Figueras-Gourgues et al. Porcine Health Management (2019) 5:21 https://doi.org/10.1186/s40813-019-0128-7 RESEARCH Open Access Effect of Porcine circovirus 2 (PCV-2) maternally derived antibodies on performance and PCV-2 viremia in vaccinated piglets under field conditions S. Figueras-Gourgues1* , L. Fraile3, J. Segalés4,5, I. Hernández-Caravaca1, R. López-Úbeda6,2, F. A. García-Vázquez1,2, O. Gomez-Duran7 and B. Grosse-Liesner7 Abstract Background: Nowadays, the most common presentation of PCV-2 is the subclinical infection in piglets after weaning. The success of PCV-2 vaccination is associated with the control of the clinical disease as well as the improvement of production parameters. In consequence, the objective of the present study was to analyse the effect of PCV-2 maternally derived antibody (MDA) levels on vaccine efficacy in piglets vaccinated at three weeks of age with a commercial PCV-2 subunit vaccine. The study was performed analysing a database with 6112 wean-to- slaughter piglets from 4 different European regions. Results: Results showed that the use of the vaccine was able to decrease the PCV-2 viremia calculated as area under the curve (AUC = 60.29 ± 3.73), increase average daily weight gain (ADWG = 0.65 ± 0.01 kg/day) and reduce mortality (7%) in vaccinated piglets compared to non-vaccinated ones (AUC of 198.27 ± 6.14, 0.62 ± 0.01 kg/day and 11% respectively). The overall difference of ADWG between both groups was close to 30 g per day (p < 0.05), also when they were split for low and high levels of MDA titres. Moreover, the animals with the highest ADWG were observed in the group of piglets vaccinated with high or extremely high antibody titres (0.66 and 0.65 kg/day respectively). Considering only animals with extremely high antibody titres, both study groups performed similar, however there was a numerical difference of 10 g/day in favour of vaccinated piglets. Likewise, lack of correlation between ADWG and MDA was observed suggesting that no maternal antibody interference was present with the tested vaccine because the vaccinated animals grew faster compared to unvaccinated control animals, regardless of the level of maternal antibodies present at the time of vaccination. Conclusions: The results of the present study demonstrated that the MDA against PCV-2 transferred through the colostrum intake has a protective effect against this viral infection. The vaccine used in the present study (Ingelvac CircoFLEX®) was effective when applied at three weeks of age and was not affected by the level of MDA at the time of vaccination. Keywords: Porcine circovirus type 2, Piglets, Vaccine * Correspondence: sebastian-jose.figueras@boehringer-ingelheim.com 1 Department of Physiology, Faculty of Veterinary, Campus Mare Nostrum, University of Murcia, 30100 Murcia, Spain Full list of author information is available at the end of the article © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Figueras-Gourgues et al. Porcine Health Management (2019) 5:21 Page 2 of 11 Background might pose a risk to vaccination efficacy. Therefore, it Porcine circovirus type 2 (PCV-2) has been associated may be necessary to refine the vaccination timing sched- with a number of clinical conditions in swine that can ules commonly established around weaning for piglets. cause high economic losses for the pig industry [1, 2]. Different laboratory tests may be used for the detec- The most common clinical manifestation of these dis- tion of PCV-2 antibodies in serum. Therefore, serology eases has been post-weaning multisystemic wasting syn- can be regarded as a useful tool for monitoring presence drome (PMWS), which was first described in Canada in of antibodies in pigs and sows, and potentially assess 1991 [3, 4]. The term porcine circovirus diseases their levels at the time of vaccination. Traditionally, the (PCVDs) has been used to compile all conditions linked IPMA (immunoperoxidase monolayer assay) method has to PCV-2 [5], and subsequently these clinical syndromes been used to detect PCV-2 antibodies quantitatively. were reviewed and some new names proposed [6]. Now- However, Guedes et al. [26] showed that the IFAT (in- adays the most common presentation of PCV-2 is the direct fluorescence antibody test) method for the diag- subclinical infection (PCV-2-SI) in piglets after weaning. nosis of the porcine proliferative enteropathy was able to Other described syndromes are: PCV-2 systemic disease offer very similar values to the IPMA (98,6% of agree- (PCV-2-SD, the former PMWS) [7–9], PCV-2 lung dis- ment). PCV-2 antibody levels have been described to be ease (PCV-2-LD), PCV-2 enteric disease (PCV-2-ED) moderate to high in piglets when above 1:320 or its [10–13], PCV-2 reproductive disease (PCV-2-RD) [14– equivalent 2.5 log10 using indirect fluorescence antibody 16] and porcine dermatitis and nephropathy syndrome titration (IFAT) [27]. (PDNS) [17]. However, PCV-2-LD and PCV-2-ED have The objective of the present study was to analyse the