Evolution and virulence of porcine epidemic diarrhea virus following in vitro and in vivo propagation.

Practice of inoculating porcine epidemic diarrhea virus (PEDV) in piglets generating feedback material might influence the genetic evolution and attenuation of PEDV. The study was conducted to evaluate evolutionary rate and attenuation following serial in vitro and in vivo propagation. In the study, PED-JPFP0-PJ, Passage 0 (P0), was isolated from infected pigs and serially passaged in Vero cells for 5 consecutive times, P1-P5. P0, P2 and P5 were then subjected to orally inoculate 3-day-old piglets. At 24 h post inoculation, intestines of each passage (F1), were collected, and subsequently sub-passaged in piglets for 2 additional passages (F2-F3). Virus titration, PEDV genomic copies number, VH:CD ratios, and immunohistochemistry were evaluated. S and ORF3 genes were characterized. The results of the study demonstrated that virus titer and virulence were negatively correlated with increased passages, both in vitro and in vivo. Increased substitution rate was observed in higher passages. The evolutionary rate of S gene was higher than that of ORF3. Seven aa changes at positions 223, 291, 317, 607, 694, 1114 and 1199, with reduced N-linked glycan were observed in P5F3. In conclusion, serial passage of PEDV, both in vitro and in vivo, influence the genetic development and the attenuation of PEDV.

The immune response of pregnant sow after vaccination with crude fimbriae (F4) extracts vaccine and immunoprotection of nursery pig against pathogenic E. coli (F4+ETEC).

BACKGROUND: Neonatal and post-weaning diarrhea is a concern disease caused by enterotoxigenic Escherichia coli fimbriae F4 (F4+ETEC) in pig farms. Diarrhea outbreaks are often severe and costly due to the high prevalence and spread of the disease within the same herd. Vaccine is one of strategic solution in protecting pig against F4+ETEC infection in particular pig farm. In present study, we conducted two trials of vaccination with crude F4 fimbriae extract vaccine in pregnant sow and nursery pigs. METHODS: In experiment 1 (20 sows; non-vaccinated control, n=10), we vaccinated pregnant sows (n=10) twice at 4 wk and 2 wk before farrowing and evaluated impact of vaccination on maternal immunity. The sow serum and colostrum were collected before vaccination, 2 and 4 weeks after vaccination, 6 hours after farrowing, respectively, and the piglet's serum from both groups (2 piglet/sow, 10 piglets from each group) were also collected on 3 days old to measure F4 specific IgG, F4 specific IgA using in house ELISA kit. In experiment 2, to optimize doses and dosage of candidate vaccine in piglets, 18 piglets (3 piglets/group) were allocated into five immunized groups and one control group (unimmunized group), we immunized piglets twice at 4 and 6 weeks old with difference doses (i.e., 0, 50, 100, 150, 200 µg), and for a dose 150 µg, we immunized with two dosages at 1 ml and 2 ml. Piglets were challenged with a 3 ml dose of 3 × 109 CFU/ml bacterial culture of enterotoxigenic Escherichia coli (F4+ETEC) in order to evaluate the efficacy of vaccine. After challenging, the clinical sign of the piglets was daily observed and the rectal swab was performed every day for investigation of the fecal shedding of Escherichia coli (F4+ETEC) by using PCR technique. Serum were collected before, 2 and 4 weeks after vaccination and 1 week after challenge to measure F4 specific IgG, F4 specific IgA using in house ELISA kit and cytokines levels (i.e., IL-1 beta, IL-6, IL-8 and TNF alpha) before and 1 week after challenge using commercial ELISA kit. RESULTS: The levels of antibody results showed that in experiment 1, the anti-F4 antibody levels both F4 specific IgG and F4 specific IgA in serum and colostrum of vaccinated sow increased significantly after vaccination. The piglets of immunized sows have antibody level both F4 specific IgG and F4 specific IgA in their serum higher than those piglets of unimmunized sows significantly (p < 0.01). In experiment 2, irrespective of different doses and dosage, there is no difference in term of F4 specific IgG and F4 specific IgA levels among immunized groups. However, all of vaccinated piglets showed F4 specific IgG and F4 specific IgA levels higher and the elimination of Escherichia coli (F4+ETEC) in feces post challenge faster (< 3 days) than unvaccinated group (> 5 days). For cytokines levels, a higher level of IL-1 beta, IL-6, IL-8 and TNF alpha at 1 week after challenge in vaccinated groups was found when compared with the levels in non-vaccinated group. CONCLUSIONS: Our results suggest that crude F4 fimbriae extract autogenous vaccine is a candidate vaccine for protecting piglets against diarrhea disease caused by enterotoxigenic Escherichia coli (F4+ETEC) and vaccination the pregnant sow twice before farrowing is one of strategies to provide maternal derived antibody to the newborn piglets for against enterotoxigenic Escherichia coli (F4+ETEC) during early life.

