Oral fluid samples used for PRRSV acclimatization program and sow performance monitoring in endemic PRRS-positive farms.

An effective gilt acclimatization program is one of the most important management strategies for controlling porcine reproductive and respiratory syndrome virus (PRRSV) infection. Recently, oral fluid samples have been used as alternative diagnostic samples for various swine diseases. This study utilized oral fluids for PRRSV monitoring during the gilt acclimatization period in PRRSV endemic farms. The study was performed in two selected commercial breeding herds (farm A and farm B). PRRSV RNA and PRRSV-specific antibodies were monitored using oral fluid and serum samples. Sow performance parameters related to PRRSV infection were recorded and assessed. After PRRSV exposure during acclimatization, viral RNA was demonstrated in oral fluids from 1 to 10 weeks post-exposure (WPE). PRRSV RNA was detected in serum at 1 and 4 WPE in farm A and at 1, 4, 8, and 12 WPE in farm B. Prolonged viremia of gilts from farm B was possibly due to re-infection (within the herd) and later, reproductive problems were found in the breeding herd. The correlation of PRRSV RNA concentration in oral fluids and serum was evident. The S/P ratio values of PRRSV antibodies in oral fluid samples were higher and had similar patterns of antibody responses to the serum samples. The results suggest that the use of oral fluid samples for PRRSV monitoring during gilt acclimatization in endemic farms is effective, convenient, practical, and economical and would be most beneficial when used with other parameters.

Efficacy of a type 2 PRRSV modified live vaccine (PrimePac™ PRRS) against a Thai HP-PRRSV challenge.

The Chinese highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) has caused a severe threat to the pig population in Southeast Asian countries. The purpose of this study was to investigate the efficacy of a type 2 PRRSV modified live vaccine (PrimePac™ PRRS, lineage 7) against a Thai HP-PRRSV (10PL01, lineage 8). Three-week-old PRRSV-free pigs were randomly assigned into three groups. Vaccinated challenged group (group 1, n = 16) was immunized with PrimePac™ PRRS vaccine at 3 weeks old. The unvaccinated challenged group (group 2, n = 16) was injected with PBS at 3 weeks old, and unvaccinated unchallenged group (group 3, n = 10) was served as a negative control. At 9 weeks old, all groups, except the negative control group, were challenged with the Thai HP-PRRSV. All pigs were monitored daily during 10 days post-infection (dpi) and were necropsied at 10 and 17 dpi. The results revealed that vaccinated challenged pigs showed significantly lower (p < 0.05) mean rectal temperatures, clinical respiratory scores, lung lesion scores, and levels of virus load in serum and lung tissue compared with the unvaccinated challenged pigs. Moreover, vaccinated challenged pigs exhibited PRRSV-specific serum neutralizing antibodies at the end of the experiment. Our findings indicated that the studied type 2 PRRSV vaccine provided partial protection against the Thai HP-PRRSV infection based on the body temperature, levels of viremia, and the severity of lung lesions. These results demonstrated that partial protection of PrimePac™ PRRS vaccine might be useful for controlling HP-PRRSV infection in the endemic area.

Efficacy of Fostera® PRRS modified live virus (MLV) vaccination strategy against a Thai highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) infection.

Recently, the Chinese highly pathogenic porcine reproductive and respiratory syndrome virus (PRRSV) (HP-PRRSV) belonging to lineage 8 causes severe symptom with high morbidity and high mortality rates to the Asian pig industry. A recent study showed that pigs immunized with Fostera® PRRS modified live virus (MLV) of lineage 8 could provide a degree of protection against a Vietnamese HP-PRRSV infection. It should be noted that PRRSV commonly found after weaning causes porcine respiratory disease complex (PRDC). Vaccination strategy should be evaluated in each farm scenario. Eighty-one PRRSV-free piglets obtained from a PRRS-free herd were divided into two experiments with the major difference of infection timing after vaccination, 42 days in experiment 1 (n = 42) and 28 days in experiment 2 (n = 39). Each experiment had similar protocol containing three groups including a negative control, unvaccinated challenged, and vaccinated challenged groups. Pigs in vaccination groups were immunized with Fostera® PRRS MLV vaccine at 3 weeks of age. Then, unvaccinated challenged and vaccinated challenged groups were intranasally inoculated with a Thai HP-PRRSV (10PL01). Vaccinated challenged pigs showed significantly lower levels of mean rectal temperatures, clinical severity, lung lesion scores, and viral titers in serum and lung tissue compared to the unvaccinated challenged pigs (p < 0.05). Vaccinated challenged pigs had higher survival rate than those of unvaccinated challenged pigs in both experiments. It should be noted that pigs challenged 42 days after vaccination showed a better performance than pigs challenged 28 days after vaccination. In conclusion, Fostera® PRRS MLV vaccine was able to improve the survival rate against the Thai HP-PRRSV infection in both 42- and 28-day vaccination-to-infection protocols.

