Serotype diversity and antimicrobial susceptibility profiles of Actinobacillus pleuropneumoniae isolated in Italian pig farms from 2015 to 2022.

Actinobacillus pleuropneumoniae (APP) is a bacterium frequently associated with porcine pleuropneumonia. The acute form of the disease is highly contagious and often fatal, resulting in significant economic losses for pig farmers. Serotype diversity and antimicrobial resistance (AMR) of APP strains circulating in north Italian farms from 2015 to 2022 were evaluated retrospectively to investigate APP epidemiology in the area. A total of 572 strains isolated from outbreaks occurring in 337 different swine farms were analysed. The majority of isolates belonged to serotypes 9/11 (39.2%) and 2 (28.1%) and serotype diversity increased during the study period, up to nine different serotypes isolated in 2022. The most common resistances were against tetracycline (53% of isolates) and ampicillin (33%), followed by enrofloxacin, florfenicol and trimethoprim/sulfamethoxazole (23% each). Multidrug resistance (MDR) was common, with a third of isolates showing resistance to more than three antimicrobial classes. Resistance to the different classes and MDR varied significantly depending on the serotype. In particular, the widespread serotype 9/11 was strongly associated with florfenicol and enrofloxacin resistance and showed the highest proportion of MDR isolates. Serotype 5, although less common, showed instead a concerning proportion of trimethoprim/sulfamethoxazole resistance. Our results highlight how the typing of circulating serotypes and the analysis of their antimicrobial susceptibility profile are crucial to effectively manage APP infection and improve antimicrobial stewardship.

Regional-Scale Analysis of Antimicrobial Usage in Smallholder Cattle Herds (Aosta Valley, Italy): Why Surveillance Matters.

Optimising antimicrobial usage (AMU) in livestock is pivotal to counteract the emergence of antimicrobial resistance. We analysed AMU in more than 1000 cattle herds over 11 years (2008-2018) in the Aosta Valley (Italy), a region where 80% of farms house less than 50 cattle. Dairy cows accounted for over 95% of AMU. AMU was estimated using the defined daily dose animal for Italy (DDDAit) per biomass for the whole herd and a treatment incidence 100 (TI100) for cows. Average annual herd-level AMU was low, with 3.6 DDDAit/biomass (range: 3.2-4.0) and 1.2 TI100 in cows (range: 1.1-1.3). Third and fourth generation cephalosporins, which are critical for human medicine, represented almost 10% of usage, and intramammary antimicrobials accounted for over 60%. We detected significant downward temporal trends in total AMU, as well as a positive relationship with herd size. The magnitude of such effects was small, leaving scant room for further reduction. However, the frequent use of critical antimicrobials and intramammary products should be addressed, following the principles of prudent AMU. Our findings highlight the importance of monitoring AMU even in low-production, smallholding contexts where a low usage is expected, to identify any deficiencies and implement interventions for further AMU optimisation.

PRRS Monitoring by Processing Fluids on Italian Swine Breeding Farms.

The porcine reproductive and respiratory syndrome (PRRS) control strategy within swine breeding farms is based on herd classification relative to PRRSV infection status. This study aims to assess the efficacy of a monitoring plan based on processing fluids (PFs) by comparing it with the classification of herds based on the analysis of blood serum. Twenty-five breeding herds were enrolled in the study, with at least five consecutive batches sampled from each herd. Each batch was tested for PRRSV by RT-PCR performed on (i) pre-weaning blood serum from 30 piglets and (ii) PFs from all the male piglets in the batch. PRRS categories following the Holtkamp classification were assigned based on the results of each testing protocol. The two protocols assigned the same category to 18 out of 25 herds: while they showed perfect agreement in identifying positive unstable and stable herds, we observed some discrepancy in discriminating between low- and high-prevalence classes within unstable herds. PFs are thus a reliable sample to assign PRRS categories in Italian breeding herds characterized by widespread PRRSV circulation. However, in case of an unstable epidemiological scenario, we recommend the adoption of an integrated monitoring strategy that combines blood sampling with PFs.

