Immune response of gilts to single and double infection with porcine epidemic diarrhea virus

Transcription

1 Arch Virol (2017) 162: DOI /s BRIEF REPORT Immune response of gilts to single and double infection with porcine epidemic diarrhea virus Anchalee Srijangwad 1 Christopher James Stott 1 Gun Temeeyasen 1 Raweewan Senasuthum 2 Wanchai Chongcharoen 2 Angkana Tantituvanont 2 Dachrit Nilubol 1 Received: 27 July 2016 / Accepted: 28 February 2017 / Published online: 7 March 2017 Ó Springer-Verlag Wien 2017 Abstract Immune response of gilts following single and double infection with porcine epidemic diarrhea virus (PEDV) at gilt acclimatization and prepartum were investigated. One hundred PEDV-naïve gilts were divided into two groups: negative (Neg) and feedback (FB) groups. Antibody responses in serum, colostrum, and milk samples were measured by IgG/IgA ELISA and virus neutralization assay (VN). Fecal shedding was investigated using RT- PCR. In summary, a single infection at gilt acclimatization resulted in slightly increased serum antibody titers as determined by VN assay and IgG ELISA, but not by IgA ELISA. Viral RNA was detected in fecal samples up to 6 days post-exposure. A double infection at prepartum resulted in significantly increased IgA and VN titers in milk samples compared to the single-infection group. No fecal shedding was detected following the double infection. Keywords Porcine epidemic diarrhea virus Lactogenic immunity Oral exposure Thailand Electronic supplementary material The online version of this article (doi: /s ) contains supplementary material, which is available to authorized users. & Dachrit Nilubol dachrit@gmail.com 1 2 Department of Veterinary Microbiology, Faculty of Veterinary Science, Chulalongkorn University, Henry Dunant Road, Pathumwan, Bangkok 10330, Thailand Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok 10330, Thailand Introduction Porcine epidemic diarrhea (PED) is a devastating enteric infectious disease characterized by vomiting and acute, severe, watery diarrhea with high mortality in young pigs [14]. The causative agent is PED virus (PEDV), an enveloped, single-stranded RNA virus belonging to the genus Alphacoronavirus, family Coronaviridae, order Nidovirales. Since its first recognition in Belgium and the United Kingdom in [14, 19], PEDV has been reported in several countries [1, 4, 7, 8, 10, 12, 18]. PED was reported in Thailand in 2007 [17]. The PEDV variant responsible for the outbreaks and continuing to circulate in Thailand is in the genogroup 2a. The virus shares close genetic similarities to Chinese isolates, which possess two insertions and one deletion in the spike protein gene [2]. Following outbreaks, all sows in outbreak herds are orally exposed to minced intestines of PEDVinfected pigs (feedback) to stop the outbreak [11], and the sows then deliver healthy live-born pigs 3-4 weeks after oral exposure. However, the side effects of feedback, including an increased percentage of mummified fetuses and the risk of contamination by other pathogens including porcine reproductive and respiratory syndrome virus, have been recognized. Following the cessation of an outbreak, the herd remains at risk for pathogen re-introduction. In herds experiencing repetitive outbreaks, clinical disease is evident mainly in primiparous sows, suggesting that it would be advantageous to induce immunity in replacement gilts using feedback prior to introduction to the breeding herd. However, feedback during gilt acclimatization has raised some concerns regarding fecal shedding, the type of antibody present in serum, colostrum, and milk samples following oral exposure, and the level of virus needed in the feedback to