been considered as negligible conditions, since the virus effect of PCV-2 MDA levels on vaccine efficacy (mea- linked with lung and intestine inflammation is usually sured by ADWG and PCV-2 viremia) in piglets vacci- found within the framework of PCV-2-SD [18, 19]. nated at three weeks of age with a commercial PCV-2 The success of PCV-2 vaccination is associated with subunit vaccine. The study was performed in four differ- the control of the clinical disease as well as prevention ent farms in Europe. of tissue lesions [1, 20–23]. Even when no overt clinical signs are observed in PCV-2-SI scenarios, several field Results studies have demonstrated that PCV-2 piglet vaccination Descriptive analysis of the effect of vaccination is able to improve production parameters [average daily The first objective was to analyse the effect of the vac- weight gain (ADWG)], percentage of runts, body condi- cine on each of the parameters studied independently tion and carcass weight) [24]. In consequence, the use of (Fig. 1). The mean ADWG was significantly higher in vaccination in farms with clinical PCVD and PCV-2-SI the vaccinated animals (0.65 ± 0.01 kg/day) compared has shifted the general pig health from a period of with the placebo-treated animals (0.62 ± 0.01 kg/day), worldwide severe clinical outbreaks (1997–2007) to self- corresponding to an overall ADWG difference between limiting subclinical infections with occasional outbreaks. the groups of 30 g per day (Fig. 1a; p < 0.05). The same In this widespread vaccination scenario in piglets and result was observed in all farms used in this study; in all the increasing use of PCV-2 vaccines in sows, the mater- of them ADWG was significantly higher in the vacci- nally derived antibody (MDA) levels and their potential nated animals (Table 1; p < 0.05), with a range of ADWG interference with vaccines applied in their offspring [25] differences between groups between 20 and 50 g per day. Fig. 1 Effect of vaccination on average daily weight gain (ADWG), PCV-2 viral load (represented as area under curve-AUC) and PCV-2 antibody titre. a) Comparison of the average daily weight gain (ADWG, kg/day) in placebo-treatment (n = 2728) and vaccinated animals (n = 2835), b) comparison of the PCV-2 viral load as area under curve (AUC) in placebo-treatment (n = 484) and vaccinated animals (n = 472) and c) comparison of the PCV-2 antibody titre (log10) in placebo-treatment (n = 2728) and vaccinated animals (n = 2835). Different letters (a, b) indicate significant differences (P < 0.05) Figueras-Gourgues et al. Porcine Health Management (2019) 5:21 Page 3 of 11 Table 1 Mean of ADWG (kg/day) ± SEM for each of the four studies analysed Treatment Southern Germany [1] Northern Germany [28] United Kingdom [29] France [30] Placebo animals (n = 2728) 0.61 ± 0.01 a (n = 654) 0.65 ± 0.01 a (n = 706) 0.56 ± 0.01 a (n = 657) 0.66 ± 0.01 a (n = 711) b b b Vaccinated animals (n = 2835) 0.64 ± 0.01 (n = 677) 0.67 ± 0.01 (n = 718) 0.61 ± 0.01 (n = 718) 0.68 ± 0.01 b (n = 722) ADWG difference between groups (g/day) 30 20 50 20 a,b n: number of animals. Different letters in the same column indicate significant differences (p < 0.05). Only the surviving animals per treatment group were used The mean value of area under the curve (AUC) in vac- differences (p > 0.05) were found between the experi- cinated animals (60.29 ± 3.73) was significantly (p < 0.05) mental groups (vaccinated vs non-vaccinated) after ana- lower than that of unvaccinated animals (198.27 ± 6.14). lysing the population of animals with extremely high The profile of PCV-2 viral load as AUC at the end of the PCV-2 antibody titres (≥3.7 log10). However, a numerical study is illustrated in Fig. 1b. The overall AUC in vacci- difference (10 g) was observed in favour of vaccinated nated animals was 3.3 times lower than that of placebo- animals (0.65 vs. 0.64 kg/day, respectively) and it must treated animals (p < 0.05). be taken into account the low number of animals in this MDA titres were determined from all animals the day group that decreases significantly the statistical potency. of vaccination. Prior to vaccination PCV-2 antibody ti- It is important to note that for both study groups the tres of both treatment groups were statistically equal in lowest levels of ADWG appear in the group of low both study groups with a mean geometric value of 2.7 PCV-2 antibody titres. log10 (Fig. 1c; p > 0.05). The same result was observed in After analysing the ADWG for the different levels of all farms used in this study; in all of them MDA was sig- PCV-2 antibody titres (low, high and extremely high) in nificantly similar in the vaccinated animals vs. placebo- each one of the study groups (placebo vs. vaccinated ani- treated animals (Table 2; p > 0.05). mals), it is observed that in the case of the placebo animals there are statistical differences between all the levels (0.61, Average daily weight gain (ADWG) according to MDA 0.62 and 0.64) whereas in the vaccinated animals only