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Altres ajuts: Instituto Nacional de Investigación y Tecnologia Agraria y Alimentaria RTA2017-00007-00-00 Porcine circovirus 3 (PCV-3) was discovered in 2015 using next-generation sequencing (NGS) methods. Since then, the virus has been detected worldwide in pigs displaying several clinical-pathological outcomes as well as in healthy animals. The objective of this review is to critically discuss the evidence existing so far regarding PCV-3 as a swine pathogen. In fact, a significant number of publications claim PCV-3 as a disease causal infectious agent, but very few of them have shown strong evidence of such potential causality. The most convincing proofs of disease association are those that demonstrate a clinical picture linked to multisystemic lymphoplasmacytic to lymphohistiocytic perivascular inflammation and presence of viral nucleic acid within these lesions. Based on these evidence, individual case definitions for PCV-3-reproductive disease and PCV-3-systemic disease are proposed to standardize diagnostic criteria for PCV-3-associated diseases. However, the real frequency of these clinical-pathological conditions linked to the novel virus is unknown, and the most frequent outcome of PCV-3 infection is likely subclinical based on its worlwide distribution.

Glaesserella (Haemophilus) parasuis, an early colonizer of the nasal cavity in piglets, is a highly heterogeneous species, comprising both commensal and virulent strains. Virulent G. parasuis strains can cause fibrinous polyserositis called Glässer's disease. Colostrum is a source of passive immunity for young piglets. When vaccinating sows, protective antibodies are transferred to their offspring through the colostrum. Here, sow vaccination was performed with a protein fragment, F4, from the outer membrane trimeric autotransporters VtaAs exclusively found in virulent G. parasuis. Piglets were allowed to suckle for 3 weeks, following which a challenge with two virulent strains of G. parasuis was performed. A group of nonvaccinated sows and their piglets were included as a control. Antibodies against F4 were confirmed using ELISA in the vaccinated sows and their offspring before the G. parasuis challenge. Compared to the control group, F4-vaccination also resulted in an increased level of serum TGF-β both in vaccinated sows and in their offspring at early time points of life. After the challenge, a lower body temperature and a higher weight were observed in the group of piglets from vaccinated sows. One piglet from the non-vaccinated group succumbed to the infection, but no other significant differences in clinical signs were noticed. At necropsy, performed 2 weeks after the virulent challenge, the level of surfactant protein D (SP-D) in bronchoalveolar lavage was higher in the piglets from vaccinated sows. Vaccination did not inhibit the nasal colonization of the piglets by the challenge strains. info:eu-repo/semantics/publishedVersion

This study evaluated different gilt vaccination protocols againstMycoplasma (M.) hyopneumoniaeat acclimationand their effect on the genetic diversity. A total of 180M. hyopneumoniaenaïve gilts were selected 1 week post-entry (wpe) at the acclimation barn in a clinically affectedM. hyopneumoniaefarm. Gilts were distributed ac-cording to theM. hyopneumoniaeantibodies levels into three different vaccination schedules: A) four doses of aM. hyopneumoniaecommercial vaccine at 2, 4, 6 and 8 wpe; B) two vaccine doses at 2 and 6 wpe and PBS at 4 and8 wpe; and C) four PBS doses at the same wpe. Detection ofM. hyopneumoniae(rt-PCR) and antibodies (ELISA)were assessed in gilts at 1, 14, 27 and 34 wpe and in 6 of their piglets at weaning. Rt-PCR positive gilts weredetected at 14 wpe, being the proportion significantly lower in groups A and B (3/120, 3%) than C (27/60, 45%).Seroconversion was detected at 14 wpe, showing significant differences in percentage of inhibition (PI) betweengroups A (median 4.9, range 3.1–19.9) and B (5.5, 3.7–13.5), and C (14.3, 3.3–53.2). Gilts remained seropositiveover the study and significant differences in PI were detected between groups A and B versus C. All piglets werert-PCR negative, but the proportion of seropositive piglets coming from vaccinated gilts was significantly higherthan the non-vaccinated group.M. hyopneumoniaecharacterization showed high variability. Hence, gilt vacci-nation with 2 or 4 doses significantly decreased the pathogen infectious pressure, variability, and provided highantibody levels to gilts and their offspring. info:eu-repo/semantics/acceptedVersion

Porcine circovirus 3 (PCV-3) has been widely detected in healthy and diseased pigs, among different pathologic conditions, the strongest evidence of association comes from reproductive disease cases. However, simple viral detection does not imply the causality of the clinical conditions. Detection of PCV-3 within lesions may provide stronger evidence of causality. Thus, this study aimed to assess the frequency of PCV-3 detection in tissues from fetuses/stillborn piglets in cases of reproductive problems in domestic swine, as well as the histopathologic assessment of fetal tissues. Fetuses or stillborn piglets from 53 cases of reproductive failure were collected and analyzed by PCV-3 qPCR. The presence of porcine reproductive and respiratory syndrome virus (PRRSV), porcine circovirus 2 (PCV-2), and porcine parvovirus 1 (PPV1) was also checked. PCV-3 qPCR positive samples with a high viral load were tested by PCV-3 in situ hybridization (ISH), sequenced, and phylogenetically analyzed. PCV-3 DNA was detected in 18/53 (33.9%) reproductive failure cases and in 16 of them PCV-3 was the only pathogen found. PCV-2 DNA was found in 5/53 (9.4%), PRRSV RNA in 4/53 (7.5%) and PPV1 was not detected. Four out of the six PCV-3 qPCR-positive cases with Ct value &lt, 30 were positive when tested by ISH. In these samples, PCV-3 was detected within mild histopathologic lesions, such as arteritis and periarteritis in multiple tissues. The present work emphasizes the need to include PCV-3 as a potential causative agent of reproductive failure in swine.

Altres ajuts: acords transformatius de la UAB Porcine circovirus 3 (PCV-3) has been detected in diseased and healthy pigs of different ages. Several reports have associated the agent with reproductive failure and mummified and stillborn piglets. One report from North America has proposed a consistent potential association with postweaning disorders. Thus, the present case report aimed to describe the histopathological lesions and their association with the presence of PCV-3 genome in postweaning pigs showing growth-retardation and thrown-back ears. All affected animals displayed multi-organic lymphoplasmacytic periarteritis, lymphocytic myocarditis and/or lymphoplasmacytic meningoencephalitis. PCV-3 genetic material was detected by in situ hybridization within the lesions and confirmed by PCV-3 real-time quantitative PCR detection in tissues. This study represents the first report of PCV-3 associated with clinical disease in postweaning pigs in Europe.

Vaccination against porcine circovirus 2 (PCV-2) is a common practice all over the world. Vaccines can prevent PCV-2-systemic disease (PCV-2-SD) outbreaks but not PCV-2 infection, which can be detectable in a percentage of vaccinated animals. Occasionally, PCV-2-SD is diagnosed in vaccinated farms. The objective of this study was to genotype the PCV-2 strains detected in vaccinated animals diagnosed with PCV-2-SD. Additionally, the evolution of the frequency of PCV-2 genotype detection at Spanish, European, and world levels was assessed. Fifty cases diagnosed as PCV-2-SD between 2009 and 2020 were included in this study. PCV-2 genotype was determined by sequencing the Cap gene region. Among them, only PCV-2b (23/50, 46%) and PCV-2d (27/50, 54%) genotypes were detected. Although the frequency of detection of these two genotypes was similar, their temporal distribution was different. Whereas most PCV-2b sequences (17/23, 74%) were detected between 2009 and 2012, PCV-2d sequences were obtained from 2013 to 2020. Indeed, a predominance of the PCV-2d genotype was observed from 2013 onwards, a trend also noticed at European and world levels. The results suggest that detection of particular genotypes in vaccinated animals probably reflects the general prevalence of the genotypes over time rather than genotype-specific vaccine-immunity escaping. info:eu-repo/semantics/publishedVersion

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.