The Effect of Antimicrobial Peptide (PA-13) on Escherichia coli Carrying Antibiotic-Resistant Genes Isolated from Boar Semen.

A major global public health concern is antimicrobial resistance (AMR). Antimicrobial peptides (AMPs) are a potentially appropriate replacement for conventional antibiotics. The purpose of this research was to investigate the potential of the antimicrobial peptide PA-13, a synthetic AMP with 13 amino acids, to inhibit E. coli isolated from boar semen expressing antibiotic-resistant genes, as well as to determine the mechanism of action of this antimicrobial peptide on the bacterial membrane. The effectiveness of the bacterial inhibitory activity of PA-13 was tested at different concentrations by two fold serial dilutions in the range 0.488-500 µg/mL using the MIC and MBC methods. The impact of PA-13 on the bacterial membrane was examined at different concentrations of 0×, 0.5×, 1×, 2× and 4× of MIC using DNA leakage assay and electron microscopy. The PA-13 antibacterial activity result exhibited the same MIC and MBC values at a concentration of 15.625 µg/mL. When comparing DNA leakage at different MIC values, the results revealed that the maximum amount of DNA concentration was found two and three hours after incubation. For the results of SEM and TEM, the bacterial membrane disruption of this E. coli was found in the PA-13-treated group when compared with the negative control. In conclusion, synthetic PA-13 with its antibacterial properties is an alternative antimicrobial peptide to antibiotics in the pig industry.

Camellia oil with its rich in fatty acids enhances post-thawed boar sperm quality.

BACKGROUND: Boar sperm are highly susceptible to specific conditions during cryopreservation, leading to a significant decrease in their fertilizing potential due to damage to their membranes. Camellia oil, known for its fatty acids with antioxidant and biological properties, has not been previously explored for the cryopreservation of boar semen. This study aimed to examine the effects of camellia oil on post-thawed boar sperm quality. Boar semen ejaculates (n = 9) were collected and divided into six equal aliquots based on camellia oil concentrations (0, 0.5, 1, 1.5, 2 and 2.5% v/v) in the freezing extender. Semen samples were processed and cryopreserved using the liquid nitrogen vapor method. Thereafter, frozen semen samples were thawed at 50 °C for 12 s and evaluated for sperm morphology by scanning electron microscope, sperm motility using a computer-assisted sperm analyzer, sperm viability, acrosome integrity, mitochondrial function, MDA level and total antioxidant capacity. RESULTS: The results demonstrated that the supplementation of 1.5% (v/v) camellia oil showed superior post-thaw sperm qualities such as improved sperm morphology, motility, acrosome integrity and mitochondrial function by 14.3%, 14.3% and 11.7%, respectively, when compared to the control group. Camellia oil at a concentration of 1.5% (v/v) showed the lowest level of MDA (18.3 ± 2.1 µmol/L) compared to the other groups. CONCLUSIONS: In conclusion, adding 1.5% (v/v) camellia oil in the freezing extender reduced the oxidative damage associated with cryopreservation and resulted in a higher post-thawed sperm quality.

The effects of using multi-species probiotics in late-pregnant and lactating sows on milk quality and quantity, fecal microflora, and performance of their offspring.