First Detection of mcr-9 in a Multidrug-Resistant Escherichia coli of Animal Origin in Italy Is Not Related to Colistin Usage on a Pig Farm.

The emergence of colistin resistance raises growing concerns because of its use as a last-resort antimicrobial for the treatment of severe gram-negative bacterial infections in humans. Plasmid-borne mobile colistin resistance genes (mcr) are particularly worrisome due to their high propensity to spread. An mcr-9-positive Escherichia coli was isolated from a piglet in Italy, representing the first isolation of this gene from an E. coli of animal origin in the country. Whole genome sequencing (WGS) revealed that mcr-9 was borne by an IncHI2 plasmid carrying several other resistance genes. The strain was indeed phenotypically resistant to six different antimicrobial classes, including 3rd and 4th generation cephalosporins. Despite the presence of mcr-9, the isolate was susceptible to colistin, probably because of a genetic background unfavourable to mcr-9 expression. The lack of colistin resistance, coupled with the fact that the farm of origin had not used colistin in years, suggests that mcr-9 in such a multidrug-resistant strain can be maintained thanks to the co-selection of neighbouring resistance genes, following usage of different antimicrobials. Our findings highlight how a comprehensive approach, integrating phenotypical testing, targeted PCR, WGS-based techniques, and information on antimicrobial usage is crucial to shed light on antimicrobial resistance.

The hazard of carbapenemase (OXA-181)-producing Escherichia coli spreading in pig and veal calf holdings in Italy in the genomics era: Risk of spill over and spill back between humans and animals.

Carbapenemase-producing Enterobacterales (CPE) are considered a major public health issue. In the frame of the EU Harmonized AMR Monitoring program conducted in Italy in 2021, 21 epidemiological units of fattening pigs (6.98%; 95% CI 4.37-10.47%; 21/301) and four epidemiological units of bovines <12 months (1.29%; 95% CI 0.35-3.27%, 4/310) resulted positive to OXA-48-like-producing E. coli (n = 24 OXA-181, n = 1 OXA-48). Whole Genome Sequencing (WGS) for in-depth characterization, genomics and cluster analysis of OXA-181-(and one OXA-48) producing E. coli isolated, was performed. Tracing-back activities at: (a) the fattening holding of origin of one positive slaughter batch, (b) the breeding holding, and (c) one epidemiologically related dairy cattle holding, allowed detection of OXA-48-like-producing E. coli in different units and comparison of further human isolates from fecal samples of farm workers. The OXA-181-producing isolates were multidrug resistant (MDR), belonged to different Sequence Types (STs), harbored the IncX and IncF plasmid replicons and multiple virulence genes. Bioinformatics analysis of combined Oxford Nanopore Technologies (ONT) long reads and Illumina short reads identified bla OXA-181 as part of a transposon in IncX1, IncX3, and IncFII fully resolved plasmids from 16 selected E. coli, mostly belonging to ST5229, isolated during the survey at slaughter and tracing-back activities. Although human source could be the most likely cause for the introduction of the bla OXA-181-carrying IncX1 plasmid in the breeding holding, concerns arise from carbapenemase OXA-48-like-producing E. coli spreading in 2021 in Italian fattening pigs and, to a lesser extent, in veal calf holdings.

Wastewater-based surveillance in Italy leading to the first detection of mcr-10-positive Klebsiella quasipneumoniae.

Wastewater-based surveillance enabled the first detection of the mobile colistin resistance gene mcr-10 in Italy. This plasmid-borne resistance gene was found in strains of Klebsiella quasipneumoniae isolated from samples of human raw sewage collected over several months. Although the isolates were phenotypically susceptible to colistin, the emergence of mcr-10 is concerning due to the highly variable expression of the gene and the potential for horizontal transfer to other species. In addition, the strains also carried an extended-spectrum ß-lactamase gene and were phenotypically resistant to several beta-lactams. This study highlights the value of wastewater-based surveillance as an effective tool to monitor the emergence of antimicrobial resistance in strains circulating in the community and the environment.

