Accepted Manuscript Title: Can Porcine circovirus type 2 (PCV2) infection be eradicated by mass vaccination? Author: H. Feng G. Blanco J. Segal´es M. Sibila PII: DOI: Reference:

S0378-1135(14)00239-9 http://dx.doi.org/doi:10.1016/j.vetmic.2014.05.003 VETMIC 6609

To appear in:

VETMIC

Received date: Revised date: Accepted date:

4-12-2013 25-4-2014 4-5-2014

Please cite this article as: Feng, H., Blanco, G., Sibila, J.S., , M., Can Porcine circovirus type 2 (PCV2) infection be eradicated by mass vaccination?, Veterinary Microbiology (2014), http://dx.doi.org/10.1016/j.vetmic.2014.05.003 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Can Porcine circovirus type 2 (PCV2) infection be eradicated

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by mass vaccination?

H. Feng1, G. Blanco2, J. Segalés1,3 M. Sibila1*

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Autònoma de Barcelona, 08193 Bellaterra, Barcelona, Spain.

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Departament de Producció Animal. ETSEA. Universitat de Lleida, 25198 Lleida, Spain.

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Departament de Sanitat i d’Anatomia Animals, Universitat Autònoma de Barcelona, 08193

Bellaterra, Barcelona, Spain

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Centre de Recerca en Sanitat Animal (CReSA), UAB-IRTA, Campus de la Universitat

*Email address of corresponding author: [email protected]

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HIGHLIGHTS

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Exploring the feasibility to eradicate PCV2 by vaccinating both sows and piglets One year of mass vaccination application in a conventional farm During the strategy, PCV2 infectious pressure was reduced to undetectable levels Stopping mass-vaccination lead to the re-appearance of PCV2 infection

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Abstract:

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The feasibility to eradicate Porcine circovirus type 2 (PCV2) in a conventional PCV2

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infected farm by vaccinating both sows and piglets using a commercially subunit vaccine

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was assessed. Vaccination strategy implied that all sows, boars and gilts of the farm were

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vaccinated every four months, and all piglets vaccinated and revaccinated with the same

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vaccine at 4 and 7 weeks of age, respectively. This vaccination strategy was applied during

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12 consecutive months. Blood samples from 15 piglets of 4, 8, 12, 16, 20 and 24 weeks of

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age and 15 sows were taken monthly PRE, DURING and POST mass vaccination strategy.

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From all the collected sera (n=1796), a representative part of them (n=1235, 69%) were

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analysed (n=1121 from piglets and n=114 from sows). All these samples were tested by

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PCV2 ELISA and PCV2 PCR (and quantitative-PCR when PCR positive). All tested sows

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were negative by PCR but seropositive. ELISA mean OD values of sows decreased

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throughout the study. Percentages of PCV2 PCR positive samples in piglets were 8%

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(12/150), 0.9% (6/659) and 3.5% (11/312) PRE, DURING and POST application of the

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mass vaccination program, respectively. ELISA mean OD values of PCV2 seropositive

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animals progressively decreased at the end of the mass vaccination period, but a clear

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seroconversion was observed after stopping such strategy. In conclusion, one year period of

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mass PCV2 vaccination (without implementing further farm management practices or

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biosafety measures) was not able to clear out PCV2 infection, and the virus became

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detectable again when vaccination was stopped.

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Key words: Porcine circovirus type 2 (PCV2), vaccine; eradication; viraemia;

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seroconversion

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1. Introduction Porcine circovirus type 2 (PCV2), a circular single-strand DNA virus of the Circoviridae

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family, is the etiologic agent of a number of swine diseases collectively named as porcine

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circovirus diseases (PCVD) (Segalés, 2012). The most significant conditions included as

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PCVDs are the PCV2-systemic disease (PCV2-SD) (also known as postweaning

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multisystemic wasting syndrome [PMWS]), porcine dermatitis and nephropathy syndrome

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(PDNS) and PCV2-reproductive disease (Rose et al., 2012; Segalés, 2012). PCV2-SD is

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considered the most economically significant condition within PCVDs (Segalés et al.,

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2012).

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Traditionally, PCV2-SD control was based on preventing risks or triggering factors by

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means of management improvement, control of co-infections and changes of the boar

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genetic background (Fraile et al., 2012a). Nowadays, the disease control is mainly based on

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vaccination. The vaccines currently available in the international market have shown to be

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very effective in controlling PCV2 infection and PCV2-SD under both experimental and

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field conditions (Cline et al., 2008; Opriessnig et al., 2010 , Fachinger et al., 2008). Such

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vaccines are able to improve production parameters (mortality and average daily gain) and

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reduce viraemia, viral shedding, PCV2-SD associated microscopic lesions and the

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likelihood of co-infection with other viruses (Fort et al., 2008; Fraile et al., 2012b; Gerber

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et al., 2011; Kixmoller et al., 2008; Martelli et al., 2011; Pejsak et al., 2010; Segalés et al.,

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2009).