Background and Aim: The dietary probiotics in sows during gestation to lactation period have gained considerable attention with respect to their beneficial effects on sows and their piglets' performance and health. This study aimed to evaluate the effects of using probiotics in late-pregnant and lactating sows on milk quality, quantity, fecal microflora of sows, and growth performance of their offspring until weaning. Materials and Methods: Thirty-four sows were equally divided into two groups (control and treatment). Only those in the treatment group were fed 5 g of probiotics at 12 weeks of pregnancy, once daily for 7 weeks, until their piglets were weaned. Colostrum samples were collected at 3, 6, 12, and 24 h after farrowing and measured for immunoglobulin concentration. Percentages of fat, protein, and lactose in colostrum, colostrum production, total intake of immunoglobulin A (IgA), immunoglobulin G (IgG), fat, protein, and lactose, the change of fecal microflora of sows, and average daily gain of piglets were measured. Results: The results showed that there were no significant differences in the concentrations of IgA, IgG, and IgM in colostrum and the percentages of fat, protein, lactose, solid-not-fat, and total solid in colostrum between the groups; however, the colostrum production at 24 h in the treatment group (6,075.29 mL) was higher than in the control group (4,809.54 mL). Higher total intakes of IgA and IgG as well as total intake of fat, protein, and lactose, particularly at 3 h after farrowing, were found in the treatment group. Probiotic supplementation remarkably altered the microbiota community at the phylum level. We found that Firmicutes and Bacteroidetes are the dominant phyla, present in the gut of more than 90% of pregnant and lactating sows. Changes in microbial proportions were observed due to the changes of pig production stage. The weaning weight of the treatment group was higher than in the control group (6.34 ± 1.71 vs. 4.84 ± 1.29 kg, respectively). Conclusion: Feeding of multi-species probiotic BACTOSAC-P™ during late pregnancy and lactation in sows positively influenced colostrum production. In this experiment, the use of BACTOSAC-P™ improved the yield of colostrum production. The high immunoglobulin concentration and high yield of the colostrum of sows with a diet supplemented with BACTOSAC-P™ significantly reduced piglet mortality during the suckling period. Furthermore, the probiotic diet induced changes in the fecal microbial population in sows by increasing the number of microorganisms from the Firmicutes phylum, which had positive effects on sow health and their piglets, leading to better piglet growth performance.

Feasibility of crude F4 fimbriae extract as a vaccine candidate for preventing Escherichia coli-induced diarrhea in piglets.

Background and Aim: Enterotoxigenic Escherichia coli (ETEC) poses a substantial risk of neonatal diarrhea and post-weaning diarrhea among piglets, with F4+ ETEC strains emerging as a particularly challenging issue within the pig farming industry. This study aimed to introduce a straightforward approach for generating a crude extract of F4 fimbriae that shows promise as an antigenic determinant for potential vaccination strategies. Materials and Methods: A crude F4 fimbriae extract was obtained from F4+ ETEC using a combination of heat shock and homogenization techniques. Subsequently, three 4-week-old piglets were immunized with a primary dose of 150 µg and a booster dose 2 weeks later. Blood samples were collected to evaluate the level of serum F4-specific antibodies using an enzyme-linked immunosorbent assay. Results: Analysis using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and liquid chromatography tandem-mass spectrometry techniques unveiled crucial insights into the composition of the crude F4 fimbriae extract. Notably, a distinct prominent band (~24 kDa) was identified, corresponding to the size of FaeG, the major subunit of F4 fimbriae. Regarding antibody response, there was a remarkable disparity between the levels of serum immunoglobulin (Ig)G and IgA antibodies targeting F4 compared with other E. coli strains (F18+ ETEC, F41+ ETEC, and F4-F18-F41- EC), as well as with the unvaccinated control group (p < 0.01). Specifically, the levels of IgG antibodies against other E. coli strains were also significantly higher than those observed in the unvaccinated control group (p < 0.05). Conclusion: Our findings suggest that the crude F4 fimbriae extracts obtained using our simple extraction method induce specific immune responses against F4+ E. coli and stimulate cross-immunity against other E. coli strains. Therefore, our method shows potential for use in future vaccine development against diarrhea in pigs caused by E. coli.

Palm Kernel Meal Protein Hydrolysates Enhance Post-Thawed Boar Sperm Quality.

