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Maternally-derived antibodies (MDAs) impair piglets’ humoral and cellular immune responses to vaccination against porcine reproductive and respiratory syndrome (PRRS)
Veterinary Microbiology 192 (2016) 175–180
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Veterinary Microbiology journal homepage: www.elsevier.com/locate/vetmic
Short communication
Maternally-derived antibodies (MDAs) impair piglets’ humoral and cellular immune responses to vaccination against porcine reproductive and respiratory syndrome (PRRS) C. Fableta,e,* , P. Rensonb,c,e, F. Eonoa,e, S. Mahéb,e , E. Evenoa,e , M. Le Dimnab,e , V. Normandd , A. Lebretd , N. Rosea,e , O. Bourryb,e a
Agence Nationale de Sécurité Sanitaire (ANSES),ty -2 Unité Epidémiologie et Bien-Etre du Porc, B.P. 53, 22440 Ploufragan, France Agence Nationale de Sécurité Sanitaire (ANSES), Unité Virologie Immunologie Porcines, B.P. 53, 22440 Ploufragan, France c Union des Groupements de Producteurs de Viande de Bretagne (UGPVB), 104 rue Eugène Pottier, CS 26553, 35065 Rennes, France d Porc. Spective, Groupe Vétérinaire Chêne Vert Conseil, 56920 Noyal-Pontivy, France e Université Bretagne Loire, France b
A R T I C L E I N F O
Article history: Received 25 January 2016 Received in revised form 11 July 2016 Accepted 22 July 2016 Keywords: PRRS Vaccination Immune response Maternal neutralizing antibody
A B S T R A C T
The influence of maternally-derived antibodies (MDAs) on the post-vaccination humoral and cellular immune responses in piglets vaccinated against PRRS was studied. The piglets came from a vaccinated breeding herd. Thirty piglets with a low (A ) or high level (A+) of PRRSV-neutralizing MDAs were vaccinated (V+) with a modified live vaccine at 3 weeks of age. Blood samples were collected before vaccination and then at 2, 4, 8 and 14 weeks post-vaccination (WPV). The samples were analysed to detect the vaccine viraemia (RT-PCR) and quantify the post-vaccination humoral (ELISA and virus neutralisation test) and cellular (ELISPOT IFNg) immune responses. PRRSV vaccine strain was detected in 60%, 64%, 36% and 0% of A V+ piglets 2, 4, 8 and 14 WPV respectively. No virus was detected in A+V+ piglets during the first four WPV but 32% and 6% of A+V+ piglets were PCR-positive at 8 and 14 WPV. Eighty-five percent of A-V+ piglets and 0% of A+V+ piglets seroconverted (ELISA) between 2 and 4 WPV. Neutralising antibodies appeared 4 WPV in the A-V+ piglets and 14 WPV in the A+V+ piglets. The number of PRRSV-specific IFNg-secreting cells was significantly higher in A V+ piglets at 2 and 4 WPV than in A +V+ piglets. These results show that MDAs can affect both post-vaccination humoral and cellular immune responses in piglets. Further studies are required to assess the impact of MDAs on vaccine efficacy following a PRRSV challenge and its ability to reduce viral transmission. ã 2016 Elsevier B.V. All rights reserved.
1. Introduction Porcine reproductive and respiratory syndrome (PRRS) is a disease affecting pigs worldwide, leading to huge economic losses in the swine industry (Neumann et al., 2005). PRRS is caused by a virus (PRRSV) that belongs to the family Arteriviridae, genus Arterivirus (Cavanagh, 1997). PRRSV is responsible for reproductive disorders in sows and respiratory problems in pigs. Interacting with other infectious pathogens, the virus is also a factor triggering
* Corresponding author at: ANSES, Unité d’Epidémiologie et Bien Etre du Porc, B. P.53, 22440 Ploufragan, France. E-mail address: [email protected] (C. Fablet). http://dx.doi.org/10.1016/j.vetmic.2016.07.014 0378-1135/ã 2016 Elsevier B.V. All rights reserved.
production diseases (Rose et al., 2003; Fablet et al., 2012) as well as food-borne diseases (Beloeil et al., 2004; Salines et al., 2015). Vaccination is one of the main and commonly used tools to control the disease in swine operations. Modified live vaccines (MLVs) have shown their efficacy in limiting the clinical consequences of the infection (Martelli et al., 2009). Recently, these vaccines have also demonstrated their ability to significantly reduce transmission of the virus under experimental conditions (Pileri et al., 2015; Rose et al., 2015). With such theoretical performances, MLVs could be used to control PRRSV propagation within a chronically infected herd and even to eradicate the virus at both herd and regional levels. However, in the field, the ability of MLVs to control PRRSV transmission appears to be much lower than under experimental conditions (Geldhof et al., 2013; Pileri et al., 2015).
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The factors impairing vaccine efficacy under field conditions are not yet well known. One of the putative explanation of this divergence relates to maternally derived antibodies (MDAs), present in piglets born in infected herds and lacking in the SPF pigs used in experimental trials. The influence of maternal immunity has been described for several swine viral diseases (Tielen et al., 1981; Kitikoon et al., 2006; Huang et al., 2014). Interestingly, interference between MDAs and a vaccine was also proven for Equine Viral Arteritis, caused by a virus belonging to the same family as PRRSV (McCollum, 1976). Moreover, PRRSV-specific neutralising antibodies (PRRSV-NAs) transferred from the dam to her piglets by the colostrum can delay their infection by PRRSV (Geldhof et al., 2013). PRRSV-NAs were also found to be able to prevent PRRSV infection in piglets submitted to the passive transfer of sufficient amounts of NA (Lopez et al., 2007). Taken altogether, it thus seems likely that MDAs (in particular PRRSV-NA) present in piglets from infected herds could interfere with PRRS MLVs. However, up to now, no data were available on the impact of passive immunity on PRRS MLVs in piglets. Therefore, the main aim of the current field study was to investigate the influence of MDAs on the development of post-vaccination humoral and cellular immune responses in piglets vaccinated against PRRS. The study also investigated the decay of PRRSV-specific MDAs in nonvaccinated piglets. 2. Material and methods 2.1. Animals and experimental setting The study was performed in accordance with current legislation on ethical and welfare recommendations. ANSES-Ploufragan is certified for animal experimentation and is registered under certification number C-22-745-1 delivered by the official French veterinary services. The experiment was carried out in a PRRS-positive farrow-tofinish field herd which initiated a PRRS control programme six years ago. Based on serological monitoring results from the vets (blood samples from finishing pigs), the herd has been free from PRRSV circulation since 2010. PRRS vaccination is maintained for the breeding herd by security (MLV Porcilis PRRS1, MSD, Beaucouzé, France), the whole breeding herd being vaccinated every 4 months (i.e. blitz vaccination). This negative PRRSV circulation status was confirmed before the study began by ELISAs (PRRS X3 Ab, Idexx, Eragny sur Oise, France) on the sera from 50 market-age pigs. A batch of gestating sows was selected (7 weeks after the last PRRS vaccination). All were bled 4 weeks before the expected farrowing time. Serum samples were tested for antibodies against PRRSV by ELISA (PRRS X3 Ab, Idexx, Eragny sur Oise, France) and the virus neutralisation test (VNT). The six sows with the highest PRRSV-NA titres (mean titre (log2) = 7.1; s = 0.4) and the four with the lowest levels of PRRSV-NA (mean titre (log2) = 2.9; s = 0.5) were selected. All the piglets born to the selected sows were eartagged and weighed at birth. They were bled at 1 week of age. The sera were titrated for PRRSV-NA by VNT. The NA titres established 1
