An investigation of the transmission of Actinobacillus pleuropneumoniae within vertically integrated systems using whole genome sequencing.

Actinobacillus pleuropneumoniae (APP) causes significant economic losses to the swine industry. Antibiotic treatment can be challenging due to its clinical urgency and the turnover of antimicrobial susceptibility results from the diagnostic laboratory. The aim of this study was to evaluate the vertical transmission of APP within integrated systems as a criterion for optimising antimicrobial treatment in the field, using whole genome sequencing (WGS). Additionally, the genetic variability of Spanish APP isolates has been assessed to decipher antimicrobial resistance (AMR) determinants, toxin presence, serotype, and phenotype/genotype concordance of AMR. A total of 169 isolates from clinical cases of porcine pleuropneumonia with known antimicrobial susceptibility profiles were sequenced. Additionally, 48 NCBI assemblies were included to perform a phylogenetic analysis. Phylogenetic analysis revealed high association between phylogenetic clusters, serotypes, and presence of toxins that are associated within vertically integrated systems by its epidemiological link. Concordance between presence of AMR determinants (genotype) vs in-vitro antimicrobial susceptibility pattern (phenotype) was acceptable for amoxicillin, florfenicol, oxytetracycline, and enrofloxacin using epidemiological cut-off values (ECOFFs), but low concordance was observed for doxycycline and trimethoprim-sulfamethoxazole (T/S). On the other hand, using CLSI clinical breakpoints (CBPs), concordance was acceptable for florfenicol and enrofloxacin and not evaluated for doxycycline, oxytetracycline, trimethoprim-sulfamethoxazole (T/S), and amoxicillin because no CBP are available for them. Finally, WGS has demonstrated the clonality between isolates that shared a common origin (grandmother's farm) and resistance phenotype, suggesting vertical transmission of this pathogen and supporting the use of the epidemiological approach as a good criterion to optimise the antimicrobial use.

The Susceptibility Trends of Respiratory and Enteric Porcine Pathogens to Last-Resource Antimicrobials.

Monitoring the antimicrobial susceptibility of last-resource antimicrobials for veterinary pathogens is urgently needed from a one-health perspective. The objective of this study was to analyze the antimicrobial susceptibility trends of Spanish porcine bacteria to quinolones, cephalosporins, and polymyxins. Isolates of Actinobacillus pleuropneumoniae, Pasteurella multocida, and Escherichia coli were isolated from sick pigs from 2019 to 2022. An antimicrobial susceptibility test was determined based on the minimal inhibitory concentration (MIC) following an internationally accepted methodology. The MIC categorization was based on distributing the range of MIC values in four categories, with category one being the most susceptible (lowest MIC value) and category four the least susceptible (highest MIC value). Moreover, clinical susceptibility (susceptible/non-susceptible) was also determined according to the CLSI and EUCAST clinical breakpoints. A logistic and multinomial logistic regression model was used to analyze the susceptibility data for dichotomized and categorized MIC data, respectively, for any pair of antimicrobial/microorganism. In general terms, the antimicrobial susceptibility of pig bacteria to these antimicrobials remained stable or increased in the last four years in Spain. In the case of A. pleuropneumoniae and quinolones, a significant temporal trend was observed where isolates from 2020 had significantly increased odds of being more susceptible than isolates from 2019. In the case of E. coli and polymyxins, a significant temporal trend was observed where isolates from 2020 and 2021 had significantly increased odds of being more susceptible than isolates from 2019 and 2020, respectively. Finally, significant odds of being less susceptible were only observed for cephalosporins and E. coli for 2020 versus 2019, stagnating for the rest of study period. These results provide sound data on critically important antimicrobials in swine medicine.

Susceptibility trends of swine respiratory pathogens from 2019 to 2022 to antimicrobials commonly used in Spain.

