Update on Mycoplasma hyopneumoniae infections in pigs : knowledge gaps for improved disease control

Mycoplasma hyopneumoniae (M. hyopneumoniae) is the primary pathogen of enzootic pneumonia, a chronic respiratory disease in pigs. Infections occur worldwide and cause major economic losses to the pig industry. The present paper reviews the current knowledge on M. hyopneumoniae infections, with emphasis on identification and analysis of knowledge gaps for optimizing control of the disease. Close contact between infected and susceptible pigs is the main route of M. hyopneumoniae transmission. Management and housing conditions predisposing for infection or disease are known, but further research is needed to better understand M. hyopneumoniae transmission patterns in modern pig production systems, and to assess the importance of the breeding population for downstream disease control. The organism is primarily found on the mucosal surface of the trachea, bronchi and bronchioles. Different adhesins and lipoproteins are involved in the adherence process. However, a clear picture of the virulence and pathogenicity of M. hyopneumoniae is still missing. The role of glycerol metabolism, myoinositol metabolism and the Mycoplasma Ig binding protein-Mycoplasma Ig protease system should be further investigated for their contribution to virulence. The destruction of the mucociliary apparatus, together with modulating the immune response, enhances the susceptibility of infected pigs to secondary pathogens. Clinical signs and severity of lesions depend on different factors, such as management, environmental conditions and likely also M. hyopneumoniae strain. The potential impact of strain variability on disease severity is not well defined. Diagnostics could be improved by developing tests that may detect virulent strains, by improving sampling in live animals and by designing ELI-SAs allowing discrimination between infected and vaccinated pigs. The currently available vaccines are often cost-efficient, but the ongoing research on developing new vaccines that confer protective immunity and reduce transmission should be continued, as well as optimization of protocols to eliminate M. hyopneumoniae from pig herds

Neutralizing antibodies against porcine circovirus type 2 in liquid pooled plasma contribute to the biosafety of commercially manufactured spray-dried porcine plasma

Neutralizing antibodies (NA) inherently present in pooled plasma collected at commercial abattoirs may provide some protection against potential porcine circovirus type 2 (PCV2) infectivity of plasma. Moreover, these NA may also contribute to the biosafety of spray-dried porcine plasma (SDPP). The objective of the study was to characterize and quantify the PCV2 antibody neutralizing capacity in pooled liquid porcine plasma and SDPP samples collected from industrial spray-drying facilities located in the Southeast and Midwest regions of the United States and the Northeast region of Spain. In the United States, PCV2 NA was determined in 1 sample of pooled liquid plasma from commercial spray-drying plants in the Southeast and 1 from the Midwest region. Obtained results were compared with those of a plasma sample from a PCV2 vaccinated sow and 1 from a PCV2 antibody negative sow. In Spain, 15 pooled liquid porcine plasma samples and 10 SDPP samples were collected at a commercial spray-drying plant total and NA against PCV2 were determined. Results with pooled liquid porcine plasma from commercial spray-drying facilities in the United States indicated that NA titers against PCV2 in these samples (log2 8.33 ± 0.41 and 9.0 ± 0.0) were similar or greater than the plasma from the PCV2-vaccinated sow (log2 6.33 ± 0.41). The analysis of U.S. samples indicated that liquid plasma diluted to 1:256 (10–2.41) was able to neutralize between 100 to 200 PCV2 virus particles or about 4 logs10 median tissue culture infective dose (TCID50) per milliliter. Similarly, samples from the Spanish pooled liquid plasma and the SDPP samples indicated an increased amount of NA activity against PCV2. Specifically, a dilution of 10–2.47 ± 0.33 of plasma was able to inactivate 100 PCV2 virus particles; therefore, the inactivation capacity of commercial liquid plasma was greater than 104 TCID50/mL. The calculated 90% reduction in infected cells because of NA in pooled plasma samples (log2 8.2 ± 0.38) was less (P < 0.05) than in its concentrate form of SDPP (mean, log2 10.2 ± 0.85). In conclusion, PCV2 NA contained in liquid pooled plasma from market pigs was detected at greater concentrations than that from a vaccinated sow and that after spray-drying biological neutralizing activity was conserved, which implies that the inherent NA in liquid plasma may have an important role in the biosafety of commercially produced SDPP.info:eu-repo/semantics/publishedVersio