Boar sperm is sensitive to particular conditions during cryopreservation, resulting in an extreme reduction in fertilizing ability due to damage to the sperm membranes. PKMPH contains bioactive peptides that have antioxidant and antimicrobial activities. There is no information on the use of palm-kernel-meal-derived bioactive peptides for boar semen cryopreservation. This study aimed to examine the effects of bioactive peptides from PKMPH on post-thawed boar sperm quality. Boar semen ejaculates (n = 17) were collected and divided into six equal aliquots based on PKMPH concentrations (0, 1.25, 2.5, 5, 10, and 15 µg/mL) in a freezing extender. Semen samples were processed and cryopreserved using the liquid nitrogen vapor method. Thereafter, the frozen semen samples were thawed at 50 °C for 12 s and evaluated for sperm motility using a computer-assisted sperm analyzer and for sperm viability, acrosome integrity, mitochondrial function, and lipid peroxidation by measuring the level of malondialdehyde (MDA). The results demonstrate that the supplementation of PKMPH with 2.5 µg/mL afforded superior post-thawed sperm qualities, such as increased total motility, viability, acrosome integrity, and mitochondrial function by 10.7%, 12.3%, 18.3%, and 12.7%, respectively, when compared to the control group. PKMPH at a concentration of 2.5 µg/mL showed the lowest level of MDA (40.6 ± 2.0 µMol/L) compared to the other groups. In conclusion, adding PKMPH peptides at 2.5 µg/mL to the freezing extender reduced the oxidative damage associated with cryopreservation and resulted in higher post-thawed sperm quality.

The Beneficial Effect of Resveratrol on the Quality of Frozen-Thawed Boar Sperm.

This study aimed to determine the effect of resveratrol and its optimal concentration on the quality of frozen-thawed (FT) boar sperm. Semen ejaculates were obtained from 13 Duroc boars aged between 1.5 and 3 years. The sperm sample was separated into 7 groups based on the concentrations of resveratrol in the freezing extender, which were 0 (control), 25, 50, 75, 100, 125, and 250 µM, respectively. The sperm was frozen using liquid nitrogen vapor and thawed at 50 °C for 12 s. After thawing, total motility, progressive motility, viability, intact acrosomes, mitochondrial membrane potential and level of MDA were assessed. The supplementation of 50-100 µM resveratrol improved the sperm motility and viability of FT sperm in comparison to the control group (p < 0.05). Furthermore, the 50 µM resveratrol group was significantly more protective than the control group in terms of intact acrosome, mitochondrial membrane potential, and level of MDA (p < 0.05). Nonetheless, the detrimental effect of resveratrol was found at a concentration of 250 µM. In conclusion, the addition of 50-100 µM resveratrol to a freezing extender is the optimal concentration for enhancing the quality of cryopreserved boar sperm.

Antimicrobial activity of cell free supernatants from probiotics inhibits against pathogenic bacteria isolated from fresh boar semen.

The use of antibiotics with semen extender appears to be a practical solution to minimise bacterial growth in fresh boar semen preservation. Unfortunately, the excessive use of antibiotics promotes antimicrobial resistance (AMR). This becomes a worldwide concern due to the antimicrobial resistance genes transmitted to animals, environment, and humans. Probiotics are one of the alternative methods to reduce antibiotic use. They could inhibit pathogenic bacteria by producing antimicrobial substances in cell free supernatants (CFS). Nevertheless, there is no comprehensive study undertaken on inhibitory activity against pathogenic bacteria isolated from boar semen origin. Our study investigated the efficacy of CFS produced from selected probiotics: Bacillus spp., Enterococcus spp., Weissella spp., Lactobacillus spp., and Pediococcus spp. inhibiting pathogenic bacteria isolated from fresh boar semen. Besides, the semen-origin pathogenic bacteria are subjected to identification, antimicrobial resistance genes detection, and antibiotic susceptibility test (AST). Pseudomonas aeruginosa, Escherichia coli, and Proteus mirabilis are the most common pathogens identified in boar semen with resistance to numerous antibiotics used in pig industry. The CFS with its antimicrobial peptides and/or bacteriocin constituent derived from selected probiotics could inhibit the growth of pathogenic bacteria carrying antimicrobial resistance genes (mcr-3 and int1 genes). The inhibition zones for Pseudomonas aeruginosa, Escherichia coli, and Proteus mirabilis provided more efficient results in the CFS derived from Lactobacillus spp. and Pediococcus spp. than those of the CFS produced from Enterococcus spp., Weissella spp. and Bacillus spp., respectively. It is worth noted that as the incubation time increased, the antibacterial activity decreased conversely. Our results on CFS with its antimicrobial peptides and/or bacteriocin constituent inhibits semen-origin pathogenic bacteria guide the direction as a promising alternative method used in the semen extender preservation of the pig industry.