stat- This analysis was done in order to investigate whether istical differences appear between the low level and the the presence of MDA against PCV-2 had any influence rest, there being no differences between the somewhat on the efficacy of vaccination, in terms of average daily high and extremely high levels (0.66 vs. 0.65). weight gain (ADWG). Figure 2 shows the ADWG de- Because of these results, two further analyses were car- pending on the MDA titres in vaccinated and placebo- ried out to evaluate in depth the group of extremely high treated animals. No significant correlation (p > 0.05) was antibody titres. For the first one, those animals with PCV- observed between these two variables for both treatment 2 antibody titres ≥4.31 log10 (highest antibody titres) were groups. compared for both study groups (placebo-treatment n = The levels of MDA recorded for all animals on the day 158 and vaccinated animals n = 163), without finding sig- of inclusion (3 weeks of age), were used to evaluate the nificant differences between them (p > 0.05). For the sec- distribution of antibody levels in the study population. ond analysis, animals with highest antibody titres (≥4.31 The animals were split in low (< 2.5 log10), high (≥2.5 log10; n = 163) were compared to animals with lowest anti- log10) and extremely high (≥3.7 log10) PCV-2 titres at body titres (≤1.90 log10; n = 202) and only within the vac- the time of vaccination. Figure 3 shows a lack of correl- cinated group finding numerically superior (10 g; p > 0.05) ation between ADWG and MDA of vaccinated or non- differences for the highest antibody titre group. vaccinated animals according to this grouping (p > 0.05). When the ADWG was analysed comparing vaccinated PCV-2 AUC level according to MDA and non-vaccinated animals for low (< 2.5 log10) and When de AUC was analysed for the different levels of high (≥2.5 log10) PCV-2 antibody titres at vaccination PCV-2 antibody titres (low, high and extremely high) in (Table 3), ADWG was significantly higher in the vacci- each one of the study groups (placebo vs. vaccinated ani- nated animals vs the control ones for both groups (low: mals) (Table 4), it is observed that in the case of the pla- 0.64 vs. 0.61 kg/day, respectively; high: 0.66 vs. 0.62 kg/ cebo animals only statistical differences appear between day, respectively; p < 0.05). In contrast, no significant low and extremely high levels (225.0 vs. 147.6) whereas in Table 2 Mean of MDA (PCV-2 antibody titre (log10)) ± SEM for each of the four studies analysed Treatment Southern Germany [1] Northern Germany [28] United Kingdom [29] France [30] Placebo animals (n = 2728) 2.80 ± 0.03 (n = 654) 2.87 ± 0.03 (n = 706) 2.42 ± 0.02 (n = 657) 2.56 ± 0.02 (n = 711) Vaccinated animals (n = 2835) 2.83 ± 0.03 (n = 677) 2.86 ± 0.03 (n = 718) 2.39 ± 0.02 (n = 718) 2.60 ± 0.02 (n = 722) n: number of animals. No significant differences found (p > 0.05). Only the surviving animals per treatment group were used Figueras-Gourgues et al. Porcine Health Management (2019) 5:21 Page 4 of 11 a) b) Fig. 2 Bivariate adjustment between the average daily weight gain (ADWG) and the PCV-2 antibody titre. Graphs show dates for placebo- treatment (n = 2728) a) and vaccinated (n = 2835) animals b). The ADWG of each animal was calculated as the difference in body weight between two measurement periods (4 and 25 weeks of age) divided by the number of days between them the vaccinated animals no differences were found between AUC viremia, the lower the ADWG. This correlation the different levels (63.4, 55.4 and 75.5). After comparing was not observed in vaccinated population (p = 0.06). the study groups for each of the antibody levels, it is ob- served that in all cases the viremia is statistically higher for the placebo animals compared to the vaccinated ani- Mortality mals (low: 225.0 vs. 63.4; high: 188.5 vs. 55.4 and ex- The mortality within vaccinated animals was always tremely high: 147.6 vs. 75.5, respectively; p < 0.05). lower than in control animals at farm level, showing statistical significant differences in two of the studies (p < 0.05) (Table 5). Moreover, merging data from the Average daily weight gain (ADWG) according to PCV-2 four studies, the mortality in vaccinated animals was sig- AUC level nificantly lower than in placebo pigs (7% vs. 11%, re- A significant (p < 0.05) negative correlation (R = − spectively; p < 0.05) (Table 5). However, it was not 0.3748) between ADWG and AUC (Fig. 4) was observed observed a significant association between PCV-2 MDA for non-vaccinated pigs. Thus, the higher the PCV-2 and mortality between farms. a) b) c) d) e) f) Fig. 3 Bivariate adjustment between average daily weight gain (ADWG) and PCV-2 antibody titre divided by PCV-2 titres. Graphs show dates for placebo-treatment (n = 2728) and vaccinated animals (n = 2835). Analysis divided by PCV-2 titres a) placebo low titre (< 2.5 log10; n = 811), b) placebo high titre (≥2.5 log10; n = 1917), c) placebo extremely high