The genus Circovirus includes several species and mostly causes asymptomatic infections. Porcine circovirus 2 (PCV-2) and, with increasing evidence, Porcine circovirus 3 (PCV-3), have been associated with different clinical conditions all over the world. In 2019, a new porcine circovirus (PCV-4) was identified from diseased animals in China. Because of the lessons learned from PCV-2 and PCV-3, it appears mandatory to investigate the actual distribution of this new virus and its potential association with clinical outcomes. To this purpose, an exploratory study to detect PCV-4 by molecular methods was performed in Italy and Spain by testing more than 300 samples of different types (serum and tissues), collected from both healthy and diseased pigs and wild boar as well. All samples, independently from the country, type, health status and host, tested PCV-4 negative. Therefore, no evidence of PCV-4 presence was found in Italy and Spain through this exploratory study. Considering the dense pig trade among European countries, its presence in the continent can similarly be considered unlikely. The reasons behind the restricted PCV-4 distribution compared to other porcine circoviruses will require further investigations. Careful surveillance might nevertheless be important since prompt recognition of PCV-4 would allow the implementation of effective countermeasures to prevent its spreading and potential economic losses. info:eu-repo/semantics/publishedVersion

Funding: Instituto Nacional de Investigación y Tecnologia Agraria y Alimentaria E-RTA2017-00007-00-00 INIA Project i Programa CERCA, Generalitat de Catalunya. Porcine circovirus 3 (PCV-3) is the third member of the family Circoviridae, genus Circovirus, able to infect swine. A high prevalence of viral DNA has been recorded in wild boars. Recently, PCV-3 DNA was identified in Italian wild ruminants. Based on these previous results, this study assessed the frequency of PCV-3 DNA detection in free-ranging ruminants and Lagomorpha species in Spain. In addition, the genetic characterization of the PCV-3 PCR-positive samples was performed. A total of 801 serum samples, including red deer (Cervus elaphus, [CE]; n = 108), roe deer (Capreolus capreolus, [CC]; n = 87), Pyrenean chamois (Rupicapra pyrenaica, [RP]; n = 133), Iberian ibex (Capra pyrenaica, [CP]; n = 92), mouflon (Ovis aries, [OA]; n = 91), fallow deer (Dama dama, [DD]; n = 104), European rabbit (Oryctolagus cuniculus, [OC]; n = 101), and European hare (Lepus europaeus, [LE]; n = 85) from Catalonia (northeast Spain) were tested by conventional polymerase chain reaction (PCR) and, when positive, sequenced. Overall, PCV-3 DNA was found in three out of 801 analyzed sera (0.37%) corresponding to one red deer (1/108, 0.9%), one mouflon (1/91, 1.1%), and one fallow deer (1/104, 0.96%). None of the samples collected from Lagomorpha species resulted PCR positive. The partial genome sequences detected in positive samples displayed high identity with some PCV-3 sequences detected in wild boars and domestic pigs (99.7% and 100%, respectively). In conclusion, the present study indicated that free-ranging ruminant and Lagomorpha species are not relevant in the epidemiology of PCV-3 in Spain.

Made available in DSpace on 2020-12-12T02:13:37Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-01-01 Centres de Recerca de Catalunya Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria Porcine circovirus 3 (PCV-3) has been suggested as a putative causal agent of swine reproductive disease. A number of different studies have pointed out this association, but there is still a lack of information regarding the normal rates of PCV-3 infection in farms with normal reproductive parameters. The objective of the present study was to assess the frequency of PCV-3 detection in primiparous and multiparous sows and in tissues from their respective fetuses from farms with average reproductive parameters. Sera from 57 primiparous and 64 multiparous sows from 3 different farms were collected at two time points. Brain and lung tissues from 49 mummies and 206 stillborn were collected at farrowing. Samples were tested by PCR, and when positive, quantified by quantitative PCR. Thirty-nine complete genomes were obtained and phylogenetically analyzed. All sera from multiparous sows were negative, while 19/57 (33.3%) primiparous sows were PCV-3 PCR positive. From the 255 tested fetuses, 86 (33.7%) had at least one tissue positive to PCV-3. The frequency of detection in fetuses from primiparous sows (73/91, 80.2%) was significantly higher than those from multiparous ones (13/164, 7.9%). It can be concluded that PCV-3 is able to cause intrauterine infections in absence of overt reproductive disorders. IRTA Centre de Recerca en Sanitat Animal (CReSA IRTA-UAB) Campus de la Universitat Autònoma de Barcelona, Bellaterra OIE Collaborating Centre for the Research and Control of Emerging and Re-emerging Swine Diseases in Europe (IRTA-CReSA), Bellaterra Zoetis Manufacturing & Research Spain S.L., Ctra. Camprodon s/n, La Riba Department of Immunology and Microbiology Institute of Biosciences São Paulo State University (Unesp) Departament de Sanitat i Anatomia Animals Universitat Autònoma de Barcelona (UAB) UAB Centre de Recerca en Sanitat Animal (CReSA IRTA-UAB) Campus de la Universitat Autònoma de Barcelona, Bellaterra Department of Immunology and Microbiology Institute of Biosciences São Paulo State University (Unesp) FAPESP: 2017/26649-4 Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria: E-RTA2017-00007-00-00

Altres ajuts: INITA/E-RTA2017-00007-00-00 PCV-2 is considered one of the most economically important viral agents in swine worldwide. Recently, PCV-3 has been found in pigs affected by different disorders and in healthy animals. The objective of this epidemiological work was to describe the frequency of detection of PCV-2 and PCV-3 in pig farms of 9 European countries. Moreover, a second aim was to assess the most frequent PCV-2 genotypes found in the studied farms. Sera from 5 to 10 pigs per farm were collected from 2 to 11 farms per studied country. A total of 624 sera of fattening pigs (10-25 week old) from 64 farms from Spain (n = 11), Belgium (n = 10), France (n = 8), Germany (n = 8), Italy (n = 7), Denmark (n = 8), the Netherlands (n = 5), Ireland (n = 5) and Sweden (n = 2) were analysed by conventional PCR. In addition, one or two PCV-2-positive samples per farm were genotyped by sequencing the ORF2 gene. PCV-3 PCR-positive samples with relatively low Ct values were also sequenced and phylogenetically analysed. PCV-2 DNA was detected in pig sera from all European tested countries, but Sweden. A total of 132 out of 624 (21%) sera were positive for PCV-2 PCR, corresponding to 30 out of the 64 (47%) tested farms. PCV-3 DNA was detected in 52 out of 624 (8%) sera, corresponding also to 30 out of the 64 (47%) studied farms from all tested countries. A total of 48 PCV-2 PCR-positive samples were successfully sequenced and genotyped, being PCV-2d the most frequently genotype found (n = 28), followed by PCV-2b (n = 11) and PCV-2a (n = 9). These results pointed out PCV-2d as the most prevalent genotype currently in Europe. The PCV-3 phylogenetic analysis showed high identity (>98%) among sequences from all the analysed countries. The relatively low co-infection (3%), likely suggest an independent circulation patterns of PCV-2 and PCV-3.

Porcine circovirus 2 (PCV2) is a single stranded DNA virus with one of the highest mutation rates among DNA viruses. This ability allows it to generate a cloud of mutants constantly providing new opportunities to adapt and evade the immune system. This pig pathogen is associated to many diseases, globally called porcine circovirus diseases (PCVD) and has been a threat to pig industry since its discovery in the early 90’s. Although 11 ORFs have been predicted from its genome, only two main proteins have been deeply characterized, i.e. Rep and Cap. The structural Cap protein possesses the majority of the epitopic determinants of this non-enveloped virus. The evolution of PCV2 is affected by both natural and vaccine-induced immune responses, which enhances the genetic variability, especially in the most immunogenic Cap region. Intra-host variability has been also demonstrated in infected animals where long-lasting infections can take place. However, the association between this intra-host variability and pathogenesis has never been studied for this virus. Here, the within-host PCV2 variability was monitored over time by next generation sequencing during an experimental infection, demonstrating the presence of large heterogeneity. Remarkably, the level of quasispecies diversity, affecting particularly the Cap coding region, was statistically different depending on viremia levels and clinical signs detected after infection. Moreover, we proved the existence of hyper mutant subjects harboring a remarkably higher number of genetic variants. Altogether, these results suggest an interaction between genetic diversity, host immune system and disease severity.

