Analysis of the spike, ORF3, and nucleocapsid genes of porcine epidemic diarrhea virus circulating on Thai swine farms, 2011–2016

Porcine epidemic diarrhea virus (PEDV) outbreaks on pig farms have caused significant economic loss in the swine industry since it was first reported in Thailand a decade ago. Anecdotal evidence suggests that PEDV is now endemic in this region, therefore genome information of circulating PEDV is important for molecular surveillance and evaluation of potential benefits of field vaccination. Here, we characterized PEDV infection on commercial Thai swine farms by screening 769 samples of feces and small intestinal contents from pigs with diarrhea between 2011 and 2016. Using reverse-transcription polymerase chain reaction targeting the spike (S) gene, 153 PEDV-positive samples were further subjected to analysis of the open reading frame 3 and nucleocapsid (N) genes. Comparison of 95 samples in which nucleotide sequencing was successfully obtained for all three genes revealed evolutionary diversity among the Thai PEDV strains. Phylogenetic analyses suggest that although some Thai strains changed little from years past, others resembled more closely to the recent strains reported in China. Interestingly, eight Thai PEDV strains possessed amino acid deletions in the N protein. The PEDV sequence divergence may be responsible for driving periodic outbreaks and continued persistence of PEDV on commercial swine farms. Our findings provide important insight into regional PEDV strains in circulation, which may assist future inclusions of suitable strains for future PEDV vaccines

Evaluation of synbiotics as gut health improvement agents against Shiga toxin-producing Escherichia coli isolated from the pig

Colibacillosis is one of the major health problems in young piglets resulting in poor health and death caused by Escherichia coli producing F18 pili and Shiga toxin 2e. It is pivotal to reduce colibacillosis in weaned piglets to enhance production performance. In this study, we evaluated synbiotics as the gut health improvement agents in the mouse model challenged with Shiga toxin-producing E. coli (STEC) isolated from piglets. Prebiotic lactulose was formulated with each 5.0 × 106 CFU/mL of Pediococcus acidilactici GB-U15, Lactobacillus plantarum GB-U17, and Lactobacillus plantarum GB 1-3 to produce 3 combinations of synbiotics. A total of 40 three weeks old BALB/c mice were randomly assigned to 4 groups (n = 10): a control group and 3 synbiotics treated groups. Each treatment groups were daily administrated with 5.0 × 106 CFU/mL of one synbiotics for the first week, and every 3 days during the second week. All the mice were challenged with 8.0 × 108 CFU/mL of STEC 5 days after animals began to receive synbiotics. Mice treated with synbiotics based on Pediococcus acidilactici GB-U15 and Lactobacillus plantarum GB-U17 significantly improved daily weight gain compared to mice in other groups. While mice treated with GB-U15 showed better fecal index, no significant differences were observed among groups. Gross lesion and histopathological evaluations showed that mice treated with GB-U15 moderately improved recovery from STEC infection. In conclusion, our results suggest that the synbiotics formulated with lactulose and Pediococcus acidilactici GB-U15 have potential benefits to prevent and improve colibacillosis in weaned piglets

Differential innate immune response of endometrial cells to porcine reproductive and respiratory syndrome virus type 1 versus type 2.

Modification of cellular and immunological events due to porcine reproductive and respiratory syndrome virus (PRRSV) infection is associated with pathogenesis in lungs. PRRSV also causes female reproductive dysfunction and persistent infection which can spread to fetus, stillbirth, and offspring. In this study, changes in cellular and innate immune responses to PRRSV type 1 or type 2 infection, including expression of PRRSV mediators, mRNA expression of Toll-like receptors (TLRs) and cytokine, and cytokine secretion, were examined in primary porcine glandular endometrial cells (PGE). Cell infectivity as observed by cytopathic effect (CPE), PRRSV nucleocapsid proteins, and viral nucleic acids was detected as early as two days post-infection (2 dpi) and persisted until 6 dpi. A higher percentage of CPE and PRRSV-positive cells were observed in type 2 infections. PRRSV mediator proteins, CD151, CD163, sialoadhesin (Sn), integrin and vimentin, were upregulated following type 1 and type 2 infection. CD151, CD163 and Sn were upregulated by type 2. In both PRRSV types, mRNA expression of TLR1 and TLR6 was upregulated. However, TLR3 was upregulated by type 1, but TLR4 and TLR8 mRNA and protein were downregulated by type 2 only. Interleukin (IL)-1β, IL-6 and tumor necrotic factor (TNF)-α were upregulated by type 2, but IL-8 was upregulated by type 1. Both PRRSV type 1 and 2 stimulated IL-6 but suppressed TNF-α secretion. In addition, IL-1β secretion was suppressed only by type 2. These findings reveal an important mechanism underlying the strategy of PRRSV infection in the endometrium and associated with the viral persistence

Differential innate immune response of endometrial cells to porcine reproductive and respiratory syndrome virus type 1 versus type 2

