Pleguezuelos et al. Porcine Health Management (2021) 7:35 https://doi.org/10.1186/s40813-021-00213-2 RESEARCH Open Access Exploratory field study on the effects of porcine circovirus 2 (PCV-2) sow vaccination at different physiological stages mimicking blanket vaccination Patricia Pleguezuelos1,2* , Marina Sibila1,2, Raúl Cuadrado1,2, Rosa López-Jiménez1,2, Diego Pérez1,2, Eva Huerta1,2, Anna M. Llorens1,2, José Ignacio Núñez1,2, Joaquim Segalés2,3,4 and Sergio López-Soria1,2 Abstract Background: The objective of the present study was to explore the benefits of Porcine circovirus 2 (PCV-2) blanket vaccination in a sow herd on productive parameters, PCV-2 infection and immune status in sows and their progeny. For this purpose, 288 sows were distributed among four balanced experimental groups. One group remained as negative control group and the other three received 1 mL of PCV-2 Ingelvac Circoflex® intramuscularly at different productive cycle moments: before mating, mid gestation (42–49 days post-insemination) or late gestation (86–93 days post-insemination); phosphate buffered saline (PBS) was used as negative control item. Reproductive parameters from sows during gestation and body weight of their progeny from birth to weaning were recorded. Additionally, blood was collected from sows at each vaccination time and piglets at 3 weeks of age. Moreover, up to 4 placental umbilical cords (PUC) per sow were taken at peri-partum. Sera from sows and piglets were analysed for PCV-2 antibody detection using an enzyme-linked immunosorbent assay (ELISA). Sera from sows and PUC were tested to quantify viraemia using a real time quantitative polymerase chain reaction (qPCR) assay. Results: Globally, results indicated that vaccinated sows showed heavier piglets at birth and at weaning, less cross- fostered piglets, lower viral load at farrowing as well as in PUC, and higher antibody levels at farrowing, compared to non-vaccinated ones. When all groups were compared among them, sows vaccinated at mid or late gestation had heavier piglets at birth than non-vaccinated sows, and lower proportion of PCV-2 positive PUC. Also, cross- fostering was less frequently practiced in sows vaccinated at pre-mating or mid gestation compared to non- vaccinated ones. Conclusions: In conclusion, the present study points out that PCV-2 sow vaccination at different time points of their physiological status (mimicking blanket vaccination) offers benefits at production and serological and virological levels. Keywords: Porcine circovirus type 2, Blanket vaccination, Sow, Piglet, Production parameters * Correspondence: patricia.pleguezuelos@irta.cat 1 IRTA, Centre de Recerca en Sanitat Animal (CReSA, IRTA-UAB), Campus de la Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain 2 OIE Collaborating Centre for the Research and Control of Emerging and Re-emerging Swine Diseases in Europe (IRTA-CReSA), Bellaterra, Spain Full list of author information is available at the end of the article © The Author(s). 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. 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Porcine Health Management (2021) 7:35 Page 2 of 12 Background applied on the second cycle at 2 weeks before farrowing, Porcine circovirus 2 (PCV-2) is a small, circular, non- reported higher number of live-born piglets per litter at enveloped, single-stranded DNA virus of 20 nm in diam- the second cycle and higher vitality score from the fist eter approximately, belonging to the genus Circovirus, cycle [13] compared to the non-vaccinated (NV) group. family Circoviridae [1]. This virus is ubiquitous in the Nevertheless, the effect of PCV-2 sow vaccination strat- pig population [2–6] and is the etiologic agent of clinical egies on reproductive parameters has been, up to now, diseases and subclinical infection comprised under the scarcely studied [13, 26]. Furthermore, practices in the term of porcine circovirus diseases [7]. field have explored the option of sow blanket vaccination Intrauterine PCV-2 infections in different stages of (personal communication of field veterinarians); however, pregnancy may cause different reproductive disorders no contrasted data has been scientifically described. depending on the foetal immunological competence [8], Hence, the objective of the present work was to evalu- the so-called PCV-2 reproductive disease. Intrauterine ate the effects of sow vaccination against PCV-2 applied PCV-2 infection of foetuses, via insemination or trans- at different stages of the production cycle (before mat- placental, may cause late-term abortions, mummified, ing, mid gestation and late gestation), mimicking a blan- stillborn and weak born piglets [9–14]. Furthermore, ket vaccination fashion, on productive parameters as pigs may be born PCV-2 viraemic due to transplacental well as on virological and serological parameters in sows infection that may subsequently act as an infection focus and their progeny. for their pen mates. All these situations imply that in- fected sows have a very important role in PCV-2 infec- Results tion maintenance and dissemination in the herd due to Productive parameters horizontal and vertical