Co-infection of porcine deltacoronavirus and porcine epidemic diarrhea virus induces early TRAF6-mediated NF-κB and IRF7 signaling pathways through TLRs.

Porcine deltacoronavirus (PDCoV) and porcine epidemic diarrhea virus (PEDV) infect the small intestine and cause swine enteric coronavirus disease. The mucosal innate immune system is the first line of defense against viral infection. The modulatory effect of PDCoV and PEDV coinfection on antiviral signaling cascades of the intestinal mucosa has not been reported. Here, we investigate the gene expression levels of pattern recognition receptors, downstream inflammatory signaling pathway molecules, and associated cytokines on the intestinal mucosa of neonatal piglets either infected with a single- or co-infected with PDCoV and PEDV using real-time PCR. The results demonstrate that single-PEDV regulates the noncanonical NF-κB signaling pathway through RIG-I regulation. In contrast, single-PDCoV and PDCoV/PEDV coinfection regulate proinflammatory and regulatory cytokines through TRAF6-mediated canonical NF-κB and IRF7 signaling pathways through TLRs. Although PDCoV/PEDV coinfection demonstrated an earlier modulatory effect in these signaling pathways, the regulation of proinflammatory and regulatory cytokines was observed simultaneously during single viral infection. These results suggested that PDCoV/PEDV coinfection may have synergistic effects that lead to enhanced viral evasion of the mucosal innate immune response.

Development of a Rapid Reverse Transcription-Recombinase Polymerase Amplification Couple Nucleic Acid Lateral Flow Method for Detecting Porcine Epidemic Diarrhoea Virus.

Porcine epidemic diarrhea virus (PEDV) infection is an important acute diarrheal disease of swine that results in economic and industrial losses worldwide. The clinical manifestations in infected piglets are severe diarrhea, dehydration with milk curd indigestion, leading to death. The diagnosis of PEDV is essential for monitoring and managing the disease. PEDV can be detected and identified by serology and the nucleic acid of the virus in clinical samples. Therefore, a novel isothermal amplification and detection technique, reverse transcription-recombinase polymerase amplification couple nucleic acid lateral flow (RT-RPA-NALF) was developed for the rapid detection of PEDV. Qualitative reverse transcription-polymerase chain reaction (RT-qPCR) was established as the gold standard assay to compare results. Specific primer pairs and probes were designed, and RT-RPA conditions were optimized to amplify the M gene of PEDV. The established RT-RPA-NALF assay could finish in 25 min at a temperature of 42 °C and the amplicon interpreted by visual detection. The developed RT-RPA-NALF assay was specific to the M gene of PEDV, did not detect other common swine diarrhea pathogens, and showed minimal detection at 102 TCID50/mL PEDV. The RT-RPA-NALF assay can detect PEDV in 5 simulated fecal samples. Furthermore, in 60 clinical fecal samples, the results of RT-RPA-NALF correlated with RT-qPCR assay, which provides sensitivity of 95.65% and specificity of 100%, with a coincident rate of 98.33%. The rapid RT-RPA-NALF is simple and rapid, increases high sensitivity, and can be used in the field.

Antibiotic resistant Escherichia coli from diarrheic piglets from pig farms in Thailand that harbor colistin-resistant mcr genes.

Antibiotic-resistant Escherichia coli is one of the most serious problems in pig production. This study aimed to determine the antibiotic susceptibility and genotypes profiles of diarrhoeagenic E. coli that causes diarrhea in piglets. Thirty-seven pathogenic E. coli strains were used in this study. These were isolated from rectal swabs of diarrheic piglets from farms in Thailand from 2018 to 2019. Escherichia coli isolates were highly resistant to amoxicillin (100%), followed by oxytetracycline (91.9%), enrofloxacin (89.2%), trimethoprim/sulfamethoxazole (86.5%), amoxicillin: clavulanic acid (81.1%), colistin and gentamicin (75.7%), ceftriaxone and ceftiofur (64.9%), ceftazidime (35.1%) and 97.3% showed multidrug-resistance (MDR). There were 8 (21.6%) mcr-1 carriers, 10 (27.0%) mcr-3 carriers and 10 (27.0%) co-occurrent mcr-1 and mcr-3 isolates. The phenotype-genotype correlation of colistin resistance was statistically significant (performed using Cohen's kappa coefficient (κ = 0.853; p < 0.001)). In addition, PCR results determined that 28 of 37 (75.7%) isolates carried the int1 gene, and 85.7% int1-positive isolates also carried the mcr gene. Genetic profiling of E. coli isolates performed by ERIC-PCR showed diverse genetics, differentiated into thirteen groups with 65% similarity. Knowledge of the molecular origins of multidrug-resistant E. coli should be helpful for when attempting to utilize antibiotics in the pig industry. In terms of public health awareness, the possibility of transmitting antibiotic-resistant E. coli from diarrheic piglets to other bacteria in pigs and humans should be of concern.