Effectiveness of gilt acclimatization - improvement procedures in a farm with recurrent outbreaks of porcine epidemic diarrhea.

Background and Aim: Porcine epidemic diarrhea (PED) is a severe infectious disease that causes very high mortality in newborn piglets up to 2-3 weeks age. The main cause of repeated outbreaks of PED in infected farms is the continuing circulation of the PED virus (PEDV). Improper gilt management, including inappropriate gut feedback, commingling, and inadequate immunization, causes a prolonged virus circulation in breeding herds. Moreover, insufficient transfer of passive immunity through the colostrum to newborn piglets can also increase infection risk. Therefore, a gilt management program that controls infection should focus on infection monitoring and acclimatization. We investigated the source of recurrent PEDV outbreaks and examined how the effect of immunization methods, specifically using gut feedback mechanism and vaccination, can reduce PEDV circulation and improve immune responses in replacement gilts. Materials and Methods: The study site was a segregated commercial production farm with endemic PEDV. The acclimatization methods included gut feedback and vaccination. This longitudinal study evaluated two strategies of gilt acclimatization against PEDV: Program 1 (routine farm management) and Program 2 (early feedback program and all-in-all-out system). Levels of PED RNA in fecal samples were measured using quantitative reverse transcription-polymerase chain reaction, and the PEDV S gene was sequenced. Porcine epidemic diarrhea-specific immune responses were assessed using enzyme-linked immunosorbent assay and the serum neutralization test. Results: Porcine epidemic diarrhea outbreaks occurred in the farrowing, nursery, and finishing units and farrowed litters 5-10 days old were symptomatic of PED. Phylogenetic analyses of the S gene showed PEDV sequence divergence between PEDV field strains and vaccine strain, which may contribute to periodic outbreaks and continued persistence of PEDV in the farm. After gut feedback and acclimatization, replacement gilts from Program 1 continued to shed PEDV before being introduced to sow herds, while those from Program 2 did not shed PEDV before being introduced to sow herds. However, the components of the immune response against PEDV in serum samples, including specific immunoglobulin (Ig)G, specific IgA, and neutralizing antibodies were lower in gilts of Program 2 than those in Program 1. Conclusion: We speculate that implementing the appropriate gilt acclimatization program can control PEDV circulation in farm. However, the acclimatization methods in Program 2 did not induce a strong and adequate immune response in replacement gilts. Therefore, maternal immunity levels and the degree of protection against PEDV require further study.

Bacteriophage efficacy in controlling swine enteric colibacillosis pathogens: An in vitro study.

Background and Aim: Swine enteric colibacillosis caused by Escherichia coli is a major problem in the swine industry, causing diarrhea among swine and resulting in substantial financial losses. However, efforts to counter this disease are impeded by the increase in antimicrobial resistance (AMR) worldwide, so intensive research is being conducted to identify alternative treatments. This study isolated, characterized, and evaluated the efficacy of bacteriophages to control pathogens causative of swine enteric colibacillosis. Materials and Methods: Five sewage samples were collected from different areas of a swine farm in Suphanburi province, Thailand and the bacteriophages were enriched and isolated, followed by purification by the agar overlay method using E. coli RENR as the host strain. The selected phages were characterized by evaluating their morphology, while their specificity was verified by the host range test. The efficiency of plating and multiplicity of infection (MOI) were also determined. Results: Four selected phages, namely, vB_Eco-RPNE4i3, vB_Eco-RPNE6i4, vB_Eco-RPNE7i1, and vB_Eco-RPNE8i3, demonstrated different patterns of host range and phage efficiency. They significantly decreased E. coli concentration at the tested MOIs (0.01-100) from 1 h onward. However, bacterial regrowth was observed in all phage treatments. Conclusion: This study shows the potential of using phages as an alternative treatment for swine enteric colibacillosis. The obtained results demonstrated that the selected phages had a therapeutic effect against pathogens causative of swine enteric colibacillosis. Therefore, phages could be applied as an alternative treatment to control specific bacterial strains and reduce AMR arising from the overuse of antibiotics.

