basis of preliminary clinical diagnosis. The virus isolate was identified by gene sequence analysis and Immunoperoxidase monolayer assay (IPMA). The bacterial isolate was identified by biochemical tests, 16S rDNA sequence analysis, and species- and serotype-specific PCR, and the pathogenicity was analyzed. Porcine circovirus type 2a (PCV2a) genotype from the lungs, spleen, and lymph nodes and Pasteurella (P.) multocida capsular serotypes D from the lungs were found. The PCV2a isolates could specifically bound the anti-PCV2-Cap polyclonal antibody. The 16S rDNA sequence of P. multocida isolates had 99.9% identity with that of the strain from cattle, and the isolate was highly pathogenic to mice. The results showed that the co-infection of PCV2a and P. Multocida capsular serotypes D should be responsible for the disease. The uncommon PCV2a is still prevalent in some pig farms besides the dominant PCV2d genotype. This study could provide important etiological information for effective control and treatment of the disease in pig farms.

The changes of immune-related molecules within the ileal mucosa of piglets infected with porcine circovirus type 2.

BACKGROUND: Enteritis is one of the most frequently reported symptoms in piglets infected with porcine circovirus type 2 (PCV2), but the immunopathogenesis has not been reported. OBJECTIVES: This study examined the effect of a PCV2 infection on the intestinal mucosal immune function through morphological observations and immune-related molecular detection. METHODS: Morphological changes within the ileum of piglets during a PCV2 infection were observed. The expression of the related-molecules was analyzed using a gene chip. The immunocyte subsets were analyzed by flow cytometry. The secretory immunoglobulin A (SIgA) content was analyzed by enzyme-linked immunosorbent assay. RESULTS: The PCV2 infection caused ileal villus damage, intestinal epithelial cells exfoliation, and an increase in lymphocytes in the lamina propria at 21 days post-infection. Differentially expressed genes occurred in the defense response, inflammatory response, and the complement and coagulation cascade reactions. Most of them were downregulated significantly at the induction site and upregulated at the effector site. The genes associated with SIgA production were downregulated significantly at the induction site. In contrast, the expression of the Toll-like receptor-related genes was upregulated significantly at the effector site. The frequencies of dendritic cells, B cells, and CD8⁺T cells were upregulated at the 2 sites. The SIgA content decreased significantly in the ileal mucosa. CONCLUSIONS: PCV2 infections can cause damage to the ileum that is associated with changes in immune-related gene expression, immune-related cell subsets, and SIgA production. These findings elucidated the molecular changes in the ileum after a PCV2 infection from the perspective of intestinal mucosal immunity, which provides insights into a further study for PCV2-induced enteritis.

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.

Isolation and characteristics of multi-drug resistant Streptococcus porcinus from the vaginal secretions of sow with endometritis.

BACKGROUND: Sow endometritis is a common disease in pig breeding farms after artificial insemination, which leads to gray-green vaginal secretions and decreased conception rates. It is important to perform an etiologic diagnosis for effective treatments and control of diseases. The aim of this study was to carry out a pathogenic detection in five specimens of vaginal secretions collected from sick pigs with endometritis, implement identification of the pathogens by phenotypic detection and 16 s rDNA sequence and phylogeny analysis, and determinate antibiotic susceptibility of the isolates. RESULTS: A Streptococcus strain was isolated and identified from all of the five specimens. The isolate was positive for Voges-Proskauer (V-P) and for the hydrolysis of arginine, esculin and myelin-associated glycoprotein (MAG). Acid formation was observed for sorbitol, mushroom sugar, sucrose, and glucose. The 16S rDNA sequence of the isolate possessed 99.93% similarity to that of Streptococcus porcinus. The phylogenetic analysis of 16S rDNA sequence showed that the isolate belonged to the same clade as the S. porcinus strains from humans, pigs, and other animals. The isolate exhibited multi-drug resistance to aminoglycosides, quinolones, macrolides and tetracyclines except being sensitive to some ß- lactams such as penicillin G, cephalothin, cefazolin, cephradine and cefuroxime. CONCLUSIONS: A S. porcinus isolate with multi-drug resistance was identified from vaginal secretions of sows with endometritis in one pig breeding farm, which suggests that the sow endometritis was caused by S. porcinus infection during artificial insemination. This study indicates that sensitive antibiotics such as penicillin G or some cephalosporins could be used for treatment of the diseases. In addition, the study hints that bacterial multi-drug resistance is a tough problem for disease treatment in pig farms.

