Genetic diversity and antimicrobial susceptibility of Streptococcus suis from diseased Swiss pigs collected between 2019 - 2022.

Streptococcus suis is an important pathogen causing severe disease in pigs and humans, giving rise to economic losses in the pig production industry. Out of 65 S. suis isolates collected from diseased pigs in Switzerland between 2019 and 2022, 57 isolates were thoroughly examined by phenotypic and whole genome sequence (WGS) based characterization. The isolates' genomes were sequenced allowing for a comprehensive analysis of their distribution in terms of serovar, sequence type (ST), clonal complex (CC), and classical virulence markers. Antimicrobial resistance (AMR) genes were screened, and phenotypic susceptibility to eight classes of antimicrobial agents was examined. Serovar 6, devoid of any resistance genes, was found to be most prevalent, followed by serovars 1, 3, 1/2, and 9. Thirty STs were identified, with ST1104 being the most prevalent. Serovar 2 and serovar 1/2 were associated with CC1, potentially containing the most virulent variants. Based on single nucleotide polymorphism (SNP) analyses, fifteen isolates belonged to one of seven putative transmission clusters each consisting of two or three isolates. High phenotypic AMR rates were detected for tetracyclines (80%) and macrolides (35%) and associated with the resistance genes tet(O) and erm(B), respectively. In contrast, susceptibility to ß-lactam antibiotics and phenicols was high. Determination of phenotypic AMR profiling, including the minimum inhibitory concentrations (MICs) of the tested antimicrobial agents, sets a baseline for future studies. The study provides valuable insights into the genetic diversity and antimicrobial susceptibility of Swiss S. suis isolates, facilitating the identification of emerging clones relevant to public health concerns.

Development of a new multiplex quantitative PCR for the detection of Glaesserella parasuis, Mycoplasma hyorhinis, and Mycoplasma hyosynoviae.

Glaesserella parasuis, Mycoplasma hyorhinis, and Mycoplasma hyosynoviae are important porcine pathogens responsible for polyserositis, polyarthritis, meningitis, pneumonia, and septicemia causing significant economic losses in the swine industry. A new multiplex quantitative polymerase chain reaction (qPCR) was designed on one hand for the detection of G. parasuis and the virulence marker vtaA to distinguish between highly virulent and non-virulent strains. On the other hand, fluorescent probes were established for the detection and identification of both M. hyorhinis and M. hyosynoviae targeting 16S ribosomal RNA genes. The development of the qPCR was based on reference strains of 15 known serovars of G. parasuis, as well as on the type strains M. hyorhinis ATCC 17981T and M. hyosynoviae NCTC 10167T . The new qPCR was further evaluated using 21 G. parasuis, 26 M. hyorhinis, and 3 M. hyosynoviae field isolates. Moreover, a pilot study including different clinical specimens of 42 diseased pigs was performed. The specificity of the assay was 100% without cross-reactivity or detection of other bacterial swine pathogens. The sensitivity of the new qPCR was demonstrated to be between 11-180 genome equivalents (GE) of DNA for M. hyosynoviae and M. hyorhinis, and 140-1200 GE for G. parasuis and vtaA. The cut-off threshold cycle was found to be at 35. The developed sensitive and specific qPCR assay has the potential to become a useful molecular tool, which could be implemented in veterinary diagnostic laboratories for the detection and identification of G. parasuis, its virulence marker vtaA, M. hyorhinis, and M. hyosynoviae.

Strain diversity in Mycobacterium avium subsp. paratuberculosis-positive bovine fecal samples collected in Switzerland.