In vivo assessment of bacteriophages specific to multidrug resistant Escherichia coli on fecal bacterial counts and microbiome in nursery pigs.

Escherichia coli is the most common cause of economic loss in swine industry. Nowadays, bacteriophages have been proven as good candidates for controlling bacterial infections. In this study, 6 phages were isolated and selected based on their high efficacy against 11 stains of E. coli isolated from diarrheal pigs. Six groups of weaned piglets were assigned (control, bacterial control (BC), two phage control (PC) and two phage treatment (PT) groups). Two titers (2 × 109 PFU/animal and 2 × 1010 PFU/animal) of phage cocktails consisting of these phages were tested in the PC and PT groups via oral gavage at 24, 48, and 72 h against an E. coli cocktail (2 × 109 CFU/animal) that was given to the piglets at 0, 12, 24, and 48 h of the trial. A significant reduction of fecal E. coli counts was observed in both PT groups from day 1 to 7 following the final phage dosage when compared to those of the BC group. Microbiomes in feces obtained 24 h after the final phage administration revealed phage therapy with both dosages could restore the gut's bacterial composition. Moreover, the given phage cocktails resulted in a significantly higher average daily gain of piglets during the first few weeks in both PC groups and the PT group receiving a higher phage dosage. These findings suggest that bacteriophages might be a potential alternative to antibiotics in the treatment of pathogens. In addition, they could also be utilized to improve pig growth performance.

PCV3 in Thailand: Molecular epidemiology and relationship with PCV2.

Porcine circovirus type 3 has been circulating throughout the world and since their first report, various clinical signs and disease developments have been documented. The virus is similar to the closely related PCV2 and is associated with several clinical signs called porcine circovirus-associated diseases (PCVAD). PCV2 or PCV3 is occasionally reported with clinical signs such as PDNS, respiratory signs and reproductive failure. Retrospective research conducted in Thailand revealed that both PCV2 and PCV3 have been circulation for decades. However, awareness about PCV3 infection has just arisen in recent years because of the similarities observed in disease circulation and clinical signs that have led to concerns. This study was conducted to find the relationship between the quantity of PCV2 and PCV3 in Thai pigs displaying the clinical signs related to PCVAD. A total of 479 serum samples with different production phases and clinical signs were sent to Kamphaeng Saen Veterinary Diagnostic Center (KVDC) for qPCR to detect the presence of PCV2 or PCV3. There was no relationship between the PCV3 and PCVAD-related clinical signs. Also, the relationship between PCV2 and PCV3 with no clinical signs suggested that both viruses might come from the same reservoir or have been circulating in Thailand for a long time, leading to common incidents in finding. The viral load of PCV2 was significantly different among the pig groups with and without clinical signs. The capsid sequence analysis of PCV3 revealed that 22 capsid sequences obtained from this study were found as clusters within PCV3a with a minor variation. Additional control measures are further needed to reduce the findings of the viruses. A future study with a control experiment may be needed to clarify the pathogenesis of PCV3.

The retrospective identification and molecular epidemiology of porcine circovirus type 3 (PCV3) in swine in Thailand from 2006 to 2017.

Porcine circovirus type 3 (PCV3) has recently been detected in pigs worldwide, with similar clinical manifestations to porcine circovirus-associated disease (PCVAD) from porcine circovirus type 2 (PCV2) infection. Here, we report the identification and molecular epidemiology of PCV3 in swine in Thailand from clinical samples retrieved from 2006 to 2017. The epidemiological data revealed co-infection with PCV2, PRRSV, and PCV2/PRRSV was common in our samples. Circulating PCV3 from this study shared a high similarity of nucleotide and deduced amino acid sequences of the partial capsid gene (96.7%-100% and 96.7%-100% respectively), indicated the genetic stability of PCV3 in Thailand. Phylogenetic analysis based on the capsid gene revealed scatter clustering with current PCV3 having no relation to the geographical origin of the virus strains. In this retrospective study, results have demonstrated that PCV3 has spread extensively within Thai swine from as early as 2006 and may also be involved in PRDC and PCVAD.

Porcine circovirus type 3 (PCV3) infection in grower pigs from a Thai farm suffering from porcine respiratory disease complex (PRDC).

Porcine circovirus type 3 (PCV3) is a newly emerging virus with unknown pathogenesis. The major objective of this study was to investigate the presence of PCV3 in pigs from a farm in Thailand suffering from porcine respiratory disease complex (PRDC). Initially, a Thai PCV3 strain (PCV3/Thailand/PB01/17) was identified from a pig originated from a farm with PRDC problem during grower period and whole genome analysis showed that the Thai PCV3 shared highest nucleotide identity of 99.60% with the South Korean strain PCV3/KU-1602. The presence of PCV3 infection in PRDC-affected pigs was then investigated in this farm. Serum samples from clinically healthy pigs and pigs showing PRDC-related clinical signs during 5-18 weeks were used in PCV3 detection by PCR. The results showed that the PRDC-affected pigs exhibited higher prevalence of PCV3 infection and higher PCV3 titers comparing with the clinically healthy pigs. These results confirmed the presence of PCV3 in a Thai farm with PRDC problem. The pathogenesis of PCV3 on PRDC should be clarified in further studies.

Porcine circovirus type 3 (PCV3) shedding in sow colostrum.

The major objective of this work was to investigate the shedding of porcine circovirus type 3 (PCV3) in sow colostrum. PCV3 titers in the serum and colostrum samples of 38 sows were determined using qPCR. Interestingly, this is the first report regarding the identification of PCV3 from the colostrum samples. In the studied farm, the prevalence of PCV3 in the colostrum samples was 44.74% (17/38). When sows were grouped based on the PCV3 titers in the serum into the "High-viremic", "Low-viremic" and "Non-viremic" sows, it was shown that the High-viremic sows showed significantly higher PCV3 colostrum prevalence (100%; 9/9) with the PCV3 titers ranging from 4.01 to 7.33 genomic copies/mL. The results indicated that PCV3 in the colostrum might be partly influenced by the viremic stage of the infection. However, the results also showed that approximately 41% of sows shedding PCV3 with low titers in the colostrum (7/17) were non-viremic sows. In conclusion, this study identified the presence of PCV3 in sow colostrum. Clinical impacts and mechanisms of colostrum shedding of PCV3 should be further investigated.

Protection of human influenza vaccines against a reassortant swine influenza virus of pandemic H1N1 origin using a pig model.

Since the pandemic H1N1 emergence in 2009 (pdmH1N1), many reassortant pdmH1N1 viruses emerged and found circulating in the pig population worldwide. Currently, commercial human subunit vaccines are used commonly to prevent the influenza symptom based on the WHO recommendation. In case of current reassortant swine influenza viruses transmitting from pigs to humans, the efficacy of current human influenza vaccines is of interest. In this study, influenza A negative pigs were vaccinated with selected commercial human subunit vaccines and challenged with rH3N2. All sera were tested with both HI and SN assays using four representative viruses from the surveillance data in 2012 (enH1N1, pdmH1N1, rH1N2 and rH3N2). The results showed no significant differences in clinical signs and macroscopic and microscopic findings among groups. However, all pig sera from vaccinated groups had protective HI titers to the enH1N1, pdmH1N1 and rH1N2 at 21DPV onward and had protective SN titers only to pdmH1N1and rH1N2 at 21DPV onward. SN test results appeared more specific than those of HI tests. All tested sera had no cross-reactivity against the rH3N2. Both studied human subunit vaccines failed to protect and to stop viral shedding with no evidence of serological reaction against rH3N2. SIV surveillance is essential for monitoring a novel SIV emergence potentially for zoonosis.

Positive immunomodulatory effects of heterologous DNA vaccine- modified live vaccine, prime-boost immunization, against the highly-pathogenic PRRSV infection.

Porcine reproductive and respiratory syndrome virus (PRRSV) infection is one of the most important swine pathogens, and causes a major economic impact worldwide. Recently, a new variant type 2 PRRSV, highly pathogenic PRRSV (HP-PRRSV) has emerged and continued to circulate in Southeast Asia region. Currently, commercially available PRRSV vaccines, modified live PRRS vaccines (MLV) are not able to provide complete protection against HP-PRRSV and been reported to induce negative immunomodulatory effects. Interestingly, a novel DNA vaccine was developed and successfully used to improve PRRSV-specific immune responses following MLV vaccination. To investigate the efficacy of a heterologous DNA-MLV prime-boost immunization against the HP-PRRSV infection, an experimental vaccinated-challenged study was conducted. Two-week-old, PRRSV-seronegative, crossbred pigs (5-8 pigs/group) were allocated into 5 groups. At day -14 (D-14), the treatment group (DNA-MLV) was immunized with a DNA vaccine encoding PRRSV-truncated nucleocapsid protein (pORF7t), followed by a commercial modified live type 2 PRRS vaccine (MLV) at D0. The other groups included the group that received PBS at D-14 followed by MLV at D0 (MLV), pORF7t at D-14 (DNA), PBS at D0 (PBS) and the negative control group. At D42, all groups, except the negative control group, were challenged with HP-PRRSV (strain 10PL1). The results demonstrated that pigs that received MLV, regardless of the DNA priming, exhibited less clinical signs and faster viral clearance. Following HP-PRRSV challenge, the DNA-MLV group exhibited improved PRRSV-specific immunity, as observed by increased neutralizing antibody titers and PRRSV-specific IFN-γ production, and reduced IL-10 and PRRSV-specific Treg productions. However, neither the prime-boost immunization nor the MLV was able to induce complete clinical protection against HP-PRRSV infection. In conclusion, improved immunological responses, but not clinical protection, were achieved by DNA-MLV prime-boost immunization. This study highlights the potential use of heterologous prime-boost vaccination regimen, where DNA can be incorporated with other vaccine candidates, for improving anti-PRRSV immunity that may eventually lead induction of complete PRRSV protection.

Determination of current reference viruses for serological study of swine influenza viruses after the introduction of pandemic 2009 H1N1 (pdmH1N1) in Thailand.

Since the introduction of pandemic H1N1 2009 virus (pdmH1N1) in pigs, the status of Thai swine influenza virus (SIV) has changed. The pdmH1N1 and its reassortant viruses have become the predominant strain circulating in the Thai swine population based on the surveillance data from 2012 to 2014. For this reason, the reference viruses for serological assays using the hemagglutination inhibition (HI) test needed to be updated. Six anti-sera against reference viruses from 2006 to 2009 (enH1N1-06, enH1N1-09, enH1N2-09, pdmH1N1-09, enH3N2-07 and enH3N2-09) were used for the HI test with four contemporary viruses (enH1N1-10, pdmH1N1-10, rH1N2 and rH3N2) and the selected reference viruses were tested with sera collected from the field to determine the current SIV status. The results showed that anti-sera of swH1N1-06 had the highest titers against enH1N1-10. Anti-sera of pdmH1N1-09 had the highest titers against pdmH1N1-10 and rH1N2, whereas, anti-sera of enH3N2-09 had the highest titers against rH3N2. The results demonstrated that enH1N1-06, pdmH1N1-09 and enH3N2-09 should be selected as reference viruses for contemporary serological studies and HI tests. The seroprevalence results from 410 samples revealed enH1N1 (37.79%), pdmH1N1 (37.32%) and H3N2 (35.86%), respectively. The present study indicated that pdmH1N1 was widespread and commonly found in the Thai pig population increasing the risk of novel reassortant viruses and should be added as a reference virus for HI test. SIV surveillance program and serological studies should be conducted for the benefits of SIV control and prevention as well as monitoring for zoonotic potential.