infection transmission. Productive parameters from gilts/sows and piglets from Nowadays, vaccination is a very effective tool to con- the three vaccinated (V) groups taken together and the trol PCV-2 infection. From 2007 to present, four major non-vaccinated (NV) group are shown in Table 1. The PCV-2 vaccines have been marketed worldwide [15], but comparison of these parameters among each treatment only two are licensed in several countries for their use in group are detailed in Table 2. In both tables, statistically sows to protect their progeny. Therefore, current prod- significant differences and tendencies are indicated. ucts allow applying different vaccination regimes com- bining piglet and/or sow vaccination [16, 17]. These abovementioned vaccines are an inactivated PCV-2a vac- PCV-2 antibody values in serum samples of sows and cine, a recombinant vaccine based on an inactivated piglets PCV-1/PCV-2a chimeric virus or subunit vaccines based PCV-2 IgG and IgM ELISA optical density (OD) values of sows on a PCV-2a Cap protein. Specifically, the vaccine tested Sow blood samples collected at the three vaccine/PBS in this study, Ingelvac Circoflex®, is a PCV-2a subunit application times as well as at farrowing were used to vaccine based on the product of the ORF2 gene determine the dynamics of IgG antibodies against PCV- expressed in a baculovirus system [15]. However, cross- 2 (Fig. 1). Each of the different V treatments showed sig- protection between the major genotypes worldwide nificantly higher IgG values than the NV group from the (PCV-2a, PCV-2b and PCV-2d) has been observed in ex- sampling after their vaccine application until farrowing. perimental trials and field studies [18–20]. At this point, no statistically significant differences be- Different studies have shown the capacity of the sow tween sows vaccinated at mid (V MG) and late (V LG) vaccination to induce an immune response and the trans- nor between sows vaccinated at pre-mating (V PM) and fer of passive immunity to the offspring. Sow vaccination V MG group were observed. before mating stabilizes and homogenizes the PCV-2 im- In general terms, the PCV-2 IgM OD values of the mune status of the sow population during gestation [21– four groups were very low (mean OD values between 24]. Sow vaccination administered before farrowing can 0.40 ± 0.14 and 0.51 ± 0.20 at the different sampling confer protection through maternally derived immunity to points, data not shown) and no statistical differences in their offspring against PCV-2 systemic disease by reducing OD values between each experimental group at any time viraemia [16], lesions and viral load in tissues [25] as well point were found. as increasing their average daily weight gain in PCV-2 subclinical infection [22]. When this vaccination strategy PCV-2 IgG ELISA S/P values of weaned pigs is boosted in the following reproductive cycles at 3 weeks Blood samples from 4 to 6 randomly selected piglets per before farrowing (for 3 years), an improvement of the each sow were taken at weaning and used to detect reproduction rate, number of piglets born alive, birth PCV-2 antibodies. Mean PCV-2 S/P values per treatment weight of piglets and number of piglets weaned per a litter groups are represented in Fig. 2. Piglets from V sows was achieved [26]. Another study, where the boost was showed significantly higher PCV-2 S/P values than Pleguezuelos et al. Porcine Health Management (2021) 7:35 Page 3 of 12 Table 1 Productive parameters (mean ± SD or proportion [percentage] plus confidence interval [CI]) of V and NV sows V NV Total born piglets /sow 14.1 ± 3.0a 13.6 ± 3.6a a Live born piglets/sow 13.4 ± 3.0 12.9 ± 3.7a Weaning-to-mate interval (days) 4.6 ± 5.1a 4.6 ± 5.0a a Weaning-to-fecundation interval (days) 30.7 ± 7.0 30.2 ± 5.3a Weaned piglets/sow 12.1 ± 2.7a 11.8 ± 3.7a a Abortions 4/180 (2.2%) 2/53 (3.8%)a CI: 0.1–4.4% CI: − 1.4–8,9% Mummies 34/2424 (1.4%)a 15/695 (2.2%)a CI: 0.9–1,9% CI: 1.1–3.2% Stillbirth 105/2424 (4.3%)a 24/695 (3.5%)a CI: 3.5–5.1% CI: 2.1–4.8% Dead suckling piglets 213/2295 (9.3%)a 58/658 (8.8%)a CI: 8.1–10.5% CI: 6.6–11.0% Cross-fostered piglets 127/2079 (6.1%)a 53/600 (8.8%)b CI: 5.1–7.1% CI: 6.6–11.1% Piglets birth weight (Kg) 1.64 ± 0.39a 1.58 ± 0.38b Weaned piglet weight (Kg) 6.51 ± 1.48a 6.37 ± 1.48b Different letters in superscript in a row indicate significant differences (p ≤ 0.05) between V and NV groups (highlighted in bold) V Vaccinated, NV Non-vaccinated piglets from NV sows. The highest values were obtained assess the proportion of PCV-2 qPCR positive sera from in piglets from V MG and V LG sows. V and NV animals (Table 3). Only three sows were positive to qPCR through the study: One V animal from V PM group was positive at qPCR to detect PCV-2 mid gestation sampling point (104.2 PCV-2 copies/mL), PCV-2 DNA in sow serum samples and two NV sows were positive at farrowing (one with Blood samples from sows taken at the three vaccine/PBS 105.3 PCV2 copies/mL and another one below the limit application times as well as at farrowing were used to of quantification (LOQ = 104.00 PCV-2 genome copies/ Table 2 Productive parameters (mean ± SD or proportion [percentage] plus CI) of the four studied experimental groups V PM V MG V LG NV Total born piglets /sow 14.1 ± 2.8a 13.9 ± 2.5a 14.3 ± 3.5a 13.6 ± 3.6a Live born piglets/sow 13.3 ± 2.7a 13.5 ± 2.6a 13.4 ± 3.6a 12.9 ± 3.7a a a a Weaning-to-mate interval (days) 4.2 ± 3.6 4.7 ± 5.9 4.8 ± 5.7 4.6 ± 5.0a Weaning-to-fecundation interval (days) 30.7 ± 7.7a 30.6 ± 6.7a 30.7 ± 6.6a 30.2 ± 5.3a a a a Weaned piglets/sow 12.0 ± 2.5 12.2 ± 2.4 12.1 ± 3.1 11.8 ± 3.7a Abortions 1/60 (1.7%)a 3/59 (5.1%)a 0/61 (0.0%)a 2/53 (3.8%)a CI: −1.6-4.9% CI: −0.5-10.7% CI: 0.0% CI: −1.4–8,9% Mummies 9/815 (1.1%)A 10/753 (1.3%)A,B 15/856 (1.8%)A,B 15/695 (2.2%)B CI: 0.4–1.8% CI: 0.5–2.1% CI: 0.9–2.6% CI: 1.1–3.2% Stillbirth 37/815 (4.5%)a 27/753 (3.6%)a 41/856 (4.8%)a 24/695 (3.5%)a CI: 3.1–6.0% CI: 2.3–4.9% CI: 3.4–6.2% CI: 2.1–4.8% Dead suckling piglets 74/769 (9.6%)a 67/727 (9.2%)a 72/799 (9.0%)a 58/658 (8.8%)a CI: 7.5–11.7% CI: 7.1–11.3% CI: 7.0–11.0% CI: 6.6–11.0% Cross-fostered piglets 40/694 (5.8%)a,A,B 33/659 (5.0%)a, A 54/726 (7.4%)a,b,B 53/600 (8.8%)b,A,B CI: 4.0–7.5% CI: 3.3–6.7% CI: 5.5–9.3% CI: 6.6–11.1% Piglets birth weight (Kg) 1.62 ± 0.40a,b 1.66 ± 0.38a 1.64 ± 0.39a 1.58 ± 0.38b Weaned piglet weight (Kg) 6.49 ± 1.50a 6.52 ± 1.48a 6.52 ± 1.45a 6.37 ± 1.48a Different letters in superscript in a row indicate significant differences (p ≤ 0.05 for lower case letters) or tendency (p ≤ 0.10 for capital letters) among experimental groups (highlighted in bold) V PM Vaccinated pre-mating, V MG Vaccinated at mid gestation, V LG Vaccinated at late gestation, NV Non-vaccinated Pleguezuelos et al. Porcine Health Management (2021) 7:35 Page 4 of 12 Fig. 1 PCV-2 IgG OD results (mean ± SD) form sows’ serum samples of the four experimental groups. V PM: vaccinated pre-mating; V MG: vaccinated at mid gestation; V LG: vaccinated at late gestation; NV: non-vaccinated. Different letters indicate statistically significant differences (p ≤ 0.05) among experimental groups for a given sampling point mL) with 103.9 PCV2 copies/ml). At farrowing sampling significantly lower (p = 0.01) in V sow groups (12/142 pools, point, a lower (p = 0.01) proportion of positive PCV-2 9% [CI: 3.9–13.0%]) compared to NV group (10/42 pools, qPCR in serum was detected in V sows (0 out of 172) 24% [CI: 10.9–36.7%]). When the comparison between each compared to NV ones (2 out of 49). experimental group was performed, animals from V LG group had a significantly lower (p = 0.01) proportion of PCV-2 DNA in serum samples from placental umbilical cord PCV-2 qPCR positive pools of PUCs (2/45 pools, 4% [CI: − pools 1.6-10.5%]) compared to NV sows (10/42 pools, 24% [CI: Blood from 3 (ranging 1 to 4) porcine umbilical cords 10.9–36.7%]). Additionally, a tendency (p = 0.08) in the pro- (PUCs) per sow were individually collected at farrowing to portion of PCV-2 qPCR positive pools of PUCs were noted quantify virus in serum samples. All these samples were in dams from V MG group (4/47 pools, 9% [CI: 0.5– processed as a pool of 2–3 PUC sera/sow at farrowing (n = 16.5%]) when compared to NV sows (10/42 pools, 24% [CI: 171), except when only one PUC was collected (n = 13). 10.9–36.7%]). Moreover, a lower (p = 0.01) PCV-2 load in The number of PCV-2 qPCR positive pools of PUCs was PUC pools was observed in sows from the V groups (below Fig. 2 PCV-2 IgG S/P values (mean ± SD) from weaned piglets’ serum samples of each experimental group. V PM: vaccinated pre-mating; V MG: vaccinated at mid gestation; V LG: vaccinated at late gestation; NV: non-vaccinated. Different letters indicate significant differences among experimental groups (p ≤ 0.05) Pleguezuelos et al. Porcine Health Management (2021) 7:35 Page 5 of 12 Table 3 Proportion of PCV-2 qPCR positive sera in sows and CI genome copies/mL) were sequenced to ascertain the main from V and NV groups PCV-2 genotype/s circulating in the farm. Specifically, one Sampling point positive serum sample from the V PM group collected at Pre-mating Mid gestation Late gestation Farrowing mid gestation sampling point and 7 individual PUC sam- V 0/186 (0.0%) 1/178 (0.6%) 0/177 (0.0%) 0/172 (0.0%)a ples recovered from 3 pools (three from the NV, two from CI: 0.0% CI: −0.5-1.7% CI: 0.0% CI: 0.0% the V MG and two from the V PM sow groups). The NV 0/60 (0.0%) 0/52 (0.0%) 0/51 (0.0%) 2/49 (4.1%)b phylogenetic tree including the relationships among the CI: 0.0% CI: 0.0% CI: 0.0% CI: −1.5-9.6% ORF2 sequences determined in this study and reference Different letters in superscript indicate significant differences (p ≤ 0.05) among strains is presented in Fig. 3. Genotype PCV-2b was found experimental groups V Vaccinated, NV Non-vaccinated in the V PM sow serum sample from the mid gestation sampling point (MT572494 S 4525) and in 3 PUC from the same NV sow, all three with identical sequence (MT572495 PUC 3978). These two sequences showed 99, limit of detection [LOD] [100.42 ± 101.43 PCV-2 genome 54% of nucleotide identity between them. Genotype PCV- copies/mL]) compared to NV ones (below LOD [101.12 ± 2d was found in two PUC from a V PM sow (both with 102.11 PCV-2 genome copies/mL]). No statistically signifi- identical sequence, MT572497 PUC 4182) and