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X.D. was funded by a grant from the National Institute for Health Research (NIHR) Health Protection Research Unit in Genomics and Enabling Data (no. NIHR200892). M.A.H. was supported by grants from the Medical Research Council (nos. G1001787/1, MR/N002660/1 and MR/P007201/1) and the Economic and Social Research Council (no. ES/S000186/1). E.M.H. was supported by a UK Research and Innovation (UKRI) Fellowship (no. MR/S00291X/1). The discovery of antibiotics more than 80 years ago has led to considerable improvements in human and animal health. Although antibiotic resistance in environmental bacteria is ancient, resistance in human pathogens is thought to be a modern phenomenon that is driven by the clinical use of antibiotics1. Here we show that particular lineages of methicillin-resistant Staphylococcus aureus—a notorious human pathogen—appeared in European hedgehogs in the pre-antibiotic era. Subsequently, these lineages spread within the local hedgehog populations and between hedgehogs and secondary hosts, including livestock and humans. We also demonstrate that the hedgehog dermatophyte Trichophyton erinacei produces two β-lactam antibiotics that provide a natural selective environment in which methicillin-resistant S. aureus isolates have an advantage over susceptible isolates. Together, these results suggest that methicillin resistance emerged in the pre-antibiotic era as a co-evolutionary adaptation of S. aureus to the colonization of dermatophyte-infected hedgehogs. The evolution of clinically relevant antibiotic-resistance genes in wild animals and the connectivity of natural, agricultural and human ecosystems demonstrate that the use of a One Health approach is critical for our understanding and management of antibiotic resistance, which is one of the biggest threats to global health, food security and development. Publisher PDF

Campylobacter infection is a leading cause of ovine abortion worldwide. Campylobacter fetus and C. jejuni are the major species involved. We report herein on abortion storms in 4 Danish sheep flocks. Initially, no pathogenic bacteria were isolated from placental and fetal tissues on aerobic and selective media despite the presence of severe suppurative and necrotizing placentitis with numerous bacteria located intracellularly in trophoblasts. Fluorescence in situ hybridization (FISH) was then applied on abortion material from 13 cases; species-specific oligonucleotide probes directed against either C. fetus or C. jejuni were used in combination with a general bacterial probe. C. fetus was detected as the only lesion-associated bacterial species in 4 cases from 2 flocks, and C. jejuni in 6 cases from the other 2 flocks, thereby establishing the likely etiology of the abortion storms in all 4 flocks. FISH is a useful detection tool in culture-negative cases with tissue lesions suggestive of bacterial infection. Furthermore, FISH is a fast and economical method to detect and identify the zoonotic agent Campylobacter within ovine abortion material.

Actinobacillus pleuropneumoniae is the etiological agent of porcine pleuropneumonia, a disease of major impact on pig health, welfare, and productivity globally. Serovar 8 (APP) is the predominant clinical serovar in Norway and the United Kingdom (UK), and has been isolated from clinical cases in Denmark. The primary objective of this study was to characterize the genetic variability of isolates of A. pleuropneumoniae APP8 in the Norwegian population. The secondary objectives were to determine the within-host variability of APP8; to compare the APP8 bacterial populations in Norway, Denmark, and the UK, including antimicrobial resistance (AMR) gene profiles and to assess the effect of national differences in antimicrobial drug use and restricted animal movement on the occurrence of resistance. Isolates of APP8 from the UK (n=67), Denmark (n=22), and Norway (n=123) collected between 1983 and 2020 were compared using whole genome sequencing. To investigate genetic variability within individual hosts, an additional 104 APP8 isolates from the lungs of six Norwegian pigs were compared. Very low within-host variation was observed (≤ 2 single nucleotide polymorphisms). The phylogeny of 123 Norwegian APP8 isolates from 76 herds revealed some within-herd genetic variation, but substantial geographical clustering. When inferring the relatedness of the three international APP8 collections, the topology highlighted the existence of two distinct monophyletic branches characterized by the Norwegian and UK isolates, respectively. Three Danish isolates were scattered across the UK branch, whereas the remaining 19 Danish isolates clustered in two monophyletic groups nested in the Norwegian branch. Coalescence analysis, performed to estimate the divergences from a common ancestor, indicated a last common ancestor several centuries ago. The phylogenetic analyses also revealed striking differences in occurrence of AMR genes, as these were 23-times more prevalent among the UK isolates than among the Norwegian isolates. An increased understanding of the effects of population strategies is helpful in surveillance and control of infectious diseases.

Actinobacillus pleuropneumoniae (APP), the causative agent of porcine pleuropneumonia, is highly contagious and responsible for high morbidity, mortality, and economic losses in the swine industry worldwide, but quick serotyping and diagnosis are still not widely available. In this study, we sought to validate the use of Whatman FTA® cards for collection and processing of A. pleuropneumoniae isolates, or porcine lung tissue samples, for direct use in diagnostic multiplex PCRs. We have optimized the processing of 3-mm discs punched from FTA® cards loaded with cultured A. pleuropneumoniae, or imprinted on lesioned regions of lung tissue, with only three distilled water washes before addition into our APP-multiplex PCR (mPCR) assay for rapid, low-cost identification and serotyping. DNA captured on FTA® cards generated the same diagnostic PCR results as DNA extracted using commercial kits for 85 A. pleuropneumoniae clinical isolate cultures and 22 lung samples. Additionally, bacterial DNA bound to FTA® cards was detectable by PCR after 6 months of storage at 37°C. This study provides simple, efficient, rapid, and practical sample processing for detection and molecular serotyping of A. pleuropneumoniae.

