Sequence variants associated with resilient responses in growing pigs.

The current work aimed to identify genomic regions and candidate genes associated with resilience in pigs. In previous work, we proposed the body weight deviation from the expected growth curve (ΔBW) and the increase of the positive acute-phase protein haptoglobin (ΔHP) after a vaccine challenge as resilience indicators which may be improved through selective breeding in pigs. Individuals with steady growth rate and minor activation of haptoglobin (high ΔBW and low ΔHP values) were considered resilient. In contrast, pigs with perturbed growth rate and high activation of haptoglobin (low ΔBW and high ΔHP values) were considered susceptible. Both ∆BW and ∆HP were simultaneously considered to select the most resilient (N = 40) and susceptible (N = 40) pigs. A genome-wide association study was carried out for the pigs' response classification to the challenge test using whole-genome sequence data (7,760,720 variants). Eleven associated genomic regions were identified, harbouring relevant candidate genes related to the immune response (such as pro- and anti-inflammatory responses) and growth pathways. These associated genomic regions harboured 41 potential functional mutations (frameshift, splice donor, splice acceptor, start loss and stop loss/gain) in candidate genes. Overall, this study advances our knowledge about the genetic determinism of resilience, highlighting its polygenic nature and strong relationship with immunity and growth.

How Streptococcus suis escapes antibiotic treatments.

Streptococcus suis is a zoonotic agent that causes sepsis and meningitis in pigs and humans. S. suis infections are responsible for large economic losses in pig production. The lack of effective vaccines to prevent the disease has promoted the extensive use of antibiotics worldwide. This has been followed by the emergence of resistance against different classes of antibiotics. The rates of resistance to tetracyclines, lincosamides, and macrolides are extremely high, and resistance has spread worldwide. The genetic origin of S. suis resistance is multiple and includes the production of target-modifying and antibiotic-inactivating enzymes and mutations in antibiotic targets. S. suis genomes contain traits of horizontal gene transfer. Many mobile genetic elements carry a variety of genes that confer resistance to antibiotics as well as genes for autonomous DNA transfer and, thus, S. suis can rapidly acquire multiresistance. In addition, S. suis forms microcolonies on host tissues, which are associations of microorganisms that generate tolerance to antibiotics through a variety of mechanisms and favor the exchange of genetic material. Thus, alternatives to currently used antibiotics are highly demanded. A deep understanding of the mechanisms by which S. suis becomes resistant or tolerant to antibiotics may help to develop novel molecules or combinations of antimicrobials to fight these infections. Meanwhile, phage therapy and vaccination are promising alternative strategies, which could alleviate disease pressure and, thereby, antibiotic use.

A genome-wide screen for resilient responses in growing pigs.

BACKGROUND: There is a growing interest to decipher the genetic background of resilience and its possible improvement through selective breeding. The objective of the present study was to provide new insights into the genetic make-up of resilience in growing pigs by identifying genomic regions and candidate genes associated with resilience indicators. Commercial Duroc pigs were challenged with an attenuated Aujeszky vaccine at 12 weeks of age. Two resilience indicators were used: deviation from the expected body weight at 16 weeks of age given the growth curve of non-vaccinated pigs (∆BW) and the increase in acute-phase protein haptoglobin at four days post-vaccination (∆HP). Genome-wide association analyses were carried out on 445 pigs, using genotypes at 41,165 single nucleotide polymorphisms (SNPs) and single-marker and Bayesian multiple-marker regression approaches. RESULTS: Genomic regions on pig chromosomes 2, 8, 9, 11 (∆BW) and 8, 9, 13 (∆HP) were found to be associated with the resilience indicators and explained high proportions of their genetic variance. The genomic regions that were associated explained 27 and 5% of the genetic variance of ∆BW and ∆HP, respectively. These genomic regions harbour promising candidate genes that are involved in pathways related to immune response, response to stress, or signal transduction (CD6, PTGDR2, IKZF1, RNASEL and MYD88), and growth (GRB10 and LCORL). CONCLUSIONS: Our study identified novel genomic regions that are associated with two resilience indicators (∆BW and ∆HP) in pigs. These associated genomic regions harbour potential candidate genes involved in immune response and growth pathways, which emphasise the strong relationship between resilience and immune response.

Salmonella Infection in Mesenteric Lymph Nodes of Breeding Sows.