week after birth were designed to confirm the MDA transfer level to offspring. On the basis of these VNT results, 93 piglets were further selected and allocated to four groups (Table 1). A group of 30 piglets with a low level of PRRSV-NAs (A ) and a group of 30 piglets (A+) with a high level of PRRSV-NAs were thus formed. All 60 piglets were vaccinated (V+) at 3 weeks of age with 0.2 ml/pig of Porcilis PRRS1 (MSD, Beaucouzé, France) vaccine by the intradermal route using the IDAL1 system specifically designed for this purpose. These litters were located at the back of the farrowing room and were not in direct contact with the other three selected litters (not vaccinated; A+V ; detailed above). However, indirect transmission of the virus may not be excluded. At weaning (4 weeks of age), the piglets were moved to four pens at the back of a nursery room (three pens with commingled A +V+ and A V+ piglets and one pen of A+V+ piglets). A sample size of 30 animals per group was chosen in order to have 80% chance of showing a significant difference of 35% between A+V+ and A V+ groups (i.e. 20% responding in one group versus 55% in the other one) at a level of 5%. A third group of 21 piglets with intermediate levels of PRRSVNAs was not vaccinated (A+V ). These litters were separated from the vaccinated pigs in the farrowing and nursery rooms (kept at the front of the room, whereas vaccinated litters were at the back). This group was used to investigate the natural waning of MDAs over time. In addition, a group of 12 piglets with low PRRSV-NA titres was not vaccinated (A V ) and used as a sentinel. During the nursery phase, they were in direct contact with A+V+ and A V+ piglets (i.e. three sentinels per pen for all four pens) to assess vaccine strain transmission under field conditions. The batch was moved to the finishing section at 11 weeks of age, keeping the contact structure of the nursery room during the first 2 weeks. Half of the pigs were then moved to a second room. The whole batch of piglets was vaccinated against Mycoplasma hyopneumoniae (Ingelvac Mhyo, Boehringer Ingelheim; 1 and 4 weeks of age) and PCV-2 (Circoflex, Boehringer Ingelheim; 5 weeks old). When doing his daily routine, the farmer followed biosecurity measures to reduce the odds of transmitting the PRRS vaccine strain to non-vaccinated piglets. 2.2. Collection and processing of samples Blood samples were collected 2 days before vaccination (W0) to confirm from serum the PRRSV-negative status of the animals by PCR tests and to evaluate the level of MDAs (ELISA and VNT). The pigs were repeatedly bled at 2, 4, 8 and 14 weeks (W2, W4, W8, W14) post-vaccination (PV). Blood was sampled by jugular vein puncture, using evacuated tubes (Vacuette, Dutscher SAS, Brumath, France) without additives or with heparin to collect either serum or whole blood. Adhesive dressings were applied at the blood sampling site to soak up blood during the clotting process and thus prevent contamination of the environment with the blood of potentially viraemic piglets. Serum was purified by centrifugation for 10 min at 3500 x g. Heparinised blood samples were taken from W0 to W8 in vaccinated pigs in order to isolate peripheral blood mononuclear cells (PBMC). Piglets were weighed at birth and W14 (17 weeks of age).
Table 1 Description of the four groups used in the study (93 piglets; one herd). Group
Mean PRRSV-neutralising antibody titres (log2) at 1 week old (s)
Vaccination
Number of piglets
A V+ A+V+ A V A+V
1.4 (1.5) 7.0 (0.8) <2.3a 4.9 (0.9)
YES YES NO NO
30 30 12 21
a
All titres (log2) were <2.3.
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2.3. Laboratory analyses 2.3.1. Virological analysis PRRSV RNA was isolated from sera using the NucleoSpin 1 RNA virus kit (Macherey-Nagel, Düren, Germany) and amplified using the AdiavetTM PRRS real-time RT-PCR kit (BioMerieux, Marcy l’Etoile, France) in keeping with the instructions of both manufacturers. An animal was considered positive when the Ct was below 45. Sera from A+V piglets were not tested at W4 and W14. 2.3.2. Evaluation of the immune response 2.3.2.1. ELISA tests to detect PRRSV-specific IgG. Sera were tested for the presence of PRRSV-IgG antibodies (targeting the nucleocapsid antigens) (ELISA PRRS X3 Ab, IDEXX Laboratory, Eragny sur Oise, France) according to the kit’s instructions. Results were expressed as sample to positive control (S/P) optical density ratios. A sample was considered positive when the S/P ratio 0.4. 2.3.2.2. Virus neutralisation tests to titrate PRRSV-specific NAs. PRRSV-NAs were quantified in sera from vaccinated (A+V+ and A V+) and non-vaccinated A+V piglets on MARC145 cells against the vaccine strain according to the VNT method described by Charpin et al. (2012). In addition, an indirect immunofluorescence method using a PRRS-hyperimmune serum followed by a fluorescein-labelled anti-porcine IgG antibody (BioRad, Hercules, CA, USA) was used to confirm the NA titres obtained by cytopathogenic effect observation. The NA titres were obtained using the Karber method and expressed as Log2-transformed values. The vaccine strain was obtained by suspending the lyophilised Porcilis PRRS strain on EMEM, then propagated once and titrated on MARC145 cells. 2.3.2.3. ELISPOT assay to quantify PRRSV-specific IFNg -secreting cells. PBMCs were isolated by Ficoll-Paque plus density gradient media (GE Healthcare, Little Chalfont, UK) using LeucoSep centrifuge tubes (Greiner Bio One, Les Ulis, France). PRRSVspecific IFNg-secreting cells (IFNg-SC) were then quantified in triplicate as previously described by Gerner et al. (2006) using a 16-h stimulation of 4 105 PBMCs with a multiplicity of infection of 0.2 for the PRRS vaccine strain (obtained as described above). In addition, each sample was stimulated in triplicate with culture media or 10 mg/ml of PHA (Eurobio, Les Ulis, France) as controls. The number of spots per well were counted by an ELR04 XL ELISPOT reader (AID, Strassberg, Germany). The number of IFNg-SC was calculated by subtracting the mean number of spots obtained for the triplicate vaccine stimulation from the mean of potential unspecific spots obtained for the triplicate negative stimulation, then expressed per 106 PBMC. 2.4. Data analysis The average daily gain between birth and 17 weeks of age was calculated. An analysis of variance was used to compare growth performance between groups (p < 0.05). Post-hoc pairwise comparisons were then performed using the Tukey test to adjust the pvalues of these comparisons given the number of tests conducted (p < 0.05). The PRRSV-NA titres and number of PRRSV-specific IFNg SC in the A+V+ and A V+ groups were compared with a Mann and Whitney test (p < 0.05). The kinetics of PRRSV-specific IgG seroconversion was analysed by survival analysis. The time-toevent for a given animal was the onset of PRRSV-specific IgG in MDA-negative piglets (by ELISA) or an increase in the S/P ratio after a decline in MDA-positive (ELISA+) piglets. The midpoint between the ages at observation for these two measurements was selected