BACKGROUND: Antimicrobial resistance is one of the most important health challenges in humans and animals. Antibiotic susceptibility determination is used to select the most suitable drug to treat animals according to its success probability following the European legislation in force for these drugs. We have studied the antibiotic susceptibility pattern (ASP) of Actinobacillus pleuropneumoniae (APP) and Pasteurella multocida (PM) isolates, collected during the period 2019-2022 in Spain. ASP was measured by determining minimum inhibitory concentration using standardized laboratory methods and its temporal trend was determined by logistic regression analysis of non-susceptible/susceptible isolates using clinical breakpoints. RESULTS: It was not observed any significant temporal trends for susceptibility of Actinobacillus pleuropneumoniae to ceftiofur, florfenicol, sulfamethoxazole/trimethoprim, tulathromycin and tildipirosin during the study period (p > 0.05). Contrarily, a significant temporal trend (p < 0.05) was observed for quinolones (enrofloxacin and marbofloxacin), tetracyclines (doxycycline and oxyteracycline), amoxicillin, tiamulin and tilmicosin. On the other hand, it was not observed any significant temporal trends for susceptibility of Pasteurella multocida to quinolones (enrofloxacin and marbofloxacin), amoxicillin, ceftiofur, florfenicol and macrolides (tildipirosin, tulathromycin and tilmicosin) during the study period (p > 0.05). Contrarily, a significant temporal trend (p < 0.05) was observed for tetracyclines (oxyteracycline), tiamulin and sulfamethoxazole/trimethoprim. CONCLUSIONS: In general terms, pig pathogens (APP and PM) involved in respiratory diseases analysed herein appeared to remain susceptible or tended to increase susceptibility to antimicrobials over the study period (2019-2022), but our data clearly showed a different pattern in the evolution of antimicrobial susceptibility for each combination of drug and microorganism. Our results highlight that the evolution of antimicrobial susceptibility must be studied in a case-by-case situation where generalization for drug families and bacteria is not possible even for bacteria located in the same ecological niche.

Antimicrobial Susceptibility Testing of Porcine Bacterial Pathogens: Investigating the Prospect of Testing a Representative Drug for Each Antimicrobial Family.

Antimicrobial susceptibility testing is necessary to carry out antimicrobial stewardship but a limited number of drugs belonging to each antimicrobial family has to be tested for technical limitations and economic resources. In this study, we have determined the minimal inhibitory concentration, using microdilution following international standards (CLSI), for 490 Actinobacillus pleuropneumoniae, 285 Pasteurella multocida, 73 Bordetella bronchiseptica, 398 Streptococcus suis and 1571 Escherichia coli strains from clinical cases collected in Spain between 2018 and 2020. The antimicrobial susceptibility pattern was deciphered using a principal component analysis for each bacterium and a matrix correlation (high > 0.8, medium 0.5−0.8 and low < 0.5) was obtained for each pair of antimicrobials. No significant associations were observed between MIC patterns for different antimicrobial families, suggesting that co-selection mechanisms are not generally present in these porcine pathogens. However, a high correlation was observed between the fluroquinolones (marbofloxacin and enrofloxacin) for all mentioned pathogens and for ceftiofur and cefquinome for E. coli and S. suis. Moreover, a significant association was also observed for tetracyclines (doxycycline and oxytetracycline) and B. bronchiseptica and tildipirosin/tulathromycin for P. multocida. These results suggest that generally, a representative drug per antimicrobial class cannot be selected, however, for some drug−bug combinations, MIC values from one representative drug could be extrapolated to the whole antimicrobial family.

Porcine Reproductive and Respiratory Syndrome Surveillance in breeding Herds and Nurseries Using Tongue Tips from Dead Animals.

The detection capacity of Porcine Reproductive and Respiratory Syndrome virus (PRRSV) in tongues from dead animals in breeding herds (stillborns and piglets dying during the lactating period) and nursery farms (naturally dead animals) for PRRSV surveillance was evaluated. The samples were selected if pairs of serum and tongues were available from 2018 to 2020. Serum (pools of five) and exudate from tongues (one bag) were analyzed by PRRSV RT-PCR. The agreement between the serum sample procedure versus tongues exudate was assessed using a concordance test (Kappa statistic) at batch level. A total of 32 submissions, corresponding to 14 farms, had PRRSV diagnostic information for serum and tongues exudate. The overall agreement of batch classification as positive or negative, based on RT-PCR PRRSV results, between serum and tongue exudate of the 32 pairs was 76.9%. Cohen's Kappa was 0.55. The main discrepancy came from the presence of positive samples in tongues exudate and not in serum, suggesting that tongue exudate to monitor PRRSV seems to be more sensitive than serum. These results suggest that this sample procedure could be also used for PRRSV surveillance and monitoring.