Salmonellosis is one of the main foodborne diseases worldwide. Breeding sows asymptomatically infected with Salmonella can transmit the pathogen to piglets and humans. The isolation of Salmonella from mesenteric lymph nodes (MLNs) is considered a demonstration of asymptomatic infection in swine. As previous breeding sow studies have been performed using feces, the aim of this work was to study the occurrence of Salmonella infections by sampling MLNs, in comparison to their serological status. First, Salmonella fecal shedding was studied in 12/16 large breeding farms to establish the framework of study. Then, MLN (n = 264) and blood (n = 237) samples were obtained at an abattoir from sows of 15 of these 16 breeding farms. Additionally, risk factors associated with Salmonella MLN infection were analyzed. A total of 6.1% (16/264) sows, distributed in 40% (6/15) of the farms, had the pathogen in MLN. Salmonella Typhimurium was the most frequent serovar isolated. Interestingly, 43.8% (7/16) of MLN isolates were susceptible to all the antimicrobials tested and were found distributed throughout all farms with at least one sow positive. As well, one isolate of the emerging DT195 clone was detected and found to be resistant to six antibiotic families (ASSuTNx-Cfx). The serovars and the resistance profiles of the Salmonella isolates from feces were completely different to those obtained from MLNs. The seroprevalence (41.8% of sows and 100% of farms) was higher than that of MLN infections, showing no concordance (k = 0.15) between these two diagnostic tests in sows. Strategies directed to correct two risk factors (i.e., administration of dry food and old premises) would most likely help to reduce Salmonella infections in breeding sows.

Treatment with etamsylate reduces haemolactia in lactating dairy cows.

This Research Communication describes the efficacy of etamsylate to reduce haemolactia in dairy cows. A dairy cow with haemolactia produces milk that is reddish or pinkish due to the presence of blood. Haemolactia causes economic loss because bloody milk is rejected by the industry and the consumers. A total of 58 dairy cows with haemolactia were included in the study and randomly divided into treated (n = 31) and control (n = 27) groups. Treatment consisted of three consecutive daily doses of etamsylate at 15 mg/kg, delivered intramuscularly. Milk production was recorded daily for 7 d, whether or not blood was detected in milk. The mean number of days with the presence of blood in milk in the treatment group was significantly lower (3·4 d) than in the control group (4·9 d). Treatment with etamsylate did not significantly affect milk yield. In conclusion, treatment with etamsylate reduces the number of days blood is observed in milk and it does not have any negative effect on milk production.

African swine fever virus infection in Classical swine fever subclinically infected wild boars.

BACKGROUND: Recently moderate-virulence classical swine fever virus (CSFV) strains have been proven capable of generating postnatal persistent infection (PI), defined by the maintenance of viremia and the inability to generate CSFV-specific immune responses in animals. These animals also showed a type I interferon blockade in the absence of clinical signs. In this study, we assessed the infection generated in 7-week-old CSFV PI wild boars after infection with the African swine fever virus (ASFV). The wild boars were divided in two groups and were infected with ASFV. Group A comprised boars who were CSFV PI in a subclinical form and Group B comprised pestivirus-free wild boars. Some relevant parameters related to CSFV replication and the immune response of CSFV PI animals were studied. Additionally, serum soluble factors such as IFN-α, TNF-α, IL-6, IL-10, IFN-γ and sCD163 were analysed before and after ASFV infection to assess their role in disease progression. RESULTS: After ASFV infection, only the CSFV PI wild boars showed progressive acute haemorrhagic disease; however, the survival rates following ASFV infection was similar in both experimental groups. Notwithstanding, the CSFV RNA load of CSFV PI animals remained unaltered over the study; likewise, the ASFV DNA load detected after infection was similar between groups. Interestingly, systemic type I FN-α and IL-10 levels in sera were almost undetectable in CSFV PI animals, yet detectable in Group B, while detectable levels of IFN-γ were found in both groups. Finally, the flow cytometry analysis showed an increase in myelomonocytic cells (CD172a+) and a decrease in CD4+ T cells in the PBMCs from CSFV PI animals after ASFV infection. CONCLUSIONS: Our results showed that the immune response plays a role in the progression of disease in CSFV subclinically infected wild boars after ASFV infection, and the immune response comprised the systemic type I interferon blockade. ASFV does not produce any interference with CSFV replication, or vice versa. ASFV infection could be a trigger factor for the disease progression in CSFV PI animals, as their survival after ASFV was similar to that of the pestivirus-free ASFV-infected group. This fact suggests a high resistance in CSFV PI animals even against a virus like ASFV; this may mean that there are relevant implications for CSF control in endemic countries. The diagnosis of ASFV and CSFV co-infection in endemic countries cannot be ruled out and need to be studied in greater depth.