Optimal transportation of bacteria is important for accurate clinical interpretation, quantitative assays, and microbiome studies. A transport medium should ideally keep the bacteria alive without supporting growth or altering the relative proportions of the constituent species. We investigated the effect of nasal mucus and mucin on the growth and survival of two Staphylococcus aureus strains in liquid Amies transport medium at room temperature and 4 °C for 14 days. The study showed that the presence of nasal mucus in microbiological samples stimulated undesired S. aureus growth at room temperature in a dose-dependent manner. These findings underscore that microbiological samples from humans and animals should be stored at 4 °C until analysis to avoid undesired S. aureus growth.

Two serologically and molecularly non-typeable isolates of the porcine lung pathogen Actinobacillus pleuropneumoniae have been identified from diseased swine in two different continents. Genome sequencing was carried out to identify their diagnostically relevant genotypes. Both isolates are biovar 1 and encode genes for production of ApxIV and ApxII (apxIICA structural genes, and apxIBD export genes). They both possess the same novel type II capsule locus (most similar to serovar 1, but with two capsule genes not previously found in A. pleuropneumoniae) but differ in their O-Ag loci. Strain 7213384-1 from Denmark, which we propose as the reference strain for serovar 19, has a serogroup 3/6/8/15 O-Ag locus; the Canadian isolate A08-013 has a serogroup 4/7 O-Ag locus. We have expanded the second of our two previously described A. pleuropneumoniae mPCRs to include capsule gene-specific primers for definitive detection of serovars 13–14 and 16–19.

This work was supported by a Longer and Larger (LoLa) grant from the Biotechnology and Biological Sciences Research Council (grant numbers BB/G020744/1, BB/G019177/1, BB/G019274/1, BB/G018553/1, BB/S002103/1, and BB/S005897/1), the UK Department for Environment, Food and Rural Affairs, and Zoetis (formerly Pfizer Animal Health) awarded to the Bacterial Respiratory Diseases of Pigs-1 Technology (BRaDP1T) consortium. Funding for LL provided by the ‘National Natural Science Foundation of China’ (No.31520103917). MTGH and DH were supported by the Wellcome Trust (grant number 098051). We report here the complete genome sequence of the widely studied Actinobacillus pleuropneumoniae serovar 8 reference strain 405, generated using the Pacific Biosciences (PacBio) RS II platform. Furthermore, we compared draft sequences generated by Illumina sequencing of six stocks of this strain, including the same original stock used to generate the PacBio sequence, held in different countries and found little genetic variation, with only three SNPs identified, all within the degS gene. However, sequences of two small plasmids, pARD3079 and p405tetH, detected by Illumina sequencing of the draft genomes were not identified in the PacBio sequence of the reference strain. Publisher PDF

Species-specific detection of Chlamydia psittaci is challenging and all published PCR tests have so far shown deficiencies in specificity or sensitivity. The present investigation reports on the development of a species-specific real-time PCR assay for C. psittaci. The test is based on an 84 bp indel in a gene of unknown function that is unique to C. psittaci. The Cps-indel84-PCR assay was validated on a wide range of chlamydial and other bacterial strains as well as on clinical samples from animals and humans in two different diagnostic laboratories in Germany and Denmark. Furthermore, the test was employed for investigating samples from racing pigeon flocks in Denmark. The evaluation showed that the Cps-indel84-PCR assay has excellent test characteristics and is a highly reliable method for identifying C. psittaci in clinical samples both from humans and animals.

Campylobacter infection is a leading cause of ovine abortion worldwide. Campylobacter fetus and C. jejuni are the major species involved. We report herein on abortion storms in 4 Danish sheep flocks. Initially, no pathogenic bacteria were isolated from placental and fetal tissues on aerobic and selective media despite the presence of severe suppurative and necrotizing placentitis with numerous bacteria located intracellularly in trophoblasts. Fluorescence in situ hybridization (FISH) was then applied on abortion material from 13 cases; species-specific oligonucleotide probes directed against either C. fetus or C. jejuni were used in combination with a general bacterial probe. C. fetus was detected as the only lesion-associated bacterial species in 4 cases from 2 flocks, and C. jejuni in 6 cases from the other 2 flocks, thereby establishing the likely etiology of the abortion storms in all 4 flocks. FISH is a useful detection tool in culture-negative cases with tissue lesions suggestive of bacterial infection. Furthermore, FISH is a fast and economical method to detect and identify the zoonotic agent Campylobacter within ovine abortion material.

Serotyping has been used to categorize most pathogenic members of Pasteurellaceae of human or animal importance. In particular, serotyping of isolates from vaccine breaks is of primary importance to ensure that vaccines used are covering outbreak isolates in the region. Traditional serotyping methods are available for Pasteurella multocida, Actinobacillus pleuropneumoniae, Glaesserella parasuis (older name Haemophilus parasuis), Mannheimia haemolytica, Avibacterium paragallinarum, Haemophilus influenzae, and Aggregatibacter actinomycetemcomitans. PCR-based methods to predict the serotypes have been developed for all agents with the exception of Avibacterium paragallinarum. For several of the species, work is in progress to predict serotypes from whole genomic sequences. The combination of population structures determined by whole genomic sequencing and the prediction of serotypes provides a strong synergy in epidemiological investigations of the species of Pasteurellaceae involved in disease in humans and animals.