Rapid displacement of SARS-CoV-2 variant Delta by Omicron revealed by allele-specific PCR in wastewater.

On November 26, 2021, the B.1.1.529 COVID-19 variant was classified as the Omicron variant of concern (VOC). Reports of higher transmissibility and potential immune evasion triggered flight bans and heightened health control measures across the world to stem its distribution. Wastewater-based surveillance has demonstrated to be a useful complement for clinical community-based tracking of SARS-CoV-2 variants. Using design principles of our previous assays that detect SARS-CoV-2 variants (Alpha and Delta), we developed an allele-specific RT-qPCR assay which simultaneously targets the stretch of mutations from Q493R to Q498R for quantitative detection of the Omicron variant in wastewater. We report their validation against 10-month longitudinal samples from the influent of a wastewater treatment plant in Italy. SARS-CoV-2 RNA concentrations and variant frequencies in wastewater determined using these variant assays agree with clinical cases, revealing rapid displacement of the Delta variant by the Omicron variant within three weeks. These variant trends, when mapped against vaccination rates, support clinical studies that found the rapid emergence of SARS-CoV-2 Omicron variant being associated with an infection advantage over Delta in vaccinated persons. These data reinforce the versatility, utility and accuracy of these open-sourced methods using allele-specific RT-qPCR for tracking the dynamics of variant displacement in communities through wastewater for informed public health responses.

The role of co-infections in M. hyopneumoniae outbreaks among heavy fattening pigs: a field study.

Little is known about how co-infections and genotype dynamics affect Mycoplasma hyopneumoniae infection in fattening pigs. This study was aimed at assessing the role of co-infections in M. hyopneumoniae outbreaks, their influence on the presence of M. hyopneumoniae genotypes and their impact on consequent lung lesions. Tracheobronchial swabs (TBS) from 300 finishers were collected from 10 farms at the onset of enzootic pneumonia outbreaks and 1 month later, sampling of 3 groups per farm: Group A showed clinical signs first, Group B was housed near Group A, and Group C was located in a different building. Pigs' lungs were scored at the slaughterhouse. TBS were tested for the main pathogens involved in respiratory diseases, and samples positive for M. hyopneumoniae were genotyped by multiple-locus variable-number tandem repeat analysis (MLVA). Pigs in Group A showed the highest prevalence and load of M. hyopneumoniae. A positive association was detected between M. hyopneumoniae and Mycoplasma hyorhinis, whereas Actinobacillus pleuropneumoniae was more frequent when the M. hyopneumoniae load was higher. Nevertheless, co-infection had no effect on lung lesion scores. The presence of multiple MLVA types (mixed infections) increased in time only in pigs from Group C and was positively associated with porcine reproductive and respiratory syndrome virus infection. Lung lesions were more severe in pigs with at least one TBS positive for M. hyopneumoniae and in pigs with a history of mixed infections. The central role of M. hyopneumoniae and relevance of mixed infections suggest that increased biosecurity might be beneficial for lung lesion sequelae.

Preliminary Evidence of Endotoxin Tolerance in Dairy Cows during the Transition Period.