Piglet nasal microbiota at weaning may influence the development of Glässer's disease during the rearing period.

BACKGROUND: The microbiota, the ensemble of microorganisms on a particular body site, has been extensively studied during the last few years, and demonstrated to influence the development of many diseases. However, these studies focused mainly on the human digestive system, while the populations in the respiratory tract have been poorly assessed, especially in pigs. The nasal mucosa of piglets is colonized by an array of bacteria, many of which are unknown. Among the early colonizers, Haemophilus parasuis also has clinical importance, since it is also the etiological agent of Glässer's disease. This disease produces economical losses in all the countries with pig production, and the factors influencing its development are not totally understood. Hence, the purpose of this work was to characterize the nasal microbiota composition of piglets, and its possible role in Glässer's disease development. RESULTS: Seven farms from Spain (4 with Glässer's disease and 3 control farms without any respiratory disease) and three farms from UK (all control farms) were studied. Ten piglets from each farm were sampled at 3-4 weeks of age before weaning. The total DNA extracted from nasal swabs was used to amplify the 16S RNA gene for sequencing in Illumina MiSeq. Sequencing data was quality filtered and analyzed using QIIME software. The diversity of the nasal microbiota was low in comparison with other body sites, showing a maximum number of operational taxonomic units (OTUs) per pig of 1,603, clustered in five phyla. Significant differences were found at various taxonomical levels, when the microbiota was compared regarding the farm health status. Healthy status was associated to higher species richness and diversity, and UK farms demonstrated the highest diversity. CONCLUSIONS: The composition of the nasal microbiota of healthy piglets was uncovered and different phylotypes were shown to be significantly altered in animals depending on the clinical status of the farm of origin. Several OTUs at genus level were identified over-represented in piglets from control farms, indicating their potential as probiotics. Although we provide relevant data, fully metagenomic approaches could give light on the genes and metabolic pathways involved in the roles of the nasal microbiota to prevent respiratory diseases.

Cross-Species Infectivity of H3N8 Influenza Virus in an Experimental Infection in Swine.

UNLABELLED: Avian influenza A viruses have gained increasing attention due to their ability to cross the species barrier and cause severe disease in humans and other mammal species as pigs. H3 and particularly H3N8 viruses, are highly adaptive since they are found in multiple avian and mammal hosts. H3N8 viruses have not been isolated yet from humans; however, a recent report showed that equine influenza A viruses (IAVs) can be isolated from pigs, although an established infection has not been observed thus far in this host. To gain insight into the possibility of H3N8 avian IAVs to cross the species barrier into pigs, in vitro experiments and an experimental infection in pigs with four H3N8 viruses from different origins (equine, canine, avian, and seal) were performed. As a positive control, an H3N2 swine influenza virus A was used. Although equine and canine viruses hardly replicated in the respiratory systems of pigs, avian and seal viruses replicated substantially and caused detectable lesions in inoculated pigs without previous adaptation. Interestingly, antibodies against hemagglutinin could not be detected after infection by hemagglutination inhibition (HAI) test with avian and seal viruses. This phenomenon was observed not only in pigs but also in mice immunized with the same virus strains. Our data indicated that H3N8 IAVs from wild aquatic birds have the potential to cross the species barrier and establish successful infections in pigs that might spread unnoticed using the HAI test as diagnostic tool. IMPORTANCE: Although natural infection of humans with an avian H3N8 influenza A virus has not yet been reported, this influenza A virus subtype has already crossed the species barrier. Therefore, we have examined the potential of H3N8 from canine, equine, avian, and seal origin to productively infect pigs. Our results demonstrated that avian and seal viruses replicated substantially and caused detectable lesions in inoculated pigs without previous adaptation. Surprisingly, we could not detect specific antibodies against hemagglutinin in any H3N8-infected pigs. Therefore, special attention should be focused toward viruses of the H3N8 subtype since they could behave as stealth viruses in pigs.