Since its emergence in the early 2000s, livestock-associated methicillin-resistant Staphylococcus aureus clonal complex 398 (LA-MRSA CC398) has led to an increasing number of human infections in Denmark and other European countries with industrial pig production. LA-MRSA CC398 is primarily associated with skin infections among pig farm workers but is also increasingly recognized as a cause of life-threatening disease among elderly and immunocompromised people. Pig farm workers may serve as vehicles for the spread of LA-MRSA CC398 and other farm-origin bacteria between farms and into the general population. Yet, little is known about the bacterial community dynamics in pig farm workers and other persons with long- and short-term exposure to the pig farm environment. To gain insight into this, we investigated the nasal microbiomes in pig farm workers during a workweek on four LA-MRSA CC398-positive pig farms, as well as in short-term visitors two hours before, immediately after, and 48 hours after a 1-hour visit to another LA-MRSA CC398-positive pig farm. S. aureus and LA-MRSA CC398 carriage was quantified by means of culture, and the composition of the bacterial communities was investigated through sequencing of the 16S rRNA gene. Pig farm workers often carried LA-MRSA CC398 and other bacteria from the pig farm environment, both at work and at home, although at lower levels at home. In contrast, short-term visitors were subject to a less dramatic and rapidly reversible change in the nasal bacterial community composition. These results suggest that pig farm workers may be an important source of LA-MRSA CC398 and perhaps other pathogens of human and veterinary relevance.

Dust is suspected to be an important factor in transmission of livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) between pigs and pig farmers and their families. The aim of this study was to determine the rate of decay for Staphylococcus aureus and LA-MRSA in dust from swine farms. Electrostatic dust fall collectors (EDCs) were used for passive sampling of settling airborne dust in 11 stable sections from six swine farms. Extraction, plating, identification, and enumeration of cultivable S. aureus and LA-MRSA from the EDCs were performed after storage for 0–30 days postsampling. The survival of S. aureus was measured in 196 dust samples from all farms, and data were used to estimate the decay constant λ according to a model for exponential decay: N(t) = N0 × e−λt. The number of S. aureus colonies was up to 600-fold higher than the number of LA-MRSA colonies on MRSA selective agar. The data showed a good fit to the model (λ = 0.13, r2 = 0.86) even with a large difference in initial concentrations of S. aureus between stables. The loads of S. aureus and LA-MRSA in the dust were significantly reduced by storage time, and the half-life was 5 days for both S. aureus and LA-MRSA. In dust samples with high initial concentrations, LA-MRSA and S. aureus could still be cultivated 30 days after sampling. On all farms MRSA isolates belonged to the clonal complex (CC) 398, and at one farm some isolates also belonged to CC30. A screening for other Staphylococcus species in the farm dust revealed 13 different species numerically dominated by Staphylococcus equorum. Based on the exponential decay model, S. equorum had a half-life of 4 days. In conclusion, the presence of MRSA in airborne dust from five of six farms indicates that dust might be an important vehicle for transmission of LA-MRSA. LA-MRSA and S. aureus was found to survive well in farm dust with half-lives of 5 days, and dependent on the initial concentration they could be found in farm dust for weeks. The 99.9% die-off rate was 66 days for LA-MRSA. Thus, farm dust can pose an exposure risk for humans in the farm environment, but also when transported to other environments. On the other hand, the risk will decrease by time. These results provide important knowledge to diminish spread from farm environments to other environments on, e.g., tools or clothing, and in relation to cleaning of emptied LA-MRSA-positive stables.

Farm animals have been identified as an emerging reservoir for transmission of livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) to humans. The low incidence of MRSA in humans and farm animals in Norway has led to the implementation of a national strategy of surveillance and control of LA-MRSA aiming to prevent livestock becoming a domestic source of MRSA to humans. In 2015, MRSA clonal complex 1 spa-type t177 was identified in nine Norwegian pig herds in two neighboring counties. An outbreak investigation was undertaken, and measures of control through eradication were imposed. We performed a register-based cohort study including pig herds and MRSA-positive persons in Norway between 2008 and 2016 to investigate the livestock-association of MRSA CC1, the transmission of the outbreak strain to humans before and after control measures, and the effect of control measures imposed. Data from the Norwegian Surveillance System of Communicable Diseases were merged with data collected through outbreak investigations for LA-MRSA, the National Registry and the Norwegian Register for Health Personnel. Whole-genome sequencing was performed on isolates from livestock and humans identified through contact tracing, in addition to t177 and t127 isolates diagnosed in persons in the same counties. It is likely that a farm worker introduced MRSA CC1 to a sow farm, and further transmission to eight fattening pig farms through trade of live pigs confirmed the potential for livestock association of this MRSA type. The outbreak strain formed a distinct phylogenetic cluster which in addition to the pig farms included one sheep herd and five exposed persons. None of the investigated isolates from possible cases without direct contact to the MRSA positive farms were phylogenetically related to the outbreak strain. Moreover, isolates of t177 or t127 from healthcare and community-acquired cases were not closely related to the outbreak cluster. Eradication measures imposed were effective in eliminating MRSA t177 from the positive pig holdings, and the outbreak strain was not detected in the national pig population or in persons from these counties after control measures. © 2019 Elstrøm, Grøntvedt, Gabrielsen, Stegger, Angen, Åmdal, Enger, Urdahl, Jore, Steinbakk and Sunde. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