The blastogenic response of bovine peripheral blood mononuclear cells (PBMCs) to lipopolysaccharides (LPS) has been investigated for a long time in our laboratories. In particular, a possible correlation between the blastogenic response to LPS and the disease resistance of dairy cows has been suggested in previous studies. Isolated PBMCs from eight cows at three different time points during the transition period (T0 = 15 days before calving; T1 = 7 days post-calving; T2 = 21 days post-calving) were cultured in the presence or absence of LPS, and the blastogenic response was assayed 72 h after in vitro stimulation. Moreover, the gene expression of proinflammatory cytokines and kynurenine pathway molecules was investigated by real-time RT-PCR on both unstimulated and stimulated PBMCs. The cows were retrospectively divided into healthy and diseased, based on the development of peripartum diseases (subclinical ketosis and placenta retention). The comparison between healthy and diseased cows suggested that healthy animals seemed to better control the response to LPS. On the contrary, diseased animals showed a much higher inflammatory response to LPS. Moreover, cows were retrospectively classified as high and low responders based on the in vitro proliferative response of PBMCs to LPS, using the median value as a threshold. Unstimulated PBMCs of low responders showed higher expression of the proinflammatory cytokines Interleukin 1-ß (IL-1ß), Interleukin 6 (IL-6) and Tumor Necrosis Factor-α (TNF-α), compared to high responders. Our preliminary data suggest that, during the peripartum period, high responders seem to be more tolerant to endotoxins and develop a lower inflammatory response to different stressors. Instead, low responders could be more prone to the development of unwanted inflammatory conditions in response to mild/moderate stressors.

Extended-Spectrum-ß-Lactamase- and AmpC-Producing Escherichia coli in Domestic Dogs: Spread, Characterisation and Associated Risk Factors.

In veterinary medicine, the issue of antimicrobial resistance was mainly addressed in food-producing animals (although companion animals also deserve attention). Indeed, these species may be reservoir of resistant microorganisms, such as extended-spectrum ß-lactamase and AmpC (ESBL/AmpC)-producing bacteria. Dogs in particular may transmit them to close-contact humans. Overall 266 faecal samples of healthy dogs were microbiologically and molecularly analyzed to investigate ESBL/AmpC-producing Escherichia coli and the effects of host and environmental factors on their spread. A prevalence of 25.9% of ESBL/AmpC-producing E. coli, supported by blaCTX-M (79.7%), blaTEM (47.8%), blaCMY (13%), and blaSHV (5.8%) gene detection, emerged. Dogs frequenting extra-urban environments showed significantly higher odds of being positive to ESBL/AmpC E. coli (30.2%) compared to urban dogs (16.7%) identifying the environment as a risk factor. About 88.4% of isolates were resistant to cephalosporins, 8.7% to cephalosporins and carbapenems, and 2.9% to cephalosporins, carbapenems, and penicillins. ESBL/AmpC-producing E. coli expressing blaCMY were significantly more resistant to cefoxitin, cefotaxime/clavulanic acid and ceftazidime/clavulanic acid, highlighting its negative effects. Our results suggest the role of domestic dogs as a maintenance host of ESBL/AmpC-producing E. coli leading to a constant health monitoring. The recorded resistances to carbapenems implies attention and further investigations.

Non-Assembled ORF2 Capsid Protein of Porcine Circovirus 2b Does Not Confer Protective Immunity.

Porcine Circovirus 2 (PCV2) vaccines are based on either inactivated whole virion, or recombinant ORF2 capsid protein assembled into Virus-like Particles (VLPs). No data are available about the immunizing properties of free, non-assembled capsid protein. To investigate this issue, ORF2 of a reference PCV2b strain was expressed in a Baculovirus-based expression system without assembly into VLPs. The free purified protein was formulated into an oil vaccine at three distinct Ag payloads: 10.8/3.6/1.2 micrograms/dose. Each dose was injected intramuscularly into five, 37-day old piglets, carefully matched for maternally-derived antibody. Five control piglets were injected with sterile PBS in oil adjuvant. Twenty-eight days later, all the pigs were challenged intranasally with 105.3 TCID50 of PCV2b strain DV6503. After challenge infection, all the pigs remained in good clinical conditions. The recombinant vaccine did not induce significant antibody and PCV2-specific IFN-γ responses. ELISPOT and lymphocyte proliferation data confirmed poor induction of cell-mediated immunity. In terms of PCV2 viremia, there was no significant difference between vaccinated and control animals. The histological data indicated the absence of a detectable viral load and of PCVAD lesions in both vaccinated and control animals, as well as of histiocytes and multi-nucleated giant cells. We conclude that free, non-assembled ORF2 capsid protein does not induce protective immunity.