2 2030 A. Srijangwad et al. effectively induce immunity. In addition, the first oral exposure is performed during the gilt acclimatization period, which is at least 6 months prior to parturition. This management practice raisesconcernsastowhether the first infection would confer protection 6 months later to piglets born to primiparous sows. Questions remain as to whether the second oral exposure at a late stage of gestation would increase the PEDV antibody titers in colostrum and milk or not. Therefore, the objectives of the study were to investigate the immune response and fecal shedding in sows following oral exposure to PEDV during gilt acclimatization and at prepartum. To perform the study, one hundred gilts at approximately 23 weeks of age were procured from a herd free of PEDV. All animal procedures were conducted in accordance with the Care and Use of Laboratory Animals of the National Research Council of Thailand under the protocol approved by the Chulalongkorn University IACUC (# ). The naïve status of the incoming gilts was confirmed serologically using a virus neutralization (VN) assay (Supplementary Material 1) and by RT-PCR analysis of fecal samples using previously described protocols [17]. Upon arrival, one hundred gilts were randomly assigned, based on stratification by weight, to two treatment groups, Neg and FB each consisting of 50 gilts (Supplementary Material 2). Both groups were housed in a separate facility, with separate air space and separate workers. Fifty gilts were individually tagged and housed in five pens with 10 gilts each. Following a 7-day acclimatization, all gilts were fasted before the oral administration of feedback. To perform feedback, 10 grams of frozen intestines from PEDVinfected suckling piglets collected from a herd during an acute PED outbreak was homogenized into small pieces. The source herd for the feedback material tested negative for porcine deltacoronavirus (PDCoV) and transmissible gastroenteritis virus (TGEV) according to previously described protocols [15]. The gilts in the Neg group served as a negative control and were orally administered PEDVnegative homogenized intestines. The gilts in the FB group (single infection) were orally administered 10 grams of homogenized intestines from PEDV-infected piglets with an infective dose of TCID 50 /gram. The presence of clinical diarrhea was scored individually on a daily basis, with 0 for no diarrhea or 1 for pigs with diarrhea following the oral exposure. Fecal samples were collected daily for 2 consecutive weeks after feedback and assayed for the presence of PEDV by RT-PCR using a previously described method [17]. Blood was collected at -7, 14, 28, and 56 days post-exposure (DPE) and assayed for the presence of PEDV-specific antibody using IgG/IgA ELISA, and VN assay (Supplementary Material 1). All gilts were left with no activities for 8 consecutive weeks, and their estrus cycle began to synchronize. The gilts were artificially inseminated, and pregnancy checking via ultrasound was performed to confirm their pregnancy status. Following the first oral exposure, gilts in the Neg group had no diarrhea, and all fecal samples collected were RT- PCR negative throughout the study. In the FB group (single infection), 10 out of 50 (20%) gilts displayed diarrhea at 2 DPE. The percentage of diarrheic gilts continuously increased and reached the highest percentage of 84% (42 out of 50 gilts) at 6 DPE (Fig. 1). The percentage of diarrheic gilts then declined, and no gilts exhibited signs of diarrhea after 10 DPE. PEDV RNA was detected in 50% of the fecal samples of the FB group at 1 DPE. The percentage of RT-PCR-positive fecal samples increased and reached the highest level of 100% at 3 DPE (Fig. 1). The percentage then declined, and no fecal samples were RT- PCR positive after 6 DPE. The period of time in which virus shedding in fecal samples was detected was shorter than that in which clinical diarrhea was observed. The period of fecal shedding in this study was shorter than in a previous study in which PEDV RNA was detected in fecal samples for days after exposure [5]. This discrepancy could be due to the exposure dose. A previous study demonstrated that the duration of fecal shedding depends on the exposure dose. It showed that high-dose-exposed gilts (5.7 log 10 plaque-forming units [PFU]) shed the virus one day earlier than low-dose-exposed gilts (2.7 log 10 PFU) and also shed the virus longer [5]. It is also notable that the gilts in this study were orally exposed to feedback containing PEDV of genogroup 2. Although a different virus variant was used, the virus shedding results are consistent with those in a previous report [13]. In that study, pigs were orally exposed to PEDV of genogroup 1 and PEDV was detected in fecal samples from 3 to 11 DPE, with the highest virus excretion from 4 to 5 DPE. Following the primary exposure, pigs in the Neg group had no detectable antibody in their serum as measured by Fig. 1 The percentage of gilts displaying diarrhea and having positive RT-PCR fecal samples following oral administration with PEDV-infected intestines