in other cant differences were observed in PCV-2 load in PUC pools two PUC from a V MG sow (also with the same sequence, when the comparison was done between each experimental MT572496 PUC 4035). These two sequences were identi- group by separate. cal between them as well. PCV-2 genotyping Discussion PCV-2 ORF2 from serum and PUC samples with some of The present study deals with a poorly described topic such the highest PCV-2 loads (between 104.19 and 108.06 PCV-2 as the effects of sow vaccination against PCV-2 on Fig. 3 Phylogenetic tree derived from PCV-2 capsid protein (ORF2) sequences. The tree was constructed by using Maximum-Likelihood method with 1000 bootstrap replicates. Bootstrap values higher than 70 are indicated. Sequences from this study are highlighted in red and labelled with the accession number + PUC (for PUC samples) or S (for sow sample) + sow number Pleguezuelos et al. Porcine Health Management (2021) 7:35 Page 6 of 12 productive and infectious parameters of sows and their sows were vaccinated pre-mating and no differences progeny. To accomplish this task, sows and gilts were vac- were observed in weaned piglet weight at 4 weeks of age. cinated at three different reproductive periods (before Reasons for such differences may be attributable to the mating, mid gestation and late gestation). When all the different vaccine product or schedule used and/or lack vaccinated sow groups were joined (independently of the of power due to the limited sample size of the study. moment of vaccine application), mimicking a blanket Moreover, there are many inter-farm factors (animal fashion vaccination strategy, significant improvement of genetics, farm facilities, management practices, treat- productive parameters in terms of piglets’ weight at birth ments, nutrition and vaccination schedule) that may in- and at weaning and cross-fostering practice reduction fluence the vaccination outcome. were achieved. Moreover, a significant reduction in the Piglets coming from V sows were significantly less proportion of PCV-2 infected sows and their viral loads at cross-fostered compared to the NV group; more specif- farrowing and in PUC were also observed when compared ically, significantly less cross-fostering was performed in to NV sows, although a low PCV-2 infection pressure was V PM and V MG dams compared to the NV group. detected. When the period of vaccine application was con- Also, a tendency to practice less cross-fostering in pig- sidered, a tendency in reduction of mummies in the V PM lets from V MG dams was noted compared to that in V group compared to NV one was observed. LG sows. Cross-fostering is a practice frequently used to Few published studies describe the benefits of sow vac- increase piglet survival and to organize litters with uni- cination on reproductive parameters under field condi- form body weight [28]. However, biosecurity procedures tions. This is probably due to the low frequency of recommend minimizing the number of cross-fostered reproductive disorders associated with PCV-2 [26], and animals [29]; therefore, reduction of this practice may their rather unknown impact. Currently, only five peer- help diminishing PCV-2 transmission among piglets. reviewed published studies recorded reproductive pa- A tendency of a lower proportion of mummies was rameters in vaccinated sow farms. In one study [22], observed in the V PM sow group compared with the NV dams were vaccinated against PCV-2 at 4 and 2 weeks group. Considering that mummification is an outcome before artificial insemination and 4 weeks pre-partum. In of late reproductive problems [30], the potential benefits another one [16], sows were vaccinated 1 week prior to of PCV-2 vaccination at pre-mating on mummies reduc- mating. In the third one [26], PCV-2 sow vaccination tion was expected. This result is in contrast with Kur- was implemented for 3 years; dams were vaccinated at 6 mann et al. [22] and Sibila et al. [24], where sows were and 3 weeks before farrowing in the first reproductive vaccinated pre-mating, and with Oliver-Ferrando et al. cycle and boosted in the following reproductive cycles at [13], where sows were vaccinated at mid-late gestation. 3 weeks before farrowing. In the fourth study [13], dams Further studies with a higher number of tested sows were immunized against PCV-2 at 6 and 3 weeks pre- would be needed to confirm the data obtained herein. farrowing on the first cycle and sows received a boost 2 In relation to stillborn, no statistical differences were weeks pre-farrowing on the second cycle. Finally, in the observed in the current study, in accordance with last one [27], three groups of sows were PCV-2 vacci- Cybulski et al. study [27]. Oliver-Ferrando et al. [13] nated 1 day after weaning, 28 days after weaning and found an inconsistent situation, where a higher number non-vaccinated, respectively. In the present study, in of stillbirths/litter in the V group was observed in the comparison with the abovementioned works, the sow first reproductive cycle but not in the second one. vaccination scheme considered PCV-2 vaccination at dif- Nevertheless, proportions of stillborn detected in the ferent