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Viral load reduction and lower percentage of infected pigs are perceived as corner-stones

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in order to control the clinical outcome of the infection. In such respect, results of several

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PCV2 experimental studies have shown a complete elimination or clearance of PCV2 4

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infection by using two (Fort et al., 2008) and one doses of different PCV2 commercial

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vaccines (Hemann et al., 2012; O'Neill et al., 2011). These results opened the question

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whether the use of vaccination may be an efficiently way to eventually eliminate or

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eradicate PCV2 infection. Considering that with the normal vaccination strategy (one dose

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for most of the commercial vaccines), PCV2 infectious pressure is reduced (Fort et al.,

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2009; Opriessnig et al., 2009), one might speculate that a more extensive vaccination

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program could be used to potentially eradicate the infection.

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In the worldwide pig production, there are some examples of pathogen eradication

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programs based on the combination of vaccination and management strategies. Among

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them, the eradication program of Aujeszky’s disease virus (ADV) is one of the most

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successfully and extended programs. Its strategy is mainly based on a mass vaccination

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program (vaccination of the entire population) combined with animal movement

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restrictions. In Spain, ADV eradication program included compulsory vaccination of

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breeding sows (at least three times per year done in a blanket fashion), fatteners (at least

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two times separated by 3-4 weeks) and gilts (three times before entering the reproductive

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cycle) (Allepuz et al., 2009). Such intense efforts to control this disease concluded with the

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classification of Spain in 2011 as an ADV officially free country (Vicente-Rubiano et al.,

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2012).

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In the present study, and based on previous data, the feasibility to eradicate PCV2

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infection in a conventional farm by vaccinating both sows and piglets in a 12 consecutive

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month period was explored. Besides, the humoral immunological response after vaccination

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of sows and piglets in different batches of the same farm was monitored.

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2. Materials and methods 5

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2.1. Farm selection The present study was conducted in a 390-sow, two-site Spanish farm without previous

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history of PCVD. Site one was composed by breeding, lactating and nurseries units. Site

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two was located 40.5 km far from site one and was composed by two finishing units (700

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animals per unit). Nursery (in site one) was managed all-in-all-out (AIAO) by room

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(without mixing animals of different ages) whereas site two was managed in continuous

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flow. The tested farm was 2.5 km away from the nearest pig farm. This herd was

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conveniently selected mainly due to the willingness of the producer and the practitioner to

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participate in the project and the previous evidence of PCV2 infection. The farm used a

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self-replacement (sows) strategy and at the moment of the study there were 2 boars in the

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farm. No animals (sows or boars) coming from outside sources entered into the facilities

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during the study.

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Before starting mass vaccination strategy, a PCV2 vaccination program was already in

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place: sows were vaccinated twice with 2 ml of an inactivated vaccine (Circovac®, Merial)

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(at 6 and 3 weeks before farrowing) at the first gestation cycle and once (at 3 weeks before

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farrowing) in the following cycles. Piglets were vaccinated off-label (0.5 ml per piglet) at 3

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weeks of age with a subunit vaccine (Ingelvac Circoflex®, Boehringer Ingelheim).

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2.2. Study design

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Study design is summarised in Table 1. The initial PCV2 infection and serological status

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of the farm was assessed by PCR and ELISA, respectively, on serum samples of pigs and

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sows in two consecutive months before starting (December 2010 and January 2011) the

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mass vaccination program (February 2011 to January 2012). These two months prior the

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mass vaccination strategy was named as the “PRE” period for the purpose of the study. The 6

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mass vaccination strategy consisted of the vaccination of all sows, boars and gilts of the

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farm with 1 ml of Ingelvac Circoflex® every four months (3 doses/animal/year) in a

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blanket fashion (all animals were vaccinated at the same day, irrespectively of their

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physiological status). In addition, all piglets were vaccinated with 1 ml of the same vaccine

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at 4 and 7 weeks of age. The first-dose of piglet PCV2 vaccine was administrated later than

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the usually recommended age (3 weeks) to avoid a putative interference of maternal

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immunity resulting from vaccinated sows (Fort et al., 2009; Fraile et al., 2012a; Fraile et

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al., 2012b). This strategy was applied during 12 consecutive months (from February 2011

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to January 2012). This twelve-month period was named as the “DURING” period for the

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purpose of the study. To evaluate whether the eradication of PCV2 infection by mass

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vaccination was successful, the program was stopped after 12 months (February 2012).

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Subsequently, piglets and sows from the six following monthly batches were followed up

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(July 2012). This six month period was named as the “POST” period.