3 Immune response to porcine epidemic diarrhea virus 2031 IgG/IgA ELISA and the VN assay throughout the study (Fig. 2B). In contrast, the antibody responses in the serum of pigs of the FB group increased at 14, 28 and 56 DPE compared to -7 DPE. However, the levels of IgG and IgA antibodies were below or close to the cutoff levels (Fig. 2A). The VN titer in serum was relatively low and observed in only 30 and 42% of the gilts in the FB group (single infection) at 14 (0.6) and 28 DPE (0.8), respectively. The VN titer, however, increased slightly at 56 DPE (1.5), and VN antibodies were detected in all gilts. The results of the study suggest that oral PEDV exposure induced antibody response in serum mainly toward the production of IgG, rather than IgA, and the increased antibody level correlated with the neutralizing activity. Compared to a previous report in which sows were intramuscularly administered S1 recombinant protein [9], the levels of IgG/IgA ELISA in serum of the present study Fig. 2 Immune response as measured by IgG/IgA ELISA and viral neutralization assay in serum samples of sows following in primary oral PEDV exposure. A) Antibody response as measured by IgG and IgA ELISA. B) Serum virus-neutralizing antibody titer). Variation is expressed as the standard deviation. Different letters in superscript indicate statistically significant differences (p \ 0.05) between groups at each time point were relatively low. The discrepancy could be due to the route of administration. Gilts in the present study were orally exposed to the virus. However,, the results of the present study are in accordance with a previous study by Langel et al., in which a similar route of administration was used[5]. In that study, it was demonstrated that the exposure dose did not correlate with the serum neutralizing titer. Gilts orally exposed to two different doses of PEDV, either low (2.7 log 10 PFU) or high (5.7 log 10 PFU) doses, induced relatively low levels of serum neutralizing antibody. A previous study demonstrated that protection against PEDV could last up to 6 months postinfection [3]. In the present study, the first oral exposure was during gilt acclimatization, which was at least 6 months prior to parturition. Therefore, the antibody titer in the serum and colostrum could potentially wane to an undetectable level. Furthermore, we are uncertain whether the antibody titer at this level would provide sufficient protection to piglets. A question remains as to whether the oral administration of feedback at the late stage of gestation would increase the PEDV antibody in colostrum and milk during the postpartum period. We therefore investigated whether a second oral PEDV exposure at a later stage of gestation would enhance the antibody response in serum, colostrum, and milk. To answer this question, a second oral exposure prior to parturition was performed. Eighty-two pregnant gilts were moved to farrowing houses at -28 days post-parturition (DPP). Forty-three pregnant gilts were of the Neg group. The remaining 39 pregnant gilts in the FB group were randomly assigned to two subgroups, FB-A and FB-B, and housed in separate farrowing rooms. Nineteen gilts in the FB-A group were left as a mock control (primary exposure at gilt acclimatization, and no pre-farrow exposure). At -14 DPP, twenty gilts in the FB-B group were orally administered feedback at a dosage similar to that of the first oral feedback (primary exposure at gilt acclimatization and prefarrow exposure). All gilts were observed daily for 14 consecutive days for the evidence of clinical diarrhea. Sera were collected at -14, -7 and 0 DPP and assayed for antibodies by IgG/IgA ELISA and a VN assay. Fecal samples were collected daily from all gilts in the FB-A and FB-B groups for 7 consecutive days and assayed for the presence PEDV by RT-PCR. Following parturition, colostrum and milk samples were collected from all farrowed sows of all three treatment groups. Colostrum was collected within 3 hours post-parturition, and milk samples were collected at 7, 14, and 21 DPP and assayed for the presence of PEDVspecific antibody using IgG/IgA ELISA and VN assay. Following the secondary oral exposure at prepartum, none of the gilts in the FB-B group had clinical diarrhea, and all fecal samples were negative for PEDV RNA by RT- PCR. This could be due to effective protection induced by