reproductive time points in the same herd, resem- present study and in Oliver-Ferrando et al. [13] are bling a blanket fashion vaccination strategy. aligned with regular values expected in the average In the present study, piglets from V sows showed a Spanish pig farm (8.6%) according to national records significantly higher mean birth weight. Among all vac- (www.bdporc.irta.es). cination groups, the highest body weights were obtained Although Oliver-Ferrando et al. [13] and Pejsak et al. in piglets coming from V MG and V LG groups. Consid- [26] reported an improvement in number of liveborn ering the short period between vaccination at late gesta- piglets, in the present case only a positive (numerically, tion and the delivery, the difference in weight between but non-significant) effect was obtained similar to the piglets from V LG and NV groups was unexpected. This study of Kurmann et al. [22] and in 28 days after wean- result, however, would be similar to the one obtained in ing vaccination group from Cybulski et al. study [27]. one study in which sows were also vaccinated at late ges- In addition, no significant differences in the proportion tation [26]. Moreover, the weight of weaned piglets (at 3 of abortions were detected in the present study similarly weeks of age approximately) coming from V sows was to results obtained in Kurmann et al. study [22]. Consid- higher compared to those from NV dams. These results ering the low frequency of abortions detected in the are in contrast with the ones of Fraile et al. [16], where present study, a larger sample size would be necessary to Pleguezuelos et al. Porcine Health Management (2021) 7:35 Page 7 of 12 analyse this parameter and determine their effect. Never- place piglets from V sows in a better immune position theless, the design of our study was not within the scope to counteract PCV-2 infection at early ages [16]. As ex- of detecting the presence of significant differences for pected, the highest MDA transfer was observed in this latter parameter. weaned piglets from V MG and V LG groups, followed Similarly, Pejsak et al. [26] and Oliver-Ferrando et al. by V PM. On the other hand, considering that all piglets [13] detected a higher number of weaned piglets/litter (be- were vaccinated at 3 weeks of age against PCV-2, certain ing only statistically significant in Pejsak) in V dams com- concerns regarding MDA interference with vaccination pared to NV dams, respectively. These two latter studies could arise. Although in this study MDA interference indicated that the repeated use of PCV-2 sow vaccination with pig vaccination was not explored, several studies can improve reproductive parameters. So, these positive have reported evidence of such interference with vaccine effects (piglets born alive and weaned piglets/litter) may humoral immune response [16, 35–38]. However, the ef- probably be more evident after several gestational cycles ficacy of the same vaccine used in this study has not vaccinating the sows against PCV-2 rather than one single shown to be jeopardized by high values of MDA [39]. vaccination of sows as in the present study. Two PCV-2 genotypes, PCV-2b and PCV-2d, circulated The antibody profile from sows immunized against in the herd, being the most prevalent ones in the field PCV-2 revealed seroconversion after each vaccination, worldwide [40, 41]. The co-circulation of two or more conferring a stronger herd immunity against PCV-2 PCV-2 genotypes in the same farm is not unusual [42– compared to the NV [10, 13, 22, 24]. Besides, the NV 44]. Current vaccines based on PCV-2a strains appear to group maintained low ELISA OD values during all gesta- be able to cope with major circulating strains worldwide tion period. In this sense, despite sow infection remained due to cross-protection among genotypes [3, 18–20]. low or non-detectable in all studied groups during the PCV2 vaccines are the most sold preventive product gestation period, no V sows were found infected at far- in the porcine industry and one of the biological rowing while 4% of NV ones were qPCR positive. Al- products in swine with highest return of inversion though significant differences at the farrowing period in [15]. Besides, the combined vaccination of sows and terms of percentage of viraemic sows was detected, the piglets is an increasing practice since provides the op- number of PCV-2 qPCR positive sera in sows was low. timal performance in animals [16, 45], and although These results, however, should not be considered sur- there is little information about gilt/sow prising since the number of infected sows tend to be low immunization, vaccination of pigs and sows has been in most farms where subclinical infection is taking place estimated cost-efficient [15]. However, each farm [8, 13, 31, 32]; moreover, batch differences may also ac- should be analysed case by case to study the cost count for certain variability. Therefore, further studies /benefit of PCV-2 vaccination [46]. would be desirable to further confirm this finding. The virological results obtained in the study were in Conclusions fairly contrast with those obtained in the screening prior Vaccination