The aim of the investigation was to determine the protective effect of wearing dust masks to prevent Methicillin-resistant Staphylococcus aureus (MRSA) contamination during a short-term visit in a MRSA-positive swine farm. A total of 118 human volunteers were randomly allocated to a group wearing masks and to the control group. On average, 9% of the participants wearing masks were MRSA-positive when leaving the farm compared to 62% of the participants not wearing masks. At day 1, 2, and 7 after the farm visit, none of those wearing masks were MRSA-positive. An odds ratio of 18.9 (CI: 6.4–56.2) for being MRSA-positive was found for those not wearing masks compared to those wearing masks when including the farm as random effect. None of the covariates (gender, age, and smoking) influenced the OR for being MRSA-positive when leaving the farm. This study shows that the use of dust masks gives a significant protection against nasal MRSA contamination after a short-term visit to a swine farm. In addition, 106 volunteers not wearing masks were investigated in order to provide an estimate of a contamination dose of airborne MRSA. Due to the high variation in the MRSA measurements, it was not possible to establish a stable estimate for this. Out of the 106 volunteers not wearing masks, 6, 7, and 3% tested MRSA-positive 1, 2, and 7 days after the farm visit, respectively.

Background The majority of antimicrobials given during the production of pigs are given to nursery pigs. The influence of antimicrobial use on the levels of antimicrobial resistant (AMR) genes is important to quantify to be able to assess the impact of resistance on the food chain and risk to human and animal health. Results This study investigated the response on the levels of nine AMR genes to five different treatment strategies with oxytetracycline, and the dynamics of gene abundance over time by following 1167 pigs from five different farms in Denmark. The results showed no significant difference between treatments and an increase in abundance for the efflux pump encoding tet(A) gene and the genes encoding the ribosomal protection proteins tet(O) and tet(W) tetracycline resistant genes following treatment, while tet(M) showed no response to treatment. However, it was also observed that the levels of tet(O), tet(W), and ermB in some farms would drift more over time compared to a single treatment-course with antibiotic. Conclusion This study underlines the large variation in AMR levels under natural conditions and the need for increased investigation of the complex interactions of antimicrobial treatment and other environmental and managerial practices in swine production on AMR gene abundance.

OBJECTIVES We unexpectedly identified MRSA isolates carrying mecC (mecC-MRSA) from a Danish swine farm located in eastern Zealand. The objective of the present study was to investigate the origin of these isolates and their genetic relatedness to other mecC-MRSA isolates from Zealand. METHODS WGS was used to infer the phylogenetic relationship between 19 identified mecC-MRSA isolates from the swine farm and 34 additional epidemiologically unrelated human isolates from the same geographical region of Denmark. Variations in the accessory genome were investigated by bioinformatics tools, and antibiotic susceptibility profiles were assessed by MIC determination. RESULTS mecC-MRSA was isolated from a domestic swine farm, but not from cattle reared at the same farm. Phylogenetic analysis revealed that all mecC-MRSA isolates from both farm animals and workers formed a separate cluster, whereas human isolates from the same municipality belonged to a closely related cluster. Analysis of the accessory genome supported this relationship. CONCLUSIONS To the best of our knowledge, this is the first report of mecC-MRSA isolated from domestic swine. The investigation strongly indicates that transmission of mecC-MRSA has taken place on the swine farm between the farmers and swine. The close clustering of farm isolates and isolates from the same municipality suggests a local transmission of mecC-MRSA.

The primary objective of this study was to determine the minimum number of individual fecal samples to pool together in order to obtain a representative sample for herd level quantification of antimicrobial resistance (AMR) genes in a Danish pig herd, using a novel high-throughput qPCR assay. The secondary objective was to assess the agreement between different methods of sample pooling. Quantification of AMR was achieved using a high-throughput qPCR method to quantify the levels of seven AMR genes (ermB, ermF, sulI, sulII, tet(M), tet(O) and tet(W)). A large variation in the levels of AMR genes was found between individual samples. As the number of samples in a pool increased, a decrease in sample variation was observed. It was concluded that the optimal pooling size is five samples, as an almost steady state in the variation was observed when pooling this number of samples. Good agreement between different pooling methods was found and the least time-consuming method of pooling, by transferring feces from each individual sample to a tube using a 10μl inoculation loop and adding 3.5ml of PBS, approximating a 10% solution, can therefore be used in future studies.

Haemophilus parasuis causes Glässer's disease and pneumonia in pigs. Indirect hemagglutination (IHA) is typically used to serotype this bacterium, distinguishing 15 serovars with some nontypeable isolates. The capsule loci of the 15 reference strains have been annotated, and significant genetic variation was identified between serovars, with the exception of serovars 5 and 12. A capsule locus and in silico serovar were identified for all but two nontypeable isolates in our collection of >200 isolates. Here, we describe the development of a multiplex PCR, based on variation within the capsule loci of the 15 serovars of H. parasuis , for rapid molecular serotyping. The multiplex PCR (mPCR) distinguished between all previously described serovars except 5 and 12, which were detected by the same pair of primers. The detection limit of the mPCR was 4.29 × 10 5 ng/μl bacterial genomic DNA, and high specificity was indicated by the absence of reactivity against closely related commensal Pasteurellaceae and other bacterial pathogens of pigs. A subset of 150 isolates from a previously sequenced H. parasuis collection was used to validate the mPCR with 100% accuracy compared to the in silico results. In addition, the two in silico -nontypeable isolates were typeable using the mPCR. A further 84 isolates were analyzed by mPCR and compared to the IHA serotyping results with 90% concordance (excluding those that were nontypeable by IHA). The mPCR was faster, more sensitive, and more specific than IHA, enabling the differentiation of 14 of the 15 serovars of H. parasuis .