Antimicrobial Resistance of Escherichia coli in Dairy Calves: A 15-Year Retrospective Analysis and Comparison of Treated and Untreated Animals.

The health problem of antimicrobial resistance (AMR) involves several species. AMR surveillance is essential to identify its development and design control strategies; however, available data are still limited in some contexts. The AMR profiles of 2612 E. coli strains isolated over a period of 15 years (2002-2016) from calf enteric cases were analyzed to determine the presence of resistance and their temporal dynamics. Furthermore, the AMR profiles and the presence of the major virulence genes of 505 E. coli strains isolated from 1-week- and 2-week-old calves, 406 treated with antimicrobials and 99 untreated, were analyzed and compared to investigate the potential effects of treatment on AMR and strain pathogenicity. Resistance to tetracycline (90.70%) was the most common, followed by resistance to sulfamethoxazole/trimethoprim (77.70%) and flumequine (72.10%). The significantly higher percentage of AMR and virulence gene expression recorded in treated calves, combined with the statistically higher resistance to sulfamethoxazole/trimethoprim in E. coli with K99, corroborates the notion of resistance being induced by the frequent use of antimicrobials, leading to treatments potentially becoming ineffective. The significantly higher resistance to amoxicillin/clavulanic acid, enrofloxacin, and florfenicol in isolates from 1-week-old calves suggests the role of the environment as a source of contamination that should be investigated further.

ESBL/AmpC-Producing Escherichia coli in Wild Boar: Epidemiology and Risk Factors.

The complex health problem of antimicrobial resistance (AMR) involves many host species, numerous bacteria and several routes of transmission. Extended-spectrum ß-lactamase and AmpC (ESBL/AmpC)-producing Escherichia coli are among the most important strains. Moreover, wildlife hosts are of interest as they are likely antibiotics free and are assumed as environmental indicators of AMR contamination. Particularly, wild boar (Sus scrofa) deserves attention because of its increased population densities, with consequent health risks at the wildlife-domestic-human interface, and the limited data available on AMR. Here, 1504 wild boar fecal samples were microbiologically and molecularly analyzed to investigate ESBL/AmpC-producing E. coli and, through generalized linear models, the effects of host-related factors and of human population density on their spread. A prevalence of 15.96% of ESBL/AmpC-producing E. coli, supported by blaCTX-M (12.3%), blaTEM (6.98%), blaCMY (0.86%) and blaSHV (0.47%) gene detection, emerged. Young animals were more colonized by ESBL/AmpC strains than older subjects, as observed in domestic animals. Increased human population density leads to increased blaTEM prevalence in wild boar, suggesting that spatial overlap may favor this transmission. Our results show a high level of AMR contamination in the study area that should be further investigated. However, a role of wild boar as a maintenance host of AMR strains emerged.

Dataset of immune responses induced in swine by an inactivated Porcine Circovirus 2b vaccine.

A whole virus, inactivated, Porcine Circovirus 2b (PCV2b) vaccine was submitted to a quantal assay of potency, as explained in detail in our companion paper [1]. To this purpose, twenty, 45-day old piglets, checked for maternally-derived antibody (MDA), were allocated to four groups of 5 animals each; these were vaccinated with 800/266/88/0 nanograms, respectively, of an inactivated PCV2b strain, consisting of two distinct virion populations. Twenty-six days later, all the pigs were challenged intranasally with the homologous PCV2b strain. In the presence of a clear dose-dependent protection in terms of viremia, no such effect was observed in terms of weight gain after challenge. The 800 and 266-ng payloads were associated with neutralizing antibody titers above the MDA levels in oral fluids. Higher levels of viremia in control and 88-ng groups [1] coincided with a higher Natural Killer activity of tracheobronchial lymph node cells from PCV2-infected pigs. The PCV2 ORF2-specific ELISPOT assay for IFN-g- secreting cells showed very few (2-4) ORF2-specific cells/105 peripheral blood mononuclear cells beyond the basal levels under our experimental conditions (non-significant differences among groups). Also, no significant differences were observed in the degree of lymphoid tissue hyperplasia among the different groups.