of the breeding herd (gilts and sows) against to the start of the study, where PCV-2 DNA was detected PCV-2, mimicking a blanket fashion schedule in a sub- in serum samples from 18 out of 30 clinically healthy clinical infection scenario, improved immune status of sows. Moreover, PCV-2 genome was also detected in 24 dams and the progeny against the virus and reduced out of 30 PUCs from the same sows. These initial results virus circulation at farrowing in sows and vertical infec- would fit well with those observed by other research tion to foetuses, although a low infectious pressure was groups in some PCV-2-SI farms in absence of vaccination detected during the study. PCV-2 sow vaccination also [33]. It is very likely that differences on percentage of in- improved piglets’ weight at birth and at weaning and fected sows among batches and the PCV-2 vaccination in reduced cross-fostering practice. Also, a tendency in ¾ of the studied groups could explain the variability and reduction of mummies from V PM group compared reduction of the PCV-2 infection pressure within the same to NV was observed. Due to the limited PCV-2 circu- farm when the large study was carried out. It is speculated lation detected in the dams during the present ex- that the reduction of the PCV-2 infection pressure prob- ploratory study, the impact of PCV-2 vaccination on ably caused a reduction of transplacental transmission of the productivity was considered of low impact. Fur- PCV-2 to foetuses, evidenced by the lower proportion of ther studies should be needed to confirm the cur- PUC PCV-2 qPCR positive samples and viral load de- rently obtained results. tected compared to the farm previous screening. The higher antibody levels of V sows than NV ones at Materials and methods farrowing suggests a higher transfer of maternally de- Farm selection rived antibodies (MDA) to piglets of V sows via colos- Inclusion criteria for farm selection were: a) sows/gilts trum as also described [16, 22, 34]. This scenario should housed in a single conventional farm (site I), b) no PCV- Pleguezuelos et al. Porcine Health Management (2021) 7:35 Page 8 of 12 2 vaccination schedule in gilts/sows, c) allowance of con- involved in study design and monitoring, vaccine dispen- trolled cross-fostering practices, and d) evidence of sation and for personnel involved in body weight and PCV-2 infection by means of viral genome detection in sample collection, whereas it was masked for farm sows and PUCs in site I. Indeed, prior to the start of the personnel (routine management, daily observation and study, PCV-2 DNA was detected in serum samples from routine reproductive data recording) as well as for la- 18 out of 30 (60%, viral load range 103.50 and 107.91 gen- boratory technicians. Study design is summarized in ome copies/mL) clinically healthy sows from different Fig. 4. Sows were bled before mating and tested for parity number and in 24 out of 30 (80%, viral load range PCV-2 IgG and IgM ELISAs. Animals were randomly al- 103.50 and 107.06. genome copies/ml) PUCs from the located into 4 different treatment groups blocking by same sows, indicating a PCV-2-SI scenario. Inclusion parity (from 0 to 8) and OD values for PCV-2 IgG and criteria for gilts/sows selection were: a) healthy animals, PCV-2 IgM. A total of 288 healthy sows were selected b) not pregnant, c) from the same genetic line, d) of all through six consecutive breeding batches and distributed parities (gilts, primiparous and multiparous). in the following treatments groups: vaccination of 73 at The study was conducted in a two-site commercial pre-mating (PM), 72 at mid gestation (MG), 73 at late farm located in Catalonia (Spain). The farm had 1400 gestation (LG) and 70 were kept as NV sows. These sows (including gilts) and used a weekly farrowing batch three specific immunization times were chosen to mimic system; piglet weaning was performed at 25 days of age a blanket vaccination fashion, which is performed in approximately. The farm was Mycoplasma hyopneumo- sows at all physiological status at a given time point. niae positive, Porcine reproductive and respiratory syn- Gestation was monitored until farrowing and then their drome virus (PRRSV) positive stable, and negative for piglets were followed up until weaning. Sows from all Aujeszky’s disease virus. Gilts and sows were crossbred treatment groups were housed comingled during the (Duroc x Landrace) and were artificially inseminated study (in same pens when they were housed in groups, with Pietrain boar semen. The vaccination routine of the and in same room when they were individualized). farm included gilt and sow immunization against Cross-fostering of piglets was only allowed among sows PRRSV, Porcine parvovirus, Erysipelothrix rhusiopathiae, of the same experimental group. Escherichia coli and Clostridium perfringens. Piglets were All sows included in the study were injected (with vaccinated against PCV-2 and Mycoplasma hyopneumo- phosphate buffered saline [PBS] or PCV-2 vaccine) three niae before weaning. times before farrowing. Sows were vaccinated intramus- cularly (neck muscle) with 1 mL of PCV-2 Ingelvac Cir- Study design coflex® or