Serum-derived exosomes from non-viremic animals previously exposed to the porcine respiratory and reproductive virus contain antigenic viral proteins.

PRRSV is the etiological agent of one of the most important swine diseases with a significant economic burden worldwide and limitations in vaccinology. Exosomes are 30-100 nm vesicles of endocytic origin. Remarkably, immunizations with exosomes containing antigens from tumors or pathogens are capable of eliciting protective immune responses, albeit variably, in cancer and infectious diseases. Here we describe the isolation, molecular composition and immunogenicity of serum-derived exosomes from naïve animals, from PRRSV viremic animals and from animals previously PRRSV infected but already free of viruses (non viremic). Exosomes were isolated through size exclusion chromatography and characterized by different methodologies. Exosome-enriched fractions from naïve and natural infected animals contained classical tetraspanin exosomal markers (CD63 and CD81) and high concentrations of particles in the size-range of exosomes as detected by nanoparticle tracking analysis and cryo-TEM. NanoLC-MS/MS was used to identify viral antigens associated to exosomes. PRRSV-proteins were detected in serum samples from only viremic animals and from animals previously infected already free of viruses (non-viremic), but not in controls. Moreover, immune sera from pigs previously exposed to PRRSV specifically reacted against exosomes purified from non-viremic pig sera in a dose-dependent manner, a reactivity not detected when naïve sera was used in the assay. To facilitate future studies, a scaling-up process was implemented. To the best of our knowledge, this is the first molecular characterization of serum-derived exosomes from naïve pigs and pigs actively or previously infected with PRRSV. The presence of antigenic viral proteins in serum-derived exosomes free of virus, suggest their use as a novel vaccine approach against PRRSV.

Biochemical and proteomic analyses of the physiological response induced by individual housing in gilts provide new potential stress markers.

BACKGROUND: The objective assessment of animal stress and welfare requires proper laboratory biomarkers. In this work, we have analyzed the changes in serum composition in gilts after switching their housing, from pen to individual stalls, which is generally accepted to cause animal discomfort. RESULTS: Blood and saliva samples were collected a day before and up to four days after changing the housing system. Biochemical analyses showed adaptive changes in lipid and protein metabolism after the housing switch, whereas cortisol and muscular markers showed a large variability between animals. 2D-DIGE and iTRAQ proteomic approaches revealed variations in serum protein composition after changing housing and diet of gilts. Both techniques showed alterations in two main homeostatic mechanisms: the innate immune and redox systems. The acute phase proteins haptoglobin, apolipoprotein A-I and α1-antichymotrypsin 3, and the antioxidant enzyme peroxiredoxin 2 were found differentially expressed by 2D-DIGE. Other proteins related to the innate immune system, including lactotransferrin, protegrin 3 and galectin 1 were also identified by iTRAQ, as well as oxidative stress enzymes such as peroxiredoxin 2 and glutathione peroxidase 3. Proteomics also revealed the decrease of apolipoproteins, and the presence of intracellular proteins in serum, which may indicate physical injury to tissues. CONCLUSIONS: Housing of gilts in individual stalls and diet change increase lipid and protein catabolism, oxidative stress, activate the innate immune system and cause a certain degree of tissue damage. We propose that valuable assays for stress assessment in gilts may be based on a score composed by a combination of salivary cortisol, lipid metabolites, innate immunity and oxidative stress markers and intracellular proteins.

Colostrum and milk pasteurization improve health status and decrease mortality in neonatal calves receiving appropriate colostrum ingestion.