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as the seroconversion time. The groups’ survival distributions were compared (log-rank test, p < 0.05). Statistical analyses were performed using SAS 9.1 and R. 3. Results 3.1. Clinical observations Of the 93 piglets, eight died during the study (6 A+V+, 1 A V+ and 1 A V [sentinel]). Death was mainly related to oedema disease occurring at around 6–7 weeks of age. The average daily gain from birth to 17 weeks was not statistically different in the different groups when using post hoc pairwise comparisons (p > 0.05). 3.2. Viraemia All pigs were RT-PCR negative before vaccination. Vaccine viraemia occurred from W2 to W8 PV in A V+ piglets (Fig. 1). Conversely, viraemia was not detected in A+V+ piglets during the first four weeks PV. The onset of viraemia was observed at W8 PV in this group and 6.3% of A+V+ piglets were viraemic 14 weeks PV. Non-vaccinated piglets were also viraemic at W8 PV (sentinels (A V ) in direct contact with vaccinated piglets as well as A+V piglets in indirect contact). In the group of sentinels, 9.1% were RTPCR positive W14 PV. 3.3. Humoral immune response 3.3.1. PRRSV-specific IgG (targeting the nucleocapsid antigens) In non-vaccinated piglets (A+V ), 10.5% of the animals were found to be still ELISA-positive at 7 weeks old. All the piglets were seronegative 4 weeks later (11 weeks old). The course of PRRSV-IgG seroconversion was significantly different in each group (p < 0.05, log-rank test) (Fig. 2). A V+ piglets seroconverted earlier and at a higher frequency than A+V+ animals and non-vaccinated piglets. In the A V+ group, 85.7% of the animals seroconverted between W0 and W4 PV. In contrast, seroconversion did not occur during the first 4 weeks PV in the other three groups, i.e. A+V+, sentinels and A +V piglets. Thereafter, seroconversion was detected at W8 PV in 28.0% of A+V+ piglets and 36.4% of sentinels. All the remaining pigs from the four groups were ELISA-positive at W14 PV. 3.3.2. PRRSV-specific NAs Before vaccination, PRRSV-NA titres were significantly lower in A V+ piglets (mean titre (log2) < 2.3) than in A+V+ (mean titre (log2) = 4.0) or A+V piglets (mean titre (log2) = 2.2). NA titres increased from W4 to W8 PV in A V+ piglets. On the contrary, those of pigs with MDAs, whether vaccinated against PRRS or not (A+V+ or A+V ), steadily decreased until W8 PV, i.e. 11 weeks of age. At W8 PV, the titre of PRRSV-NAs became significantly higher in A V+ piglets (mean titre (log2) = 2.6; s = 2.2) than in A+V+ (mean titre (log2) < 2.3) or A+V piglets (mean titre (log2) < 2.3) (p < 0.05). The level of PRRSV-NAs was similar in A V+ (mean (log2) = 3.3; s = 1.9) and A+V+ (mean (log2) = 2.9; s = 1.7) pigs at W14 PV (p > 0.05). 3.4. Cell-mediated immune response Piglets in the A+V+ group had a significantly higher number of PRRSV-IFNg-SC before vaccination (mean = 17.6 cells/106 PBMC; s = 17.2) than piglets in the A V+ group (mean = 8.2 cells/106 PBMC; s = 9.2) (p < 0.05). Conversely, the number of PRRSV-IFNgSC increased in A V+ animals from W2 to W8 PV (mean at W8 PV = 124.6 cells/106 PBMC; s = 144.3) and became significantly
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Fig. 1. Course of PRRSV vaccine viraemia over time in groups A-V+ (piglets with a low level of PRRSV-NAs and vaccinated against PRRS; n = 30), A+V+ (piglets with a high level of PRRSV-NAs and vaccinated against PRRS; n = 30), sentinel (piglets with a low level of PRRSV-NAs and not vaccinated against PRRS; n = 12, in direct contact with vaccinated piglets) and A+V (piglets with PRRSV-NAs and not vaccinated against PRRS; n = 21; not in direct contact with vaccinated piglets). Data are expressed as the percentage of RT-PCR positive pigs per group (nt: not tested for A+V ).
Fig. 2. Survival distribution function of time-to-seroconversion (PRRSV-specific IgG) in group A V+ (piglets with a low level of PRRSV-NAs and vaccinated against PRRSV; n = 30), A+V+ (piglets with a high level of PRRSV-NAs and vaccinated against PRRSV; n = 30), sentinel (piglets with a low level of PRRSV-NAs and not vaccinated against PRRSV, in direct contact with vaccinated pigs; n = 12) and A+V (piglets with PRRSV-NAs and not vaccinated against PRRSV, not in direct contact with vaccinated pigs, n = 21).
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higher than in A+V+ piglets (mean at W8 PV = 26 cells/106 PBMC; s = 67.7) (p < 0.05). 4. Discussion Our study was designed to investigate, under field conditions, the immune response following vaccination with an MLV of piglets with or without PRRSV-MDAs. It was furthermore designed to fit in with common pig farming practices whereby piglets are routinely vaccinated at approximately 3 weeks old with an MLV frequently used in French pig herds. The study was carried out in a herd without wild PRRSV circulation among growers and finishers and where PRRSV vaccination was reserved for the breeding herd. The vaccination of dams with an MLV provides piglets with high PRRSV-MDA levels. Furthermore, the absence of PRRSV circulation in the growing and finishing sections facilitates the follow-up of the post-vaccination immune response of MDA and MDA+ piglets without natural PRRSV infection, which is likely to occur in an infected herd. Even though the growing and finishing pigs were regularly monitored for PRRSV to ensure the herd’s continued negative status, the absence of any wild virus circulation was also confirmed on the batch under investigation. The distinction between MDA and MDA+ piglets was based on VNT results at 1 week of age, i.e. on the level of PRRSV-NAs, and not on ELISA results showing a response targeting the nucleocapsid antigens. Since the main function of PRRSV-NAs consists in neutralising the virus (Lopez and Osorio, 2004), which potentially applies to the vaccine strain, VN results were deemed to be relevant and used as the criteria for classifying the piglets according to their level of MDAs. Although an antibody response can be rapidly detected by ELISA after infection, these antibodies (which target nucleocapsid antigens) are not effective in protecting the animal against further infection, and should only be considered as indicators of past exposure to the virus or a passive transfer via the colostrum (Lopez and Osorio, 2004; Lopez et al., 2007). In the group of A- piglets, there was a sub-population of piglets positive by ELISA (data not shown). The post-vaccination humoral and cellular responses of this sub-population were both similar to those of other piglets in the A- group which were ELISA-negative. Furthermore, their immune responses differed from those of A+ and ELISA-positive piglets. Altogether, these data validate the criteria chosen to distinguish groups according to the PRRSV-NA level. After vaccination, the course of vaccine viraemia and the humoral and cellular immune responses of piglets with few MDAs (A V+) were in accordance with those previously described (Martelli et al., 2007; Zuckermann et al., 2007). As early as 2 weeks post-vaccination, the virus was detected in the blood of a majority of piglets from this group. At the same time, an early antibody response was detected by ELISA. The post-vaccination cellular immune response, characterised by the number of IFNgSC, also appeared rapidly. Conversely, PRRSV-NAs were detected later, around 4 weeks after vaccination as previously reported (Lopez and Osorio, 2004; Pileri et al., 2015). In contrast to this group, neither viraemia nor immune response were detected for 4 weeks post-vaccination in the group of piglets having the highest level of maternal PRRSV-NAs at vaccination time. Since Lopez et al. (2007) showed that a sufficient amount of PRRSV-NAs at the time of infection may protect against viraemia, we speculate that the level of maternal NAs at vaccination was sufficient to neutralise viral replication, thus inhibiting the afferent immune response during this time period. As previously described, not only antibodies, but also other immune factors and components (such as cytokines and immune cells) can be transferred in the colostrum (Salmon et al., 2009). In