Paratuberculosis or Johne's disease is a chronic intestinal disease in domestic and wild ruminants. It affects global dairy economy and is caused by Mycobacterium avium subsp. paratuberculosis (MAP). The objective of this study was to analyze strain diversity in MAP-positive fecal samples by using a particular single nucleotide polymorphism (SNP) distinguishing between cattle (C-) and sheep (S-) type MAP and analysis of SNPs within gyrA and gyrB genes differentiating between Types I, II, and III. Moreover, mycobacterial interspersed repetitive unit and variable-number tandem repeat (MIRU-VNTR) analysis using eight established loci was performed. A total of 90 fecal samples from diseased animals presenting diarrhea and/or weight loss, originating from 59 bovine herds across 16 cantons of Switzerland were screened by PCR for the MAP-specific F57 and IS900 genes and were further subtyped. 96.7% and 3.3% of the samples contained C- and S-type MAP, respectively. Ten INRA Nouzilly MIRU-VNTR (INMV) profiles, with a discriminatory index of 0.802, calculated based on 65 epidemiological independent genotypes, were detected: INMV 1 (33.8%), INMV 2 (23.1%), INMV 6 (16.9%), INMV 9 (9.2%), INMV 116 (4.6%), INMV 3 (3.1%), INMV 5 (3.1%) and INMV 72 (1.5%), including two novel INMV profiles, namely INMV 253 (3.1%; S-type III) and INMV 252 (1.5%; C-type). INMV 1, INMV 2, and INMV 6 comprised almost 75% of the F57- and IS900-positive samples. Typing data from 11 herds suggest that there are some herds with intra-herd diversity of genotypes. The results of this study indicate a heterogeneity of MAP in Switzerland.

Isolation of Mycobacterium avium ssp. paratuberculosis and other non-tuberculous mycobacteria from head lymph nodes of wild ruminants and badgers in Switzerland.

Introduction: The family Mycobacteriaceae contains over 188 species, most of which are saprophytic non-tuberculous mycobacteria (NTM). In wildlife, a variety of different NTM can be found, with different reports about their pathogenic potential. A pathogenic member of NTM is Mycobacterium avium ssp. paratuberculosis (MAP), which can infect farmed and wild ruminants. It causes paratuberculosis which is an economically important chronic disease. Infected farm animals are considered to be the source of infection in wild animals. Wildlife, on the other hand, is thought to be a reservoir for certain members of the Mycobacterium tuberculosis complex (MTBC), such as M. caprae, which causes tuberculosis in cattle and red deer. Methods: Switzerland implemented a surveillance program for tuberculosis in wild animals in 2014. Here, we describe the results from the mycobacterial culture of lymph node samples collected from red deer, roe deer, chamois, ibex, and badgers collected within this surveillance program from 2020 to 2022. Overall, samples from 548 animals were checked macroscopically for tuberculosis-like lesions. Results: In total, 88 animals (16.1%), which either had lesions in their lymph nodes or were male and aged older than 5 years, were investigated using mycobacterial culture. In total, 25 animals (28.4%) were positive for NTM, while no MTBC was detected. The most often identified NTM was M. vaccae, followed by M. avium. Most animals positive for NTM did not show any macroscopic lesions. Furthermore, MAP was isolated from the head lymph nodes of two male red deer. Neither of the two MAP-positive animals had any macroscopic lesions in their head lymph nodes or any other signs of disease. Discussion: The shooting sites of the two MAP-positive animals were located in Alpine pastures used for grazing of cattle during summer, which confirms that species transmission can occur when contaminated pastures are used by different species. In agreement with other studies, the occurrence of MAP in red deer was quite low. However, so far, MAP was mostly isolated from feces and intestinal lymph nodes of wild animals. This is the first detection of MAP in the head lymph nodes of red deer in Switzerland.

Identification of Glaesserella parasuis and Differentiation of Its 15 Serovars Using High-Resolution Melting Assays.

Glaesserella parasuis is the etiological agent of Glässer's disease, which is associated with polyserositis and arthritis and has a significant impact on the economy of the pig production industry. For the optimal surveillance of this pathogen, as well as for the investigation of G. parasuis-associated diseases, it is crucial to identify G. parasuis at the serovar level. In this work, we designed and developed new high-resolution melting (HRM) approaches, namely, the species-specific GPS-HRM1 and two serovar-specific HRM assays (GPS-HRM2 and GPS-HRM3), and evaluated the sensitivity and specificity of the assays. The HRM assays demonstrated good sensitivity, with 12.5 fg-1.25 pg of input DNA for GPS-HRM1 and 125 fg-12.5 pg for GPS-HRM2 and GPS-HRM3, as well as a specificity of 100% for the identification of all recognized 15 G. parasuis serovars. Eighteen clinical isolates obtained between 2014 and 2022 in Switzerland were tested by applying the developed HRM assays, which revealed a heterogeneous distribution of serovars 2, 7, 4, 13, 1, and 14. The combination with virulence marker vtaA (virulence-associated trimeric autotransporters) allows for the prediction of potentially virulent strains. The assays are simple to execute and enable a reliable low-cost approach, thereby refining currently available diagnostic tools.