Low pathogen diarrhoea is a group-level diagnosis, characterised by non-haemorrhagic diarrhoea. In the current study, the apparent prevalence of low pathogen diarrhoea outbreaks in Danish herds was investigated along with the clinical utility of a laboratory examination for intestinal disease, agreement between three consecutive herd examinations from the same herd and agreement between quantitative PCR results from pooled faecal samples and sock samples. Twenty-four veterinarians submitted faecal and sock samples for quantitative PCR testing from outbreaks of diarrhoea in nursery pigs ( n = 38 herds) where the farmer or veterinarian had decided that antimicrobial treatment was necessary. The veterinarians were asked to fill in a questionnaire and participate in telephone interviews. The apparent prevalence of low pathogen diarrhoea was 0.18 (95% CL: 0.08–0.34). Agreement between the veterinarians’ clinical aetiological diagnosis and the pooled faecal sample was 0.18 (95% CL: 0.08–0.34), and Cohen’s Kappa was 0.03 (95% CL: −0.08 to 0.14). Antibiotic treatment or prevention strategies were changed in 0.63 (95% CL: 0.46–0.78) of the herds, and the veterinarians indicated that, for 0.32 (95% CL: 0.18–0.50) of the herds, changes were related to the diagnostic results from the laboratory examination performed in the study. In 0.16 (95% CL: 0.05–0.36) of the herds, the same infections were demonstrated at all three consecutive examinations. No herds had three consecutive diarrhoea outbreaks classified as low pathogen diarrhoea. For the quantitative results (log 10 of the summed amounts of Lawsonia intracellularis , Brachyspira pilosicoli , Escherichia coli F4 and F18) agreement between pooled faecal samples and sock samples was evaluated. Lin’s concordance correlation coefficient was 0.69 (95% CL: 0.48–0.82), and the mean difference between the two types of samples was −0.38 log 10 bacteria/g faeces (SD = 1.59 log 10 bacteria/g faeces; 95% CI: −0.90 to 0.14 log 10 bacteria/g faeces). Agreement for the dichotomised results was 0.89 (95% CI: 0.75–0.97) when test results were classified as low pathogen diarrhoea or not, and Cohen’s Kappa was 0.61 (95% CI: 0.26–0.95). In relation to detection of the individual infections, agreement was 0.63 (95% CI: 0.46–0.78), and Cohen’s Kappa was 0.53 (95% CI: 0.34–0.71). In conclusion, low pathogen diarrhoea is a common finding amongst diarrhoea outbreaks that are subjected to antibiotic batch treatment in Danish nursery pigs. Sock samples seem to offer a reliable diagnostic method with impact on clinical decisions for treatment and prevention. However, both the diarrhoea type and the aetiology change with time in the majority of herds, indicating a potential need for frequent diagnostic examinations.

Haemophilus parasuis is a diverse bacterial species that is found in the upper respiratory tracts of pigs and can also cause Glässer's disease and pneumonia. A previous pangenome study of H. parasuis identified 48 genes that were associated with clinical disease. Here, we describe the development of a generalized linear model (termed a pathotyping model) to predict the potential virulence of isolates of H. parasuis based on a subset of 10 genes from the pangenome. A multiplex PCR (mPCR) was constructed based on these genes, the results of which were entered into the pathotyping model to yield a prediction of virulence. This new diagnostic mPCR was tested on 143 field isolates of H. parasuis that had previously been whole-genome sequenced and a further 84 isolates from the United Kingdom from cases of H. parasuis- related disease in pigs collected between 2013 and 2014. The combination of the mPCR and the pathotyping model predicted the virulence of an isolate with 78% accuracy for the original isolate collection and 90% for the additional isolate collection, providing an overall accuracy of 83% (81% sensitivity and 93% specificity) compared with that of the “current standard” of detailed clinical metadata. This new pathotyping assay has the potential to aid surveillance and disease control in addition to serotyping data.

We evaluated a widely used culture-based method for detection of livestock-associated meticillin-resistant Staphylococcus aureus (LA-MRSA) in samples collected from pigs and the environment inside pig stables in Denmark and Norway. Selective enrichment in tryptic soy broth containing cefoxitin and aztreonam led to a high ratio of false-negative results (26%; 57/221). On this basis, we recommend reconsidering the use of selective enrichment for detection of LA-MRSA in animal and environmental samples.

Transmission of methicillin-resistant Staphylococcus aureus (MRSA) from animals to humans is of great concern due to the implications for human health and the health care system. The objective was to investigate the frequency and duration of MRSA carriage in human volunteers after a short-term exposure in a swine farm. The experimental study included 34 human volunteers staying 1 h in a MRSA-positive swine farm in four trials. In two of the trials, the influence of farm work involving pig contact was studied using a crossover design. The quantities of MRSA in nasal swabs, throat swabs, and air samples were measured at different time points and analyzed in relation to relevant covariates. This investigation showed that, overall, 94% of the volunteers acquired MRSA during the farm visit. Two hours after the volunteers left the stable, the nasal MRSA count had declined to unquantifiable levels in 95% of the samples. After 48 h, 94% of the volunteers were MRSA-negative. Nasal MRSA carriage was positively correlated to personal exposure to airborne MRSA and farm work involving pig contact and negatively correlated to smoking. No association was observed between MRSA carriage and face touching behavior, nasal methicillin-susceptible Staphylococcus aureus (MSSA) carriage, age, or gender. The increase in human MRSA carriage among the volunteers with pig contact seems to be dependent on the increased concentration of airborne MRSA of the surrounding air and not directly on physical contact with pigs. MRSA was not detected in any of the throat samples. IMPORTANCE The experimental approach made it possible to elucidate the contributions of airborne MRSA levels and farm work to nasal MRSA carriage in a swine farm. Short-term exposure to airborne MRSA poses a substantial risk for farm visitors to become nasal carriers, but the carriage is typically cleared within hours to a few days. The risk for short-term visitors to cause secondary transmissions of MRSA is most likely negligible due to the observed decline to unquantifiable levels in 95% of the nasal samples after only 2 h. The MRSA load in the nose was highly correlated to the amount of MRSA in the air and interventions to reduce the level of airborne MRSA or the use of face masks might consequently reduce nasal contamination.