The objective of the study was to evaluate if on-farm heat treatment of colostrum and bulk tank milk can improve calf health status and morbidity and mortality rates during the first 21d of life in neonatal Holstein calves receiving appropriate colostrum ingestion. A total of 587 calves were randomly assigned to 2 groups of males and females over 18mo. The nonpasteurized group (n=287, 143 males and 144 females) was fed frozen (-20°C) colostrum (6-8L during the first 12h of life) that was previously reheated up to 40°C. They were also fed refrigerated (4°C) raw milk from the bulk tank that was also reheated up to 40°C (1.8L every 12h). The pasteurized group (n=300, 150 males and 150 females) was also fed colostrum and milk, but both were pasteurized before freezing. Blood samples were drawn from all calves to obtain serum at 2 to 5d of life. Serum total protein (g/dL) was determined using a commercially available refractometer. Colostrum and milk underwent routine bacteriological analysis to determine total plate counts (cfu/mL) and total coliform counts (cfu/mL). All the calves underwent clinical examination every 24h during the first 21d of life. Every day, calves were clinically diagnosed either as being healthy or suffering from respiratory disease, neonatal calf diarrhea, or suffering other diseases. On-farm heat treatment for colostrum and milk reduced total plate counts and total coliform counts between 1 and 2 log10. Pasteurization of colostrum and milk significantly decreased the morbidity and mortality (5.2 and 2.8%) in comparison with calves receiving nonpasteurized colostrum and milk (15.0 and 6.5%), respectively, during the first 21d of life, even in animals receiving appropriate colostrum ingestion.

Impact of the use of ß-lactam antimicrobials on the emergence of Escherichia coli isolates resistant to cephalosporins under standard pig-rearing conditions.

The aim of this study was to evaluate if the treatments with ceftiofur and amoxicillin are risk factors for the emergence of cephalosporin resistant (CR) E. coli in a pig farm during the rearing period. One hundred 7-day-old piglets were divided into two groups, a control (n = 50) group and a group parenterally treated with ceftiofur (n = 50). During the fattening period, both groups were subdivided in two. A second treatment with amoxicillin was administered in feed to two of the four groups, as follows: group 1 (untreated, n = 20), group 2 (treated with amoxicillin, n = 26), group 3 (treated with ceftiofur, n = 20), and group 4 (treated with ceftiofur and amoxicillin, n = 26). During treatment with ceftiofur, fecal samples were collected before treatment (day 0) and at days 2, 7, 14, 21, and 42 posttreatment, whereas with amoxicillin, the sampling was extended 73 days posttreatment. CR E. coli bacteria were selected on MacConkey agar with ceftriaxone (1 mg/liter). Pulsed-field gel electrophoresis (PFGE), MICs of 14 antimicrobials, the presence of cephalosporin resistance genes, and replicon typing of plasmids were analyzed. Both treatments generated an increase in the prevalence of CR E. coli, which was statistically significant in the treated groups. Resistance diminished after treatment. A total of 47 CR E. coli isolates were recovered during the study period; of these, 15 contained blaCTX-M-1, 10 contained blaCTX-M-14, 4 contained blaCTX-M-9, 2 contained blaCTX-M-15, and 5 contained blaSHV-12. The treatment with ceftiofur and amoxicillin was associated with the emergence of CR E. coli during the course of the treatment. However, by the time of finishing, CR E. coli bacteria were not recovered from the animals.

Clinical response to pandemic H1N1 influenza virus from a fatal and mild case in ferrets.

BACKGROUND: The majority of pandemic 2009 H1N1 (A(H1N1)pdm09) influenza virus (IV) caused mild symptoms in most infected patients, however, a greater rate of severe disease was observed in healthy young adults and children without co-morbid conditions. The purpose of this work was to study in ferrets the dynamics of infection of two contemporary strains of human A(H1N1)pdm09 IV, one isolated from a patient showing mild disease and the other one from a fatal case. METHODS: Viral strains isolated from a patient showing mild disease-M (A/CastillaLaMancha/RR5661/2009) or from a fatal case-F (A/CastillaLaMancha/RR5911/2009), both without known comorbid conditions, were inoculated in two groups of ferrets and clinical and pathological conditions were analysed. RESULTS: Mild to severe clinical symptoms were observed in animals from both groups. A clinical score distribution was applied in which ferrets with mild clinical signs were distributed on a non-severe group (NS) and ferrets with severe clinical signs on a severe group (S), regardless of the virus used in the infection. Animals on S showed a significant decrease in body weight compared to animals on NS at 4 to 7 days post-infection (dpi). Clinical progress correlated with histopathological findings. Concentrations of haptoglobin (Hp) and serum amyloid A (SAA) increased on both groups after 2 dpi. Clinically severe infected ferrets showed a stronger antibody response and higher viral titres after infection (p = 0.001). CONCLUSIONS: The severity in the progress of infection was independent from the virus used for infection suggesting that the host immune response was determinant in the outcome of the infection. The diversity observed in ferrets mimicked the variability found in the human population.