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the current study, A+V+ animals not only differed from the A-V+ piglets in their humoral but also their cellular immune statuses at vaccination time. Hence, we cannot rule out the possibility that these cells play a complementary role against the vaccine virus in addition to the neutralising function of their PRRSV-NAs. The results of our study indicate that viraemia was detected 8 weeks PV and onwards in a non-negligible proportion of piglets with a high level of MDAs at vaccination time. Meanwhile, there was an increase in the number of IFNg-SC, testifying to the activation of a cell-mediated immune response specific to the vaccine strain. Furthermore, a humoral immune response developed as indicated by the ELISA and VNT results at W14 PV. Together with these findings, a high proportion of sentinel pigs in direct contact with vaccinated and viraemic pigs were detected as viraemic for the first time. The results of the analysis of the ORF5 sequence of A+V+ piglets and sentinels show perfect homology (100%) with the vaccine strain (data not shown), thus confirming vaccine viraemia 8 and 14 weeks PV. Putative explanations of the delayed viraemia in A+V+ piglets compared to A V+ piglets tend to take two directions, though both are related to the loss of MDAs. One hypothesis is based on the prolonged and silent ability of the virus to persist in the host. Several studies have previously shown that infected or vaccinated pigs may harbour the virus in organs such as tonsils without being viraemic (Lopez et al., 2007; Rose et al., 2015). The vaccine viral strain may thus have persisted in the tissues of A+V+ piglets and have further replicated once PRRSVNAs had waned sufficiently. Another explanation may be that the virus was cleared from the body following the action of PRRSV-NAs but that these animals—in direct contact with A V+ piglets—were then re-infected by infectious A V+ piglets still excreting the vaccine strain when maternal immunity had declined. Interestingly, the results of our study indicate that the vaccine strain may be transmitted to susceptible pigs by either direct or indirect contact. Indeed, sentinels in direct contact with vaccinated pigs became viraemic, as did non-vaccinated pigs housed in the same room but not in direct contact with vaccinated pigs. All of them seroconverted by the end of the study, revealing exposure to the virus. Viral transmission when using MLV has already been reported both for North American and European vaccine strains (Mortensen et al., 2002; grosse Beilage et al., 2009). Pigs not directly exposed to a vaccinated pig became viraemic at the same time as piglets in direct contact with the vaccinated pig. As the farmer applied the same biosecurity measures throughout the study, an aerosol transmission of the virus is likely to have occurred, although this cannot be proved because the virus was not sought in the air. In practical terms, the findings of this study under field conditions singularly indicate that for a proportion of piglets vaccinated against PRRS with a MLV at 3 weeks old, the postvaccination immune response may be hampered for at least 4 weeks due to maternal immunity. In order to gain insight into the prevalence of piglets that may be affected, further studies are needed to assess the distribution of the level of PRRSV-NAs in a large sample of 3-week-old piglets. Furthermore, the duration of passive immunity may also be an important point to consider when looking at the appropriate window time for vaccination. Thus, the study also aimed at describing the persistence of PRRSVspecific maternal immunity. Humoral maternal immunity was detected by ELISA in non-vaccinated piglets up to 7 weeks old and by VNT up to 11 weeks old (one pig). Considering that the duration of maternal immunity depends on the initial level acquired by colostrum intake and that the group of piglets had intermediate MDA levels, these results are within the range of values previously published (Nodelijk et al., 1997; Geldhof et al., 2013). The results of the current study show that maternal immunity influences the course of the humoral and cell-mediated responses
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after vaccination against PRRS with an MLV at 3 weeks old. In the current field study, the animals were not naturally or experimentally challenged by a wild virus strain after vaccination. These findings therefore raise the question of the vaccine’s efficacy under such conditions. Further experimental studies are therefore required to test whether the effect of MDAs observed on three immune response parameters could also impair vaccine efficacy after an infectious challenge. It is important to know the impact of MDAs on a vaccine’s ability to protect against infection in terms of clinical and virological parameters. More specifically, from an epidemiological point of view, it is crucial to assess the influence of the level of MDAs on the virus’s transmission and spread within such a vaccinated and challenged population in order to implement a more effective control programme. Acknowledgements The authors are grateful to the pig farmers involved. They would also like to thank the CEA’s TIPIV laboratory for the ELISPOT plate scanning. They are furthermore indebted to the Regional Council of Brittany, the “Union des Groupements de Producteurs de Viande de Bretagne’’, and the European PRRS Research Award 2014 for their financial support. References Beloeil, P.A., Fravalo, P., Fablet, C., Jolly, J.P., Eveno, E., Hascoet, Y., Chauvin, C., Salvat, G., Madec, F., 2004. Risk factors for Salmonella enterica subsp. enterica shedding by market-age pigs in French farrow-to-finish herds. Prev. Vet. Med. 63, 103– 120. Cavanagh, D., 1997. Nidovirales: a new order comprising Coronaviridae and Arteriviridae. Arch. Virol. 142, 629–633. Charpin, C., Mahé, S., Keranflec’h, A., Belloc, C., Cariolet, R., Le Potier, M.F., Rose, N., 2012. Infectiousness of pigs infected by the Porcine Reproductive and Respiratory Syndrome virus (PRRSV) is time-dependent. Vet. Res. 43, 69. Fablet, C., Marois-Créhan, C., Simon, G., Grasland, B., Jestin, A., Kobisch, M., Madec, F., Rose, N., 2012. Infectious agents associated with respiratory diseases in 125 farrow-to-finish pig herds: a cross-sectional study. Vet. Microbiol. 157, 152–163. Geldhof, M.F., Van Breedam, W., De Jong, E., Lopez Rodriguez, A., Karniychuk, U.U., Vanhee, M., Van Doorsselaere, J., Maes, D., Nauwynck, H.J., 2013. Antibody response and maternal immunity upon boosting PRRSV-immune sows with experimental farm-specific and commercial PRRSV vaccines. Vet. Microbiol. 167, 260–271. Gerner, W., Denyer, M.S., Takamatsu, H.H., Wileman, T.E., Wiesmuller, K.H., Pfaff, E., Saalmuller, A., 2006. Identification of novel foot-and-mouth disease virus specific T-cell epitopes in c/c and d/d haplotype miniature swine. Virus Res. 121, 223–228. Huang, Y.-L., Deng, M.-C., Wang, F.-I., Huang, C.-C., Chang, C.-Y., 2014. The challenges of classical swine fever control: modified live and E2 subunit vaccines. Virus Res. 179, 1–11. Kitikoon, P., Nilubol, D., Erickson, B.J., Janke, B.H., Hoover, T.C., Sornsen, S.A., Thacker, E.L., 2006. The immune response and maternal antibody interference to a heterologous H1N1 swine influenza virus infection following vaccination. Vet. Immunol. Immunopathol. 112, 117–128.