Diagnostic accuracy of two DNA‐based molecular assays for detection of porcine circovirus 3 in swine population using Bayesian latent class analysis

Molecular‐based tools sometimes are the only laboratory techniques available to detect a recently discovered agent, and their validation without the existence of previously described ‘gold standard’ methods poses a challenge for the diagnosticians. A good example within this scenario is the recently described porcine circovirus 3 (PCV‐3) in the swine population worldwide, from which only few PCR methods have been described. Therefore, the primary objective of this study was to estimate the diagnostic accuracy of a direct PCR (dPCR) and a real‐time qPCR (qPCR) for detection of PCV‐3 in Italian swine population. Bayesian latent class analysis approach was used to rigorously assess their features and applicability in routine diagnostic activity. Data on dPCR and qPCR were available from 116 domestic pigs, which were randomly selected from 55 farms located at different regions in Northern Italy. The sensitivity (Se) estimates of dPCR (94%; posterior credible interval (PCI%) 84–100) and qPCR (96%; PCI% 90–100) were high and similar. The estimated specificity (Sp) of both dPCR and qPCR assays was around 97%. dPCR and qPCR assays showed a high and comparable Se and Sp estimates for the detection of PCV‐3 in Italian swine population.info:eu-repo/semantics/acceptedVersio

UV-C irradiation is able to inactivate pathogens found in commercially collected porcine plasma as demonstrated by swine bioassay

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.info:eu-repo/semantics/publishedVersio

Combined effects of spray‐drying conditions and postdrying storage time and temperature on Salmonella choleraesuis and Salmonella typhimurium survival when inoculated in liquid porcine plasma

The objective of this study was to determine the effectiveness of the spray-drying process on the inactivation of Salmonella choleraesuis and Salmonella typhimurium spiked in liquid porcine plasma and to test the additive effect of immediate postdrying storage. Commercial spray-dried porcine plasma was sterilized by irradiation and then reconstituted (1:9) with sterile water. Aliquots of reconstituted plasma were inoculated with either S. choleraesuis or S. typhimurium, subjected to spray-drying at an inlet temperature of 200°C and an outlet temperature of either 71 or 80°C, and each spray-drying temperature combinations were subjected to either 0, 30 or 60 s of residence time (RT) as a simulation of residence time typical of commercial dryers. Spray-dried samples were stored at either 4·0 ± 3·0°C or 23·0 ± 0·3°C for 15 days. Bacterial counts of each Salmonella spp., were completed for all samples. For both Salmonella spp., spray-drying at both outlet temperatures reduced bacterial counts about 3 logs at RT 0 s, while there was about a 5·5 log reduction at RT 60 s. Storage of all dried samples at either 4·0 ± 3·0°C or 23·0 ± 0·3°C for 15 days eliminate all detectable bacterial counts of both Salmonella spp.info:eu-repo/semantics/publishedVersio

Detection of MERS-CoV antigen on formalin-fixed paraffin-embedded nasal tissue of alpacas by immunohistochemistry using human monoclonal antibodies directed against different epitopes of the spike protein

Middle East respiratory syndrome (MERS) represents an important respiratory disease accompanied by lethal outcome in one third of human patients. In recent years, several investigators developed protective antibodies which could be used as prophylaxis in prospective human epidemics. In the current study, eight human monoclonal antibodies (mAbs) with neutralizing and non-neutralizing capabilities, directed against different epitopes of the MERS-coronavirus (MERS-CoV) spike (MERS-S) protein, were investigated with regard to their ability to immunohistochemically detect respective epitopes on formalin-fixed paraffin-embedded (FFPE) nasal tissue sections of MERS-CoV experimentally infected alpacas. The most intense immunoreaction was detected using a neutralizing antibody directed against the receptor binding domain S1B of the MERS-S protein, which produced an immunosignal in the cytoplasm of ciliated respiratory epithelium and along the apical membranous region. A similar staining was obtained by two other mAbs which recognize the sialic acid-binding domain and the ectodomain of the membrane fusion subunit S2, respectively. Five mAbs lacked immunoreactivity for MERS-CoV antigen on FFPE tissue, even though they belong, at least in part, to the same epitope group. In summary, three tested human mAbs demonstrated capacity for detection of MERS-CoV antigen on FFPE samples and may be implemented in double or triple immunohistochemical methods.info:eu-repo/semantics/acceptedVersio