Porcine circovirus 3 (PCV‐3) prevalence has been minimally investigated in wild boar; dynamics of infection and viral tissue distribution are currently unknown. In this study, serum samples from 518 wild boar (from years 2004 to 2018) were used to study frequency of infection. Also, serum samples from 19 boar captured and recaptured at least two times for a period of time from 1 month to 1 year were collected to determine PCV‐3 infection dynamics. Finally, to elucidate PCV‐3 DNA organic distribution, sera, different tissues and faeces were obtained from 35 additional wild boar. PCV‐3 DNA was extracted and amplified with a conventional PCR. For the PCV‐3 PCR‐positive sera from the longitudinally sampled and different tissue types, a quantitative PCR was performed. Genome sequence was obtained from a number of PCV‐3 PCR‐positive samples from different years, different time‐points of infection and tissues. Obtained results confirmed the susceptibility of wild boar to the virus, showing high frequency of PCV‐3 detection (221 out of 518, 42.66%) and demonstrating circulation at least since 2004. Compiled data indicate the possibility of long‐term infections, since 5 out of 10 PCV‐3 PCR‐positive boars longitudinally sampled showed positivity in samplings separated for more than 5 months. All tested tissue types' harboured PCV‐3 genome, with the highest percentage of PCR positivity in submandibular lymph node, tonsil, lung, liver, spleen and kidney. The amount of DNA in all tested PCV‐3 PCR‐positive samples was moderate to low. All partial and complete PCV‐3 sequences obtained from wild boar displayed high nucleotide identity, higher than 98%. In conclusion, this study further confirms that wild boar is susceptible to PCV‐3 infection, showing high frequency of detection in this animal species. Furthermore, PCV‐3 can be found in different tissues of wild boar and is apparently able to cause persistent infection. Instituto Nacional de Investigación y Tecnologia Agraria y Alimentaria. Grant Number: E‐RTA2017‐00007‐00‐00

This study was aimed to evaluate the effect of Porcine circovirus 2 (PCV2) sow vaccination on cell-mediated immune responses in sows and their progeny. At 7 weeks before farrowing, fifteen PCV2 PCR negative pregnant sows with medium-low antibody values were selected and randomly distributed in two groups according to the antibody levels. Seven sows were vaccinated with a commercial PCV2 vaccine and eight were injected with phosphate-buffered saline at 6 and 3 weeks before farrowing. Blood samples were taken from sows and their piglets (n = 90) during the study duration. PCV2 DNA and antibodies were tested in sera, and cytokine (IFN-α, IFN-γ, IL-12p40, TNF-α, IL-1β, IL-8, IL-4, IL-6 and IL-10) levels were assessed in supernatant from cultured peripheral blood mononuclear cells. All sows and piglets were negative by PCV2 PCR throughout the study. Significantly higher PCV2 antibody levels were detected in vaccinated sows after vaccination and in their offspring after colostrum ingestion compared to the non-vaccinated counterparts. Vaccinated sows did not show significant differences in cytokine secretion levels at farrowing compared to unvaccinated dams. In contrast, piglets from vaccinated sows had significantly higher levels of cytokines linked to Th1 memory cells (IFN-γ and TNF-α) in comparison to the ones from non-vaccinated dams. In conclusion, PCV2 sow vaccination, apart from triggering a humoral immunity response in sows and their progeny, might be associated to an increased transfer of cell-mediated immunity from the dam to the piglet. info:eu-repo/semantics/acceptedVersion

Mycoplasma hyopneumoniae(M. hyopneumoniae) is the primary pathogen of enzooticpneumonia, a chronic respiratory disease in pigs. Infections occur worldwide andcause major economic losses to the pig industry. The present paper reviews the cur-rent knowledge onM. hyopneumoniaeinfections, with emphasis on identificationand analysis of knowledge gaps for optimizing control of the disease. Close contactbetween infected and susceptible pigs is the main route ofM. hyopneumoniaetrans-mission. Management and housing conditions predisposing for infection or diseaseare known, but further research is needed to better understandM. hyopneumoniaetransmission patterns in modern pig production systems, and to assess the impor-tance of the breeding population for downstream disease control. The organism isprimarily found on the mucosal surface of the trachea, bronchi and bronchioles. Dif-ferent adhesins and lipoproteins are involved in the adherence process. However, aclear picture of the virulence and pathogenicity ofM. hyopneumoniaeis still missing.The role of glycerol metabolism, myoinositol metabolism and theMycoplasmaIgbinding protein—MycoplasmaIg protease system should be further investigated fortheir contribution to virulence. The destruction of the mucociliary apparatus,together with modulating the immune response, enhances the susceptibility ofinfected pigs to secondary pathogens. Clinical signs and severity of lesions dependon different factors, such as management, environmental conditions and likely alsoM. hyopneumoniaestrain. The potential impact of strain variability on disease sever-ity is not well defined. Diagnostics could be improved by developing tests that maydetect virulent strains, by improving sampling in live animals and by designing ELI-SAs allowing discrimination between infected and vaccinated pigs. The currentlyavailable vaccines are often cost-efficient, but the ongoing research on developingnew vaccines that confer protective immunity and reduce transmission should becontinued, as well as optimization of protocols to eliminateM. hyopneumoniaefrompig herds. info:eu-repo/semantics/acceptedVersion

Mycoplasma hyopneumoniae (M. hyopneumoniae) is the aetiologic agent of enzootic pneumonia in swine, a prevalent chronic respiratory disease worldwide. Mycoplasma hyopneumoniae is a small, self‐replicating microorganism that possesses several characteristics allowing for limited biosynthetic abilities, resulting in the fastidious, host‐specific growth and unique pathogenic properties of this microorganism. Variation across several isolates of M. hyopneumoniae has been described at antigenic, proteomic, transcriptomic, pathogenic and genomic levels. The microorganism possesses a minimal number of genes that regulate the transcription process. Post‐translational modifications (PTM) occur frequently in a wide range of functional proteins. The PTM by which M. hyopneumoniae regulates its surface topography could play key roles in cell adhesion, evasion and/or modulation of the host immune system. The clinical outcome of M. hyopneumoniae infections is determined by different factors, such as housing conditions, management practices, co‐infections and also by virulence differences among M. hyopneumoniae isolates. Factors contributing to adherence and colonization as well as the capacity to modulate inflammatory and immune responses might be crucial. Different components of the cell membrane (i.e. proteins, glycoproteins and lipoproteins) may serve as adhesins and/or be toxic for the respiratory tract cells. Mechanisms leading to virulence are complex and more research is needed to identify markers for virulence. The utilization of typing methods and complete or partial‐gene sequencing for M. hyopneumoniae characterization has increased in diagnostic laboratories as control and elimination strategies for this microorganism are attempted worldwide. A commonly employed molecular typing method for M. hyopneumoniae is Multiple‐Locus Variable number tandem repeat Analysis (MLVA). The agreement of a shared terminology and classification for the various techniques, specifically MLVA, has not been described, which makes inferences across the literature unsuitable. Therefore, molecular trends for M. hyopneumoniae have been outlined and a common terminology and classification based on Variable Number Tandem Repeats (VNTR) types has been proposed. info:eu-repo/semantics/acceptedVersion

Highlights • UV-C light as an additional safety step for spray-dried plasma production is proposed. • Swine bioassay is a sensitive technique able to detect infectivity of raw liquid plasma. • Inoculated pigs with UV-C irradiated liquid porcine plasma did not seroconvert or become PCR positive against tested viruses. • UV irradiation at 3000 J/L or more (9000 J/L) was enough to avoid transmission of any of the tested viruses to the animals. • UV-C treatment is useful step to inactivate potential pathogen contamination of commercially collected animal plasma., Liquid porcine plasma is an animal origin raw material for the manufacturing process of spray-dried porcine plasma that is used in pig nutrition worldwide. In previous studies we found that the application of ultraviolet light C (UV-C) in liquid plasma that was inoculated with a variety of bacteria or viruses of importance in the swine industry can be considered as redundant safety steps because in general achieve around 4 logs reduction for most of these pathogens. However, the final validation of the UV-C light as safety feature should be conducted with commercial liquid plasma and using the pig bioassay model. As a first objective, the potential infectivity of a raw liquid plasma product collected from an abattoir was tested by means of a swine bioassay. We used Porcine circovirus 2 (PCV-2), a ubiquitous virus that has been systematically detected by PCR in porcine plasma at abattoirs as selection criteria for commercial liquid plasma lot. As a second aim of the study, the effects of different doses of UV-C irradiation on the selected raw liquid plasma were assayed in the animal bioassay. Moreover, other swine infecting agents, including Porcine reproductive and respiratory syndrome virus (PRRSV), were also determined in the original plasma and monitored in the inoculated animals. Pigs negative for PCV-2 and PRRSV genome and antibodies were allotted to one of five groups (6 to 8 pigs/ group) and injected intra-peritoneally with 10 mL of their assigned inoculum at 50 d of age. Negative control pigs (group 1) were injected with PBS. Positive control pigs (group 5) were injected with a PCV-2 inoculum. Groups 2, 3 and 4 were injected with liquid porcine plasma that had been subjected to 0 (raw plasma), 3000 or 9000 J/L UV-C irradiation, respectively. Group 2 pigs (0 J/L UV-C) got infection by PRRSV but no PCV-2 infection or seroconversion. However, one pig from group 2 seroconverted to Rotavirus A (RVA) and Hepatitis E virus (HEV) and three group 2 pigs seroconverted to Porcine parvovirus (PPV). Groups 1, 3 and 4 pigs showed no evidence of infection or seroconversion associated with the tested viruses or any other pathogens found in the liquid plasma before UV-C irradiation. Group 5 pigs developed PCV-2 infectivity as expected. UV-C irradiation of liquid plasma at 3000 and 9000 J/L was effective in preventing PRRSV and other pathogens transmission. Moreover, raw liquid plasma was non-infectious for PCV-2 in naïve pigs.