Lopez, O.J., Osorio, F.A., 2004. Role of neutralizing antibodies in PRRSV protective immunity. Vet. Immunol. Immunopathol. 102, 155–163. Lopez, O.J., Oliveira, M.F., Garcia, E.A., Kwon, B.J., Doster, A., Osorio, F.A., 2007. Protection against porcine reproductive and respiratory syndrome virus (PRRSV) infection through passive transfer of PRRSV-neutralizing antibodies is dose dependent. Clin. Vaccine Immunol. 14, 269–275. Martelli, P., Cordioli, P., Alborali, L.G., Gozio, S., De Angelis, E., Ferrari, L., Lombardi, G., Borghetti, P., 2007. Protection and immune response in pigs intradermally vaccinated against porcine reproductive and respiratory syndrome (PRRS) and subsequently exposed to a heterologous European (Italian cluster) field strain. Vaccine 25, 3400–3408. Martelli, P., Gozio, S., Ferrari, L., Rosina, S., De Angelis, E., Quintavalla, C., Bottarelli, E., Borghetti, P., 2009. Efficacy of a modified live porcine reproductive and respiratory syndrome virus (PRRSV) vaccine in pigs naturally exposed to a heterologous European (Italian cluster) field strain: clinical protection and cellmediated immunity. Vaccine 27, 3788–3799. McCollum, W.H., 1976. Studies of passive immunity in foals to equine viral arteritis. Vet. Microbiol. 1, 45–54. Mortensen, S., Stryhn, H., Søgaard, R., Boklund, A., Stärk, K.D.C., Christensen, J., Willeberg, P., 2002. Risk factors for infection of sow herds with porcine reproductive and respiratory syndrome (PRRS) virus. Prev. Vet. Med. 53, 83–101. Neumann, E.J., Kliebenstein, J.B., Johnson, C.D., Mabry, J.W., Bush, E.J., Seitzinger, A. H., Green, A.L., Zimmerman, J.J., 2005. Assessment of the economic impact of porcine reproductive and respiratory syndrome on swine production in the United States. J. Am. Vet. Med. Assoc. 227, 385–392. Nodelijk, G., van Leengoed, L.A.M.G., Schoevers, E.J., Kroese, A.H., De Jong, M.C.M., Wensvoort, G., Verheijden, J.H.M., 1997. Seroprevalence of porcine reproductive and respiratory syndrome virus in Dutch weaning pigs. Vet. Microbiol. 56, 21– 32. Pileri, E., Gibert, E., Soldevila, F., Garcia-Saenz, A., Pujols, J., Diaz, I., Darwich, L., Casal, J., Martin, M., Mateu, E., 2015. Vaccination with a genotype 1 modified live vaccine against porcine reproductive and respiratory syndrome virus significantly reduces viremia, viral shedding and transmission of the virus in a quasi-natural experimental model. Vet. Microbiol. 175, 7–16. Rose, N., Larour, G., Le Diguerher, G., Eveno, E., Jolly, J.P., Blanchard, P., Oger, A., Le Dimna, M., Jestin, A., Madec, F., 2003. Risk factors for porcine post-weaning multisystemic wasting syndrome (PMWS) in 149 French farrow-to-finish herds. Prev. Vet. Med. 61, 209–225. Rose, N., Renson, P., Andraud, M., Paboeuf, F., Le Potier, M.F., Bourry, O., 2015. Porcine reproductive and respiratory syndrome virus (PRRSv) modified-live vaccine reduces virus transmission in experimental conditions. Vaccine 33, 2493–2499. Salines, M., Barnaud, E., Andraud, M., Eono, F., Renson, P., Bourry, O., Pavio, N., Rose, N., 2015. Hepatitis E virus chronic infection of swine co-infected with Porcine Reproductive and Respiratory Syndrome Virus. Vet. Res. 46, 55. Salmon, H., Berri, M., Gerdts, V., Meurens, F., 2009. Humoral and cellular factors of maternal immunity in swine. Dev. Comp. Immunol. 33, 384–393. Tielen, M.J., Van Exsel, A.C.A., Brus, D.H.J., Truijen, W.T., 1981. Aujeszky’s disease: serological responsiveness after vaccination of 6–10-week-old piglets with maternal antibody (author’s transl). Tijdschr. Diergeneeskd. 106, 739–747. Zuckermann, F.A., Garcia, E.A., Luque, I.D., Christopher-Hennings, J., Doster, A., Brito, M., Osorio, F., 2007. Assessment of the efficacy of commercial porcine reproductive and respiratory syndrome virus (PRRSV) vaccines based on measurement of serologic response, frequency of gamma-IFN-producing cells and virological parameters of protection upon challenge. Vet. Microbiol. 123, 69–85. grosse Beilage, E., Nathues, H., Meemken, D., Harder, T.C., Doherr, M.G., Grotha, I., Greiser-Wilke, I., 2009. Frequency of PRRS live vaccine virus (European and North American genotype) in vaccinated and non-vaccinated pigs submitted for respiratory tract diagnostics in North-Western Germany. Prev. Vet. Med. 92, 31– 37.
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Veterinary Microbiology journal homepage: www.elsevier.com/locate/vetmic
Short communication
Maternally-derived antibodies (MDAs) impair piglets’ humoral and cellular immune responses to vaccination against porcine reproductive and respiratory syndrome (PRRS) C. Fableta,e,* , P. Rensonb,c,e, F. Eonoa,e, S. Mahéb,e , E. Evenoa,e , M. Le Dimnab,e , V. Normandd , A. Lebretd , N. Rosea,e , O. Bourryb,e a
Agence Nationale de Sécurité Sanitaire (ANSES),ty -2 Unité Epidémiologie et Bien-Etre du Porc, B.P. 53, 22440 Ploufragan, France Agence Nationale de Sécurité Sanitaire (ANSES), Unité Virologie Immunologie Porcines, B.P. 53, 22440 Ploufragan, France c Union des Groupements de Producteurs de Viande de Bretagne (UGPVB), 104 rue Eugène Pottier, CS 26553, 35065 Rennes, France d Porc. Spective, Groupe Vétérinaire Chêne Vert Conseil, 56920 Noyal-Pontivy, France e Université Bretagne Loire, France b
A R T I C L E I N F O
Article history: Received 25 January 2016 Received in revised form 11 July 2016 Accepted 22 July 2016 Keywords: PRRS Vaccination Immune response Maternal neutralizing antibody
A B S T R A C T
The influence of maternally-derived antibodies (MDAs) on the post-vaccination humoral and cellular immune responses in piglets vaccinated against PRRS was studied. The piglets came from a vaccinated breeding herd. Thirty piglets with a low (A ) or high level (A+) of PRRSV-neutralizing MDAs were vaccinated (V+) with a modified live vaccine at 3 weeks of age. Blood samples were collected before vaccination and then at 2, 4, 8 and 14 weeks post-vaccination (WPV). The samples were analysed to detect the vaccine viraemia (RT-PCR) and quantify the post-vaccination humoral (ELISA and virus neutralisation test) and cellular (ELISPOT IFNg) immune responses. PRRSV vaccine strain was detected in 60%, 64%, 36% and 0% of A V+ piglets 2, 4, 8 and 14 WPV respectively. No virus was detected in A+V+ piglets during the first four WPV but 32% and 6% of A+V+ piglets were PCR-positive at 8 and 14 WPV. Eighty-five percent of A-V+ piglets and 0% of A+V+ piglets seroconverted (ELISA) between 2 and 4 WPV. Neutralising antibodies appeared 4 WPV in the A-V+ piglets and 14 WPV in the A+V+ piglets. The number of PRRSV-specific IFNg-secreting cells was significantly higher in A V+ piglets at 2 and 4 WPV than in A +V+ piglets. These results show that MDAs can affect both post-vaccination humoral and cellular immune responses in piglets. Further studies are required to assess the impact of MDAs on vaccine efficacy following a PRRSV challenge and its ability to reduce viral transmission. ã 2016 Elsevier B.V. All rights reserved.
1. Introduction Porcine reproductive and respiratory syndrome (PRRS) is a disease affecting pigs worldwide, leading to huge economic losses in the swine industry (Neumann et al., 2005). PRRS is caused by a virus (PRRSV) that belongs to the family Arteriviridae, genus Arterivirus (Cavanagh, 1997). PRRSV is responsible for reproductive disorders in sows and respiratory problems in pigs. Interacting with other infectious pathogens, the virus is also a factor triggering
* Corresponding author at: ANSES, Unité d’Epidémiologie et Bien Etre du Porc, B. P.53, 22440 Ploufragan, France. E-mail address: [email protected] (C. Fablet). http://dx.doi.org/10.1016/j.vetmic.2016.07.014 0378-1135/ã 2016 Elsevier B.V. All rights reserved.
production diseases (Rose et al., 2003; Fablet et al., 2012) as well as food-borne diseases (Beloeil et al., 2004; Salines et al., 2015). Vaccination is one of the main and commonly used tools to control the disease in swine operations. Modified live vaccines (MLVs) have shown their efficacy in limiting the clinical consequences of the infection (Martelli et al., 2009). Recently, these vaccines have also demonstrated their ability to significantly reduce transmission of the virus under experimental conditions (Pileri et al., 2015; Rose et al., 2015). With such theoretical performances, MLVs could be used to control PRRSV propagation within a chronically infected herd and even to eradicate the virus at both herd and regional levels. However, in the field, the ability of MLVs to control PRRSV transmission appears to be much lower than under experimental conditions (Geldhof et al., 2013; Pileri et al., 2015).