PBS. Blood samples from sows were collected The present study was a parallel group, randomised and at the three vaccine/PBS application times as well as at controlled trial. The study was unmasked for personnel farrowing. Obtained sera were tested for PCV-2 IgG and Fig. 4 Experimental study design, including sampling time points and PCV-2 vaccine/PBS application timing. V PM: vaccinated at pre-mating; V MG: vaccinated at mid gestation; V LG: vaccinated at late gestation; NV: non-vaccinated; AI: artificial insemination; PCV-2 vaccine: 1 mL of PCV-2 Ingelvac Circoflex®; PBS: phosphate buffer saline Pleguezuelos et al. Porcine Health Management (2021) 7:35 Page 9 of 12 IgM antibodies using a commercial ELISA assay and to – Results over LOQ were transformed as log10 (PCV-2 quantify virus using a qPCR assay. At pre-mating sam- genome copies/mL + 1). pling point, PCV-2 IgG and IgM ELISA assay was per- formed in 288 sow sera to randomize the sows in 4 experimental groups. ELISA assay from mid gestation, PCV-2 antibody detection by ELISA late gestation and farrowing sampling points and qPCR PCV-2 antibodies in sows were detected at the three assay from the fourth sampling points were performed vaccine/PBS application times as well as at farrowing only in those artificially inseminated sows (Additional using the ELISA kit Ingezim Circo IgG/IgM 11.PCV.K2® file 1). Blood from 3 (ranging 1 to 4) PUCs per sow were assay (Ingenasa, Madrid, Spain). ELISA results were individually collected at farrowing to quantify virus in expressed as mean OD (± SD) according to the kit serum samples using the qPCR assay. In order to instructions. minimize PCV-2 environmental contamination, gloves PCV-2 antibodies in piglets at weaning were detected were changed, and scissors were disinfected with ethanol using the ELISA kit Ingezim Circo IgG 11.PCV.K1® assay for each PUC sampling. Additionally, productive param- (Ingenasa, Madrid, Spain). Mean positive cut-off was eters from gilts/sows and piglets were recorded (de- established as OD of negative control + 0.25). ELISA re- scribed in section 7). Moreover, at weaning, sera sults from piglets were expressed as S/P ratio (OD of samples from 4 to 6 randomly selected piglets per each sample / OD of positive control for each ELISA plate) sow were taken and used to detect PCV-2 antibodies following the manufacturer’s recommendations. using an ELISA test. Once in the laboratory, blood samples were centri- fuged at 2500 rpm (1300 g) during 10 min at 4 °C to ob- PCV-2 ORF2 amplification and sequencing tain sera. All sera were stored at − 20 °C until testing. Capsid protein gene (ORF2) was sequenced from PCV-2 qPCR positive sow serum and PUC samples to deter- mine the PCV-2 genotype/s circulating in the farm. DNA extraction and qPCR DNA was extracted from serum samples by using the Presence of PCV-2 DNA by qPCR was assessed in the MagMax™ Pathogen RNA/DNA Kit (Thermo Fischer serum samples from sows and PUCs. All these samples Scientific Baltics. Vilnius, Lithuania) following the manu- were processed by pools. Indeed, pools from 2 to 3 sow facturer’s instructions. serum samples at each sampling point and pools of 2–3 PCV-2 Cap gene was amplified from nucleotide 1050 PUC sera per sow at farrowing (except when only one to 1735 (PCV-2 genome; GenBank Accession Number: PUC was collected) were created. When a pool from AY181948) using the primers PCV-2all_F (5′ GGGTCT sow sera was qPCR positive, individual serum samples TTAAGATTAAATYC 3′) and PCV-2all_R (5′ ATGA were tested by qPCR following the same protocol. CGTATCCAAGGAG 3′) [48]. PCR was performed in a DNA was extracted from 200 μL of serum (from sows) 25 μL reaction containing 5 μL of PCR Promega buffer, or pool by using the MagMAX™ Pathogen RNA/DNA Kit 2.5 μL of 25 Mm MgCl2, 1.25 μL of each primer at 10 (Thermo Fischer Scientific Baltics. Vilnius, Lithuania) fol- pmol/μL, 1 μL of 5 mM dNTPs, 0.15 U of Taq DNA lowing the manufacturer’s instructions. To quantify the polymerase, 11.35 μL of DEPC-treated water and 2.5 μL PCV-2 DNA in serum samples, a qPCR assay (LSI Vet- of extracted DNA. The PCR was performed with the fol- MAX™ Porcine Circovirus Type 2-Quantification, Applied lowing program: denaturation of 5 min at 94 °C, 40 cy- Biosystems, Lisseu, France) was performed. Each extrac- cles of 30 s at 95 °C for denaturation, 30 s at 53 °C for tion and qPCR plate included negative controls (diethyl- primer annealing and 40 s at 72 °C for elongation, with a pyrocarbonate [DEPC]-treated water) and each sample final elongation of 7 min at 72 °C. Amplified DNA was reaction had an internal positive control to monitor DNA confirmed by electrophoresis gel with 2% agarose. extraction and amplification procedures. NucleoSpin Gel and PCR Clean-up kit (Macherey– PCV-2 qPCR results were transformed as follows: Nagel, GmbH & Co. KG, Dueren, Germany) was used to purify the PCR product. BigDye Terminator v3.1 Cycle – Undetermined results and those below LOD Sequencing Kit (Applied Biosystems, Foster City, CA, (LOD = 103.50 PCV-2 genome copies/mL) were USA) and 3130 × l Genetic Analyser (Applied Biosys- transformed as log10 (0 + 1). tems, Ohio, USA) was