titre (≥3.7 log10; n = 300), d) vaccinated low titre (< 2.5 log10; n = 877), e) vaccinated high titre (≥2.5 log10; n = 1958) and f) vaccinated extremely high titre (≥3.7 log10; n = 334) Figueras-Gourgues et al. Porcine Health Management (2019) 5:21 Page 5 of 11 Table 3 Mean of ADWG (kg/day) for placebo and vaccinated animals according to the levels of PCV-2 antibody titre PCV2 titre Placebo animals (n = 2728) Vaccinated animals (n = 2835) Low titre (< 2.5 log10) 0.61 a,A (n = 811) 0.64 a,B (n = 877) High titre (≥2.5 log10) 0.62 b,A (n = 1617) 0.66 b,B (n = 1624) Extremely high titre (≥3.7 log10) 0.64 c,A (n = 300) 0.65 b,A (n = 334) a,b,c Different letters in the same column indicate significant differences (p < 0.05) A, B Different letters in the same row indicate significant differences (p < 0.05). Only the surviving animals per treatment group were used Discussion effective regardless of the level of maternal antibodies At the beginning of the 90s, PCV-2-SD emerged as a present at the time of vaccination. In consequence, the new swine disease, which was characterized by wasting, used vaccine was able to overcome usual levels of MDA, a strong reduction of ADWG and high mortality, decrease the viremia loads and increase ADWG com- appearing mainly between 5 and 12 weeks of life [7, 31]. pared to non-vaccinated piglets [20, 28], with an overall Moreover, the disease caused an increase in the cost of difference of ADWG between groups of 30 g per day production not only due to mortality and loss of both for low and high levels of MDA titres. Moreover, ADWG, but also due to an increase of medication costs according to obtained results, a maternal antibody inter- and the number of animals with no commercial value ference with the efficacy of the tested vaccine was not [32]. As a consequence, a loss of profitability was ob- observed, since irrespective of the maternal antibody served in affected farms. However, the advent of PCV-2 level present at the time of vaccination, the vaccinated vaccines by late 2000s demonstrated that the disease is animals grew faster compared to the non-vaccinated able to be counteracted, and further benefits come from control animals. the prevention of subclinical infections [33]. Likewise, the application of the vaccine was able to re- Antibodies of maternal origin are present in all piglets duce mortality in vaccinated animals. However, it was if appropriate colostrum intake takes place [34]; such not observed a significant association between PCV-2 antibodies in serum gradually decrease during the phases MDA and mortality between farms, probably because of lactation and nursery [35]. PCV-2 viremia usually ap- the mortality was due to many factors (infectious and pears between the final phase of nursery and the start of non-infectious ones) and the maternally immunity the fattening phase, coinciding with the time when against PCV2 is only one of them. MDA reach minimum levels [36, 37], and suggesting The interference of the maternally transferred immun- therefore that MDA confer some protection against the ity (MDI) against any vaccine can be described as a development of the disease. blocking of the antigen by the circulating antibodies or Since the very beginning, piglet vaccination was by other components, presumably cell mediated, ac- proven to be highly efficacious to control PCV-2-SD as quired through colostrum. This is well understood for well as PCV-2-SI [28]. However, taking into account that other swine infectious diseases like Aujeszky Disease vaccination takes place in presence of maternally derived virus (ADV) [38], Classical Swine Fever virus [39], Influ- immunity, the potential interference of MDA with vac- enza A virus (IAV) [40] or Swine Parvovirus [41]. For cine efficacy has to be considered. Interference with ma- example, pigs with significant IAV-specific MDA titres ternal antibodies could be vaccine dependent as the can have suppressed adaptive antibody responses to different vaccines available on the market are based on homologous infection or vaccination [40, 42]. For both different antigen expression systems and also differ in IAV and ADV evidence suggests that the cellular im- antigen quantity, purity, and adjuvants. As a working hy- mune response appears to be less susceptible to MDA pothesis, it was expected that the vaccine used in the [43]. The impact of MDA on active immunisation with present study might be less effective in animals with PCV2 vaccines in young pigs has been studied previously “high” MDA titres. However, the vaccine proved to be [44–46]. One of these studies found that under the Table 4 Mean of area under the curve (AUC) of PCV2 load over time for placebo and vaccinated animals according to the levels of PCV-2 antibody titre PCV2 titre Placebo animals (n = 472) Vaccinated animals (n = 484) Low titre (< 2.5 log10) 225.0 a,A (n = 169) 63.4 a,B (n = 173) High titre (≥2.5 log10) 188.5 ab,A (n = 265) 55.4 a,B (n = 262) Extremely high titre (≥3.7 log10) 147.6 