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The factors impairing vaccine efficacy under field conditions are not yet well known. One of the putative explanation of this divergence relates to maternally derived antibodies (MDAs), present in piglets born in infected herds and lacking in the SPF pigs used in experimental trials. The influence of maternal immunity has been described for several swine viral diseases (Tielen et al., 1981; Kitikoon et al., 2006; Huang et al., 2014). Interestingly, interference between MDAs and a vaccine was also proven for Equine Viral Arteritis, caused by a virus belonging to the same family as PRRSV (McCollum, 1976). Moreover, PRRSV-specific neutralising antibodies (PRRSV-NAs) transferred from the dam to her piglets by the colostrum can delay their infection by PRRSV (Geldhof et al., 2013). PRRSV-NAs were also found to be able to prevent PRRSV infection in piglets submitted to the passive transfer of sufficient amounts of NA (Lopez et al., 2007). Taken altogether, it thus seems likely that MDAs (in particular PRRSV-NA) present in piglets from infected herds could interfere with PRRS MLVs. However, up to now, no data were available on the impact of passive immunity on PRRS MLVs in piglets. Therefore, the main aim of the current field study was to investigate the influence of MDAs on the development of post-vaccination humoral and cellular immune responses in piglets vaccinated against PRRS. The study also investigated the decay of PRRSV-specific MDAs in nonvaccinated piglets. 2. Material and methods 2.1. Animals and experimental setting The study was performed in accordance with current legislation on ethical and welfare recommendations. ANSES-Ploufragan is certified for animal experimentation and is registered under certification number C-22-745-1 delivered by the official French veterinary services. The experiment was carried out in a PRRS-positive farrow-tofinish field herd which initiated a PRRS control programme six years ago. Based on serological monitoring results from the vets (blood samples from finishing pigs), the herd has been free from PRRSV circulation since 2010. PRRS vaccination is maintained for the breeding herd by security (MLV Porcilis PRRS1, MSD, Beaucouzé, France), the whole breeding herd being vaccinated every 4 months (i.e. blitz vaccination). This negative PRRSV circulation status was confirmed before the study began by ELISAs (PRRS X3 Ab, Idexx, Eragny sur Oise, France) on the sera from 50 market-age pigs. A batch of gestating sows was selected (7 weeks after the last PRRS vaccination). All were bled 4 weeks before the expected farrowing time. Serum samples were tested for antibodies against PRRSV by ELISA (PRRS X3 Ab, Idexx, Eragny sur Oise, France) and the virus neutralisation test (VNT). The six sows with the highest PRRSV-NA titres (mean titre (log2) = 7.1; s = 0.4) and the four with the lowest levels of PRRSV-NA (mean titre (log2) = 2.9; s = 0.5) were selected. All the piglets born to the selected sows were eartagged and weighed at birth. They were bled at 1 week of age. The sera were titrated for PRRSV-NA by VNT. The NA titres established 1
week after birth were designed to confirm the MDA transfer level to offspring. On the basis of these VNT results, 93 piglets were further selected and allocated to four groups (Table 1). A group of 30 piglets with a low level of PRRSV-NAs (A ) and a group of 30 piglets (A+) with a high level of PRRSV-NAs were thus formed. All 60 piglets were vaccinated (V+) at 3 weeks of age with 0.2 ml/pig of Porcilis PRRS1 (MSD, Beaucouzé, France) vaccine by the intradermal route using the IDAL1 system specifically designed for this purpose. These litters were located at the back of the farrowing room and were not in direct contact with the other three selected litters (not vaccinated; A+V ; detailed above). However, indirect transmission of the virus may not be excluded. At weaning (4 weeks of age), the piglets were moved to four pens at the back of a nursery room (three pens with commingled A +V+ and A V+ piglets and one pen of A+V+ piglets). A sample size of 30 animals per group was chosen in order to have 80% chance of showing a significant difference of 35% between A+V+ and A V+ groups (i.e. 20% responding in one group versus 55% in the other one) at a level of 5%. A third group of 21 piglets with intermediate levels of PRRSVNAs was not vaccinated (A+V ). These litters were separated from the vaccinated pigs in the farrowing and nursery rooms (kept at the front of the room, whereas vaccinated litters were at the back). This group was used to investigate the natural waning of MDAs over time. In addition, a group of 12 piglets with low PRRSV-NA titres was not vaccinated (A V ) and used as a sentinel. During the nursery phase, they were in direct contact with A+V+ and A V+ piglets (i.e. three sentinels per pen for all four pens) to assess vaccine strain transmission under field conditions. The batch was moved to the finishing section at 11 weeks of age, keeping the contact structure of the nursery room during the first 2 weeks. Half of the pigs were then moved to a second room. The whole batch of piglets was vaccinated against Mycoplasma hyopneumoniae (Ingelvac Mhyo, Boehringer Ingelheim; 1 and 4 weeks of age) and PCV-2 (Circoflex, Boehringer Ingelheim; 5 weeks old). When doing his daily routine, the farmer followed biosecurity measures to reduce the odds of transmitting the PRRS vaccine strain to non-vaccinated piglets. 2.2. Collection and processing of samples Blood samples were collected 2 days before vaccination (W0) to confirm from serum the PRRSV-negative status of the animals by PCR tests and to evaluate the level of MDAs (ELISA and VNT). The pigs were repeatedly bled at 2, 4, 8 and 14 weeks (W2, W4, W8, W14) post-vaccination (PV). Blood was sampled by jugular vein puncture, using evacuated tubes (Vacuette, Dutscher SAS, Brumath, France) without additives or with heparin to collect either serum or whole blood. Adhesive dressings were applied at the blood sampling site to soak up blood during the clotting process and thus prevent contamination of the environment with the blood of potentially viraemic piglets. Serum was purified by centrifugation for 10 min at 3500 x g. Heparinised blood samples were taken from W0 to W8 in vaccinated pigs in order to isolate peripheral blood mononuclear cells (PBMC). Piglets were weighed at birth and W14 (17 weeks of age).
Table 1 Description of the four groups used in the study (93 piglets; one herd). Group
Mean PRRSV-neutralising antibody titres (log2) at 1 week old (s)
Vaccination
Number of piglets
A V+ A+V+ A V A+V
1.4 (1.5) 7.0 (0.8) <2.3a 4.9 (0.9)
YES YES NO NO
30 30 12 21
a
All titres (log2) were <2.3.