Development of a novel high resolution melting assay for identification and differentiation of all known 19 serovars of Actinobacillus pleuropneumoniae.

Actinobacillus pleuropneumoniae is the etiological agent of porcine pleuropneumonia, a respiratory infectious disease responsible for global economic losses in the pig industry. From a monitoring perspective as well as due to the different courses of disease associated with the various serovars, it is essential to distinguish them in different herds or countries. In this study, we developed a novel high resolution melting (HRM) assay based on reference strains for each of the 19 known serovars and additional 15 clinical A. pleuropneumoniae isolates. The novel HRM comprises the species-specific APP-HRM1 and two serovar-specific HRM assays (APP-HRM2 and APP-HRM3). APP-HRM1 allowed polymerase chain reaction (PCR) amplification of apxIV resulting in an A. pleuropneumoniae specific melting curve, while nadV specific primers differentiated biovar 2 from biovar 1 isolates. Using APP-HRM2 and APP-HRM3, 13 A. pleuropneumoniae serovars can be determined by inspecting the assigned melting temperature. In contrast, serovar 3 and 14, serovar 9 and 11, and serovar 5 and 15 have partly overlapping melting temperatures and thus represent a challenge to accurately distinguish them. Consequently, to unambiguously ensure the correct assignment of the serovar, it is recommended to perform the serotyping HRM assay using a positive control for each serovar. This rapid and user-friendly assay showed high sensitivity with 1.25 fg-125 pg of input DNA and a specificity of 100% to identify A. pleuropneumoniae. Characteristic melting patterns of amplicons might allow detecting new serovars. The novel HRM assay has the potential to be implemented in diagnostic laboratories for better surveillance of this pathogen.

Draft Genome Sequences of Two Clinical Actinobacillus pleuropneumoniae Serotype 19 Strains from Pigs in Switzerland.

Actinobacillus pleuropneumoniae serotype 19 is a very recently described new serotype with a novel type II capsule synthesis locus. Here, we report the draft genome sequences of two Actinobacillus pleuropneumoniae serotype 19 strains with a serogroup 3/6/8/12/15 O-antigen locus that were isolated in 2018 and 2021 from two different pig farms in Switzerland.

Novel multiplex TaqMan assay for differentiation of the four major pathogenic Brachyspira species in swine.

A novel TaqMan 5-plex real-time PCR using a combination of locked nucleic acid-modified (LNA)- and minor groove binding (MGB)-conjugated DNA probes was developed for identification and differentiation between the four main pathogenic Brachyspira species in swine. B. hyodysenteriae, B. pilosicoli, and B. suanatina are identified using three hydrolysis probes targeting cpn60, while B. hampsonii is recognized by another nox specific probe. The assay also includes an exogenous internal control simultaneously verifying the PCR competency of the DNA samples. Validation of the novel assay was performed using DNA samples from 18 Brachyspira reference strains and 477 clinical samples obtained from porcine rectal swabs by comparing them with different PCR-based methods targeting nox, 16S rDNA, and 23S rDNA. The specificity of the assay was 100% without cross-reactivity or detection of different pathogens. Depending on the Brachyspira species, the limit of detection was between 10 and 20 genome equivalents with a cut-off threshold cycle (Ct) value of 37. The developed highly sensitive and specific 5-plex real-time PCR assay is easy to implement in routine veterinary diagnostic laboratories and enables rapid differentiation between the main four pathogenic Brachyspira species recognized in pigs using a single-tube approach.

Population structure, genetic diversity and pathotypes of Streptococcus suis isolated during the last 13 years from diseased pigs in Switzerland.