Occurrence of Salmonella Typhimurium and its monophasic variant (4, [5],12:i:-) in healthy and clinically ill pigs in northern Italy.

BACKGROUND: The serovar Typhimurium (4, [5],12:i:1,2), is the most frequently isolated serovar in case of salmonellosis in pigs in Europe and its monophasic variant (4, [5],12:i:-) has been increasingly responsible for Salmonella outbreaks in humans. A total of 25,215 samples were collected, during the years 2002-2017, from 1359 pig farms located in Northern Italy. Samples were collected from different material sources including fecal samples, rectal swabs, gut content and different organs. RESULTS: Salmonella was isolated in 15.80% of samples and, among the isolates, 733 were typed as Salmonella Typhimurium (ST) or its monophasic variant (MST). Over time, there was an increase of isolation of MST which outnumbered ST. Most of the strains were isolated in animals during the weaning stage and the growing - fattening period whereas the clinical cases were mainly present in young pigs after weaning. CONCLUSIONS: This study confirms the presence of ST and MST in pig farms although, considering the total of isolated serotypes, with lower percentages than previously reported. In the last few years, ST has increasingly been replaced by MST suggesting that MST has a competitive advantage over ST, probably due to its different antigenicity and pathogenicity which renders the infection stealthier to recognize and control.

Genomic variability of Mycoplasma hyopneumoniae within pig lung lobes.

BACKGROUND: Genotypic variability in M. hyopneumoniae has been reported within and among herds. However, information regarding VNTR types within single lung lobes is lacking. The objective of his study was to analyse M. hyopneumoniae infections and their association with VNTR types and lung lesions at the lobe level. Lungs from 300 pigs from 10 farms experiencing an enzootic pneumonia outbreak were collected and scored. M. hyopneumoniae was detected by real-time PCR and genotyped by MLVA assay in all samples. RESULTS: The results showed genotypic variability within single pigs and among lung lobes. At the lobe level, infection with one VNTR type (SN infection) was dominant. Lobes with lesion scores > 0 were associated with positive results for real-time PCR. At the lobe level, no relationship was observed between infections with more than one genotype (MX infections) and the proportion of Mycoplasma-like lesions. Lesion-free lobes presented a higher proportion of MX infections than lobes scored > 0. M. hyopneumoniae was detected more frequently in the right lobe of the lung (p < 0.05), with a similar distribution within lobes for SN and MX infections. The anatomic conformation of swine lungs led to a higher prevalence of infections in the right lobe. However, this study showed that this condition did not affect the distribution of infections with multiple VNTR types. Nevertheless, careful consideration of sample selection should be practised for M. hyopneumoniae genotype analyses, including lung lobes with no visible lesions. CONCLUSION: The results did not show a significant association between the number of detected genotypes and the severity of the lesions at the lung lobe level, but revealed the unexpected detection of M. hyopneumoniae genotypes in lesion-free lobes. These results imply that a representative sampling of all lobes may lead to an accurate identification of the VNTR-type distribution. Further studies including factors that can affect pathogenetic evolution of this bacterium could shed light on the complexity of the relationship between genotypes and the lung lesions magnitude.

Protective immunity in swine induced by Porcine Circovirus 2b inactivated vaccines with different antigen payload.