Altres ajuts: Secretaria del Departament d'Economia i Creixement de la Generalitat de Catalunya DI2013-0039 Information on the in vitro growth of pathogenic and non-pathogenic Mycoplasma hyopneumoniae (M. hyopneumoniae) strains is scarce and controversial. Despite its limitations, the colour changing units (CCU) assay is still considered the golden standard titration technique for M. hyopneumoniae culture. Thus, the aims of the present study were: (1) to describe the growth dynamics and kinetics of pathogenic and non-pathogenic M. hyopneumoniae strains, and (2) to monitor the strains' daily growth by ATP luminometry, CCU, colony forming units (CFU), and DNA quantification by real time quantitative PCR (qPCR) and by fluorescent double-stranded DNA (dsDNA) staining, to evaluate them as putative titration methodologies. The growth of the non-pathogenic J (ATCC ® 25934™) type strain and the pathogenic 11 (ATCC ® 25095™) reference strain and 232 strain was modelled by the Gompertz model. Globally, all three-strain cultures showed the same growing phases as well as similar maximal titres within a particular technique, but for CFU. However, the J strain displayed the fastest growing. During the logarithmic phase of growing, CCU, ATP and M. hyopneumoniae copy titres were strongly and linearly associated, and correlation between techniques could be reliably established. In conclusion, real-time culture titration by means of ATP or molecular assays was useful to describe the in vitro growth of the tested strains. Knowledge about the in vitro growth behaviour of a specific strain in a specific medium may provide several advantages, including information about the time required to reach maximal titres by the culture. Noteworthy, the obtained results refers to the three strains used, so extrapolation to other M. hyopneumoniae strains or culture conditions should be made cautiously.

Background This study sought to evaluate the effect of sow vaccination against Porcine circovirus 2 (PCV2) on reproductive parameters during two consecutive reproductive cycles. The study was performed in a PCV2 subclinical infected breeding herd (PCV2 circulation but absence of major reproductive problems). Ninety-four pregnant sows were primo-immunized with a commercial PCV2 vaccine and ninety-seven were injected with phosphate-buffered saline at 6 and 3 weeks before the first studied farrowing, and then boosted at 2 weeks before the second one. Blood samples were taken throughout the study to assess PCV2 DNA load and antibodies. At farrowing, main reproductive parameters and piglet vitality index were registered. In addition, in those litters with more than three mummified or stillborn piglets, microscopic examination and PCV2 antigen detection in foetal myocardium was done. Results Vaccinated sows showed significantly higher antibody levels compared to the non-vaccinated counterparts. PCV2 DNA was only detected at farrowing in 2 (4.2%) non-vaccinated sows. Vaccinated sows had 1.3 more live-born piglets per litter at the second cycle than non-vaccinated counterparts. Piglets from vaccinated sows had significantly higher (+ 12.7%) vitality score than the ones born from non-vaccinated sows. No PCV2 compatible lesions neither PCV2 antigen were detected in the tested foetal hearts. Conclusions The present study represents a first attempt to demonstrate that PCV2 sow vaccination may have a positive influence on prolificacy and vitality of the offspring in a subclinical infected breeding herd. However, since reproductive outcomes at farm level may be affected by a number of factors, further studies would be needed to confirm this association.

This paper discusses what can be expected with regard to efficacy of antibacterial vaccines used in swine, based on the present knowledge of pathogen–host interactions. First, vaccination against bacteria that mainly cause disease by production of exotoxins is considered. Vaccines containing the inactivated toxin or a non-toxic but antigenic recombinant protein derived from the exotoxin can be expected to provide protection against disease. The degree of protection induced by such vaccines varies, however, depending amongst other things on the pathogenesis of the disease. Vaccination against clostridial infections, Actinobacillus pleuropneumoniae infections, progressive atrophic rhinitis and enterotoxigenic Escherichia coli, is considered. The second part of the article deals with vaccination against extracellular bacteria. Protection against these bacteria is generally mediated by antibodies against their surface antigens and certain secreted antigens, but cellular immunity may also play a role. Efficacy of vaccines against swine erysipelas, Streptococcus suis infections, Mycoplasma hyopneumoniae infections and swine dysentery is discussed. Finally, vaccination against facultatively intracellular bacteria is considered. For protection against these bacteria cell-mediated immunity plays an important role, but antibodies may also be involved. It is generally accepted that live-attenuated vaccines are more suitable for induction of cell-mediated immunity than inactivated vaccines, although this also depends on the adjuvant used in the vaccine. As an example, vaccination against Salmonella enterica serotype Typhimurium is discussed. info:eu-repo/semantics/acceptedVersion

Porcine circovirus 3 (PCV-3) is a recently described virus belonging to the family Circoviridae. It represents the third member of genus Circovirus able to infect swine, together with PCV-1, considered non-pathogenic, and PCV-2, one of the most economically relevant viruses for the swine worldwide industry. PCV-3 was originally found by metagenomics analyses in 2015 in tissues of pigs suffering from porcine dermatitis and nephropathy syndrome, reproductive failure, myocarditis and multisystemic inflammation. The lack of other common pathogens as potential infectious agents of these conditions prompted the suspicion that PCV-3 might etiologically be involved in disease occurrence. Subsequently, viral genome was detected in apparently healthy pigs, and retrospective studies indicated that PCV-3 was already present in pigs by early 1990s. In fact, current evidence suggests that PCV-3 is a rather widespread virus worldwide. Recently, the virus DNA has also been found in wild boar, expanding the scope of infection susceptibility among the Suidae family; also, the potential reservoir role of this species for the domestic pig has been proposed. Phylogenetic studies with available PCV-3 partial and complete sequences from around the world have revealed high nucleotide identity (>96%), although two main groups and several subclusters have been described as well. Moreover, it has been proposed the existence of a most common ancestor dated around 50 years ago. Taking into account the economic importance and the well-known effects of PCV-2 on the swine industry, a new member of the same family like PCV-3 should not be neglected. Studies on epidemiology, pathogenesis, immunity and diagnosis are guaranteed in the next few years. Therefore, the present review will update the current knowledge and future trends of research on PCV-3. info:eu-repo/semantics/publishedVersion

Porcine circovirus 3 (PCV‐3) is an emerging circovirus species that has recently been reported in different countries around the world, suggesting a widespread circulation. In this study, sera samples originating from 654 pigs of different production phases and clinical/pathological conditions, submitted for diagnostic purposes between 1996 and 2017, were randomly selected. Detection of PCV‐3 genome in such samples was attempted with a previously described PCR method, and the partial genome sequence was obtained from selected PCV‐3‐positive samples from different years. Compiled data confirmed that PCV‐3 has been circulating in the Spanish pig population since 1996. The overall frequency of PCV‐3 PCR‐positive samples in the study period was 11.47% (75 of 654). Phylogenetic analysis of twelve PCV‐3 partial sequences obtained showed a high nucleotide identity with the already known PCV‐3 sequences, with minor variations among years. No significant correlation was found between the detection of PCV‐3 and any production phase nor clinical/pathological condition. These results confirm PCV‐3 circulation at least since 1996 in the Spanish pig population with a low/moderate frequency. Although the information obtained was limited, PCV‐3 did not appear to be linked to any specific pathological condition or age group. info:eu-repo/semantics/publishedVersion

Genetic heterogeneity of Mycoplasma hyopneumoniae in pigs has been reported, however there has been limited reproducibility on the molecular methods employed so far. The aim of this study was to modify and standardize a high-resolution multiple locus variable number tandem repeat analysis (MLVA), to investigate the genetic variability of M. hyopneumoniae circulating in the United States of America (USA), Brazil, Mexico and Spain. The MLVA was standardized on the basis of the number of tandem repeats in two Mycoplasma adhesins, P97 and P146, which are proteins involved in the adherence of the pathogen to cilia. A total of 355 samples obtained from the four countries were analyzed. The Simpson's diversity index for the assay was D = 0.976 when samples from all countries were combined. A large number of MLVA types ( n = 139) were identified, suggesting that multiple M. hyopneumoniae variants are circulating in swine. The locus P97 had 17 different types with 2–18 repeats. The P146 locus showed higher heterogeneity, with 34 different types, ranging from 7 to 48 repeats. MLVA types that presented more than 30 repeats in P146 were found in Spain and Brazil, while shorter repeats were observed in the USA and Mexico. This simplified MLVA method proved to be an efficient tool for typing M. hyopneumoniae with a high degree of stability, repeatability, and discriminatory power. In conclusion, M. hyopneumoniae showed a high variable number tandem repeat heterogeneity and this assay can be applied in molecular epidemiology investigations within farms and productions systems.