4 2032 A. Srijangwad et al. the primary exposure. The serum neutralizing antibody titer of the gilts in the FB-B group (primary exposure at gilt acclimatization and pre-farrow exposure) increased slightly following the secondary exposure. In contrast, the FB-A group had no detectable serum neutralizing antibodies prior to parturition (Fig. 3A). However, the serum neutralizing antibody titers at prepartum did not differ significantly between those three groups. The increase in serum neutralizing antibody titer as measured by VN assay observed in the serum of the FB-B group prior to parturition suggests that the effect is due to the second oral exposure. Although the serum neutralizing antibody titer was relatively low, this could be due to the time of re-exposure in which gilts in the present study were orally re-exposed with infected intestines at -14 DPP. Whether or not re-exposure at -28 or -21 DPP can significantly increase the titer at prepartum should be further investigated. In contrast to serum Fig. 3 Immune response as measured by IgG/IgA ELISA and virusneutralization assay in serum, colostrum (0 DPP) and milk (7, 14 and 21 DPP) samples of sows following secondary oral PEDV exposure. A) Virus-neutralizing antibody titer. B) Antibody response as measured by IgG ELISA. C) Antibody response as measured by IgA ELISA. Variation is expressed as the standard deviation. Different letters in superscript indicate statistically significant differences (p \ 0.05) between groups at each time point neutralizing titer, the increase in IgG/IgA ELISA titer was not observed in the serum samples of the FB-B group. The antibody response in serum as measured by IgG/IgA ELISA did not differ between the three groups (Fig. 3B and C), and the S/P ratio was below or close to 0.4, which is considered a negative status. Again, this could be due to the time of re-exposure or the route of exposure. Based on the immune response in serum following the single infection, the antibody response in the serum after parturition was not followed, but it was monitored in colostrum and milk samples. Following parturition, antibody titers in colostrum and milk samples as measured by IgG/IgA ELISA and VN assay were not detected in the Neg group throughout the lactation period. In contrast, the FB-A and FB-B groups both had a detectable level of neutralizing antibody in colostrum and milk samples throughout the lactation period. The neutralizing titers in colostrum and milk samples of both the FB-A and FB-B groups were significantly higher than that of the Neg group (Fig. 3A). The FB-B group had significantly higher neutralizing antibody titers than those of the FB-A group throughout the lactation period. In contrast to neutralizing antibody, the antibody response as measured by IgG and IgA ELISA was detected in colostrum samples of both the FB-A and FB-B groups. The levels of IgG and IgA in the colostrum samples did not differ, but they were significantly higher than those of the non-orally exposed group. The FB-B group had a significantly higher level of IgG as measured by ELISA compared to that of the FB-A group. In contrast to the antibody response in colostrum, the antibody response as measured by IgG ELISA was not detected in the milk samples of any of the three groups at 7, 14, and 21 DPP. The IgG levels in milk samples did not differ between the three groups and were below 0.2, which is considered a negative status. The results show that an IgG response was observed in colostrum only after farrowing and decreased thereafter to an undetectable or low level in milk samples. Following the decline of IgG antibody in colostrum, PEDV-specific IgA antibody subsequently predominated and remained consistently in the milk samples. In colostrum samples, the antibody response as measured by IgA ELISA was detected in both the FB-A and FB-B groups (Fig. 3C), and the IgA level in the FB-B group was higher than in the FB-A group. In milk samples, the IgA level of the FB-A group declined and was barely detectable at 7, 14, and 21 DPP. The level of IgA in milk samples of the FB-A group was below or close to 0.4, which is considered a negative status. In contrast, the FB-B group had significantly higher levels of IgA than that of the FB-A group on 7, 14 and 21 DPP. The results of the study suggest that, following oral PEDV exposure, the antibody response in milk samples switches