A TaqMan probe-based real-time PCR to differentiate porcine epidemic diarrhea virus virulent strains from attenuated vaccine strains.

Porcine epidemic diarrhea virus (PEDV) is an important pathogen causing severe watery diarrhea, vomiting, dehydration, and death in sucking piglets. Attenuated vaccines have been used widely in sows in order to protect piglets through passive lactogenic immunity. Rapid and sensitive detection methods for differentiating attenuated vaccine strains from virulent ones are essential and practical in PEDV prevention and control. Based on the deletion mutation in ORF3 gene sequence, a TaqMan probe-based real-time quantitative PCR (TaqMan qPCR) was developed to distinguish PEDV virulent strains from attenuated vaccine ones in this study. The TaqMan qPCR could specifically detect PEDV virulent strain but not attenuated vaccine strain and other viruses. At least 37 DNA copies and PEDV of 0.995 TCID50 could be detected by TaqMan qPCR. The reproducibility was evaluated using various dilution of plasmids carrying PEDV ORF3 gene and virulent PEDV, and the inter-assay coefficient of variation (CV) was less than 0.44%. The TaqMan qPCR was further applied to detect 38 samples including intestines and their contents, fecal swabs, and mesenteric lymph nodes. Meanwhile, indirect immunofluorescence assay (IFA) was employed to detect PEDV-specific antigen. PEDV positive rates were 31.58% (12/38) and 26.32% (10/38) by TaqMan PCR and IFA, respectively, which suggested that the former was more sensitive than the latter. The TaqMan qPCR based on PEDV ORF3 gene could be a valuable tool in diagnose of porcine epidemic diarrhea and in molecular epidemiological study of the virulent PEDV.

Effect of Booster Vaccination with Inactivated Porcine Epidemic Diarrhea Virus on Neutralizing Antibody Response in Mammary Secretions.

Porcine epidemic diarrhea virus (PEDV) causes acute diarrhea, dehydration in pigs, and high mortality rates in piglets <3 weeks of age. Maternal immunity protects piglets, but information on vaccination before or after natural infection in endemically exposed sow herds is limited. Currently, the recovery goal in sow units infected with PEDV is to become fully naive again or use natural virus infection to develop immune gilts through a feedback program before introduction into the sow herd. Since neutralizing antibodies in the gut are critical for protection against enteric viral infections such as PEDV, we evaluated the effect of a conditionally licensed, adjuvanted inactivated PEDV vaccine on neutralizing antibody levels in milk and colostrum in both naive and previously naturally exposed sow herds. The results illustrate that intramuscular vaccination increased neutralizing antibody titers, and anti-PEDV IgA and IgG in milk and colostrum of sows that were previously infected. Thus, inactivated PEDV vaccines may provide increased protection to piglets nursing on previously infected sows against exposure to PEDV through increased delivery of lactogenic neutralizing antibodies to the enteric site of infection.

Effect of porcine circovirus type 2 (PCV2) on the function of splenic CD11c+ dendritic cells in mice.

Porcine circovirus-associated disease (PCVAD) caused by porcine circovirus type 2 (PCV2) is an important disease in the global pig industry. Dendritic cells (DCs) are the primary immune cells capable of initiating adaptive immune responses as well as major target cells of PCV2. To determine whether PCV2 affects the immune functions of DCs, we evaluated the expression of endocytosis and co-stimulatory molecules on DCs (CD11c+) from PCV2-infected mouse spleen by flow cytometry (FCM). We also analyzed the main cytokines secreted by DCs (CD11c+) and activation of CD4+ and CD8+ T cells by DCs (CD11c+) through measurement of cytokine secretion, using ELISA. Compared with control mice, PCV2 did not affect the endocytic activity of DCs but it significantly enhanced TNF-α secretion and markedly decreased IFN-α secretion. Subsets of CD40+, MHCII+ CD40+ and CD137L+ CD86+ DCs did not increase obviously, but MHCII+ CD40- and CD137L- CD80+/CD86+ DCs increased significantly in PCV2-infected mouse spleen. Under the stimulation of DCs from PCV2-infected mouse, secretion of IFN-γ by CD4+ and CD8+ T cells and of IL-12 by CD8+ T cells was significantly lower than in control mice, while secretion of IL-4 by CD4+ T cells was remarkably higher. These results indicate that PCV2 modulates cytokine secretion and co-stimulatory molecule expression of DCs, and alters activation of CD4+ and CD8+ T cells by DCs. The immunomodulatory effects of PCV2 on DCs might be related to the host's immune dysfunction and persistent infection with this virus.