Comparison of four lung scoring systems for the assessment of the pathological outcomes derived from Actinobacillus pleuropneumoniae experimental infections.

BACKGROUND: In this study, four lung lesion scoring methods (Slaughterhouse Pleurisy Evaluation System [SPES], Consolidation Lung Lesion Score [LLS], Image analyses [IA] and Ratio of lung weight/body weight [LW/BW]) were compared for the assessment of the different pathological outcomes derived from an Actinobacillus pleuropneumoniae (App) experimental infection model. Moreover, pathological data was coupled with clinical (fever, inappetence and clinical score), production (average daily weigh gain [ADWG]) and diagnostic (PCR, ELISA and bacterial isolation) parameters within the four infection outcomes (peracute, acute, subclinically infected and non-infected). RESULTS: From the 61 inoculated animals, 9 were classified as peracute (presence of severe App-like clinical signs and lesions and sudden death or euthanasia shortly after inoculation), 31 as acutely affected (presence of App-like clinical signs and lesions and survival until the end of the experiment), 12 as subclinically infected (very mild or no clinical signs but App infection confirmed) and 9 as non-infected animals (lack of App-like clinical signs and lack of evidence of App infection). A significant correlation between all lung lesion scoring systems was found with the exception of SPES score versus LW/BW. SPES showed a statistically significant association with all clinical, production and diagnostic (with the exception of PCR detection of App in the tonsil) variables assessed. LLS and IA showed similar statistically significant associations as SPES, with the exception of seroconversion against App at necropsy. In contrast, LW/BW was statistically associated only with App isolation in lungs, presence of App-like lesions and ELISA OD values at necropsy. CONCLUSIONS: In conclusion, SPES, LLS and IA are economic, fast and easy-to-perform lung scoring methods that, in combination with different clinical and diagnostic parameters, allow the characterization of different outcomes after App infection.

Gut microbiome and resistome characterization of pigs treated with commonly used post-weaning diarrhea treatments.

BACKGROUND: The global burden of antimicrobial resistance demands additional measures to ensure the sustainable and conscious use of antimicrobials. For the swine industry, the post-weaning period is critical and for many years, antimicrobials have been the most effective strategy to control and treat post-weaning related infections. Among them, post-weaning diarrhea causes vast economic losses, as it severely compromises piglets' health and growth performance. In this study, 210 piglets were transferred from a farm with recurrent cases of post-weaning diarrhea to an experimental farm and divided into six different treatment groups to determine the effect of the different treatments on the growth performance and survival, the microbiome, and the resistome in a cross-sectional and longitudinal study. The different treatments included antimicrobials trimethoprim/sulfamethoxazole, colistin, and gentamicin, an oral commercial vaccine, a control with water acidification, and an untreated control. An extra group remained at the farm of origin following the implemented amoxicillin routine treatment. A total of 280 fecal samples from pigs at four different sampling times were selected for metagenomics: before weaning-treatment at the farm of origin, and three days, two weeks, and four weeks post-treatment. RESULTS: The control group with water acidification showed a reduced death risk in the survival analyses and non-significant differences in average daily weight gain in comparison to the antibiotic-treated groups. However, the growth-promoting effect among antibiotic-treated groups was demonstrated when comparing against the untreated control group at the experimental farm. After four weeks of treatment, diversity indexes revealed significantly decreased diversity for the untreated control and the group that remained at the farm of origin treated with amoxicillin. For this last group, impaired microbial diversity could be related to the continuous amoxicillin treatment carried out at the farm. Analysis of the resistome showed that both gentamicin and amoxicillin treatments significantly contributed to the emergence of resistance, while trimethoprim/sulphonamide and colistin did not, suggesting that different treatments contribute differently to the emergence of resistance. CONCLUSIONS: Overall, this shotgun longitudinal metagenomics analysis demonstrates that non-antibiotic alternatives, such as water acidification, can contribute to reducing the emergence of antimicrobial resistance without compromising pig growth performance and gut microbiome.

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.

Key performance indicators used by dairy consultants during the evaluation of reproductive performance during routine visits.