Streptococcus (S.) suis is a globally important swine pathogen, which comprises certain zoonotic serotypes. In this study, a detailed characterization of 88 porcine S. suis isolates was performed by analyzing capsular (cps) types, multilocus sequence typing (MLST) and investigation of the minimum core genome (MCG). In order to focus on the virulence potential of presumable invasive disease-associated S. suis isolates, virulence-associated gene profiles were assessed followed by screening a chosen subset of S. suis strains with a molecular pathotyping tool. Results showed a high genetic variability within this strain collection. In total, seventeen cps types were identified with a predominance of cps type 9 (15.9%) and 6 (14.8%). MLST revealed 48 sequence types (STs) including 41 novel ones. The population structure of S. suis was heterogenous and isolates belonged to eight different clonal complexes (CCs) including CC28 (9.1%), CC1109 (8%), CC13/149 (6.8%), CC1237 (5.7%), CC1 (3.4%), CC17 (3.4%), CC87 (2.3%), and CC1112 (1.1%), whereas a significant portion of isolates (60.2%) could not be assigned to any described CCs. Virulence-associated markers, namely extracellular protein factor (epf), muramidase-released protein (mrp), and suilysin (sly), showed a link with STs rather than with cps types. With this study an expanded knowledge about the population structure and the genetic diversity of S. suis could be achieved, which helps to contribute to an optimal public health surveillance system by promoting a focus on strains with an increased virulence and zoonotic potential.

Rapid high resolution melting assay to differentiate Streptococcus suis serotypes 2, 1/2, 1, and 14.

This rapid high resolution melting (HRM) assay allows distinguishing between Streptococcus suis serotype pairs 2 and 1/2 as well as 1 and 14, respectively, based on a single-nucleotide polymorphism within capsular polysaccharide synthesis gene cluster K. This assay is easy to implement and identifies potential zoonotic serotypes.

Morphological and Molecular Characterization of a New Mycobacterium avium Subsp. paratuberculosis S-Type Strain Genotype in Goats.

Paratuberculosis is a chronic bacterial disease of global importance mainly in domestic and wild ruminants, caused by Mycobacterium avium subsp. paratuberculosis (MAP). In goats, paratuberculosis is mostly caused by the "C-type" (cattle) and in a few cases by the "S-type" (sheep) strain of MAP. In 2017, a caprine S-type III isolate with a new VNTR profile was identified in a Swiss alpine region. In 2018, new caprine isolates with the same novel VNTR profile originating from a farm of a close by neighboring valley were analyzed. Here we report on this MAP S-type III outbreak in a Swiss dairy goat farm in which we investigated the pathological changes, distribution and genotype of MAP tissue homogenates. Full necropsy and histological examination were undertaken on two female adult goats with a history of weight loss and intermitting diarrhea. Routine and special stains were applied to characterize the morphological changes. DNA was extracted from 33 different tissue samples and tested for MAP by qPCR targeting IS900 and F57. Subtyping was performed, using the variable number tandem repeats (VNTR) and mycobacterial interspersed repetitive units (MIRU) approach. The goats showed moderate to marked emaciation and displayed typical clinical features of paratuberculosis. A moderate granulomatous enteritis and regional lymphadenitis with a small to moderate number of acid-fast bacteria within macrophages was detected. MAP detection was mainly restricted to the gastrointestinal tract, mesenteric and hepatic lymph nodes. Subtyping the S-type isolates using a panel of eight established MIRU-VNTR loci identified a new genotype, INMV 218.

Three-reaction high-resolution melting assay for rapid differentiation of Mycobacterium tuberculosis complex members.