Porcine circovirus type 2 (PCV2) is a ubiquitous virus that mainly affects nursery and fattening pigs causing systemic disease (PCV2-SD) or subclinical infection. A characteristic sign in both presentations is reduction of average daily weight gain (ADWG). The present study aimed to assess the relationship between PCV2 load in serum and ADWG from 3 (weaning) to 21 weeks of age (slaughter) (ADWG 3-21). Thus, three different boar lines were used to inseminate sows from two PCV2-SD affected farms. One or two pigs per sow were selected (60, 61 and 51 piglets from Pietrain, Pietrain × Large White and Duroc × Large White boar lines, respectively). Pigs were bled at 3, 9, 15 and 21 weeks of age and weighted at 3 and 21 weeks. Area under the curve of the viral load at all sampling times (AUCqPCR 3-21) was calculated for each animal according to standard and real time quantitative PCR results; this variable was categorized as “negative or low” ( 4.3 PCV2 genome copies/ml of serum), “medium” (≥10 4.3 to ≤10 5.3 ) and “high” (>10 5.3 ). Data regarding sex, PCV2 antibody titre at weaning and sow parity was also collected. A generalized linear model was performed, obtaining that paternal genetic line and AUCqPCR 3-21 were related to ADWG 3-21. ADWG 3-21 (mean ± typical error) for “negative or low”, “medium” and “high” AUCqPCR 3-21 was 672 ± 9, 650 ± 12 and 603 ± 16 g/day, respectively, showing significant differences among them. This study describes different ADWG performances in 3 pig populations that suffered from different degrees of PCV2 viraemia.

ENZOOTIC pneumonia, most commonly caused by Mycoplasma hyopneumoniae , is a chronic respiratory condition affecting pigs worldwide. It can lead to a decrease in production performance in affected pig herds, but also, importantly, has the potential to increase susceptibility to coinfections with other bacterial and viral agents, contributing to the development of the porcine respiratory disease complex (Thacker and Minion 2012). Therefore, controlling M hyopneumoniae in the field remains a high priority for pigs practitioners and producers. Means to control infections caused by M hyopneumoniae include improving management practices, using antimicrobials, and administrating vaccines (Maes and others 2008), as well as focusing strategies on eliminating the disease (Holst and others 2015). Although there is widespread use of specific management practices and antibiotic treatments to combat the disease, use of vaccine products to control M hyopneumoniae infections is by far the most commonly used strategy. Originally introduced to the market several decades ago, commercial vaccines against M hyopneumoniae are mainly bacterins, based on different bacterial strains and adjuvants (Haesebrouck and others 2004, Maes 2014). The advantages and limitations of M hyopneumoniae bacterins have been characterised over the years (Haesebrouck and others 2004, Maes and others 2008, Maes 2014), and a wealth of literature supports the use of vaccination as part of a comprehensive disease control programme (Thacker and Minion 2012), even …

Gilts are considered to play a key role in Mycoplasma hyopneumoniae (M. hyopneumoniae) transmission and control. An effective gilt acclimation program should ideally reduce M. hyopneumoniae shedding at first farrowing, decreasing pre-weaning colonization prevalence and potential respiratory problems in fatteners. However, information on gilt acclimation practices is scarce in Europe. The aim of this study was to identify current acclimation strategies for M. hyopneumoniae in Europe using a questionnaire designed to assess 15 questions focused on gilt replacement status, acclimation strategies and methods used to ascertain its effect. A total of 321 questionnaires (representing 321 farms) were voluntarily completed by 108 veterinarians (from 18 European countries). From these farms, 280 out of 321 (87.2%) were aware of the health status of gilts on arrival. From these 280 farms, 161 (57.5%) introduced M. hyopneumoniae positive replacements. In addition, 249 out of 321 (77.6%) farms applied an acclimation process using different strategies, being M. hyopneumoniae vaccination (145 out of 249, 58.2%) and the combination of vaccine and exposure to sows selected for slaughter (53 out of 249, 21.3%) the most commonly used. Notwithstanding, only 53 out of 224 (23.6%) farms, knowing the M. hyopneumoniae initial status and performing acclimation strategies against it, verified the effect of the acclimation by ELISA (22 out of 53, 41.5%), PCR (4 out of 53, 7.5%) or both (27 out of 53, 50.9%). This study showed that three fourths of the farms represented in this European survey have M. hyopneumoniae acclimation strategies for gilts, and one fifth of them verify to some extent the effect of the process. Taking into account that the assessment of acclimation efficacy could help in optimizing replacement gilt introduction into the breeding herd, it seems these practices for M. hyopneumoniae are still poorly developed in Europe.

Background: The objective of our study was to clinically and etiologically investigate acute outbreaks of respiratory disease in Finland. Our study also aimed to evaluate the clinical use of various methods in diagnosing respiratory infections under field conditions and to describe the antimicrobial resistance profile of the main bacterial pathogen(s) found during the study. Methods: A total of 20 case herds having finishing pigs showing acute respiratory symptoms and eight control herds showing no clinical signs suggesting of respiratory problems were enrolled in the study. Researchers visited each herd twice, examining and bleeding 20 pigs per herd. In addition, nasal swab samples were taken from 20 pigs and three pigs per case herd were necropsied during the first visit. Serology was used to detect Actinobacillus pleuropneumoniae (APP), swine influenza virus (SIV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine respiratory coronavirus (PRCV) and Mycoplasma hyopneumoniae antibodies. Polymerase chain reaction (PCR) was used to investigate the presence of porcine circovirus type 2 (PCV2) in serumand SIV in the nasal and lung samples. Pathology and bacteriology, including antimicrobial resistance determination, were performed on lung samples obtained from the field necropsies. Results: According to the pathology and bacteriology of the lung samples, APP and Ascaris suum were the main causes of respiratory outbreaks in 14 and three herds respectively, while the clinical signs in three other herds had a miscellaneous etiology. SIV, APP and PCV2 caused concurrent infections in certain herds but they were detected serologically or with PCR also in control herds, suggesting possible subclinical infections. APP was isolated from 16 (80%) case herds. Marked resistance was observed against tetracycline for APP, some resistance was detected against trimethoprim/sulfamethoxazole, ampicillin and penicillin, and no resistance against florfenicol, enrofloxacin, tulathromycin or tiamulin was found. Serology, even from paired serum samples, gave inconclusive results for acute APP infection diagnosis. Conclusions: APP was the most common cause for acute respiratory outbreaks in our study. SIV, A. suum, PCV2 and certain opportunistic bacteria were also detected during the outbreaks; however, viral pathogens appeared less important than bacteria. Necropsies supplemented with microbiology were the most efficient diagnostic methods in characterizing the studied outbreaks.