Background: The aim of this study was to investigate whether the syndrome New Neonatal Porcine Diarrhoea Syndrome (NNPDS) is associated with a viral aetiology. Four well-managed herds experiencing neonatal diarrhoea and suspected to be affected by NNPDS were included in a case-control set up. A total of 989 piglets were clinically examined on a daily basis. Samples from diarrhoeic and non-diarrhoeic piglets at the age of three to seven days were selected for extensive virological examination using specific real time polymerase chain reactions (qPCRs) and general virus detection methods. Results: A total of 91.7% of the animals tested positive by reverse transcription qPCR (RT-qPCR) for porcine kobuvirus 1 (PKV-1) while 9% and 3% were found to be positive for rotavirus A and porcine teschovirus (PTV), respectively. The overall prevalence of porcine astrovirus (PAstV) was 75% with 69.8% of the PAstV positive pigs infected with PAstV type 3. No animals tested positive for rotavirus C, coronavirus (TGEV, PEDV and PRCV), sapovirus, enterovirus, parechovirus, saffoldvirus, cosavirus, klassevirus or porcine circovirus type 2 (PCV2). Microarray analyses performed on a total of 18 animals were all negative, as were eight animals examined by Transmission Electron Microscopy (TEM). Using Next Generation de novo sequencing (de novo NGS) on pools of samples from case animals within all herds, PKV-1 was detected in four herds and rotavirus A, rotavirus C and PTV were detected in one herd each. Conclusions: Our detailed analyses of piglets from NNPDS-affected herds demonstrated that viruses did not pose a significant contribution to NNPDS. However, further investigations are needed to investigate if a systemic virus infection plays a role in the pathogenesis of NNPDS.

This study describes the results of a randomized clinical trial investigating the effect of oxytetracycline treatment dose and mode of administration on the selection of antibiotic-resistant coliform bacteria in fecal samples from nursery pigs. Nursery pigs (pigs of 4 to 7 weeks of age) in five pig herds were treated with oxytetracycline for Lawsonia intracellularis -induced diarrhea. Each group was randomly allocated to one of five treatment groups: oral flock treatment with a (i) high (20 mg/kg of body weight), (ii) medium (10 mg/kg), or (iii) low (5 mg/kg) dose, (iv) oral pen-wise (small-group) treatment (10 mg/kg), and (v) individual intramuscular injection treatment (10 mg/kg). All groups were treated once a day for 5 days. In all groups, treatment caused a rise in the numbers and proportions of tetracycline-resistant coliform bacteria right after treatment, followed by a significant drop by the time that the pigs left the nursery unit. The counts and proportions of tetracycline-resistant coliforms did not vary significantly between treatment groups, except immediately after treatment, when the highest treatment dose resulted in the highest number of resistant coliforms. A control group treated with tiamulin did not show significant changes in the numbers or proportions of tetracycline-resistant coliforms. Selection for tetracycline-resistant coliforms was significantly correlated to selection for ampicillin- and sulfonamide-resistant strains but not to selection for cefotaxime-resistant strains. In conclusion, the difference in the dose of oxytetracycline and the way in which the drug was applied did not cause significantly different levels of selection of tetracycline-resistant coliform bacteria under the conditions tested. IMPORTANCE Antimicrobial resistance is a global threat to human health. Treatment of livestock with antimicrobials has a direct impact on this problem, and there is a need to improve the ways that we use antimicrobials in livestock production. We hypothesized that antibiotic resistance development following treatment of diarrhea in nursery pigs could be reduced either by lowering the dose of oxytetracycline or by replacing the commonly used practice of flock treatment with individual or small-group treatments, since this would reduce the number of pigs treated. However, the study showed no significant difference between treatment groups with respect to the number or proportion of tetracycline-resistant coliforms selected. The most important conclusion is that under practical field conditions, there will be no added value, in terms of lowering resistance development, by exchanging flock treatment for individual or small-group treatment of nursery pigs. The reason for the lack of an effect of single-animal treatment is probably that such animals share the environment with treated animals and take up resistant bacteria from the environment.

Livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) clonal complex (CC) 398 is widespread in the Danish pig production with around 90% of herds being positive. Since 2009, cases of CC398 LA-MRSA infections in Danish mink farmers have been observed. The objective of the study was to examine the presence of LA-MRSA in farmed mink. The investigation comprised three different sample types 1) clinical samples from carcasses submitted to the laboratory for diagnostic examination, 2) paws and pharyngeal swabs from healthy animals collected at pelting, and 3) feed samples from mink feed producers. In clinical samples, LA-MRSA was found in 34% of submissions and was most prevalent in samples from paws (33%) and pharynx (17%), followed by nasal and intestinal samples (each 13%), whereas it was never detected in perineal samples. LA-MRSA was found in healthy animals on 40% of the investigated farms, including paw samples (29%) and pharyngeal samples (16%). Twenty out of the 108 feed samples from feed producers were positive for LA-MRSA. The dominant spa -types were t034 and t011 associated to CC398, corresponding to the dominant spa -types detected in the Danish pig production, from which slaughter offal is used for mink feed. The spa -types, the high prevalence of LA-MRSA on paws and in pharynx, and its detection in feed samples, suggest feed as a possible source of LA-MRSA in mink.