5 Immune response to porcine epidemic diarrhea virus 2033 toward the production of IgA, and the secondary oral exposure at prepartum provides an anamnestic response, as indicated by the significantly higher level of IgA compared to pigs with no secondary exposure. The antibody response in colostrum and milk samples after parturition contrasted with the response in serum samples in that the gilts orally exposed to PEDV exhibited antibody responses toward IgA rather than IgG, especially in milk samples. Unfortunately, piglets were not challenged to measure the protective efficacy of the lactogenic immunity. However, if the lactogenic immunity of PEDV is assessed based on the level of IgA, the increased IgA level in the study following oral exposure can indicate protection against PEDV. The predominance of IgA in milk samples is consistent with previous studies on the lactogenic immunity and milk antibodies to TGEV and PEDV in sows [5, 6], suggesting that oral PEDV exposure induces immunity similar to that of natural infection. It is noteworthy that although the levels of lactogenic immunity as measured by IgG/IgA ELISA were at or close to the negative cutoff level and similar to those of the negative control group, the VN titer in milk samples remained at a detectable level. The results of ELISA IgG and VN assays in the milk samples of the FB-A group are surprising. If the serum VN titer to PEDV is high, the levels of IgG should also be high. However, from this study, the results demonstrated a lower IgG response as measured by ELISA, but higher levels of VN. The assay had previously been validated and published elsewhere [16]. We repeated the tests and obtained similar results. In addition, we had been getting similar results in our other field investigations. The results therefore could be due to the nature of the immune response against oral immunization in pigs, functions of IgA, or an accumulated influence of hormones. In addition, whereas the spike protein is cleaved into S1 and S2 subunits, in the ELISA assay used in the present study, the plate was coated with a truncated S1 spike protein subunit. There are at least four identified virus-neutralizing epitopes on the spike protein [17]. The truncated S1 gene region might not cover all four epitopes, which could potentially result in contradictory results between the ELISA and VN assay. In conclusion, the single oral PEDV infection during the gilt acclimatization period results in a slightly increased serum antibody response as measured by IgG ELISA and the VN assay, but not by IgA ELISA. The results suggest that oral PEDV exposure induces an antibody response in serum, mainly toward the production of IgG rather than IgA, and this increased antibody production correlates with the neutralizing activity. An antibody response in the single-infection group was later observed in colostrum and milk, but the level was relatively low. The single infection during the gilt acclimatization period or at 24 weeks of age, which was approximately 6 months prior to parturition, might not have provided sufficient levels of protection to piglets during lactation. The double infection at prepartum is suggested to increase the antibody level in colostrum and milk, as shown by the results of the present study. The repeated oral feedback (double infection) at the late stage of gestation provides a booster effect, as demonstrated by the significantly higher level of antibody in colostrum and milk as measured by IgA ELISA and VN titration in the group FB-B (double infection) compared with that of the FB-A group (single infection). Further investigations including lactogenic immunity and protection of piglets against virus challenge are urgently needed to provide a successful vaccination protocol. Acknowledgements The authors are grateful to Agricultural Research Development Agency (public organization), National Research Council of Thailand, and the Thailand Research Fund. Partial funding was provided by the Special Task Force for Activating Research (STAR), Swine Viral Evolution and Vaccine Research (SVEVR), Chulalongkorn University. The