One of the main Mycoplasma hyopneumoniae (M. hyopneumoniae) swine experimental model objectives is to reproduce mycoplasmal pneumonia (MP). Unfortunately, experimental validated protocols to maximize the chance to successfully achieve lung lesions induced by M. hyopneumoniae are not available at the moment. Thus, the objective of this work was to identify those factors that might have a major influence on the effective development of MP, measured as macroscopic lung lesions, under experimental conditions. Data from 85 studies describing M. hyopneumoniae inoculation experiments were compiled by means of a systematic review and analyzed thereafter. Several variables were considered in the analyses such as the number of pigs in the experiment, serological status against M. hyopneumoniae, source of the animals, age at inoculation, type of inoculum, strain of M. hyopneumoniae, route, dose and times of inoculation, study duration and co-infection with other swine pathogens. Descriptive statistics were used to depict M. hyopneumoniae experimental model main characteristics whereas a recursive partitioning approach, using regression trees, assessed the importance of the abovementioned experimental variables as MP triggering factors. A strong link between the time period between challenge and necropsies and lung lesion severity was observed. Results indicated that the most important factors to explain the observed lung lesion score variability were: (1) study duration, (2) M. hyopneumoniae strain, (3) age at inoculation, (4) co-infection with other swine pathogens and (5) animal source. All other studied variables were not relevant to explain the variability on M. hyopneumoniae lung lesions. The results provided in the present work may serve as a basis for debate in the search for a universally accepted M. hyopneumoniae challenge model.

The feasibility to eradicate Porcine circovirus type 2 (PCV2) in a conventional PCV2 infected farm by vaccinating both sows and piglets using a commercially subunit vaccine was assessed. Vaccination strategy implied that all sows, boars and gilts of the farm were vaccinated every four months, and all piglets vaccinated and revaccinated with the same vaccine at 4 and 7 weeks of age, respectively. This vaccination strategy was applied during 12 consecutive months. Blood samples from 15 piglets of 4, 8, 12, 16, 20 and 24 weeks of age and 15 sows were taken monthly PRE, DURING and POST mass vaccination strategy. From all the collected sera (n=1796), a representative proportion of them (n=1235, 69%) were analysed (n=1121 from piglets and n=114 from sows). All these samples were tested by PCV2 ELISA and PCV2 PCR (and quantitative-PCR when PCR positive). All tested sows were negative by PCR but seropositive. ELISA mean OD values of sows decreased throughout the study. Percentages of PCV2 PCR positive samples in piglets were 8% (12/150), 0.9% (6/659) and 3.5% (11/312) PRE, DURING and POST application of the mass vaccination program, respectively. ELISA mean OD values of PCV2 seropositive animals progressively decreased until the end of the mass vaccination period, but a clear seroconversion was observed after stopping such strategy. In conclusion, one year period of mass PCV2 vaccination (without implementing further farm management practices or biosafety measures) was not able to clear out PCV2 infection, and the virus became detectable again when vaccination was stopped.

International audience; This study aimed to determine the porcine circovirus type 2 (PCV2) serological and virological dynamics in piglets vaccinated at different ages in a PCV2 subclinical infection (PCV2-SI) scenario. Six hundred and forty-four 2 week-old healthy piglets were selected and distributed into four treatment groups: vaccination at 3, 6 or 10 weeks of age (3W-VAC, 6W-VAC and 10W-VAC groups, respectively) and unvaccinated pigs (NON-VAC group). Blood (n = 112 pigs) and oral fluid (OF) (n = 40 pens) samples were taken throughout the study to assess PCV2 load, humoral immunity and viral genotyping. Percentage of PCV2-DNA positive sera mainly raised by 10 weeks of age, being maximum at 14 weeks of age, and then started to decrease at 18 and 25 weeks of age. Specifically, PCV2 vaccination at 3 or 6 weeks of age yielded similar results, since they produced an earlier seroconversion and reduced, at different sampling points, the proportion of viremic animals in comparison to the unvaccinated group. In contrast, PCV2 vaccination at 10 weeks of age only achieved such reduction at 25 weeks of age; in this case, vaccination coincided with the increase of the percentage of viremic pigs in the population. Both serological techniques used in sera and OF offered similar results with a high and statistically significant correlation. In contrast, a higher percentage of PCV2 DNA positivity was detected in OF in comparison with sera. In conclusion, under the present study conditions, the optimal time for PCV2 piglet vaccination was at either 3 or 6 weeks of age.

Porcine circovirus type 3 (PCV-3) is a recently discovered virus in domestic pigs and wild boar. The virus has been described in pigs with different clinical/pathological presentations and healthy animals, but the dynamics of infection is currently unknown. The aim of this study was to longitudinally monitor PCV-3 infection in 152 pigs from four different healthy farms (A, B, C and D) by means of PCR in serum. The selected animals were sampled five (farm A) or six (farms B–D) times from weaning until the end of the fattening period. PCV-3 genome was found in pigs from all tested ages and farms; few animals had an apparent long-term infection (4–23 weeks). PCV-3 frequency of detection remained fairly uniform along tested ages within farms A and C, but was more variable among sampling times in farms B and D. Eight partial genome sequences were obtained from six different animals. Phylogenetic tree and pairwise distance analysis showed high similarity among sequences and with available genomes from different countries. This is the first study on PCV-3 infection dynamics in longitudinally sampled pigs. Most pigs got infection during their life, although PCV-3 did not appear to be linked with any specific age. info:eu-repo/semantics/acceptedVersion

International audience; AbstractThe purpose of this study was to assess the effect of three different inoculation routes into mycoplasmal pneumonia (MP) in pigs challenged with Mycoplasma hyopneumoniae (M. hyopneumoniae). Thirty six-week-old M. hyopneumoniae seronegative piglets were randomly assigned to four groups: three challenged groups with experimentally inoculated pigs by either the endotracheal (ET; n = 8), intranasal (IN; n = 8) or aerosol (AE; n = 8) routes and one uninfected group (Control; n = 6). Blood samples were collected 1 day before challenge and at necropsy, 28 days post-inoculation (dpi), to assess seroconversion. Laryngeal swabs were collected at −1, 7, 14, 21 and 28 dpi in order to evaluate colonization. At necropsy, lung lesions were scored and lung tissue was collected for histopathological studies and M. hyopneumoniae DNA detection. Broncho-alveolar lavage fluid (BALF) was also obtained to detect M. hyopneumoniae DNA, specific IgA antibodies and cytokines. MP was observed in all inoculated groups, but the ET group displayed a significantly higher number of animals affected by MP as well as a higher mean lung lesion score. These results were paralleled with an earlier seroconversion and upper respiratory tract colonization of M. hyopneumoniae. Additionally, in the ET group, higher levels of pro-inflammatory cytokines and specific IgA antibodies in BALF were found. Under the conditions of the present study, MP was reproduced by the three evaluated inoculation routes. Obtained results suggest that the ET route is the most effective in order to induce MP in pigs experimentally challenged with M. hyopneumoniae.

The present study aimed to compare the efficacy of a porcine circovirus type 2 (PCV2) commercial vaccine in terms of average daily weight gain (ADWG) as well as infection dynamics in pigs with different maternally derived antibody (MDA) levels. A total of 337 animals from a PCV2 subclinically infected farm were distributed into two groups based on weight and PCV2 antibody levels (high [H] or low [L]) at 2 weeks of age. One week later, these animals were subdivided in four groups according to the treatment received. Vaccinated pigs (H-V and L-V) received 1 mL of a commercial vaccine and NV (H-NV and L-NV) received 1 mL of PBS. All piglets were subsequently bled at 7, 12, 18, 22 and 25 weeks of age and weighted at 12 and 25 weeks of age. V animals showed significantly lower PCV2 infection rates and viral load as well as higher ELISA S/P ratios and ADWG than NV ones. Compared with H-V piglets, L-V pigs showed numerically lower PCV2 infection rates, lower area under the curve of viral load, an earlier seroconversion and a numerically, but not significantly, higher ADWG. In this study, MDA did not seem to interfere with the effect of PCV2 vaccination on ADWG. However, only when a small subpopulation of pigs with the highest ELISA S/P ratios at vaccination was considered, an apparent interference of vaccine efficacy on ADWG was noticed. Therefore, the impact of the putative interference under field conditions is probably negligible for most farms. This study was funded by the European PCV2-Award 2012 sponsored by Boehringer Ingelheim. The authors wish to thank to the farmer (Álvaro Abadías) and the veterinarians (Lluís Cons and Paula Alcubierre) for their collaboration in conducting the field study and Eva Huerta, Rosa López, and Diego Pérez (CReSA) for their excellent technical assistance. Hua Feng was grant awarded by the Chinese Scholarship Council (No. 2011704032).