The possibility of introducing a reliable assay for a quick identification and differentiation of the main species of Mycobacterium tuberculosis complex (MTBC) supports the improvement of efficient tuberculosis combating strategies worldwide. Commercially available assays are often based on cultured samples; however, due to the long cultivation time of mycobacteria, results are delayed. Developed PCR approaches have been published previously, though, when testing intricate veterinary samples, the complex composition of multiplex qPCRs frequently leads to assay failure. In order to overcome those limits, a paradigm of a three-reaction high-resolution melting (HRM) assay for the simultaneous identification and differentiation of the main members of MTBC was established. The assay is based on single nucleotide polymorphisms within gyrB and gyrA, which have been used as target for the establishment of two highly specific HRM assays (HRM assays 1 and 2) discriminating M. tuberculosis/ Mycobacterium canetti, Mycobacterium bovis/M. bovis BCG, Mycobacterium caprae/rare M. caprae/M. bovis ecotypes, Mycobacterium africanum/Mycobacterium orygis/ Mycobacterium pinnipedii/Clade A1, Mycobacterium microti, and a rare subtype of M. canettii followed by a third HRM assay (HRM assay 3) allowing a further differentiation of M. bovis, M. bovis BCG, and a rare subtype of M. caprae/M. bovis, which is considered to be a novel ecotype. High-resolution melting assay 1 is described in a previously published report. High-resolution melting assay 2 showed 100% correlation of all 39 examined isolates with the results of a commercial identification kit. 96% of the clinical samples tested demonstrated concordant results. High-resolution melting assay 3 showed an accordance of 100% with the results of the commercially available identification kit of all 22 samples analyzed. The proposed strategy of the three-reaction HRM assay can be used for an accurate differentiation of up to seven groups of MTBC and potentially to identify a rare subtype of M. canettii either on isolates or on clinical samples.

Development of a new High Resolution Melting (HRM) assay for identification and differentiation of Mycobacterium tuberculosis complex samples.

The rapid identification and differentiation of members of the Mycobacterium tuberculosis complex (MTBC) is essential to assess the potential zoonotic risk. Different available molecular methods are time consuming since they depend on cultivation of mycobacteria. High Resolution Melting (HRM) is a low cost, rapid and easy to perform single-tube method not limited to cultured samples. In this study, a HRM assay specifically targeting gyrB was developed to simultaneously identify and differentiate Mycobacterium (M.) tuberculosis, M. microti and M. bovis/M. caprae. To evaluate the performance of this assay, 38 MTBC isolates and 25 directly extracted clinical specimens were analysed. HRM results of all 38 (100%) examined isolates correlated with the results obtained with the commercially available GenoType MTBC test (Hain Lifescience). From the 25 clinical specimens tested, species identification by HRM showed concordant results with the previously used identification methods in 23 samples (92%). The assay demonstrated a good analytical sensitivity, specificity and reproducibility and can be used directly on clinical specimens.

Distribution and expression of esat-6 and cfp-10 in non-tuberculous mycobacteria isolated from lymph nodes of slaughtered cattle in Switzerland.

The most commonly used tools for tuberculosis testing in cattle, the tuberculin skin test and the interferon-γ release assay, detect immune reactivity to various antigens of Mycobacterium bovis, including ESAT-6 and CFP-10. However, some non-tuberculous mycobacteria (NTM) can also harbor the cfp-10 and/or esat-6 genes, which can lead to false-positive results. We tested 77 NTM isolates belonging to 22 different species from lymph nodes of healthy slaughtered cattle for the occurrence of cfp-10 and esat-6. Most isolates did not harbor cfp-10 and esat-6. However, M. gordonae, 'M. lymphaticum', M. kansasii, and M. persicum were cfp-10 positive. The esat-6 gene was found in M. kansasii and M. persicum. Protein expression of cfp-10 and esat-6 could be detected for M. kansasii and M. persicum. An effective tuberculosis control program based on interferon-γ release assays and tuberculin skin testing is dependent on further monitoring and characterization of NTM in a cattle population.

Molecular Characterization of Mycobacterium avium subsp. hominissuis of Two Groups of Lymph Nodes, Being Intradermal Tuberculin or Interferon-Gamma Test Positive and Negative, Isolated from Swiss Cattle at Slaughter.