b,A (n = 38) 75.5 a,B (n = 49) a,b Different letters in the same column indicate significant differences (p < 0.05) A, B Different letters in the same row indicate significant differences (p < 0.05). Only 15% of randomly pre-selected study animals, chosen as representative sample animals, were used Figueras-Gourgues et al. Porcine Health Management (2019) 5:21 Page 6 of 11 Fig. 4 Bivariate adjustment between the average daily weight gain (ADWG) and the viraemia (represented as area under curve-AUC). Graphs show dates for a) placebo-treatment (n = 472) and b) vaccinated animals (n = 484) conditions of that trial that age of vaccination rather between both studies. However, the results obtained in than MDA influenced the efficacy of PCVD control. The the present study could be due to the fact that high titers authors concluded that PCV2 vaccination in the pres- of antibodies mean high level of protection to the field ence of high MDA levels is efficacious when used in 3- virus challenge [47]. These animals had an apparent week old but not in 1-week old pigs, suggesting that vac- double protection, the one provided by their own high cine efficacy was independent of the level of MDA. The MDA (passive immunity) and the one by vaccination authors speculated that other age-related factors may (active immunity which overcomes mostly MDA). It is have interfered with the efficacy of the vaccine in 1-week very likely that, in these circumstances, the antibody old piglets [44]. Another study using a different PCV2 levels remain fairly high during the whole life of the ani- vaccine found that while interference at the very highest mal and, therefore, protected against the natural MDA titres could not be excluded the authors con- challenge. cluded that under field conditions this impact is prob- The piglets with a huge amount of antibodies at time ably negligible [46]. The MDA interference effect could of vaccination (≥4.31 log10) showed the same or even result in a lack of passive immunity but also a lack of ac- numerically better growth (10 g; p > 0.05) than those ani- tive immunisation resulting in worst growing perform- mals with the lowest amount of circulating antibodies ance of the animals when challenged. In one study with (≤1.90 log10). This fact suggests that the vaccine antigen high titres in sows induced by vaccination, it was con- was not neutralized by MDA even in the highest anti- cluded that some interference of MDA on the induction body concentrations, reducing the likelihood of sus- of an efficient immune response should be considered pected interference. This observation is in line with the [45]. They concluded that an optimal PCV2 vaccination results obtained in the Feng et al. [46] study where 10 strategy needs to balance the levels of passive immunity, animals with extremely high MDA were compared to the management practices and timing of infection. Con- 157 animals with a lower MDA showing no statistical trary to this statement, this study shows that the pigs differences in growth. that had superior ADWG were those that presented high On the other hand, the present study also shows that or very high MDA titres at the time of vaccination (low: in the unvaccinated population the pigs that grew more 0.64; high: 0.66 and extremely: 0.65 kg/day). This obser- were those that presented high or very high MDA titres vation differs slightly with the results obtained by Feng (low: 0.61; high: 0.63 and extremely: 0.64 kg/day) sup- et al. [46] where the low and high MDA vaccinated pig- porting the fact that MDI can provide some degree of lets (n = 78 and 93 respectively) grew equally. This dif- protection. Similar results were found in the Feng et al. ference could be explained by the size of the groups study [46]. In the case of the groups with extremely high Table 5 Percentage of mortality for each of the four studies analysed and for the merged data Treatment Southern Germany [1] Northern Germany [28] United Kingdom [29] France [30] Merged data a a Placebo animals (%) 14 9 15 6 11 a b b b Vaccinated animals (%) 10 7 5 5 7 a,b Different letters in the same column indicate significant differences (p < 0.05). Merged data refers to the grouping of animals of all the farms studied Figueras-Gourgues et al. Porcine Health Management (2019) 5:21 Page 7 of 11 and highest MDA (≥ 3.7 and ≥ 4.31 log10 respectively), (southern and northern of Germany [1, 28], United no statistical differences in ADWG were observed be- Kingdom [29] and France [30]). A total of 6112 conven- tween the vaccinated and non-vaccinated animals. It tional commercial (1514 southern and 1542 northern of must be taken into account the low statistical power in Germany; 1536 United Kingdom and 1520 France) these groups selected for extremely high MDA. cross-bred piglets from non-vaccinated sows against These observations reinforce the fact that the MDA PCV-2 were used in this work. transferred through the intake colostrum has a protective These four studies were conducted 