Porcine circovirus type 2 (PCV2) negatively modulates the immune response in vitro. The objective of this study was to investigate if PCV2 interferes with the development of the immune response to Aujeszky's disease virus (ADV) vaccine, using an in vivo experimental subclinical model. Pigs were divided into four groups: (group CC) not infected with PCV2 and not vaccinated against ADV; (group IC) infected with PCV2 but not vaccinated against ADV; (group CV) not infected with PCV2 but vaccinated against ADV, and (group IV) infected with PCV2 and vaccinated against ADV. Pigs in groups IC and IV were inoculated intranasally with PCV2 and 14 days later, pigs in the CV and IV groups were vaccinated IM with a gE(-)tk(-) attenuated ADV vaccine. Clinical signs and weight gains were recorded from days 0 to 35 post-PCV2 inoculation (PI), at which point the pigs were euthanased and examined post-mortem. Throughout the experiment the PCV2 load was quantified in serum, antibodies to PCV2 and ADV were determined and antigen-specific cellular responses against both viruses were measured using an interferon-γ ELISPOT. PCV2 inoculated animals developed subclinical infection and had lower weight gain relative to non-infected controls. No differences were observed between the CV and IV groups in terms of the humoral or cellular immune responses to vaccination against Aujeszky's disease, suggesting that subclinical infection with PCV2 does not alter the response to this vaccine.

Torque teno viruses (TTV) are small, non-enveloped viruses with a circular single-stranded DNA genome, which are considered non-pathogenic. However, TTVs have been eventually linked to human diseases. TTVs infecting pigs, Torque teno sus virus 1 (TTSuV1) and 2 (TTSuV2), have been recently associated to porcine circovirus diseases (PCVD). To get more insights into such potential disease association, the aim of this study was to quantify TTSuV1 and TTSuV2 viral loads in serum of pigs affected by two PCVDs, postweaning multisystemic wasting syndrome (PMWS) and porcine dermatitis and nephropathy syndrome (PDNS). Such study was carried out by means of a newly developed real-time quantitative PCR (qPCR) method. Both TTSuVs were highly prevalent among studied pigs. TTSuV2 viral loads were significantly higher in PMWS affected animals, further supporting the previously suggested association between TTSuV2 and PMWS. On the contrary, TTSuV1 prevalence and loads were not related with the studied PCVDs.

The objective of the present work was to elucidate the potential relationship between Mycoplasma hyopneumoniae infection and seroconversion dynamics and climatological conditions in four groups of pigs from the same farm born in different seasons of the year. Nasal swabs and blood samples were taken from 184 pigs at 1, 3, 6, 9, 12, 15, 18, 22 and 25 (slaughter age) weeks of age. Outside climatologic parameters, including temperature (°C), relative humidity (%), precipitation (l/m(2)) and wind speed (m/s) were recorded weekly from January 2003 to June 2004. Percentage of nPCR detection of M. hyopneumoniae in nasal swabs was associated significantly with the weekly precipitation rate [P = 0.0018, OR = 1.31 (IC = 1.11-1.55)]; the higher the precipitation rate, the higher the probability of being M. hyopneumoniae nPCR-positive. On the other hand, the percentage of seropositive pigs had a significant association with mean weekly temperature rate [P = 0.0012, OR = 0.89 [IC = 0.84-0.95]); the lower the temperature, the higher the probability of being M. hyopneumoniae seropositive. Animals born in autumn (when higher precipitations rates were recorded), entering finishing units in winter (when lower temperatures were recorded), and reaching slaughter in spring, had the highest probability of being infected by M. hyopneumoniae and the highest probability of being M. hyopneumoniae seropositive.

Changes in porcine circovirus type 2 (PCV-2) genotypes were evaluated before, during and after outbreaks of post-weaning multisystemic wasting syndrome (PMWS) in (1) a retrospective study using pig sera collected in Spain from 1985 to 2008 and (2) a longitudinal study using pig sera collected from two farms in Spain over periods of 7 and 14 years. In both studies, there was a rapid genotypic shift from PCV-2a to PCV-2b that was related to the peak of PMWS epizootics in Spain and the appearance of PMWS on the two farms studied longitudinally.

The aim of the present study was to investigate the role of the capside (Cap) and replicase (Rep) proteins of Porcine circovirus type 2 (PCV2) as well as the whole PCV2 (both PCV2a and PCV2b genotypes) in the induction of cell-mediated immunity upon infection. At 6 weeks of age, six pigs were intranasally inoculated with the Stoon 1010 (Stoon) isolate (PCV2a) and seven with the Sp-7-10-54-13 (Sp) isolate (PCV2b). None of the pigs developed clinical disease but the Sp group had significantly higher proportion of pigs with PCV2-associated lesions and PCV2 load in tissues compared to the Stoon group. In both groups, development of IFN-gamma secreting cells (SC) in response to the whole PCV2 and Cap protein was detected by means of an ELISPOT from day 7 post-inoculation (PI) to the end of the study (21 days PI). Significant responses against Rep protein were only detected in Sp-inoculated pigs. No differences in ELISPOT results were seen when either PCV2a or PCV2b was used in vitro to recall peripheral blood mononuclear cells (PBMC) in any group. Stimulation of PBMC with the whole virus but not with Cap or Rep protein induced IL-10-SC in all pigs regardless of their PCV2 infection status, indicating an innate origin of this response. The results from this study demonstrate that PCV2-infected pigs developed cell-mediated immunity to Cap and Rep proteins and that, in the course of a sub-clinical infection, development and strength of such responses are possibly related to the levels of PCV2 replication.

Haemophilus parasuis is the etiologic agent of Glässer's disease in pigs, which is pathologically characterized by serofibrinous polyserositis and arthritis. H. parasuis include virulent and non-virulent strains and confirmation of virulence in H. parasuis is still dependent on experimental reproduction of the disease. Since the variability in virulence is supported by serotyping and genotyping (particularly, multilocus sequence typing [MLST]), we examined the relationship between the classification of 8 field strains by these methods and their capacity to cause disease in snatch-farrowed, colostrum-deprived piglets. The severity of clinical signs and lesions produced by the different strains correlated with the quantity of H. parasuis recovered from the lesions. However, the virulence of the strains in the animal model did not show a total correlation with their serovar or their classification by MLST. More studies are needed to identify a virulence marker that could substitute animal experimentation in H. parasuis. In addition, we reproduced disease in domestic pigs with a strain isolated from the nasal cavity of wild boars. This result indicates the existence of virulent strains of H. parasuis in wild suids, which could produce disease under appropriate circumstances, and suggests a possible source of infection for domestic pigs.

The present study describes the effects of a commercially available genetically engineered chimeric vaccine against porcine circovirus type 2 (PCV2) on clinical, pathological and virological features in three multi-site farms suffering from postweaning multisystemic wasting syndrome (PMWS). The vaccine product was able to reduce clinical signs, PCV2 viral load in lymphoid organs and/or sera, and overall mortality in nurseries and fattening units. This is the first time in which is shown that a PCV2 vaccine is able to decrease specifically PMWS-associated mortality. Another novelty of this study is the assessment of PMWS-like histological lesions in a large number of vaccinated and non-vaccinated pigs under field conditions.

Mycoplasma hyopneumoniae is the principal aetiological agent of enzootic pneumonia (EP), a chronic respiratory disease that affects mainly finishing pigs. Although major efforts to control M. hyopneumoniae infection and its detrimental effects have been made, significant economic losses in pig production worldwide due to EP continue. M. hyopneumoniae is typically introduced into pig herds by the purchase of subclinically infected animals or, less frequently, through airborne transmission over short distances. Once in the herd, M. hyopneumoniae may be transmitted by direct contact from infected sows to their offspring or between pen mates. The 'gold standard' technique used to diagnose M. hyopneumoniae infection, bacteriological culture, is laborious and is seldom used routinely. Enzyme-linked immunosorbent assay and polymerase chain reaction detection methods, in addition to post-mortem inspection in the form of abattoir surveillance or field necropsy, are the techniques most frequently used to investigate the potential involvement of M. hyopneumoniae in porcine respiratory disease. Such techniques have been used to monitor the incidence of M. hyopneumoniae infection in herds both clinically and subclinically affected by EP, in vaccinated and non-vaccinated herds and under different production and management conditions. Differences in the clinical course of EP at farm level and in the efficacy of M. hyopneumoniae vaccination suggest that the transmission and virulence characteristics of different field isolates of M. hyopneumoniae may vary. This paper reviews the current state of knowledge of the epidemiology of M. hyopneumoniae infection including its transmission, infection and seroconversion dynamics and also compares the various epidemiological tools used to monitor EP.