Antimicrobial Stewardship for Respiratory Pathogens in Swine.

The aim of this study was to set up antimicrobial stewardship for swine respiratory pathogens following the recommendation from the European Medicine Agency. The obtained antimicrobial susceptibility pattern recommended using antimicrobial stewardship for each clinical case instead of treatment guidelines focused on pathogens. Thus, the bacteria are isolated and the MIC values, the clinical interpretation for each antimicrobial (susceptible or resistant), additional information about the distance between the MIC obtained and the clinical breakpoint, and set up for each drug, are represented in the report provided for veterinarians. A graph from green (susceptible) to red (resistant) is enclosed for each antimicrobial and microorganism in the report. The greener, the more susceptible is the strain, and the redder, the less susceptible is the strain for each particular antimicrobial. This information could help veterinarians to select the most suitable antimicrobial from first, second, or last option antimicrobials. Thus, veterinarians should choose the antimicrobial, inside each option, with the best antimicrobial susceptibility pattern that corresponds with the greener status in the report. The information provided in the report could be useful for all clinical cases, caused by a certain bacterium within the same pig production system, if an epidemiological link could be established.

Antimicrobial Susceptibility Pattern of Porcine Respiratory Bacteria in Spain.

The monitoring of antimicrobial susceptibility of pig pathogens is critical to optimize antimicrobial treatments and prevent development of resistance with a one-health approach. The aim of this study was to investigate the antimicrobial susceptibility patterns of swine respiratory pathogens in Spain from 2017 to 2019. Bacterial isolation and identification were carried out following standardized methods from samples coming from sacrificed or recently deceased pigs with acute clinical signs compatible with respiratory tract infections. Minimum inhibitory concentration (MIC) values were determined using the broth microdilution method containing a total of 10 and 7-8 antimicrobials/concentrations respectively, in accordance with the recommendations presented by the Clinical and Laboratory Standards Institute (CLSI). The obtained antimicrobial susceptibility varies between pig respiratory pathogens. Actinobacillus pleuropneumoniae (APP) and Pasteurella multocida (PM) were highly susceptible (≥90%) to ceftiofur, florfenicol and macrolides (tilmicosin, tildipirosin and tulathromycin). However, the antimicrobial susceptibility was intermediate (>60% but <90%) for amoxicillin and enrofloxacin in the case of APP and sulfamethoxazole/trimethropim and tiamulin in the case of PM. Both bacteria showed low (<60%) antimicrobial susceptibility to doxycycline. Finally, Bordetella bronchiseptica was highly susceptible only to tildipirosin and tulathromycin (100%) and its susceptibility for florfenicol was close to 50% and <30% for the rest of the antimicrobial families tested. These results emphasize the need of determining antimicrobial susceptibility in pig respiratory cases in order to optimize the antimicrobial treatment in a case-by-case scenario.

Near Real-Time Monitoring of Clinical Events Detected in Swine Herds in Northeastern Spain.

Novel techniques of data mining and time series analyses allow the development of new methods to analyze information relating to the health status of the swine population in near real-time. A swine health monitoring system based on the reporting of clinical events detected at farm level has been in operation in Northeastern Spain since 2012. This initiative was supported by swine stakeholders and veterinary practitioners of the Catalonia, Aragon, and Navarra regions. The system aims to evidence the occurrence of endemic diseases in near real-time by gathering data from practitioners that visited swine farms in these regions. Practitioners volunteered to report data on clinical events detected during their visits using a web application. The system allowed collection, transfer and storage of data on different clinical signs, analysis, and modeling of the diverse clinical events detected, and provision of reproducible reports with updated results. The information enables the industry to quantify the occurrence of endemic diseases on swine farms, better recognize their spatiotemporal distribution, determine factors that influence their presence and take more efficient prevention and control measures at region, county, and farm level. This study assesses the functionality of this monitoring tool by evaluating the target population coverage, the spatiotemporal patterns of clinical signs and presumptive diagnoses reported by practitioners over more than 6 years, and describes the information provided by this system in near real-time. Between January 2012 and March 2018, the system achieved a coverage of 33 of the 62 existing counties in the three study regions. Twenty-five percent of the target swine population farms reported one or more clinical events to the system. During the study period 10,654 clinical events comprising 14,971 clinical signs from 1,693 farms were reported. The most frequent clinical signs detected in these farms were respiratory, followed by digestive, neurological, locomotor, reproductive, and dermatological signs. Respiratory disorders were mainly associated with microorganisms of the porcine respiratory disease complex. Digestive signs were mainly related to colibacilosis and clostridiosis, neurological signs to Glässer's disease and streptococcosis, reproductive signs to PRRS, locomotor to streptococcosis and Glässer's disease, and dermatological signs to exudative epidermitis.