Cell-free culture supernatants of Lactobacillus spp. and Pediococcus spp. inhibit growth of pathogenic Escherichia coli isolated from pigs in Thailand.

BACKGROUND: Pathogenic Escherichia coli (E. coli) is an important causative agent for infectious diseases in pigs and causes significant economic loss. The global concern of antimicrobial resistance of bacteria raises awareness of the alternative ways of using antimicrobial peptides (AMPs). The study was aimed to identify and test the efficacy of AMPs from Lactobacillus spp. against the growth of pathogenic E. coli isolated from pigs in Thailand. Briefly, cell-free culture supernatants (CFCS) from 3 strains of lactic acid bacteria (LAB) consisting of Lactobacillus acidophilus (strain KMP), Lactobacillus plantarum (strain KMP), and Pediococcus pentosaceus (strain KMP) were tested against pathogenic E. coli via agar well diffusion assay in quadruplicates. The presence of a zone of inhibition (ZOI) around wells was evaluated at different incubation time. Acid and bile tolerance test was performed for bacterial viability in acid and bile salt conditions. In addition, LAB cross-streaking assay was evaluated for antagonist activity. RESULTS: The study showed that CFCS from L. acidophilus KMP, L. plantarum KMP, and P. pentosaceus KMP could inhibit the growth of pathogenic E. coli isolated from pigs in a time-dependent manner. To exemplify, the ZOI of L. plantarum KMP against E. coli (ETEC) at 8, 10, 12, 14, and 16 h incubation, were 26.6 ± 1.1, 24.9 ± 1.9, 22.5 ± 2.4, 20.3 ± 2.9, and 17.9 ± 3.3 mm, respectively. The ZOI was significantly different between 8, 10, 12, 14 h incubation, and the ZOI of the CFCS from L. plantarum KMP was larger than others (P-value < 0.05). Furthermore, L. acidophilus KMP, L. plantarum KMP, and P. pentosaceus KMP showed viability in pH 3.0, 0.3, and 0.5% (w/v) bile salt concentration. They exhibited no antagonist activity among each other. CONCLUSIONS: According to the results, the CFCS from LAB including L. acidophilus KMP, L. plantarum KMP and P. pentosaceus KMP can inhibit the growth of pathogenic E. coli, isolated from pigs in Thailand. The antimicrobial activity observed was incubation time dependent.

Coinfection of porcine deltacoronavirus and porcine epidemic diarrhea virus increases disease severity, cell trophism and earlier upregulation of IFN-α and IL12.

Porcine epidemic diarrhea virus (PEDV) and porcine deltacoronavirus (PDCoV) cause an enteric disease characterized by diarrhea clinically indistinguishable. Both viruses are simultaneously detected in clinical cases, but a study involving the co-infection has not been reported. The study was therefore conducted to investigate the disease severity following a co-infection with PEDV and PDCoV. In the study, 4-day-old pigs were orally inoculated with PEDV and PDCoV, either alone or in combination. Following challenge, fecal score was monitored on a daily basis. Fecal swabs were collected and assayed for the presence of viruses. Three pigs per group were necropsied at 3 and 5 days post inoculation (dpi). Microscopic lesions and villous height to crypt depth (VH:CD) ratio, together with the presence of PEDV and PDCoV antigens, were evaluated in small intestinal tissues. Expressions of interferon alpha (IFN-α) and interleukin 12 (IL12) were investigated in small intestinal mucosa. The findings indicated that coinoculation increased the disease severity, demonstrated by significantly prolonged fecal score and virus shedding and decreasing VH:CD ratio in the jejunum compared with pigs inoculated with either PEDV or PDCoV alone. Notably, in single-inoculated groups, PEDV and PDCoV antigens were detected only in villous enterocytes wile in the coinoculated group, PDCoV antigen was detected in both villous enterocytes and crypts. IFN-α and IL12 were significantly up-regulated in coinoculated groups in comparison with single-inoculated groups. In conclusion, co-infection with PEDV and PDCoV exacerbate clinical signs and have a synergetic on the regulatory effect inflammatory cytokines compared to a single infection with either virus.