10 years ago effect against PCV-2 infection. Similar results were found when the disease was in the epizootic phase, so the in previous studies using the same vaccine used in the farms got infected approximately at the same time present trial [46, 48]. While a vaccine effect in relation to and clinical signs were observed and PCV-2-systemic ADWG could not be statistically demonstrated in this disease diagnosed according accepted criteria. In three sub-population of pigs receiving extremely high MDA, it studies [1, 29, 30] a consistent PCV-2 systemic dis- is also true that no conclusive evidence of interference ease (PCV-2 SD) with wasting as a major clinical sign could be determined. In the case of unvaccinated animals was described. In the other study [28], most of the with very and highest MDA titres, they appear to be more clinical signs were respiratory, but PCV-2-systemic protected against PCV-2 and therefore a good growth per- disease was also diagnosed. formance was observed in these groups. The results ob- tained could be explained by a better early protection in Test articles these animals [36] due to the presence of some type of im- An inactivated subunit vaccine (Ingelvac CircoFLEX®, mune factor from colostrum beside antibodies such as Boehringer Ingelheim Vetmedica GmbH) was adminis- lymphocytes, cytokines [34], which could contribute to a tered at three weeks of age for immunization against better basal immunity. On the other hand, the effect could PCV-2. The vaccine contained the ORF2 capsid protein be confounded by the broad benefit that high intake of of PCV-2 as active component and an aqueous polymer colostrum confers to piglets during their whole life [49]. (carbomer) as adjuvant. This protein has been identified This small sub-population of piglets receiving extremely as the major immunogenic antigen of PCV-2 inducing a high MDA might not need an active immunization against protective response [51, 52]. The ORF2 sequence was PCV2 in the context of a well-protected herd. subsequently inserted into a baculovirus expression sys- However, under a practical vaccination approach, tem using an insect cell line derived from ovaries of the where we cannot determine the MDA status of every pig armyworm Sodoptera frugiperda (SF+ cells) as host. The and cannot assume immunity, the entire piglet popula- placebo control consisted of insect cell culture super- tion should be vaccinated to ensure homogeneous and natant without PCV-2 capsid protein but containing car- full protection. Furthermore, these results may support bomer adjuvant. the vaccination of sows since generating high MDA [50] would protect piglets from early infection and would not interfere with active immunization against PCV-2. The Study design present data supports that vaccination against PCV-2 re- All 4 field trials were performed according to the princi- sults in a significant reduction in piglet viraemia irre- ples of “Good Clinical Practice” (GCP) and followed a spective of the initial MDA status, which confirms the randomized, negative-controlled, double-blinded, parallel positive effect of vaccination. Non-vaccinated animals study design. All piglets enrolled into the field studies had higher AUC of viral load and lower ADWG com- received a single dose (1 mL) of the PCV-2 vaccine pared to vaccinated ones, which is in accordance with Ingelvac CircoFLEX® (vaccine) or aqueous polymer adju- what has been obtained in recent study [48]. vant cell culture supernatant (placebo) by intramuscular injection into the neck around weaning (2 to 3 weeks of Conclusions age). Weaning and transfer to the nurseries were per- The results of the present study demonstrated that the effi- formed the day after vaccination (2 to 3 weeks of cacy (in terms of ADWG improvement and viraemia) of the age); pigs were transferred to the fattening units at 9 vaccine used in the present study (Ingelvac CircoFLEX®), weeks of age. All animals (vaccinated or not) were when applied at 3 weeks of age to control PCV-2, was not kept under conventional housing conditions and were affected by the level of MDA at the time of vaccination. mixed in pens to ensure that all study pigs were housed in similar conditions, received the same feed Materials and methods and were subjected to the same management prac- Farm description and situation tices. At each location change, animals were newly The present study was performed using data from four mixed and randomly assigned to the pens according field trials conducted in different locations of Europe to the usual farm procedure. Figueras-Gourgues et al. Porcine Health Management (2019) 5:21 Page 8 of 11 Sample collection and study parameters Average daily weight gain (ADWG) Blood samples were collected on the day of inclusion Weight gain was established as a primary parameter of from all piglets, coinciding with the moment of weaning efficacy. Average daily weight gain (kg/day) of each ani- (2 to 3 weeks of age), and prior to injection (vaccine