authors are thankful to Dr. Matthew Wegner, faculty of Veterinary Science, for manuscript editing. Compliance with ethical standards The study was funded by the Thailand Research Fund (grant number PHD57I0026) and Government Budget Year Conflict of interest The authors declare that they have no conflicts of interest related to this work. Ethical approval All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. References 1. Chen Q, Li G, Stasko J, Thomas JT, Stensland WR, Pillatzki AE, Gauger PC, Schwartz KJ, Madson D, Yoon KJ, Stevenson GW, Burrough ER, Harmon KM, Main RG, Zhang J (2014) Isolation and characterization of porcine epidemic diarrhea viruses associated with the 2013 disease outbreak among swine in the United States. J Clin Microbiol 52: Cheun-Arom T, Temeeyasen G, Tripipat T, Kaewprommal P, Piriyapongsa J, Sukrong S, Chongcharoen W, Tantituvanont A, Nilubol D (2016) Full-length genome analysis of two genetically distinct variants of porcine epidemic diarrhea virus in Thailand. Infect Genet Evol 44: Goede D, Murtaugh MP, Nerem J, Yeske P, Rossow K, Morrison R (2015) Previous infection of sows with a mild strain of porcine epidemic diarrhea virus confers protection against infection with a severe strain. Vet Microbiol 176: Hanke D, Jenckel M, Petrov A, Ritzmann M, Stadler J, Akimkin V, Blome S, Pohlmann A, Schirrmeier H, Beer M, Hoper D (2015) Comparison of porcine epidemic diarrhea viruses from Germany and the United States, Emerg Infect Dis 21: Langel SN, Paim FC, Lager KM, Vlasova AN, Saif LJ (2016) Lactogenic immunity and vaccines for porcine epidemic diarrhea

6 2034 A. Srijangwad et al. virus (PEDV): historical and current concepts. Virus Res 226: Lanza I, Shoup DI, Saif LJ (1995) Lactogenic immunity and milk antibody isotypes to transmissible gastroenteritis virus in sows exposed to porcine respiratory coronavirus during pregnancy. Am J Vet Res 56: Lee S, Lee C (2014) Outbreak-related porcine epidemic diarrhea virus strains similar to US strains, South Korea, Emerg Infect Dis 20: Lin CN, Chung WB, Chang SW, Wen CC, Liu H, Chien CH, Chiou MT (2014) US-like strain of porcine epidemic diarrhea virus outbreaks in Taiwan, J Vet Med Sci 76: Makadiya N, Brownlie R, van den Hurk J, Berube N, Allan B, Gerdts V, Zakhartchouk A (2016) S1 domain of the porcine epidemic diarrhea virus spike protein as a vaccine antigen. Virol J 13: Masuda T, Murakami S, Takahashi O, Miyazaki A, Ohashi S, Yamasato H, Suzuki T (2015) New porcine epidemic diarrhoea virus variant with a large deletion in the spike gene identified in domestic pigs. Arch Virol 160: Nilubol D, Khatiworavage C (2012) Comparitive efficacy of oral administration of minced piglet intestine vs intramusculor vaccination in the control porcine epidemic diarrhea in gilts. In: International Pig Veterinary Congress, Jeju, p Ojkic D, Hazlett M, Fairles J, Marom A, Slavic D, Maxie G, Alexandersen S, Pasick J, Alsop J, Burlatschenko S (2015) The first case of porcine epidemic diarrhea in Canada. Can Vet J 56: Pensaert M, Yeo S (2006) Porcine epidemic diarrhoea. In: Straw BE, Zimmerman JJ, D Allaire S, Taylor DJ (eds) Diseases of swine, 9th edn. Blackwell Publishing Professional, Ames, pp Pensaert MB, de Bouck P (1978) A new coronavirus-like particle associated with diarrhea in swine. Arch Virol 58: Saeng-Chuto K, Lorsirigool A, Temeeyasen G, Vui DT, Stott CJ, Madapong A, Tripipat T, Wegner M, Intrakamhaeng M, Chongcharoen W, Tantituvanont A, Kaewprommal P, Piriyapongsa J, Nilubol D (2017) Different lineage of porcine deltacoronavirus in Thailand, Vietnam and Lao PDR in Transbound Emerg Dis 64(1): Srijangwad A, Nilubol D, Chongcharoen W, Phoolcharoen W, Chuanasa T, Tantituvanon A (2016) Production of spike and nucleocapsid recombinant proteins of porcine epidemic diarrhea virus for antibody detection by ELISA. Asian J Pharm Sci 11: Temeeyasen G, Srijangwad A, Tripipat T, Tipsombatboon P, Piriyapongsa J, Phoolcharoen W, Chuanasa T, Tantituvanont A, Nilubol D (2014) Genetic diversity of ORF3 and spike genes of porcine epidemic diarrhea virus in Thailand. Infect Genet Evol 21: Vui DT, Thanh TL, Tung N, Srijangwad A, Tripipat T, Chuanasa T, Nilubol D (2015) Complete genome characterization of porcine epidemic diarrhea virus in Vietnam. Arch Virol 160: Wood EN (1977) Apparently new syndrome of porcine epidemic diarrhea. Vet Rec 100: