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.

Correlation between antibody response against porcine epidemic diarrhea virus in sows and their offspring under field conditions.

BACKGROUND AND AIM: Thai pig farmers have suffered huge financial losses from porcine epidemic diarrhea (PED) since 2007. PED, caused by the PED virus (PEDV), leads to severe diarrhea, vomiting, and subsequent dehydration in suckling piglets. Lactogenic immunity derived from colostrum and milk is very important because immunoglobulins (Ig) cannot cross the placenta in pregnant sows. The aim of this study was to investigate the immunological correlation of the sample-to-positive (S/P) ratios of IgA and IgG against PEDV between colostrum, sow serum, and their piglet serum. MATERIALS AND METHODS: A total of 43 sows were divided into three groups according to the experience of PEDV infection: Negative sow group (n=7) and treatment group (n=36, sows previously infected with PEDV). The treatment group was subdivided into two groups: Sows immunized with live-attenuated PEDV vaccine (n=15) and sows immunized with feedback (n=21) at 3 weeks before farrowing. The 7-day-old piglets (n=425) were obtained from negative sows (n=89), vaccinated sows (n=150), and feedback sows (n=275). Colostrum, sow serum, and their piglet serum were collected and analyzed for S/P ratios of their IgA and IgG levels against PEDV using an enzyme-linked immunosorbent assay. RESULTS: The piglets from sows immunized with live-attenuated PEDV vaccine had a higher S/P ratio of IgG against PEDV (p<0.001), whereas the piglets from the feedback group had a higher S/P ratio of IgA against PEDV (p<0.001) compared with piglets from the negative sows. In addition, the S/P ratios of PEDV-specific IgA and IgG between sow serum and colostrum showed a positive correlation (Pearson's coefficient r=0.61 and 0.75, respectively). Both S/P ratios of PEDV-specific IgA and IgG in sow serum and colostrum had a positive correlation to those in piglet serum. CONCLUSION: Overall, this study suggested that pregnant sows immunized with the live-attenuated vaccine against PEDV and feedback may provide maternal immunity against PEDV to their offspring.

Molecular characterization of bovine ephemeral fever virus in Thailand between 2013 and 2017.

Bovine ephemeral fever (BEF) is an arthropod-borne disease caused by bovine ephemeral fever virus (BEFV), a negative sense, single-stranded RNA virus. BEFV is endemic in tropical and sub-tropical regions including Thailand, a country in mainland Southeast Asia. However, there are few studies on BEFV and no available information regarding molecular characteristics of BEFV in Thailand. Therefore, the aims of this study were to genetically characterize Thai BEFVs and reveal their evolutions by phylogenetic analysis of G gene ectodomain sequences. From 2013 to 2017, blood samples were collected from bovine that matched with BEF case definition from three regions of Thailand. Thai BEFV G genes and a whole genome of an isolate, East Asia/TH/LRI0045/2016, were sequenced and characterized. Additionally, their phylogenies were constructed. This is the first report on genetics of BEFV in Southeast Asia. G ectodomain encoding region of Thai BEFV found during 2013-2017 are closely related to the second and third sub-clades of East Asia lineage. In addition, we observed mutation in the putative P' ORF of all Thai BEFVs which generated a premature stop codon. Thai G gene sequences are closely related to those of mainland Chinese and Taiwanese isolates. The whole genomic sequences of Thai BEFV and East Asia/China/JT02 L/2002 possess common characteristics, suggesting shared evolutionary relationship between East and Southeast Asian strains. Further studies on relationship between animal translocation, circulation of BEFV in Greater Mekong subregion and acquisition of more G gene sequences may improve understanding of BEFV epidemiology in mainland Southeast Asia.

Expression of foot and mouth disease virus nonstructural polyprotein 3ABC with inactive 3C(pro) in Escherichia coli.

Nonstructural 3ABC protein of foot and mouth disease virus (FMDV) was widely used to differentiate vaccinated from natural FMDV-infected animals. 3ABC is a polyprotein which is auto-processed to 3A, three copies of 3B and 3C(pro) by 3C(pro) protease. The 3ABC gene was cloned and expressed in Escherichia coli as native or mutated 3ABC (mu3ABC) forms. Cysteine residues 142 and 163 of the catalytic triad within the 3C(pro) of mu3ABC were changed to serine and glycine, respectively, to inhibit its protease activity. Both native and mutated 3ABC ORFs were cloned into BamHI and HindIII restriction sites of an expression vector, pQE80L. The expression of the recombinant native 3ABC and mu3ABC genes in E. coli BL21 was induced with 0.2mM isopropyl-beta-d-thiogalactopyranoside at 37 °C for 5h. SDS-PAGE and Western blot analysis revealed that the full length 3ABC was present in the lysate from mu3ABC but not native 3ABC transformed cells. The recombinant mu3ABC was expressed mainly in the inclusion body and presented as monomer and dimer. In addition, the mu3ABC reacted strongly with a convalescent serum from a natural FMDV-infected cattle but very weakly with a serum from vaccinated cattle. This study clearly demonstrates that successful expression of the full length 3ABC occurs only when the protease active sites within the 3C(pro) were completely abolished. This information would accelerate in house development of the 3ABC-based diagnostic test that can distinguish between vaccinated and FMDV-infected animals.

First whole genome characterization of swine influenza virus subtype H3N2 in Thailand.

H3N2 swine influenza viruses (SIV) were first detected in Asia shortly after the 1968 pandemic emerged in humans. Subsequently, human H3N2 viruses have sporadically reappeared in swine. In Thailand, a human-like H3N2 SIV was reported in 1978 although the genetic sequence of this virus is unknown. In this study, we undertook cross sectional syndromic surveillance in pigs in four provinces in Thailand. Seven genetically similar H3N2 viruses were isolated. A representative, A/SW/Thailand/KU5.1/04, was fully sequenced and shown to contain genes from human-like influenza viruses and North American and European SIV. The results restate that transmission of influenza A virus among human and swine populations is common and that genes from both American and Eurasian SIV lineages cocirculate in Thailand.

Occurrence of swine influenza virus infection in swine with porcine respiratory disease complex.

We studied the occurrence of swine influenza virus (SIV) infection in piglets with respiratory symptoms resembling porcine respiratory disease complex (PRDC). A total of 106 samples including nasal swab and lung suspension from sick piglets were collected from 30 farms of medium size in the central and eastern parts of Thailand from August 2006 to February 2007. Samples were inoculated onto Mardin-Darby Canine Kidney (MDCK) cells and SIV infection was confirmed by immunofluorescent assay (IFA) and reverse transcriptase polymerase chain reaction (RT-PCR) specific for M gene. Of 106 samples, 3 pigs from 3 different farms were found to be SIV positive on all assays. The positive samples were further identified by RT-PCR as H3N2 subtype using specific primers for hemagglutinin (HA) and neuraminidase (NA) genes. SIV infection was found in 2.8% of swine suffering from respiratory distress suggesting SIV may not be the major pathogen for PRDC in the central and eastern Thailand. SIV was present in 3 of 30 farms (10%) indicating the prevalence of SIV in these regions is considerable. Since pigs are vulnerable to infection from both human and avian influenza viruses and interspecies transmission between humans and swine occurs sporadically, it is essential to continue surveillance and monitoring of SIV infection in the swine population.