or mal was calculated as the difference between the body placebo) to determine the presence of PCV-2 antibodies weights of two weighing time points divided by the acquired from maternal colostrum (PCV-2 titre). All ani- number of days between these two weighing time points. mals were also individually weighed at inclusion and be- For each of the four trials analysed, the mean of ADWG fore slaughter (about 3 and 25 weeks of age). Only data was performed on the basis of the ADWG of only the from live/ear-tagged animals at the end of the study surviving animals per treatment group. In the same way were used to carry out further analysis (5563 animals the mean ADWG was calculated using all the data as a [91%]; 2835 and 2728 from vaccinated and control single set. As indicated above, a total of 5563 animals groups, respectively). were used (2835 and 2728 from vaccinated and control For quantification of PCV-2 viremia, blood samples groups, respectively). from 15% of randomly pre-selected study animals, chosen as representative sample animals (total of 956 piglets; 484 from the vaccinated group and 472 from the control PCV-2 viral load in serum group), were collected on weekly or bi-weekly basis Quantification of the PCV-2 viral load in serum was per- throughout the study period. formed at bioScreen GmbH, (Münster, Germany). Sam- ples were analysed in triplicate by a real-time PCV-2 maternally derived antibody (MDA) titre polymerase chain reaction method as described previ- Quantification of the titre of anti-PCV-2 antibodies in ously [55]. PCV-2 DNA quantification was achieved by porcine serum samples from the first blood sampling comparison of the unknown sample with a standard was performed at bioScreen GmbH (Münster, Germany), curve derived from known amounts of PCV-2 ORF plas- using an indirect fluorescence antibody titration (IFAT) mid DNA (104–1012 copies/ml, 10- fold dilution steps) assay. Briefly, 2–6 × 104 PCV-2 susceptible cells (VIDO- and the cut-off level for a positive sample was set as 104 R1 cells [53, 54]) were seeded onto a 96-well plate at 2– template copies per millilitre serum based on validation 6 × 104 cells/well, and inoculated with PCV-2 virus (104.5 experiments. TCID50/well) for approximately 48 h. After fixation of the cells with ethanol, serial dilutions of porcine serum Analysis of PCV-2 viremia parameters samples were added to the plates in triplicate and incu- The mean viral load for each piglet was calculated as the bated for 1 h at 37 °C, allowing antibodies to bind if arithmetic mean of individual viral loads (genomic present in the sera. Plates were washed and stained for equivalents (gE)/ml of serum) of each sampling day. 1 h at 37 °C with a goat-anti-swine FITC-labelled anti- Only animals that could consistently be followed over body (Dianova, Germany, #114–095-003), which allowed the course of the study were included in this calculation antigen detection in infected cells using fluorescence mi- (surviving pigs at the end of the study samples from 15% croscopy. The plates were read by an independent of randomly pre-selected study animals (total of 956 pig- blinded investigator and individual wells reported as lets; 484 from the vaccinated group and 472 from the positive or negative. Serum antibody titres were calcu- control group). lated by the method of Reed and Muench using the For a better understanding of the viraemia dynamics highest dilution still showing specific IFAT reactivity in the present study, PCV-2 qPCR data were also ana- and the number of positive wells per dilution. The lysed using the area under the curve (AUC), as an indi- method allowed the detection of antibody titres in a cator of the viral load over the time of this study. AUC range from 1:5 to 1:20480. For the analysis of MDA ti- was calculated using the time of sampling and the PCV- tres against PCV-2, the data were transformed to base 2 viral load quantified using the trapezoidal method, as 10 logarithm (log10) [25]. As indicated above a total of previously described [47]. In order to analyse differences 5563 animals were used (2835 and 2728 from vaccinated between study groups, the mean value of AUC was cal- and control groups, respectively). culated for each of the populations. According to the MDA titre results (those from the first sampling at 2 to 3 weeks of age), the animals were classified into two different groups: “high” (≥2.5 log10) Mortality and “low” (< 2.5 log10) at the time of vaccination. In Dead animals during the study in each of the farms were addition, a third group was established including the registered by the staff of the same. For each animal, their 10% of the piglets with the highest antibody titres, whose identification number was recorded as well as the date limit was established by the 90th percentile (≥3.7 log10). of death or withdrawal from the study. Figueras-Gourgues et al. Porcine Health Management (2019) 5:21 Page 9 of 11 Statistical analyses this study. J-P Denotte from Clinique