Porcine Circovirus 2 (PCV2) vaccines are poorly standardized in terms of antigen payload and correlates of protection. Therefore, twenty, 45-day old piglets were divided into four groups of 5 animals each and vaccinated with 800 / 266 / 88 / 0 nanograms, respectively, of an inactivated PCV2b strain formulated in the same oil adjuvant. Twenty-six days later, all the pigs were challenged intranasally with the homologous PCV2b strain. No clinical signs were observed in the pigs under study. Viremia was observed after challenge in all the control pigs, as well as in 3 pigs of the 266 and 88-ng groups (one and two, respectively). No pigs of the 800-ng group developed viremia. On the basis of post challenge viremia, the PCV2b vaccine under study had a titer of 11 Protective Doses (PD) 50 %, and 1 PD50 amounted to 74 ng of PCV2b Ag. Neutralizing and ELISA Ab titers showed no obvious correlation with protection in the single animals, even though the 800-ng group developed a significantly higher mean Ab response. All the pigs with a PCV2-specific, IFN-gamma response at 3 weeks after vaccination in whole blood samples were protected against viremia. In lymphoid tissues (mainly tonsils and ileum) the presence of sparse reactive histiocytes and multinucleated giant cells was the only PCV2-associated feature and, by immunohistochemistry, only 3 out of 20 subjects showed a low viral load.

In vitro Cytokine Responses to Virulent PRRS Virus Strains.

Porcine reproductive and respiratory syndrome (PRRS) affects farmed swine causing heavy direct and indirect losses. The infections sustained by PRRS viruses (PRRSV-1 and PRRSV-2) may give rise to severe clinical cases. This highlights the issue of PRRSV pathogenicity and relevant markers thereof. Since PRRSV strains can be discriminated in terms of immunotypes, we aimed to detect possible correlates of virulence in vitro based on the profile of innate immune responses induced by strains of diverse virulence. To this purpose, 10 field PRRSV isolates were investigated in assays of innate immune response to detect possible features associated with virulence. Tumor necrosis factor-α, interleukin (IL)-1beta, IL-8, IL-10, and caspase-1 were measured in cultures of PRRSV-treated peripheral blood mononuclear cells of PRRS-naive pigs, unable to support PRRSV replication. Two reference PRRSV strains (highly pathogenic and attenuated, respectively), were included in the screening. The PRRSV strains isolated from field cases were shown to vary widely in terms of inflammatory cytokine responses in vitro, which were substantially lacking with some strains including the reference, highly pathogenic one. In particular, neither the field PRRSV isolates nor the reference highly pathogenic strain gave rise to an IL-1beta response, which was consistently induced by the attenuated strain, only. This pattern of response was reversed in an inflammatory environment, in which the attenuated strain reduced the ongoing IL-1beta response. Results indicate that some pathogenic PRRSV strains can prevent a primary inflammatory response of PBMCs, associated with reduced permissiveness of mature macrophages for PRRSV replication in later phases.

Characterization of the IgA response to PRRS virus in pig oral fluids.

Porcine Reproductive and Respiratory Syndrome (PRRS) is a complex model of host/virus relationship. Disease control measures often includes "acclimatization", i.e. the exposure of PRRS-naïve gilts and sows to PRRSV-infected pigs and premises before the breeding period. In this respect, we had repeatedly observed an association between PRRSV-specific IgA responses in oral fluids (OF) of gilts and block of PRRSV spread. Therefore, we set out to investigate in vitro the inhibition of PRRSV replication by OF samples with different titers of PRRSV-specific IgA and IgG antibody, using Real-time RT PCR. PRRSV yield reduction in monocyte-derived macrophages was associated with the IgA content in OF samples, whereas the IgG-rich samples were sometimes associated with antibody-dependent enhancement (ADE) of replication. Accordingly, we could discriminate between ADE-positive and ADE-negative PRRSV strains. Next, we separated Ig isotypes in OF samples of PRRSV-infected pigs by means of protein A and size exclusion chromatography. The above results were confirmed by using separated Ig isotypes. Both dimeric and monomeric IgA were associated with the strongest reduction of PRRSV replication. The treatment of pig macrophages with separated OF antibodies before PRRSV infection was also associated with PRRSV yield reduction, along with clear changes of both CD163 and CD169 surface expression. Our results point at a role of mucosal IgA in the control of PRRSV replication by extra- and/or intracellular interaction with PRRSV, as well as by induction of signals leading to a reduced susceptibility of macrophages to PRRSV infection.