PCR is routinely used in detection of antibiotic resistance genes including different classes of tet and erm genes. It remains unknown how PCR conditions affect detection of resistance genes in terms of genetic diversity and prevalence. In this study, numbers of PCR cycles and MgCl2 concentrations were evaluated for their effect on the diversity and prevalence of the tet genes that encode ribosomal protection proteins (RPPs) in composted swine fecal samples using the degenerate Ribo2_new_FW/Ribo2-RV primer pair. Four MgCl2 concentrations and 3 cycle numbers were tested in a 4 × 3 factorial design. A clone library was constructed for each PCR condition combination, and randomly selected clones were sequenced to determine the genetic diversity and relative distribution of RPP tet genes. Significant differences in genetic diversity and prevalence of tet genes were found among the tested cycle numbers and MgCl2 concentration combinations. The results suggest that 35 PCR cycles and 7 mM MgCl2 allow for optimal detection of the tet genes in swine feces using the Ribo2_new_FW/Ribo2-RV primer pair and that this combination should be used for further assay optimization and validation. These results also suggest that PCR conditions should be taken into consideration when PCR conditions are chosen for ecological studies of tet genes and when the results are interpreted.

As a part of a prospective cohort study in four herds, a nested case control study was carried out. Five slow growing pigs (cases) and five fast growing pigs (controls) out of 60 pigs were selected for euthanasia and laboratory examination at the end of the study in each herd. A total of 238 pigs, all approximately 12 weeks old, were included in the study during the first week in the grower-finisher barn. In each herd, approximately 60 pigs from four pens were individually ear tagged. The pigs were weighed at the beginning of the study and at the end of the 6-8 weeks observation period. Clinical data, blood and faecal samples were serially collected from the 60 selected piglets every second week in the observation period. In the killed pigs serum was examined for antibodies against Lawsonia intracellularis (LI) and procine circovirus type 2 (PCV2) and in addition PCV2 viral DNA content was quantified. In faeces the quantity of LI cells/g faeces and number of PCV2 copies/g faeces was measured by qPCR. The objective of the study was to examine if growth rate in grower-finishing pig is associated with the detection of LI and PCV2 infection or clinical data. This study has shown that diarrhoea is a significant risk factor for low growth rate and that one log(10) unit increase in LI load increases the odds ratio for a pig to have a low growth rate by 2.0 times. Gross lesions in the small intestine and LI load>log(10)6/g were significant risk factors for low growth. No association between PCV2 virus and low growth was found.

This study provides strong, novel evidence that humans may introduce methicillin-resistant Staphylococcus aureus CC398 into closed pig populations; it also demonstrates that stringent control and eradication measures were effective and prevented dissemination from pig farms to the general human population., Background. Emerging livestock-associated methicillin-resistant Staphylococcus aureus (MRSA) persist in livestock populations and represent a reservoir for transmission to humans. Understanding the routes of introduction and further transmission is crucial to control this threat to human health. Methods. All reported cases of livestock-associated MRSA (CC398) in humans and pigs in Norway between 2008 and 2014 were included. Data were collected during an extensive outbreak investigation, including contact tracing and stringent surveillance. Whole-genome sequencing of isolates from all human cases and pig farms was performed to support and expand the epidemiological findings. The national strategy furthermore included a “search-and-destroy” policy at the pig farm level. Results. Three outbreak clusters were identified, including 26 pig farms, 2 slaughterhouses, and 36 humans. Primary introductions likely occurred by human transmission to 3 sow farms with secondary transmission to other pig farms, mainly through animal trade and to a lesser extent via humans or livestock trucks. All MRSA CC398 isolated from humans without an epidemiological link to the outbreaks were genetically distinct from isolates within the outbreak clusters indicating limited dissemination to the general population. Conclusions. This study identified preventable routes of MRSA CC398 introduction and transmission: human occupational exposure, trade of pigs and livestock transport vehicles. These findings are essential for keeping pig populations MRSA free and, from a “One Health” perspective, preventing pig farms from becoming reservoirs for MRSA transmission to humans.

Histophilus somni was proposed in 2003 as a common name for bacteria that earlier had been called "Haemophilus somni", "Haemophilus agni", "Haemophilus somnifer", and "Histophilus ovis". The species is clearly separated from other species and genera within the family Pasteurellaceae. The species is phenotypically variable, but highly uniform regarding the 16S rDNA sequence. Whole-genome sequencing has revealed distinct genetic differences between a commensal and a pathogenic strain, particularly in regard to putative virulence factors. However, broad generalizations regarding the genetics of H. somni cannot be applied to the entire species until the genomes of additional strains are sequenced.

Atypical Actinobacillus pleuropneumoniae serotype 13 strains present in North America are described here for the first time. Different from serotype 13 strains described in Europe, North America strains are biotype I and antigenically related to both, serotypes 13 and 10. Chemical and structural analysis of the capsular polysaccharide (CPS) and lipopolysaccharide (LPS) of a representative strain revealed that the CPS is almost identical to that of the reference strain of serotype 13, having a slightly higher degree of glycose O-acetylation. However, it produces an O-PS within the LPS antigenically and structurally identical with that of the reference strain of A. pleuropneumoniae serotype 10. The O-PS was characterized as a homopolymer of 1,2 linked β-d-galactofuranosyl residues, a structure unrelated to that of the O-PS produced by the reference strain of serotype 13. Strains from Canada and United States are antigenically, phenotypically and genotypically similar. Animals infected by one of these strains induced antibodies that were detected by a LPS-based ELISA diagnostic test using either the homologous antigen or that of serotype 10. Based on the LPS and toxin profile, these strains might be misidentified as A. pleuropneumoniae serotype 10.