Mycobacterium avium subsp. hominissuis (MAH) is an important zoonotic pathogen with raising global health concerns. In humans, MAH is one of the most widespread non-tuberculous mycobacterial species responsible for lung disease. In animals, MAH is frequently isolated from pigs; however, it is also an opportunistic pathogen for other mammals including cattle. To elucidate the genetic diversity of MAH in cattle, a molecular characterization of isolates (n = 26) derived from lymph nodes was performed. Fourteen isolates originated from slaughtered cattle with visible altered lymph nodes at meat inspection, whereas 12 isolates were from lymph nodes without any gross pathological changes of healthy slaughtered cattle. Variable number of tandem repeat (VNTR) analysis was performed at 20 loci to examine genetic differences of isolates and to compare to previously reported VNTR data of human isolates from different countries. Genetic elements IS901, IS1245, IS1311, LSPA17, ITS1 sequevar, and hsp65 code were determined. Interestingly, two bovine MAH isolates harbored ISMav6 and hsp65 code 15, which so far has only been observed in human isolates. We supposed that VNTR data of Swiss samples would show clustering with European samples. Minimum spanning tree and unweighted pair group method using arithmetic averages analyses based on the VNTR data indicated a specific cluster of MAH isolates obtained from lymph nodes without any gross pathological changes of healthy slaughtered cattle. Comparing Swiss isolates with isolates from different other countries, no geographical clustering was observed; however, four Swiss isolates had an identical VNTR profile as human isolates from the Netherlands, the United States, and Japan. These findings indicate a possible public health issue.

Epidemiological tracing of bovine tuberculosis in Switzerland, multilocus variable number of tandem repeat analysis of Mycobacterium bovis and Mycobacterium caprae.

BACKGROUND: After 15 years of absence, in 2013 bovine tuberculosis (bTB), caused by Mycobacterium (M.) bovis and M. caprae, reemerged in the Swiss dairy cattle population. In order to identify the sources of infection as well as the spread of the agents, molecular-epidemiologic tracing by MIRU-VNTR analysis in combination with spoligotyping was performed. A total of 17 M. bovis and 7 M. caprae isolates were cultured from tuberculous bovine lymph nodes and analyzed with a set of 49 genetic markers by using automated capillary electrophoresis. RESULTS: The outbreak in the western part of Switzerland was caused by M. bovis spoligotype SB0120. With the exception of four single-locus variations observed in MIRU 20, the MIRU-VNTR profiles of the 17 M. bovis isolates were identical, indicating a single source of infection. M. bovis detected in one archival bovine specimen from the outbreak region showed an identical MIRU-VNTR profile, suggesting persistence of the agent in a dairy herd for nearly fifteen years. The outbreak in the eastern part of Switzerland was caused by M. caprae spoligotype SB0418. All Swiss M. caprae isolates showed the Lechtal-type MIRU-VNTR profile, described as endemic in wild ruminants and in dairy cattle in Austrian bordering regions. This suggests the agent was most likely introduced by Swiss dairy cattle summering on Austrian pastures. CONCLUSIONS: The present study is the first MIRU-VNTR analysis of Swiss bTB mycobacterial isolates. The genotyping assay was found to be highly discriminating and suitable for the epidemiological tracing of further outbreaks. These findings will contribute to the development of an international MIRU-VNTR database aiming to improve bTB surveillance.

A novel multiplex qPCR targeting 23S rDNA for diagnosis of swine dysentery and porcine intestinal spirochaetosis.

BACKGROUND: A multiplex qPCR targeting a 128 bp region on the 23S rDNA gene was developed for detection of Brachyspira (B.) hyodysenteriae and B. pilosicoli, the agents of swine dysentery (SD) and porcine intestinal spirochaetosis (PIS), together with a triplet of apathogenic Brachyspira spp. (B. innocens, B. intermedia, B. murdochii) in porcine feces. The multiplex qPCR was evaluated against a duplex PCR (La et al., J Clin Microbiol 41:3372-5, 2003). RESULTS: Using DNA extracted from fecal culture, the multiplex qPCR showed excellent agreement with the duplex PCR (κ = 0.943 and 0.933). In addition, thanks to the three probes whereof one detecting the apathogenic Brachyspria spp., a more diversified overview of the brachyspiral flora in porcine fecal samples can be delivered as a part of the routine diagnostic. The multiplex qPCR with a limit of detection of 5-10 genomic equivalents (GE) per reaction (6 × 102 GE per gram) allows reliable detection of Brachyspira species directly from fecal swab DNA. In line with this, analysis of 202 fecal swabs in comparison with culture-based qPCR showed a high agreement for the causative agents of SD (B.hyodysenteriae: κ = 0.853, sensitivity 87% specificity 98%). CONCLUSION: The novel multiplex qPCR is robust and has a high analytical sensitivity and is therefore suitable for high-throughput screening of porcine fecal swabs for the causative agents of SD. This assay can therefore be used for the direct proof of the pathogenic B. spp. in fecal swabs within the scope of a monitoring program.