Modification of cellular and immunological events due to porcine reproductive and respiratory syndrome virus (PRRSV) infection is associated with pathogenesis in lungs. PRRSV also causes female reproductive dysfunction and persistent infection which can spread to fetus, stillbirth, and offspring. In this study, changes in cellular and innate immune responses to PRRSV type 1 or type 2 infection, including expression of PRRSV mediators, mRNA expression of Toll-like receptors (TLRs) and cytokine, and cytokine secretion, were examined in primary porcine glandular endometrial cells (PGE). Cell infectivity as observed by cytopathic effect (CPE), PRRSV nucleocapsid proteins, and viral nucleic acids was detected as early as two days post-infection (2 dpi) and persisted until 6 dpi. A higher percentage of CPE and PRRSV-positive cells were observed in type 2 infections. PRRSV mediator proteins, CD151, CD163, sialoadhesin (Sn), integrin and vimentin, were upregulated following type 1 and type 2 infection. CD151, CD163 and Sn were upregulated by type 2. In both PRRSV types, mRNA expression of TLR1 and TLR6 was upregulated. However, TLR3 was upregulated by type 1, but TLR4 and TLR8 mRNA and protein were downregulated by type 2 only. Interleukin (IL)-1β, IL-6 and tumor necrotic factor (TNF)-α were upregulated by type 2, but IL-8 was upregulated by type 1. Both PRRSV type 1 and 2 stimulated IL-6 but suppressed TNF-α secretion. In addition, IL-1β secretion was suppressed only by type 2. These findings reveal an important mechanism underlying the strategy of PRRSV infection in the endometrium and associated with the viral persistence

PRRSV-infected PGE upregulated IL-6 but downregulated TNF-F061 secretion.

Total RNA and protein were extracted from PGE infected with mock, type 1, or type 2 at 105 TCID50/mL at 4 dpi to observe mRNA expression of cytokines using real-time PCR. Media were collected at 2, 4 and 6 dpi to evaluate accumulated cytokine secretion using ELISA. A) Relative expression of IL-6, IL-8, IFN-F067 and TNF-α normalized to GAPDH was compared to uninfected cells using the 2-∆∆Ct calculation. Type 1 upregulated IL-8 whereas type 2 PRRSV upregulated IL-1β, IL-6 and TNF-α. B) Relative accumulated cytokine secretion from uninfected cells. IL-6 and TNF-α secretion was respectively upregulated and downregulated by both PRRSV 1 and 2. IL-1β secretion was downregulated only by type 2. Bar graphs represent mean ± SEM (n = 5 pigs). Bar graph with different letters indicates significantly different (p < 0.05) as analyzed by ANOVA and Bonferroni’s post hoc test.</p

Type 2 PRRSV-infected PGE demonstrated high cytopathic effects (CPE), PRRSV-positive cells and viral titers.

PGE monolayers infected with mock, type 1, or type 2 at 105 TCID50/mL were observed for CPE, and PRRSV positive cells were investigated by immunocytochemistry using antibody against PRRSV envelop glycoprotein GP5. A) Representative images of cellular morphological changes of uninfected, mock- and PRRSV-infected PGE at 2-, 4- and 6-days post-infection (dpi). The scale bar = 500 µm. B) Percentage of CPE at 2, 4 and 6 dpi. The CPE area was increased at 2 and 4 dpi, but not 6 dpi, by type 1 and 2 infection. C) Percentage of PRRSV positive PGE at 2, 4 and 6 dpi. At all dpi, the PRRSV positive cells was increased by 10–20% following type 1 and up to 60% following type 2 infection. D) PRRSV titers in PGE at 2, 4 and 6 dpi. The highest titers of viral (TCID50/mL) were detected in type 2 infected PGE at 6 dpi. Bar graphs represent mean ± SEM (n = 5 pigs). Difference between PRRSV type and date of infection was analyzed by ANOVA and Bonferroni’s post hoc test. Bar graph with different letters indicates significantly different (p < 0.05).</p

Type 2 PRRSV-infected PGE upregulated <i>CD151</i> and <i>CD163</i>.

Total RNA was isolated from PGE infected with mock, type 1, or type 2 at 105 TCID50/mL at 4 dpi to determine mRNA expression of PRRSV mediators using real-time PCR. A) The expression of PRRSV mediators in uninfected PGE normalized to house-keeping gene GAPDH as calculated by 2-∆Ct. B) Relative mRNA expression of PRRSV mediators compared to uninfected cells using the 2-∆∆Ct calculation. CD151, CD163 and Sn were upregulated by type 2 infection. Bar graphs represent mean ± SEM (n = 5 pigs). Bar graph with different letters indicates significantly different (p ANOVA and Bonferroni’s post hoc test.</p

PRRSV-infected PGE altered TLR expression.

Total RNA and protein were extracted from PGE infected with mock, type 1, or type 2 at 105 TCID50/mL at 4 dpi to determine mRNA expression using reverse transcription and real-time PCR and protein using semi-quantitative Western blot analysis. A) Fold change of TLRs expression in PRRSV-infected PGE compared to uninfected cells using the 2-∆∆Ct calculation. Type 1 upregulated TLR1, TLR3 and TLR6. Type 2 upregulated TLR1 and TLR6 but downregulated TLR4 and TLR8. B) Fold change of all TLRs (1–10) protein expressions normalized to β-actin in PRRSV-infected PGE compared to uninfected cells. C) Representative band intensity of TLRs and β-actin was measured by ImageJ software. TLR6 protein expression was upregulated by type 1 and type 2. Only type 2 downregulated TLR4 and TLR8 expression. The scale bar = 500 µm. Bar graphs represent mean ± SEM (n = 5 pigs). Bar graph with different letters indicates significantly different (p < 0.05) as analyzed by ANOVA and Bonferroni’s post hoc test.</p