Metritis and endometritis commonly occur in dairy cows after calving. Although numerous studies have been performed to identify the causative pathogens, a complete overview has not been done. Metagenomic studies have analyzed the bacterial populations of uterine flush samples from postpartum (pp) dairy cows, but the microbiota in the uterine luminal fluid may differ from the microbiota of the endometrium itself, and important putative pathogens may have been overlooked. In the present study, we compared the microbiota of the uterine lumen and the endometrium of healthy, metritic, and endometritic cows. Samples were collected from 68 Holstein dairy cows at 1, 4, and 7 weeks pp, and the data were analyzed by deep sequencing of the V1 and V2 hypervariable regions of the 16S ribosomal RNA gene. The results showed that Porphyromonadaceae, Fusobacteriaceae, Leptotrichiaceae, and Mycoplasmataceae may be associated with uterine disease. The microbiota of the uterine flush samples and the endometrial biopsies were correlated, but the microbiota of the biopsies was more diverse. Fusobacteriaceae and Leptotrichiaceae were not observed in the biopsies at week 7, whereas they accounted for 20% and 13%, respectively, of the bacterial populations in the flush samples. The Mycoplasmataceae family was observed in much higher quantity in the flush samples than in the biopsies of the endometritis groups at weeks 4 and 7. Our findings support the observations of previous metagenomic studies and illustrate the importance of including endometrial biopsies to obtain more detailed knowledge of the pp uterine microbiota.

Four quantitative PCR (qPCR) assays were evaluated for quantitative detection of Brachyspira pilosicoli, Lawsonia intracellularis, and E. coli fimbrial types F4 and F18 in pig feces. Standard curves were based on feces spiked with the respective reference strains. The detection limits from the spiking experiments were 10(2) bacteria/g feces for Bpilo-qPCR and Laws-qPCR, 10(3)CFU/g feces for F4-qPCR and F18-qPCR. The PCR efficiency for all four qPCR assays was between 0.91 and 1.01 with R(2) above 0.993. Standard curves, slopes and elevation, varied between assays and between measurements from pure DNA from reference strains and feces spiked with the respective strains. The linear ranges found for spiked fecal samples differed both from the linear ranges from pure culture of the reference strains and between the qPCR tests. The linear ranges were five log units for F4-qPCR, and Laws-qPCR, six log units for F18-qPCR and three log units for Bpilo-qPCR in spiked feces. When measured on pure DNA from the reference strains used in spiking experiments, the respective log ranges were: seven units for Bpilo-qPCR, Laws-qPCR and F18-qPCR and six log units for F4-qPCR. This shows the importance of using specific standard curves, where each pathogen is analysed in the same matrix as sample DNA. The qPCRs were compared to traditional bacteriological diagnostic methods and found to be more sensitive than cultivation for E. coli and B. pilosicoli. The qPCR assay for Lawsonia was also more sensitive than the earlier used method due to improvements in DNA extraction. In addition, as samples were not analysed for all four pathogen agents by traditional diagnostic methods, many samples were found positive for agents that were not expected on the basis of age and case history. The use of quantitative PCR tests for diagnosis of enteric diseases provides new possibilities for veterinary diagnostics. The parallel simultaneous analysis for several bacteria in multi-qPCR and the determination of the quantities of the infectious agents increases the information obtained from the samples and the chance for obtaining a relevant diagnosis.

Trans-tracheal aspirations from 56 apparently healthy calves and 34 calves with clinical signs of pneumonia were collected in six different herds during September and November 2002. The 90 samples were cultivated and investigated by PCR tests targeting the species Histophilus somni, Mannheimia haemolytica, Pasteurella multocida, Mycoplasma bovis, Mycoplasma dispar, and Mycoplasma bovirhinis. A PCR test amplifying the lktC-artJ intergenic region was evaluated and shown to be specific for the two species M. haemolytica and Mannheimia glucosida. All 90 aspirations were also analyzed for bovine respiratory syncytial virus (BRSV), parainfluenza-3 virus, and bovine corona virus by antigen ELISA. Surprisingly, 63% of the apparently healthy calves harbored potentially pathogenic bacteria in the lower respiratory tract, 60% of these samples contained either pure cultures or many pathogenic bacteria in mixed culture. Among diseased calves, all samples showed growth of pathogenic bacteria in the lower respiratory tract. All of these were classified as pure culture or many pathogenic bacteria in mixed culture. A higher percentage of the samples were positive for all bacterial species in the group of diseased animals compared to the clinically healthy animals, however this difference was only significant for M. dispar and M. bovirhinis. M. bovis was not detected in any of the samples. BRSV was detected in diseased calves in two herds but not in the clinically healthy animals. Among the diseased calves in these two herds a significant increase in haptoglobin and serum amyloid A levels was observed compared to the healthy calves. The results indicate that haptoglobin might be the best choice for detecting disease under field conditions. For H. somni and M. haemolytica, a higher percentage of the samples were found positive by PCR than by cultivation, whereas the opposite result was found for P. multocida. Detection of P. multocida by PCR or cultivation was found to be significantly associated with the disease status of the calves. For H. somni a similar association with disease status was only observed for cultivation and not for PCR.