Identification of resilient sows in porcine reproductive and respiratory syndrome virus-infected farms1.

The identification of resilient sows can improve reproductive performance in farms exposed to multiple challenges. A common challenge is the porcine reproductive and respiratory syndrome virus (PRRSV). A key issue to deal with disease resilience is to set up a feasible phenotyping strategy. Our aim was to develop a phenotyping criterion to discriminate susceptible from resilient sows in PRRSV-infected farms. A total of 517 Landrace x Large White gilts were classified as resilient (R) or susceptible (S) to PRRSV virus, following vaccination with MLV-PRRSV at 6 to 7 wk of age, in a PRRSV negative multiplication farm. Female piglets were phenotyped as R if their serum was negative to PRRSV at 7 and 21 d postvaccination (DPV) or as S if their serum was positive at 7 and/or 21 DPV. Amongst them, 382 gilts were transferred to a PRRSV-positive production farm, where the number of piglets born alive (NBA), stillborn (NSB), mummified (NMU), lost (NLP = NSB + NMU), and total born (NTB = NBA + NLP) were recorded for almost 3 yr. Data were collected during 2 periods according to the PRRSV farm health status, which were confirmed as either PRRSV-positive stable (endemic) or inestable (epidemic). Analyses were carried out under a Bayesian approach. The heritability for the resilience criterion was estimated using a threshold model. A linear (for NTB and NBA) and a binomial model (for NSB, NMU, and NLP) on the resilience criterion by the farm health status were used to assess the difference between R and S sows. The heritability of the resilience criterion was 0.46 (SD 0.06). The probability of a piglet being lost was greater (≥0.97) in S than in R litters, regardless of whether the delivery occurred during a PRRSV outbreak (20.5% vs. 17.0%) or not (15.8% vs. 13.7%). The lower piglet mortality rate in R sows was due to NSB, in the endemic phase (13.0% vs. 15.0% of NTB, with a posterior probability of 98% of S sows showing higher NSB than R sows), and to NMU, in the epidemic phase (4.0% vs. 8.4% of NTB, with a posterior probability of >99% of S sows showing higher NMU than R sows). During a PRRSV outbreak, the S sows were twice as likely to give birth to a mummified piglet when compared with R sows. These findings provide evidence that the described phenotyping scheme has a potential use as a PRRSV resilience criterion.

Identification of resilient sows in Porcine Reproductive and Respiratory Syndrome virus infected farms.