The objectives of the present study were to compare Mycoplasma hyopneumoniae (Mh) colonization and serologic status on Mh vaccinated and non-vaccinated sows and to assess the effect of sow vaccination on colonization and serologic status of their piglets at weaning as well as presence of enzootic pneumonia (EP) lung lesions at slaughter. Fifty sows (25 vaccinated and 25 unvaccinated) as well as five of their piglets were included in the study. Blood samples and nasal swabs from sows at 7 weeks pre-farrowing and 1 week post-farrowing and from piglets at 3-4 weeks of age were taken. Nasal swabs and sera were tested by a nested polymerase chain reaction (nPCR) to detect Mh DNA and by an enzyme-linked immunosorbent assay (ELISA) test to detect antibodies to the pathogen, respectively. Finally, at 23 weeks of age, pigs were sent to the slaughter where the extension of EP-compatible gross lesions was assessed. Vaccination with two doses of Mh vaccine resulted in a significantly higher (p0.05) percentage of seropositive sows than in the non-vaccinated group at 1 week post-farrowing. On the contrary, no statistical significant differences were found in the number of nasal nPCR positive sows among different treatments (p0.05). At 3-4 weeks of age, a significantly higher percentage (p0.001) of seropositive piglets came from vaccinated than from non-vaccinated sows. Although the number of Mh infected piglets coming from non-vaccinated sows was higher than the one from vaccinated sows, the difference was not statistically significant (p0.05). Overall, piglets from vaccinated sows had a significant lower (p0.05) mean of EP-compatible lung lesions (1.83+/-2.8) than piglets from non-vaccinated sows (3.02+/-3.6). Under the conditions described in this study, sow vaccination did not affect sow or piglet colonization but increased the percentage of seropositive sows and piglets at weaning and reduced significantly the mean EP-compatible lung lesion scoring at slaughter.

The notion that postweaning multisystemic wasting syndrome (PMWS)-affected pigs develop an impaired humoral response against porcine circovirus type 2 (PCV2) has been reported in several studies. However, little information is available regarding the presence of neutralizing antibodies (NA) in PCV2-infected pigs and their role in the pathogenesis of the disease. The aim of the present work was to further characterize the humoral response, and in particular the production of NA, in pigs with different PCV2-infection status. Seventy-two conventional pigs from different farms were classified into three groups based on PCV2 infection and clinico-pathological status, namely: PCV2-negative, non-PMWS PCV2-positive and PMWS-affected animals. In addition, 9-week old pigs from an experimental infection (6 controls and 14 PCV2-inoculated pigs) were also studied. NA and total PCV2 antibodies (TA) as well as viral load in serum were determined and correlated with the clinico-pathological status of pigs. Results indicated that PMWS-affected pigs had lower NA titres, if any, than healthy animals. NA titres were also inversely correlated with PCV2 load in serum. NA and TA titres were positively correlated; however, correlation differed among infection status, being lower in PCV2-positive pigs. Also, the diagnostic performance of each test was evaluated, indicating that the combination of viral neutralization and quantitative PCR in serum was useful to discard PMWS (specificity 92%). In experimentally infected animals, the evolution of NA paralleled the course TA, although a slight delay in NA production was seen in some animals. The increase of NA coincided with the drop in viral load. Results from this work further support that PMWS-affected pigs show an impaired humoral immune response and, particularly, an inefficient NA response against PCV2.

A field trial was conducted to study Mycoplasma hyopneumoniae (Mh) infection dynamics by nested polymerase chain reaction (nPCR) and serology in pigs of a farm affected by enzootic pneumonia (EP). Moreover, correlation of Mh detection at different respiratory tract sites with presence of EP gross and microscopic lung lesions was assessed. These parameters were studied and compared between vaccinated (two doses at 1 and 3 weeks of age versus one dose at 6 weeks of age) and non-vaccinated pigs. Animals were monitored from birth to slaughter by nPCR from nasal swabs and by serology. From 3 to 22 weeks of age, an average of three pigs per treatment and per batch were necropsied (n = 302). The remaining pigs were sent to the slaughter (n = 103). Nasal, bronchial and tonsillar swabs were taken from the necropsied/slaughtered pigs; gross and microscopic EP-suggestive lung lesions were also assessed. Single and double vaccination resulted in earlier seroconversion and higher percentage of Mh seropositive pigs compared to control group. At slaughter, double vaccinated pigs showed lower percentage of EP-compatible gross lung lesions and lower Mh prevalence at upper respiratory tract sites (nasal cavity and tonsil) than control pigs.

Mycoplasma hyopneumoniae is the primary aetiological agent of swine enzootic pneumonia (EP) and one of the major contributors to the porcine respiratory disease complex (PRDC). Gross lung lesions in pigs affected by EP consist of cranioventral pulmonary consolidation (CVPC), usually distributed bilaterally in the apical, intermediate, accessory and cranial parts of the diaphragmatic lobes. Several lung scoring methods are currently in place for the evaluation of CVPC. The aims of this study were (1) to review the lung lesion scoring systems used to assess pneumonia associated with M. hyopneumoniae infection, and (2) to evaluate eight of these scoring systems by applying them to the lungs of 76 pigs with experimentally-induced M. hyopneumoniae pneumonia. A significant correlation between all lung lesion scoring systems was observed and the coefficients of determination in a regression analysis were very high between each pair-wise comparison, except for a unique scoring system based on image analysis. A formula of equivalence between lung scoring methods was developed in order to compare the results obtained with these methods. The present review provides a basis for comparison (even retrospectively) of lesions evaluated using different lung scoring systems.

The present work describes the serum haptoglobin (Hp) dynamics in piglets vaccinated and non-vaccinated with a commercial porcine circovirus type 2 (PCV2) vaccine at 3 weeks of age, and its relationship with the average daily weight gain (ADWG). The field study was carried out on two farms (A and B) with a previous clinical history of PCV2-systemic disease (PCV2-SD). The aim of the study was to assess whether Hp could be used as a surrogate marker of PCV2 vaccine efficacy. PCV2 infection was confirmed by quantitative real time PCR (qPCR) in pigs from both farms, but PCV2-SD was only diagnosed in farm A. No statistically significant relation was found between serum Hp concentration and the percentage of qPCR positive animals and the treatment applied (PCV2 vaccination) in both farms. On the other hand, using linear regression analysis, a significant negative correlation between the area under the curve of Hp (AUCHp) and ADWG was observed for farm A (p

The present study was designed to investigate the prevalence of swine Torque teno virus (TTV) in post-weaning multisystemic wasting syndrome (PMWS)-affected and non-affected Spanish swine. Nested PCR (nPCR) assays to detect two distinct TTV genogroups were applied. A significantly higher prevalence of TTV infection was found in sera from PMWS-affected animals (97 %) than in sera from non-PMWS-affected animals (78 %). Whilst PMWS-affected pigs (91 %) were more likely to be infected with TTV from genogroup 2 than non-PMWS-affected swine (72 %), no such difference was observed with genogroup 1. Nucleotide sequences of nPCR products were 91–99 % identical between strains within a genogroup. In contrast, inter-genogroup sequence identities were 49–58 %. Phylogenetic analyses demonstrated that genogroups form different clusters without association with PMWS or porcine circovirus type 2 infection status of the animals. These results indicate a high prevalence of both swine TTV genogroups in Spain, being present more frequently in PMWS-affected animals than in non-PMWS-affected animals.

Postweaning multisystemic wasting syndrome (PMWS) diagnosis is based on the presence of characteristic histopathological lymphoid lesions and porcine circovirus type 2 (PCV2) within these lesions. Previous studies indicate that PCV2 load is higher in PMWS affected than in PCV2 infected, healthy pigs. On the other hand, PCV2 has been suggested to play a role in porcine dermatitis and nephropathy syndrome (PDNS) pathogenesis. This study describes a new TaqMan real time PCR assay and its use to quantify viral load in serum samples. Serum viral loads were related with different degrees of PMWS characteristic lesions and PDNS cases. DNA extracted from serum samples from 75 animals with mild, moderate and severe PMWS lesions and 12 animals with PDNS was used as template. PCV2 DNA was quantified in 69 of 75 PMWS cases and in 11 of 12 PDNS cases. Significant differences in PCV2 load were observed between animals with severe, moderate and mild PMWS lesions, although variability within each group was high, probably due to heterogeneity in disease progression. These results suggest that high viral load is a major feature of PMWS affected pigs. PDNS affected animals had lower PCV2 loads. No significant differences in viral load were found between animals suffering from PDNS and animals with mild PMWS characteristic lesions, which were unaffected clinically.