Quantitative proteomics analysis of lymph nodes from pigs infected by porcine circovirus type 2(PCV2) by 2-DE, 18O/16O labeling and linear ion trap mass spectrometry

Comunicaciones a congreso

Evolution of Swine Influenza Virus H3N2 in Vaccinated and Nonvaccinated Pigs after Previous Natural H1N1 Infection

Swine influenza viruses (SIV) produce a highly contagious and worldwide distributed disease that can cause important economic losses to the pig industry. Currently, this virus is endemic in farms and, although used limitedly, trivalent vaccine application is the most extended strategy to control SIV. The presence of pre-existing immunity against SIV may modulate the evolutionary dynamic of this virus. To better understand these dynamics, the viral variants generated in vaccinated and nonvaccinated H3N2 challenged pigs after recovery from a natural A(H1N1) pdm09 infection were determined and analyzed. In total, seventeen whole SIV genomes were determined, 6 from vaccinated, and 10 from nonvaccinated animals and their inoculum, by NGS. Herein, 214 de novo substitutions were found along all SIV segments, 44 of them being nonsynonymous ones with an allele frequency greater than 5%. Nonsynonymous substitutions were not found in NP; meanwhile, many of these were allocated in PB2, PB1, and NS1 proteins. Regarding HA and NA proteins, higher nucleotide diversity, proportionally more nonsynonymous substitutions with an allele frequency greater than 5%, and different domain allocations of mutants, were observed in vaccinated animals, indicating different evolutionary dynamics. This study highlights the rapid adaptability of SIV in different environments.info:eu-repo/semantics/publishedVersio

Vaccination against swine influenza in pigs causes different drift evolutionary patterns upon swine influenza virus experimental infection and reduces the likelihood of genomic reassortments

Influenza A viruses (IAVs) can infect a wide variety of bird and mammal species. Their genome is characterized by 8 RNA single stranded segments. The low proofreading activity of their polymerases and the genomic reassortment between different IAVs subtypes allow them to continuously evolve, constituting a constant threat to human and animal health. In 2009, a pandemic of an IAV highlighted the importance of the swine host in IAVs adaptation between humans and birds. The swine population and the incidence of swine IAV is constantly growing. In previous studies, despite vaccination, swine IAV growth and evolution were proven in vaccinated and challenged animals. However, how vaccination can drive the evolutionary dynamics of swine IAV after coinfection with two subtypes is poorly studied. In the present study, vaccinated and nonvaccinated pigs were challenged by direct contact with H1N1 and H3N2 independent swine IAVs seeder pigs. Nasal swab samples were daily recovered and broncho-alveolar lavage fluid (BALF) was also collected at necropsy day from each pig for swine IAV detection and whole genome sequencing. In total, 39 swine IAV whole genome sequences were obtained by next generation sequencing from samples collected from both experimental groups. Subsequently, genomic, and evolutionary analyses were carried out to detect both, genomic reassortments and single nucleotide variants (SNV). Regarding the segments found per sample, the simultaneous presence of segments from both subtypes was much lower in vaccinated animals, indicating that the vaccine reduced the likelihood of genomic reassortment events. In relation to swine IAV intra-host diversity, a total of 239 and 74 SNV were detected within H1N1 and H3N2 subtypes, respectively. Different proportions of synonymous and nonsynonymous substitutions were found, indicating that vaccine may be influencing the main mechanism that shape swine IAV evolution, detecting natural, neutral, and purifying selection in the different analyzed scenarios. SNV were detected along the whole swine IAV genome with important nonsynonymous substitutions on polymerases, surface glycoproteins and nonstructural proteins, which may have an impact on virus replication, immune system escaping and virulence of virus, respectively. The present study further emphasized the vast evolutionary capacity of swine IAV, under natural infection and vaccination pressure scenarios.This research was funded by grants AGL2016–75280-R from Ministerio de Ciencia, Innovación y Universidades from the Spanish government. ÁL-V has a pre-doctoral fellowship FPI 2017, Ministerio de Ciencia, Innovación y Universidades from the Spanish government.The authors would like to thank Laura Baioni for the sequencing libraries preparation. The authors also want to thank the IRTA-CReSA animal facilities staff for their work during the in vivo experiment. Lastly, we would like to thank Marta Pérez and all the people from CReSA who were involved in the experiment during the COVID-19 lockdowns.info:eu-repo/semantics/publishedVersio