The identification of resilient sows can improve reproductive performance in farms exposed to multiple challenges. A common challenge is the porcine reproductive and respiratory syndrome virus (PRRSV). A key issue to deal with disease resilience is to set up a feasible phenotyping strategy. Our aim was to develop a phenotyping criterion to discriminate susceptible from resilient sows in PRRSV-infected farms. A total of 517 Landrace x Large White gilts were classified as resilient (R) or susceptible (S) to PRRSV virus, following vaccination with MLV-PRRSV at 6-7 wk of age, in a PRRSV negative multiplication farm. Female piglets were phenotyped as R if their serum was negative to PRRSV at 7 and 21 d post-vaccination (DPV) or as S if their serum was positive at 7 and/or 21 DPV. Amongst them, 382 gilts were transferred to a PRRSV-positive production farm, where the number of piglets born alive (NBA), stillborn (NSB), mummified (NMU), lost (NLP=NSB+NMU) and total born (NTB = NBA+NLP) were recorded for almost three years. Data were collected during two periods according to the PRRSV farm health status, which were confirmed as either PRRSV-positive stable (endemic) or inestable (epidemic). Analyses were carried out under a Bayesian approach. The heritability for the resilience criterion was estimated using a threshold model. A linear (for NTB and NBA) and a binomial model (for NSB, NMU and NLP) on the resilience criterion by the farm health status were used to assess the difference between R and S sows. The heritability of the resilience criterion was 0.46 (SD 0.06). The probability of a piglet being lost was greater (≥0.97) in S than in R litters, regardless of whether the delivery occurred during a PRRSV outbreak (20.5% vs 17.0%) or not (15.8% vs 13.7%). The lower piglet mortality rate in R sows was due to NSB, in the endemic phase (13.0% vs 15.0% of NTB, with a posterior probability of 98% of S sows showing higher NSB than R sows), and to NMU, in the epidemic phase (4.0% vs 8.4% of NTB, with a posterior probability of >99% of S sows showing higher NMU than R sows). During a PRRSV outbreak, the S sows were twice as likely to give birth to a mummified piglet as compared to R sows. These findings provide evidence that the described phenotyping scheme has a potential use as a PRRSV resilience criterion.

Targeted-pig trial on safety and immunogenicity of serum-derived extracellular vesicles enriched fractions obtained from Porcine Respiratory and Reproductive virus infections.

The Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) is the etiological agent of one of the most important swine diseases with a significant economic burden worldwide. Unfortunately, available vaccines are partially effective highlighting the need of novel approaches. Previously, antigenic viral proteins were described in serum-derived extracellular vesicles (EVs) from pigs previously infected with PRRSV. Here, a targeted-pig trial was designed to determine the safety and immunogenicity of such extracellular vesicles enriched fractions. Our results showed that immunizations with EV-enriched fractions from convalescence animals in combination with montanide is safe and free of virus as immunizations with up-to two milligrams of EV-enriched fractions did not induce clinical symptoms, adverse effects and detectable viral replication. In addition, this vaccine formulation was able to elicit specific humoral IgG immune response in vaccinated animals, albeit variably. Noticeably, sera from vaccinated animals was diagnosed negative when tested for PRRSV using a commercial ELISA test; thus, indicating that this new approach differentiates vaccinated from infected animals. Lastly, after priming animals with EV-enriched fractions from sera of convalescence animals and boosting them with synthetic viral peptides identified by mass spectrometry, a distinctive high and specific IFN-γ response was elicited. Altogether, our data strongly suggest the use of serum EV-enriched fractions as a novel vaccine strategy against PRRSV.

A WUR SNP is associated with European Porcine Reproductive and Respiratory Virus Syndrome resistance and growth performance in pigs.

Porcine reproductive and respiratory syndrome (PRRS) causes decreased reproductive performance and respiratory problems in pigs. The goals of the current study were 1) to examine whether individual variation applies to infection with PRRSV European strains and 2) to investigate the association of a single nucleotide polymorphism (SNP) WUR10000125 (WUR) at the interferon-inducible guanylate-binding protein 1 gene (GBP1) with average daily gain (ADG) in PRRSV infected and uninfected pigs. The experimental procedure consisted of two trials in which pigs from negative PRRSV farms were infected with a wild-type (n=80) or vaccinated with an attenuated European PRRS virus strain (n=40) and then monitored after infection or vaccination. Viral load and ADG were determined for each pig. In a third trial, the ADG for PRRSV-free pigs was monitored. All pigs were genotyped for WUR at the GBP1 gene (AA and AG genotype were defined). Results indicated that there was individual variation in the viral load from pigs challenged with a wild-type or low virulent European PRRSV strain. Secondly, our data showed that WUR SNP was associated to ADG in vaccinated pigs. Thus, ADG in AG pigs was significantly higher than in AA ones after vaccinating with an attenuated PRRSV strain. However, the reverse happened in a PRRSV-free environment where the AA pigs were those that grew faster. Based on these results, there is a scope for selecting pigs according to their responses to PRRS virus infection with European strains and that WUR SNP may play a role in causing PRRSV resistance.