Dairy farms need thorough and efficient reproduction control. Consultants specialized in reproduction use key performance indicators (KPI) to monitor the reproductive performance of farms and must be able to decipher between the approach in a first visit and routine visits. A total of 49 consultants specialized in dairy reproduction from 21 countries responded to an online survey conducted to determine the most suitable parameters during routine visits every 2 to 4 weeks. The survey was comprised of 190 questions, 178 of them rated from 0 (irrelevant) to 10 (maximum importance) points. The questions were divided into five sections: (1) consultant and farm model, (2) general data of the farm, (3) cow reproduction, (4) postpartum and metabolic disease, and (5) heifer reproduction. The median, interquartile range, minimum and maximum values, and 95% confidence interval were determined for each question. Afterward, a multivariate analysis, using between-group linkage via Ward's hierarchical clustering was conducted to generate clusters of consultants according to their response pattern. Finally, a chi-square test was conducted to assess the association between years of experience of the consultant and farm size within the clusters generated in each section of the questionnaire. The majority of the consultants considered 34 parameters to be highly important (rated 8-10) to analyze during routine visits. The consultants used several KPI (in variable quantitative range) to evaluate any of the presented sections and considered that all the five sections are critical to control. They are aware of using KPI that reflect heat detection, fertility, and farming efficiency as well as KPI that can provide information on reproductive efficiency in the near future for cows, such as postpartum and metabolic diseases. However, parameters that are relatively old and ineffective, in terms of reproductive performance control, are still highly regarded by the majority of consultants in a routine-visit scenario. Farm size and years of experience of the consultant did not influence the type or number of parameters chosen as KPI during routine visits. The parameters rated with the highest importance (rate 10) that could be considered for an easy, fast, and universal use in routine visits to assess the reproductive status were: First service CR (%), Overall pregnancy rate (%) for cows, and age at first calving (d) for heifers.

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.

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.

Invasive Streptococcus suis isolated in Spain contain a highly promiscuous and dynamic resistome.

Introduction: Streptococcus suis is a major pathogen for swine and human. Here we aimed to know the rates of antimicrobial resistance (AMR) in invasive S. suis isolates recovered along Spain between 2016 - 2021 and elucidate their genetic origin. Methods: Antibiotic susceptibility testing was performed for 116 isolates of different genetic backgrounds and geographic origins against 18 antibiotics of 9 families. The association between AMR and genotypes and the origin of the isolates were statistically analyzed using Pearson´s chi-square test and the likelihood ratio. The antimicrobial resistant genes were identified by whole genome sequencing analysis and PCR screenings. Results: High AMR rates (>80%) were detected for tetracyclines, spectinomycin, lincosamides, and marbofloxacin, medium (20-40%) for sulphonamides/trimethoprim, tiamulin, penicillin G, and enrofloxacin, and low (< 20%) for florfenicol, and four additional ß-lactams. The occurrence of multidrug resistance was observed in 90% of isolates. For certain antibiotics (penicillin G, enrofloxacin, marbofloxacin, tilmicosin, and erythromycin), AMR was significantly associated with particular sequence types (STs), geographic regions, age of pigs, and time course. Whole genome sequencing comparisons and PCR screenings identified 23 AMR genes, of which 19 were previously reported in S. suis (aph(3')-IIIa, sat4, aadE, spw, aac(6')-Ie-aph(2'')-Ia, fexA, optrA, erm(B), mef(A/E), mrs(D), mph(C), lnu(B), lsa(E), vga(F), tet(M), tet(O), tet(O/W/32/O), tet(W)), and 4 were novel (aph(2'')-IIIa, apmA, erm(47), tet(T)). These AMR genes explained the AMR to spectinomycin, macrolides, lincosamides, tiamulin, and tetracyclines. Several genes were located on mobile genetic elements which showed a variable organization and composition. As AMR gene homologs were identified in many human and animal pathogens, the resistome of S. suis has a different phylogenetic origin. Moreover, AMR to penicillin G, fluoroquinolones, and trimethoprim related to mutations in genes coding for target enzymes (pbp1a, pbp2b, pbp2x, mraY, gyrA, parC, and dhfr). Bioinformatic analysis estimated traits of recombination on target genes, also indicative of gene transfer events. Conclusions: Our work evidences that S. suis is a major contributor to AMR dissemination across veterinary and human pathogens. Therefore, control of AMR in S. suis should be considered from a One Health approach in regions with high pig production to properly tackle the issue of antimicrobial drug resistance.