Immunodominant and Neutralizing Linear B-Cell Epitopes Spanning the Spike and Membrane Proteins of Porcine Epidemic Diarrhea Virus.

Porcine epidemic diarrhea virus (PEDV) is the causative agent of PED, an enteric disease that causes high mortality rates in piglets. PEDV is an alphacoronavirus that has high genetic diversity. Insights into neutralizing B-cell epitopes of all genetically diverse PEDV strains are of importance, particularly for designing a vaccine that can provide broad protection against PEDV. In this work, we aimed to explore the landscape of linear B-cell epitopes on the spike (S) and membrane (M) proteins of global PEDV strains. All amino acid sequences of the PEDV S and M proteins were retrieved from the NCBI database and grouped. Immunoinformatics-based methods were next developed and used to identify putative linear B-cell epitopes from 14 and 5 consensus sequences generated from distinct groups of the S and M proteins, respectively. ELISA testing predicted peptides with PEDV-positive sera revealed nine novel immunodominant epitopes on the S protein. Importantly, seven of these novel immunodominant epitopes and other subdominant epitopes were demonstrated to be neutralizing epitopes by neutralization-inhibition assay. Our findings unveil important roles of the PEDV S2 subunit in both immune stimulation and virus neutralization. Additionally, our study shows the first time that the M protein is also the target of PEDV neutralization with seven neutralizing epitopes identified. Conservancy profiles of the epitopes are also provided. In this study, we offer immunoinformatics-based methods for linear B-cell epitope identification and a more complete profile of linear B-cell epitopes across the PEDV S and M proteins, which may contribute to the development of a greater next-generation PEDV vaccine as well as peptide-based immunoassays.

Proteome profiling of bovine follicular fluid-specific proteins and their effect on in vitro embryo development.

The objective of this study was to determine the effect of bovine follicular fluid proteins (bFF) and their differently charged groups as maturation media supplements for in vitro embryo development. bFF was obtained by aspiration from large healthy follicles (4-10 mm in diameter) and was precipitated by 30-50% (NH4)2SO4. The precipitated protein was fractionated into basic and acidic fractions by ion-exchanger columns. In experiment 1, the oocytes were matured in TCM-199 with 1) FBS+hormones (control) and 2) 10% bFF. The oocyte maturation rate, the development to the blastocyst stage rate and blastocyst cell number were not significantly different between the groups. However, the INFα and IGF-2r expression levels in the 10% bFF were higher than in the control (P<0.05). In experiment 2, the specific charge proteins of bFF (basic and acidic) were also used as media supplements in the maturation medium. The basic fraction had higher oocyte maturation rate and blastocyst cell number when compared with addition of acidic fraction (P<0.05). The expression levels for almost all developmentally important genes in the basic fraction were greater than those in the acidic fraction, particularly INFα (P<0.05). Most of the protein in the basic fraction was associated with the immune response and mRNA processing. In conclusion, supplementation of 10% bFF alone in maturation medium can support oocyte maturation and embryo development. The basic fraction in bFF seemed to have effect on oocyte maturation rate and blastocyst cell number.

The effect of Curcuma longa extracted (curcumin) on the quality of cryopreserved boar semen.

The aim of this study was to determine the optimal concentration of curcumin needed for cryopreservation of boar semen. Semen samples (n = 9) were collected from nine Duroc boars which having proven fertility were used for routine artificial insemination. Semen samples were collected and divided into six groups (groups A-F) according to various concentrations of curcumin in freezing extender (i.e. 0, 0.125, 0.25, 0.50, 0.75 and 1.0 mmol/L, respectively). The semen was frozen by traditional liquid nitrogen vapor method and stored at -196°C in the liquid nitrogen tank. After storage, frozen semen samples were thawed at 50°C for 12 s and evaluated for progressive motility, viability and acrosome integrity. The present results indicated that the addition of curcumin at 0.25 (group C) or 0.50 mmol/L curcumin (group D) yielded the higher percentage of progressive motility (33.3 and 36.1%, respectively) (P < 0.001). A significantly higher percentage of acrosome integrity was found in groups B (29.7%), C (31.1%) and D (30.2%) than in the other groups (P < 0.01). However, there was no significant difference in percentage of viability among groups. In conclusion, addition to the freezing extender of curcumin during cryopreservation at a concentration of 0.25 or 0.50 mmol/L is the optimal concentration of curcumin for improving the quality (i.e. increased progressive motility and acrosome integrity) of cryopreserved boar semen.