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During the study period (2+12+6 months), blood samples from 15 (5 gilts, 5 from 2nd to

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5th parity and 5 older than 5th parity, respectively) sows and from 90 piglets (15 of each age-

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group; 4, 8, 12, 16, 20 and in some cases at 24 weeks of age) were taken monthly (during

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the third week of each month). This study design implied that samples from the same

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animal were taken longitudinally at 4, 8, 12, 16, 20 and 24 weeks of age. Farm boars (n=2)

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were not monitored in the present study.

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Once in the laboratory, blood samples were allowed to clot and were centrifuged at 3400 rpm for 10 min at 4ºC. All samples were frozen at−80ºC until testing.

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Animal care and study procedures were conducted in accordance with the guidelines of

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Good Experimental Practice, under the approval of the Ethical and Animal Welfare

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Committee of the Universitat Autònoma of Barcelona (Reference 665M2). Treatments, 7

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housing, and husbandry conformed to the European Union Guidelines and Good Clinical

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Practices.

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2.3. DNA extraction, PCR and real-time quantitative PCR

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DNA was extracted from 200 μL of serum using BioSprint® 96 DNA Blood kit (Qiagen,

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GmbH, D-40724 Hilden) on the Bio Sprint 96 system (Qiagen). Obtained DNA was eluted

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in to 200 μL DNA elution buffer. All the samples were processed by standard PCV2 PCR

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(Quintana et al., 2002) and those yielding positive results were subsequently tested by a real

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time quantitative PCR (Q-PCR) (Olvera et al., 2004). Results of the Q-PCR were expressed

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as the mean PCV2 DNA copy numbers per mL (±SD).

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2.4. PCV2 serology

Serum samples were tested by indirect enzyme-linked immunosorbent assay (ELISA)

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using a commercial kit (INGEZIM, Circo IgG 1.1. PCV. K.1, Spain). Results of the ELISA

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were expressed as mean OD (±SD). Mean positive OD cut-off was set at 0.4.

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2.5. Statistical analyses

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From all sera received in the laboratory (n=1796), only a representative part of them

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(69%) were analysed (n=1121 from piglets and n=114 from sows [3 sows were sampled

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twice and one sow was sampled three times]) (Table 1). Selection of tested sera was based

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on the feasibility to analyse the infectious/serological status against PCV2 before, during

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and after the mass vaccination program in two different fashions: cross-sectional (including

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animals of different ages sampled at the same moment) and longitudinal (including all the

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samples taken during the lifespan of a given animal). For the cross-sectional follow up in 8

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the DURING period, sample selection included sera collected at the beginning, in the

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middle and at the end of this period. In total, 7 cross-sectional groups (C1 to C7) and 9

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longitudinal groups (L1 to L9) were monitored. The status of vaccination of the animals

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included in the L groups was defined at the age of vaccination. The analysed samples from

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monthly sampled sows (S) were numbered from S1 to S7.

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Moreover, and in order to analyse the overall obtained information, results from C, L and

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S groups were collectively divided in three periods: PRE (n=180), DURING (n=703) and

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POST (n=352) mass vaccination periods (Table 1) periods. On the other hand, the results of

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sows were also analysed by different parities, named gilts (G1, n=46), 2nd to 5th parity (G2,

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n=49) and 6th to 8th parity (G3, n=19).

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All statistical analyses were carried out using the SPSS v.20.0 software. The significance

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level was set at 0.05. Descriptive statistics were used to summarize categorical and

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quantitative variables. Shapiro Wilk´s and Levene tests were used to evaluate the normality

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of the distribution of the examined quantitative variables and the homogeneity of variances,

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respectively. The Chi-square or Fischer exact test was applied to evaluate the proportion of

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positive and negative animals by PCR between the three periods of the study. One way

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analysis of variance (ANOVA) test was used to compare the ELISA mean OD values and

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mean viral load between the different study periods.

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3. Results

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3.1. Clinical findings

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No significant clinical signs were observed during the PRE and DURING periods,

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besides eventual cases of polyarthritis and unspecific chronic diarrhoea in lactating and

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nursery pigs. In the POST mass vaccination period, weight loss and diarrhoea were 9

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observed in nursery piglets; moreover, the farmer and the veterinarian noticed loss of

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weight homogeneity and slight increase in mortality in finishing pigs (data not recorded).

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No clinical PCV2-SD associated problems were observed during the whole study period.

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3.2. PCV2 infection and seroconversion in sows

All tested serum samples from sows (n=114) were PCR negative through the whole

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production period, but all of them were seropositive against PCV2. The mean ELISA OD

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values of the sows decreased progressively among the three periods of the study (Figure 1).

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The mean ELISA OD values was significantly lower (P

Can Porcine circovirus type 2 (PCV2) infection be eradicated by mass vaccination?

The feasibility to eradicate Porcine circovirus type 2 (PCV2) in a conventional PCV2 infected farm by vaccinating both sows and piglets using a commer...
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