vétérinaire Faubourg de Châlons, Vitry All statistical analyses were carried out using the SAS Le François, France. We are grateful to the staff of the Clinic for swine, Lud- wig Maximilians University, Munich, Germany for their support in collecting system V.9.1.3 (SAS institute Inc., Cary, NC, USA). For blood samples and weighing animals. We also acknowledge Prof. Dr. Thomas all analyses, the individual pig was considered as the ex- Blaha and the personnel of the Aussenstelle fur Epidemiologie, Bakum, perimental unit. The farm was included as a variable in Germany for necropsy and collection of organ samples. We are grateful to Christiane Fetzer, Christoph Keller, Volker Ohlinger and the technical staff of the analysis. The significance level (α) was set at 0.05. bioScreen GmbH, Münster, Germany for PCR and serologic analyses. Finally, we thank the pig farmers for their cooperation. Evaluation of the efficacy of the vaccination protocol Authors’ contributions A basal homogeneity analysis was performed to verify SF-G contributed to the design of the study, analysed the data and wrote that the treatment groups were balanced by sex and the draft of the paper. LF analysed the data and wrote the draft of the weight prior the beginning of the trial. Parameters paper. JS wrote the draft of the paper. IH-C contributed to the design of the study. RL-Ú analysed the data and wrote the draft of the paper. FAG-V con- ADWG, AUC and mortality were analysed for assump- tributed to the design of the study. OG-D wrote the draft of the paper. BG-L tion of normality and homogeneity of variances using conceived and designed the experiments. All authors read and approved the the Kolmogorov-Smirnov and Levene test, respectively. final manuscript. Differences between groups for ADWG, AUC and mor- Funding tality were compared using an ANOVA test for normally Not applicable. distributed variables and Kruskall-Wallis test for non- normally distributed ones. Availability of data and materials Not applicable. Maternally derived antibody effects on ADWG Ethics approval A linear regression was performed to evaluate the The field trials were carried out in strict accordance with the strength of association between the PCV-2 antibody titre recommendations in the Good Clinical Practice Guidance Document (VICH at the day of vaccination for vaccinated and control pig- GL9). The protocols were developed following the Legislation for the protection of animals used for scientific purposes (Directive 2010/63/EU) and lets and the ADWG during the whole rearing period all efforts were carried out to minimize suffering. (from 4 to 25 weeks of age). Moreover, a similar linear regression analysis was performed for vaccinated piglets Consent for publication Not applicable. but splitting the animals in low (< 2.5 log10), high (≥2.5 log10) and extremely high (≥3.7 log10) PCV-2 titres, as Competing interests described above. The authors declare that they have no competing interests. Author details Maternally derived antibody effects on viremia (PCV-2 AUC) 1 Department of Physiology, Faculty of Veterinary, Campus Mare Nostrum, Differences between groups (placebo-treatment and vac- University of Murcia, 30100 Murcia, Spain. 2IMIB-Arrixaca, Murcia, Spain. 3 cinated animals) for AUC were performed splitting the Departamento de Ciencia Animal, Universidad de Lleida, Lleida, Spain. 4 Departament de Sanitat i Anatomia Animals, Facultat de Veterinària, animals in low (< 2.5 log10), high (≥2.5 log10) and ex- Universitat Autònoma Barcelona, 08193 Bellaterra, Spain. 5UAB, Centre de tremely high (≥3.7 log10) PCV-2 titres, as described Recerca en Sanitat Animal (CRESA, IRTA-UAB), Campus de la Universitat above. Autònoma de Barcelona, 08193 Bellaterra, Spain. 6Department of Cell Biology and Histology, Faculty of Medicine, Campus Mare Nostrum, University of Murcia, 30100 Murcia, Spain. 7Boehringer Ingelheim Vetmedica GmbH AH PCV-2 AUC effects on ADWG Swine, Ingelheim, Germany. A linear regression was performed to evaluate the Received: 2 May 2019 Accepted: 16 July 2019 strength of association between the PCV-2 AUC for vac- cinated and control piglets and the ADWG during the whole rearing period (from 4 to 25 weeks of age). References 1. Kixmöller M, Ritzmann M, Eddicks M, Saalmüller A, Elbers K, Fachinger V. Abbreviations Reduction of PMWS-associated clinical signs and co-infections by ADWG: Average daily weight gain; AUC: Area under the curve; IAV: Influenza vaccination against PCV2. Vaccine. 2008;26(27):3443–51. A virus; IFAT: Indirect fluorescence antibody test; IPMA: Immunoperoxidase 2. Horlen KP, Schneider P, Anderson J, Nietfeld JC, Henry SC, Tokach LM, et al. monolayer assay; MDA: Maternally derived antibody; MDI: Maternally A cluster of farms experiencing severe porcine circovirus associated disease: transferred immunity; PCV-2: Porcine circovirus 2; PCV-2-ED: Porcine clinical features and association with the PCV2b genotype. 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