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2.3. Laboratory analyses 2.3.1. Virological analysis PRRSV RNA was isolated from sera using the NucleoSpin 1 RNA virus kit (Macherey-Nagel, Düren, Germany) and amplified using the AdiavetTM PRRS real-time RT-PCR kit (BioMerieux, Marcy l’Etoile, France) in keeping with the instructions of both manufacturers. An animal was considered positive when the Ct was below 45. Sera from A+V piglets were not tested at W4 and W14. 2.3.2. Evaluation of the immune response 2.3.2.1. ELISA tests to detect PRRSV-specific IgG. Sera were tested for the presence of PRRSV-IgG antibodies (targeting the nucleocapsid antigens) (ELISA PRRS X3 Ab, IDEXX Laboratory, Eragny sur Oise, France) according to the kit’s instructions. Results were expressed as sample to positive control (S/P) optical density ratios. A sample was considered positive when the S/P ratio 0.4. 2.3.2.2. Virus neutralisation tests to titrate PRRSV-specific NAs. PRRSV-NAs were quantified in sera from vaccinated (A+V+ and A V+) and non-vaccinated A+V piglets on MARC145 cells against the vaccine strain according to the VNT method described by Charpin et al. (2012). In addition, an indirect immunofluorescence method using a PRRS-hyperimmune serum followed by a fluorescein-labelled anti-porcine IgG antibody (BioRad, Hercules, CA, USA) was used to confirm the NA titres obtained by cytopathogenic effect observation. The NA titres were obtained using the Karber method and expressed as Log2-transformed values. The vaccine strain was obtained by suspending the lyophilised Porcilis PRRS strain on EMEM, then propagated once and titrated on MARC145 cells. 2.3.2.3. ELISPOT assay to quantify PRRSV-specific IFNg -secreting cells. PBMCs were isolated by Ficoll-Paque plus density gradient media (GE Healthcare, Little Chalfont, UK) using LeucoSep centrifuge tubes (Greiner Bio One, Les Ulis, France). PRRSVspecific IFNg-secreting cells (IFNg-SC) were then quantified in triplicate as previously described by Gerner et al. (2006) using a 16-h stimulation of 4 105 PBMCs with a multiplicity of infection of 0.2 for the PRRS vaccine strain (obtained as described above). In addition, each sample was stimulated in triplicate with culture media or 10 mg/ml of PHA (Eurobio, Les Ulis, France) as controls. The number of spots per well were counted by an ELR04 XL ELISPOT reader (AID, Strassberg, Germany). The number of IFNg-SC was calculated by subtracting the mean number of spots obtained for the triplicate vaccine stimulation from the mean of potential unspecific spots obtained for the triplicate negative stimulation, then expressed per 106 PBMC. 2.4. Data analysis The average daily gain between birth and 17 weeks of age was calculated. An analysis of variance was used to compare growth performance between groups (p < 0.05). Post-hoc pairwise comparisons were then performed using the Tukey test to adjust the pvalues of these comparisons given the number of tests conducted (p < 0.05). The PRRSV-NA titres and number of PRRSV-specific IFNg SC in the A+V+ and A V+ groups were compared with a Mann and Whitney test (p < 0.05). The kinetics of PRRSV-specific IgG seroconversion was analysed by survival analysis. The time-toevent for a given animal was the onset of PRRSV-specific IgG in MDA-negative piglets (by ELISA) or an increase in the S/P ratio after a decline in MDA-positive (ELISA+) piglets. The midpoint between the ages at observation for these two measurements was selected
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as the seroconversion time. The groups’ survival distributions were compared (log-rank test, p < 0.05). Statistical analyses were performed using SAS 9.1 and R. 3. Results 3.1. Clinical observations Of the 93 piglets, eight died during the study (6 A+V+, 1 A V+ and 1 A V [sentinel]). Death was mainly related to oedema disease occurring at around 6–7 weeks of age. The average daily gain from birth to 17 weeks was not statistically different in the different groups when using post hoc pairwise comparisons (p > 0.05). 3.2. Viraemia All pigs were RT-PCR negative before vaccination. Vaccine viraemia occurred from W2 to W8 PV in A V+ piglets (Fig. 1). Conversely, viraemia was not detected in A+V+ piglets during the first four weeks PV. The onset of viraemia was observed at W8 PV in this group and 6.3% of A+V+ piglets were viraemic 14 weeks PV. Non-vaccinated piglets were also viraemic at W8 PV (sentinels (A V ) in direct contact with vaccinated piglets as well as A+V piglets in indirect contact). In the group of sentinels, 9.1% were RTPCR positive W14 PV. 3.3. Humoral immune response 3.3.1. PRRSV-specific IgG (targeting the nucleocapsid antigens) In non-vaccinated piglets (A+V ), 10.5% of the animals were found to be still ELISA-positive at 7 weeks old. All the piglets were seronegative 4 weeks later (11 weeks old). The course of PRRSV-IgG seroconversion was significantly different in each group (p < 0.05, log-rank test) (Fig. 2). A V+ piglets seroconverted earlier and at a higher frequency than A+V+ animals and non-vaccinated piglets. In the A V+ group, 85.7% of the animals seroconverted between W0 and W4 PV. In contrast, seroconversion did not occur during the first 4 weeks PV in the other three groups, i.e. A+V+, sentinels and A +V piglets. Thereafter, seroconversion was detected at W8 PV in 28.0% of A+V+ piglets and 36.4% of sentinels. All the remaining pigs from the four groups were ELISA-positive at W14 PV. 3.3.2. PRRSV-specific NAs Before vaccination, PRRSV-NA titres were significantly lower in A V+ piglets (mean titre (log2) < 2.3) than in A+V+ (mean titre (log2) = 4.0) or A+V piglets (mean titre (log2) = 2.2). NA titres increased from W4 to W8 PV in A V+ piglets. On the contrary, those of pigs with MDAs, whether vaccinated against PRRS or not (A+V+ or A+V ), steadily decreased until W8 PV, i.e. 11 weeks of age. At W8 PV, the titre of PRRSV-NAs became significantly higher in A V+ piglets (mean titre (log2) = 2.6; s = 2.2) than in A+V+ (mean titre (log2) < 2.3) or A+V piglets (mean titre (log2) < 2.3) (p < 0.05). The level of PRRSV-NAs was similar in A V+ (mean (log2) = 3.3; s = 1.9) and A+V+ (mean (log2) = 2.9; s = 1.7) pigs at W14 PV (p > 0.05). 3.4. Cell-mediated immune response Piglets in the A+V+ group had a significantly higher number of PRRSV-IFNg-SC before vaccination (mean = 17.6 cells/106 PBMC; s = 17.2) than piglets in the A V+ group (mean = 8.2 cells/106 PBMC; s = 9.2) (p < 0.05). Conversely, the number of PRRSV-IFNgSC increased in A V+ animals from W2 to W8 PV (mean at W8 PV = 124.6 cells/106 PBMC; s = 144.3) and became significantly
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Fig. 1. Course of PRRSV vaccine viraemia over time in groups A-V+ (piglets with a low level of PRRSV-NAs and vaccinated against PRRS; n = 30), A+V+ (piglets with a high level of PRRSV-NAs and vaccinated against PRRS; n = 30), sentinel (piglets with a low level of PRRSV-NAs and not vaccinated against PRRS; n = 12, in direct contact with vaccinated piglets) and A+V (piglets with PRRSV-NAs and not vaccinated against PRRS; n = 21; not in direct contact with vaccinated piglets). Data are expressed as the percentage of RT-PCR positive pigs per group (nt: not tested for A+V ).
Fig. 2. Survival distribution function of time-to-seroconversion (PRRSV-specific IgG) in group A V+ (piglets with a low level of PRRSV-NAs and vaccinated against PRRSV; n = 30), A+V+ (piglets with a high level of PRRSV-NAs and vaccinated against PRRSV; n = 30), sentinel (piglets with a low level of PRRSV-NAs and not vaccinated against PRRSV, in direct contact with vaccinated pigs; n = 12) and A+V (piglets with PRRSV-NAs and not vaccinated against PRRSV, not in direct contact with vaccinated pigs, n = 21).