Conception rate and litter size in multiparous sows after intrauterine insemination using frozen-thawed boar semen in a commercial swine herd in Thailand.

The aim of the present study was to determine the conception rate and litter size in sows after fixed time intra-uterine insemination using frozen-thawed boar semen in a commercial swine herd in Thailand. Sixty-nine Landrace multiparous sows were randomly allocated into two groups, including control (n=36) and treatment (n=33). The control sows were inseminated with extended fresh semen (3 × 10(9) motile sperm/dose, 100 ml) at 24, 36 and 48 hr after the onset of estrus. The treatment sows were inseminated with frozen-thawed semen (2 × 10(9) motile sperm/dose, 20 ml) at 24 and 36 hr after induction of ovulation by human chorionic gonadotropin. All inseminations were carried out by using an intra-uterine insemination technique. The time of ovulation was determined by using transrectal real-time B-mode ultrasonography. The conception rate, farrowing rate, total number of piglets born/litter (TB) and number of piglets born alive/litter (BA) were evaluated. The sows inseminated with extended fresh semen yield a higher TB (10.8 versus 9.0 piglets/l, P=0.015) and tended to have a higher conception rate (88.9% versus 75.8%, P=0.150) than sows inseminated with frozen-thawed semen. In conclusion, insemination using frozen-thawed boar semen can be practiced with convinced fertility under field conditions by fixed-time intrauterine insemination with 2 × 10(9) sperm/ dose of 20 ml at 24 and 36 hr after the onset of estrus.

Effect of gamma-oryzanol-enriched rice bran oil on quality of cryopreserved boar semen.

The aim of this study was to determine the effect of gamma-oryzanol-enriched -rice bran oil on the quality of cryopreserved boar semen. Ten boars provided semen of proven motility and morphology for this study. The semen was divided into three portions in which lactose-egg yolk (LEY) extender used to resuspend the centrifuged sperm pellet was supplemented with 2 types of rice bran oils, at a gamma-oryzanol concentration of 0 mg/ml of lactose egg yolk (LEY) freezing extender (group A, control), 0.1 mg/ml(0.16 mMol) of freezing extender (group B) and 0.1 mg/ml of freezing extender (group C). Semen suspensions were loaded in medium straws (0.5 ml) and placed in a controlled-rate freezer. After cryopreservation, frozen semen samples were thawed and investigated for progressive motility, viability and acrosomal integrity. There was a significantly higher percentage of progressive motility (34 versus 47.0 and 48.5, P<0.001), viability (35.5 versus 48.1 and 50.1, P<0.001) and acrosomal integrity (39.8 versus 50.8 and 54.9, P<0.001) in the gamma-oryzanol-enriched rice bran oil-supplemented groups (groups B, C) than in the control group (group C), respectively. In conclusion, addition of gamma-oryzanol-enriched rice bran oil to LEY freezing extender is appropriated for improving the quality of frozen-thawed boar semen.

Cryopreservation of boar semen by egg yolk-based extenders containing lactose or fructose is better than sorbitol.

The present study determined the effect of different types of sugars (lactose, fructose, glucose and sorbitol) used in egg yolk-based extender on the post-thawed boar semen quality. Twenty-two ejaculates from 6 fertility-proven Yorkshire boars were cryopreserved by liquid nitrogen vapor method. Sperm motility, viability, acrosome integrity and intact functional plasma membrane were determined at 0, 2 and 4 hr after thawing. It was found that the lactose-based extender resulted in a higher percentage of post-thawed sperm motility, viability, intact acrosome and functional plasma membrane than sorbitol-based extender (P<0.05) and fructose-based extender yielded a higher post-thawed sperm motility and viability than sorbitol-based extender (P<0.05). It could be concluded that sorbitol was not an effective sugar for the cryopreservation in boar semen.