Characterization of anti-porcine epidemic diarrhea virus neutralizing activity in mammary secretions.

Porcine epidemic diarrhea virus (PEDV) causes a severe clinical enteric disease in suckling neonates with up to 100% mortality, resulting in devastating economic losses to the pork industry in recent years. Maternal immunity via colostrum and milk is a vital source to neonates of passive protection against diarrhea, dehydration and death caused by PEDV. Comprehensive information on neutralizing activity (NA) against PEDV in mammary secretions is critically important for assessing the protective capacity of sows. Therefore, the objectives of this study were to characterize anti-PEDV neutralizing activity in mammary secretions. Anti-PEDV NA was present in colostrum, milk and serum from PEDV-infected sows as determined both by immunofluorescence and ELISA-based neutralizing assays, with neutralization levels higher in colostrum and milk than in serum. The highest NA was observed in colostrum on day 1, and decreased rapidly in milk at day 3, then gradually declined from day 3 to day 19 post-farrowing. Notably, the NA in mammary secretions showed various patterns of decline over time of lactation that may contribute to variation in sow protective capacities. The kinetics of NA decline were associated with total IgA and IgG antibody levels. Neutralizing activity significantly correlated with specific IgA primarily to spike domain 1 (S1) and domain 2 (S2) proteins of PEDV rather than to specific IgG in colostrum. Subsequently, the NA in milk was mainly related to specific IgA to S1 and S2 during lactation.

Prevalence and genetic variation of porcine circovirus type 2 in Hebei, China from 2004 to 2014.

Porcine circovirus type 2 (PCV2), the primary causative agent of porcine circovirus-associated disease (PCVAD), causes severe economic losses to the pig industry in China since 2002. To investigate the molecular epidemic characteristics and genetic evolution of PCV2, 12 PCV2 isolates obtained from different pig farms with various clinical symptoms of PCVAD in Hebei, China from 2004 to 2014 were sequenced and analyzed. The phylogenetic analysis showed that the 12 isolates were divided into two distinct genotypes, PCV2b (7/12) and PCV2d (5/12), based on the sequences of either viral complete genome or open reading frame 2 (ORF2). Of the 7 PCV2b strains, 5 were isolated from 2004 to 2008 while all PCV2d were isolated from 2009 to 2014. This exhibited that PCV2b isolates were the most common before 2009 and then PCV2d isolates became predominant and widely distributed in pig farms. Sequence comparisons among total isolates indicated that the nucleotide identity ranged from 95.5% to 100% for complete genome and 93.1%-100% for ORF2. Compared with seven PCV2b isolates, there were thirteen amino-acid substitutions in the ORF2 region and one additional amino-acid K at this region terminal for five PCV2d isolates. The results suggest that a higher genetic variation and a distinct genotype shift occurred among the PCV2 isolates collected from 2004 to 2014 in Hebei.

Development of a nanoparticle-assisted PCR assay for detection of porcine epidemic diarrhea virus.

Porcine epidemic diarrhea virus (PEDV) is an important pig pathogen that can cause vomiting, diarrhea, and dehydration, leading to serious damage to the swine industry worldwide. In this study, a nanoparticle-assisted polymerase chain reaction (nanoPCR) assay targeting the N gene of PEDV was developed and the sensitivity and specificity were investigated. Under the optimized conditions for detection of PEDV RNA, the nanoPCR assay was 100-fold more sensitive than a conventional RT-PCR assay. The lower detection limit of the nanoPCR assay was 2.7 × 10(-6) ng/µL of PEDV RNA and no cross-reaction was observed with other viruses. This is the first report to demonstrate the application of a nanoPCR assay for the detection of PEDV. The sensitive and specific nanoPCR assay developed in this study can be applied widely in clinical diagnosis and field surveillance of PEDV-infection.