A novel quantitative real-time polymerase chain reaction method for detecting toxigenic Pasteurella multocida in nasal swabs from swine.

BACKGROUND: Progressive atrophic rhinitis (PAR) in pigs is caused by toxigenic Pasteurella multocida. In Switzerland, PAR is monitored by selective culture of nasal swabs and subsequent polymerase chain reaction (PCR) screening of bacterial colonies for the P. multocida toxA gene. A panel of 203 nasal swabs from a recent PAR outbreak were used to evaluate a novel quantitative real-time PCR for toxigenic P. multocida in porcine nasal swabs. RESULTS: In comparison to the conventional PCR with a limit of detection of 100 genome equivalents per PCR reaction, the real-time PCR had a limit of detection of 10 genome equivalents. The real-time PCR detected toxA-positive P. multocida in 101 samples (49.8%), whereas the conventional PCR was less sensitive with 90 toxA-positive samples (44.3%). In comparison to the real-time PCR, 5.4% of the toxA-positive samples revealed unevaluable results by conventional PCR. CONCLUSIONS: The approach of culture-coupled toxA PCR for the monitoring of PAR in pigs is substantially improved by a novel quantitative real-time PCR.

First screening for Brachyspira hampsonii in Swiss pigs applying a new high resolution melting assay.

A new High Resolution Melting (HRM) assay was developed for the rapid detection of Brachyspira (B.) hampsonii. B. hampsonii occurs in different European countries, however, until today it has not been encountered in Switzerland. Four B. hampsonii reference strains were used to develop the HRM assay: B. hampsonii clade I ATCC BAA2463 and clade II ATCC BAA2464 strain, as well as two isolated strains P280/1 from the UK and the German isolate 5369-1x/12. A conserved region of the nox gene was used to design B. hampsonii-specific primers. The HRM melting curves for the four reference strains showed reproducible difference graphs with distinct differences between the four strains based on a slight variation between the four amplicon sequences. In addition, DNA from 22 B. hampsonii strains representing four genetic B. hampsonii groups was used to validate the method. Melting temperatures in the interval between 73.1 and 74°C were obtained for all B. hampsonii strains and allow differentiating B. hampsonii from other Brachyspira species. In total 897 Swiss porcine fecal Brachyspira isolates, cultured between 2009 and 2015, were analysed by the HRM protocol. B. hampsonii was not detected among these Swiss Brachyspira isolates. In conclusion, the rapid and low-cost HRM approach allows a sensitive and specific identification of B. hampsonii.

Stochastic signalling rewires the interaction map of a multiple feedback network during yeast evolution.

During evolution, genetic networks are rewired through strengthening or weakening their interactions to develop new regulatory schemes. In the galactose network, the GAL1/GAL3 paralogues and the GAL2 gene enhance their own expression mediated by the Gal4p transcriptional activator. The wiring strength in these feedback loops is set by the number of Gal4p binding sites. Here we show using synthetic circuits that multiplying the binding sites increases the expression of a gene under the direct control of an activator, but this enhancement is not fed back in the circuit. The feedback loops are rather activated by genes that have frequent stochastic bursts and fast RNA decay rates. In this way, rapid adaptation to galactose can be triggered even by weakly expressed genes. Our results indicate that nonlinear stochastic transcriptional responses enable feedback loops to function autonomously, or contrary to what is dictated by the strength of interactions enclosing the circuit.