used to perform the sequencing – Results between LOD and LOQ (LOQ = 104.00 PCV- reaction and the analysis, respectively [48]. Sequences 2 genome copies/mL) were transformed following were edited and assembled by using ChromasPro Ver- the method proposed by Croghan et al. [47], where sion 2.1.8 (Technelysium). The sequences obtained were the result was calculated as log10 ((LOQ/√2) + 1). submitted to the GenBank with the following accession Therefore, the imputed value was log10 (7071 + 1). numbers MT572494-MT572497). Pleguezuelos et al. Porcine Health Management (2021) 7:35 Page 10 of 12 PCV-2 capsid ProteinG (ORF2) phylogenetic and sequence respiratory syndrome virus; PUC: Placental umbilical cord; qPCR: Real time analysis quantitative polymerase chain reaction; SD: Standard Deviation; V LG: Vaccinated at late gestation; V MG: Vaccinated at mid gestation; V To genotype the PCV-2 sequences obtained, an align- PM: Vaccination at pre-mating ment with 18 representative sequences from genotypes PCV-2a, b, c and d was carried out with Clustal Omega Supplementary Information (EMBL-EBI). The phylogenetic tree was created by using The online version contains supplementary material available at https://doi. the Maximum Likelihood method included in Mega-X org/10.1186/s40813-021-00213-2. software [49]. The best substitution model according with the Bayesian information criterion was the Additional file 1. Number of animals analysed at each sampling point. Hasegawa-Kishino-Yano model, with a discrete Gamma distribution. Bootstrap resampling test was carried out Acknowledgments The authors would like to thank to the farm animal caretakers and with 1000 replicates. Bootstrap values higher than 70 veterinarian for their willingness to collaborate in the study. were indicated in the constructed phylogenetic tree. Authors’ contributions JS, MS and SL designed the study; SL and PP coordinated the study Statistical analyses execution and performed the statistical analyses; MS coordinated the sample Statistical analyses were carried out using the software processing; DP, RC, and RL contributed to the sampling and study execution. NCSS (Kaysville, Utah, USA). Comparisons were per- AML and EH processed all the samples collected. JIN performed the PCV-2 genotyping; PP drafted the manuscript; MS, JIN, JS and SL revised the manu- formed in two ways: 1) all V groups (mimicking a blan- script. All the authors read and approved the published version of the ket fashion PCV-2 vaccination) versus the NV group, manuscript. and 2) all experimental groups among them. When comparison was performed between V and NV Funding This research was awarded in the European Porcine Circovirus (PCV-2) groups Mann-Whitney U test was used to total born Research Award by Boehringer Ingelheim. piglets per sow, live born piglets per sow, weaned piglets per sow, weaning-to-mate interval, weaning-to- Availability of data and materials The datasets used and/or analysed during the current study are available fecundation interval, PCV-2 viral load in sera of sows as from the corresponding author on reasonable request. well as viral load in PUC. Chi-square test was used to compare proportions of abortions, mummified fetuses, Declarations stillbirth piglets, dead suckling piglets, cross-fostered Ethics approval and consent to participate piglets, as well as of viraemic sows and PUC. The T – The present study was approved by the Ethics Committee for Animal test was used to analyse birth and weaning body weights. Experimentation from the Universitat Autònoma de Barcelona and the Animal When comparison was performed among all experi- Experimentation Commission from the local government (Dpt. de Medi Ambient i Habitatge from the Generalitat de Catalunya; Reference 5796). mental groups, Kruskal Wallis test (including Bonferroni test for multiple comparison) was used to analyse total Consent for publication born piglets per sow, live born piglets per sow, weaned Not applicable. piglets per sow, weaning-to-mate interval, weaning-to- Competing interests fecundation interval, PCV-2 ELISA antibody values in The authors declare that they have no competing interests. sow and piglets’ serum samples and viral load in sera of sows and PUCs. Serological parameters from sows were Author details 1 IRTA, Centre de Recerca en Sanitat Animal (CReSA, IRTA-UAB), Campus de la analysed performing only the comparison among experi- Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain. 2OIE mental groups to assess the serological effect of each Collaborating Centre for the Research and Control of Emerging and vaccination. Chi-square or Fisher’s Extact test was used Re-emerging Swine Diseases in Europe (IRTA-CReSA), Bellaterra, Spain. 3UAB, Centre de Recerca en Sanitat Animal (CReSA, IRTA- UAB), Campus de la to compare proportion of abortions, mummified piglets, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain. 4Departament stillbirth piglets, dead suckling piglets, cross-fostered de Sanitat i Anatomia Animals, Facultat de Veterinària, UAB, 08193 Bellaterra, piglets, as well as of viraemic sows and PUCs. Moreover, Spain. 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