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higher than in A+V+ piglets (mean at W8 PV = 26 cells/106 PBMC; s = 67.7) (p < 0.05). 4. Discussion Our study was designed to investigate, under field conditions, the immune response following vaccination with an MLV of piglets with or without PRRSV-MDAs. It was furthermore designed to fit in with common pig farming practices whereby piglets are routinely vaccinated at approximately 3 weeks old with an MLV frequently used in French pig herds. The study was carried out in a herd without wild PRRSV circulation among growers and finishers and where PRRSV vaccination was reserved for the breeding herd. The vaccination of dams with an MLV provides piglets with high PRRSV-MDA levels. Furthermore, the absence of PRRSV circulation in the growing and finishing sections facilitates the follow-up of the post-vaccination immune response of MDA and MDA+ piglets without natural PRRSV infection, which is likely to occur in an infected herd. Even though the growing and finishing pigs were regularly monitored for PRRSV to ensure the herd’s continued negative status, the absence of any wild virus circulation was also confirmed on the batch under investigation. The distinction between MDA and MDA+ piglets was based on VNT results at 1 week of age, i.e. on the level of PRRSV-NAs, and not on ELISA results showing a response targeting the nucleocapsid antigens. Since the main function of PRRSV-NAs consists in neutralising the virus (Lopez and Osorio, 2004), which potentially applies to the vaccine strain, VN results were deemed to be relevant and used as the criteria for classifying the piglets according to their level of MDAs. Although an antibody response can be rapidly detected by ELISA after infection, these antibodies (which target nucleocapsid antigens) are not effective in protecting the animal against further infection, and should only be considered as indicators of past exposure to the virus or a passive transfer via the colostrum (Lopez and Osorio, 2004; Lopez et al., 2007). In the group of A- piglets, there was a sub-population of piglets positive by ELISA (data not shown). The post-vaccination humoral and cellular responses of this sub-population were both similar to those of other piglets in the A- group which were ELISA-negative. Furthermore, their immune responses differed from those of A+ and ELISA-positive piglets. Altogether, these data validate the criteria chosen to distinguish groups according to the PRRSV-NA level. After vaccination, the course of vaccine viraemia and the humoral and cellular immune responses of piglets with few MDAs (A V+) were in accordance with those previously described (Martelli et al., 2007; Zuckermann et al., 2007). As early as 2 weeks post-vaccination, the virus was detected in the blood of a majority of piglets from this group. At the same time, an early antibody response was detected by ELISA. The post-vaccination cellular immune response, characterised by the number of IFNgSC, also appeared rapidly. Conversely, PRRSV-NAs were detected later, around 4 weeks after vaccination as previously reported (Lopez and Osorio, 2004; Pileri et al., 2015). In contrast to this group, neither viraemia nor immune response were detected for 4 weeks post-vaccination in the group of piglets having the highest level of maternal PRRSV-NAs at vaccination time. Since Lopez et al. (2007) showed that a sufficient amount of PRRSV-NAs at the time of infection may protect against viraemia, we speculate that the level of maternal NAs at vaccination was sufficient to neutralise viral replication, thus inhibiting the afferent immune response during this time period. As previously described, not only antibodies, but also other immune factors and components (such as cytokines and immune cells) can be transferred in the colostrum (Salmon et al., 2009). In
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the current study, A+V+ animals not only differed from the A-V+ piglets in their humoral but also their cellular immune statuses at vaccination time. Hence, we cannot rule out the possibility that these cells play a complementary role against the vaccine virus in addition to the neutralising function of their PRRSV-NAs. The results of our study indicate that viraemia was detected 8 weeks PV and onwards in a non-negligible proportion of piglets with a high level of MDAs at vaccination time. Meanwhile, there was an increase in the number of IFNg-SC, testifying to the activation of a cell-mediated immune response specific to the vaccine strain. Furthermore, a humoral immune response developed as indicated by the ELISA and VNT results at W14 PV. Together with these findings, a high proportion of sentinel pigs in direct contact with vaccinated and viraemic pigs were detected as viraemic for the first time. The results of the analysis of the ORF5 sequence of A+V+ piglets and sentinels show perfect homology (100%) with the vaccine strain (data not shown), thus confirming vaccine viraemia 8 and 14 weeks PV. Putative explanations of the delayed viraemia in A+V+ piglets compared to A V+ piglets tend to take two directions, though both are related to the loss of MDAs. One hypothesis is based on the prolonged and silent ability of the virus to persist in the host. Several studies have previously shown that infected or vaccinated pigs may harbour the virus in organs such as tonsils without being viraemic (Lopez et al., 2007; Rose et al., 2015). The vaccine viral strain may thus have persisted in the tissues of A+V+ piglets and have further replicated once PRRSVNAs had waned sufficiently. Another explanation may be that the virus was cleared from the body following the action of PRRSV-NAs but that these animals—in direct contact with A V+ piglets—were then re-infected by infectious A V+ piglets still excreting the vaccine strain when maternal immunity had declined. Interestingly, the results of our study indicate that the vaccine strain may be transmitted to susceptible pigs by either direct or indirect contact. Indeed, sentinels in direct contact with vaccinated pigs became viraemic, as did non-vaccinated pigs housed in the same room but not in direct contact with vaccinated pigs. All of them seroconverted by the end of the study, revealing exposure to the virus. Viral transmission when using MLV has already been reported both for North American and European vaccine strains (Mortensen et al., 2002; grosse Beilage et al., 2009). Pigs not directly exposed to a vaccinated pig became viraemic at the same time as piglets in direct contact with the vaccinated pig. As the farmer applied the same biosecurity measures throughout the study, an aerosol transmission of the virus is likely to have occurred, although this cannot be proved because the virus was not sought in the air. In practical terms, the findings of this study under field conditions singularly indicate that for a proportion of piglets vaccinated against PRRS with a MLV at 3 weeks old, the postvaccination immune response may be hampered for at least 4 weeks due to maternal immunity. In order to gain insight into the prevalence of piglets that may be affected, further studies are needed to assess the distribution of the level of PRRSV-NAs in a large sample of 3-week-old piglets. Furthermore, the duration of passive immunity may also be an important point to consider when looking at the appropriate window time for vaccination. Thus, the study also aimed at describing the persistence of PRRSVspecific maternal immunity. Humoral maternal immunity was detected by ELISA in non-vaccinated piglets up to 7 weeks old and by VNT up to 11 weeks old (one pig). Considering that the duration of maternal immunity depends on the initial level acquired by colostrum intake and that the group of piglets had intermediate MDA levels, these results are within the range of values previously published (Nodelijk et al., 1997; Geldhof et al., 2013). The results of the current study show that maternal immunity influences the course of the humoral and cell-mediated responses
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after vaccination against PRRS with an MLV at 3 weeks old. In the current field study, the animals were not naturally or experimentally challenged by a wild virus strain after vaccination. These findings therefore raise the question of the vaccine’s efficacy under such conditions. Further experimental studies are therefore required to test whether the effect of MDAs observed on three immune response parameters could also impair vaccine efficacy after an infectious challenge. It is important to know the impact of MDAs on a vaccine’s ability to protect against infection in terms of clinical and virological parameters. More specifically, from an epidemiological point of view, it is crucial to assess the influence of the level of MDAs on the virus’s transmission and spread within such a vaccinated and challenged population in order to implement a more effective control programme. Acknowledgements The authors are grateful to the pig farmers involved. They would also like to thank the CEA’s TIPIV laboratory for the ELISPOT plate scanning. They are furthermore indebted to the Regional Council of Brittany, the “Union des Groupements de Producteurs de Viande de Bretagne’’, and the European PRRS Research Award 2014 for their financial support. References Beloeil, P.A., Fravalo, P., Fablet, C., Jolly, J.P., Eveno, E., Hascoet, Y., Chauvin, C., Salvat, G., Madec, F., 2004. Risk factors for Salmonella enterica subsp. enterica shedding by market-age pigs in French farrow-to-finish herds. Prev. Vet. Med. 63, 103– 120. Cavanagh, D., 1997. Nidovirales: a new order comprising Coronaviridae and Arteriviridae. Arch. Virol. 142, 629–633. Charpin, C., Mahé, S., Keranflec’h, A., Belloc, C., Cariolet, R., Le Potier, M.F., Rose, N., 2012. Infectiousness of pigs infected by the Porcine Reproductive and Respiratory Syndrome virus (PRRSV) is time-dependent. Vet. Res. 43, 69. Fablet, C., Marois-Créhan, C., Simon, G., Grasland, B., Jestin, A., Kobisch, M., Madec, F., Rose, N., 2012. Infectious agents associated with respiratory diseases in 125 farrow-to-finish pig herds: a cross-sectional study. Vet. Microbiol. 157, 152–163. Geldhof, M.F., Van Breedam, W., De Jong, E., Lopez Rodriguez, A., Karniychuk, U.U., Vanhee, M., Van Doorsselaere, J., Maes, D., Nauwynck, H.J., 2013. Antibody response and maternal immunity upon boosting PRRSV-immune sows with experimental farm-specific and commercial PRRSV vaccines. Vet. Microbiol. 167, 260–271. Gerner, W., Denyer, M.S., Takamatsu, H.H., Wileman, T.E., Wiesmuller, K.H., Pfaff, E., Saalmuller, A., 2006. Identification of novel foot-and-mouth disease virus specific T-cell epitopes in c/c and d/d haplotype miniature swine. Virus Res. 121, 223–228. Huang, Y.-L., Deng, M.-C., Wang, F.-I., Huang, C.-C., Chang, C.-Y., 2014. The challenges of classical swine fever control: modified live and E2 subunit vaccines. Virus Res. 179, 1–11. Kitikoon, P., Nilubol, D., Erickson, B.J., Janke, B.H., Hoover, T.C., Sornsen, S.A., Thacker, E.L., 2006. The immune response and maternal antibody interference to a heterologous H1N1 swine influenza virus infection following vaccination. Vet. Immunol. Immunopathol. 112, 117–128.
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