Phylogenetic diversity, antimicrobial susceptibility and virulence gene profiles of Brachyspira hyodysenteriae isolates from pigs in Germany

Swine dysentery (SD) is an economically important diarrheal disease in pigs caused by different strongly hemolytic Brachyspira (B.) species, such as B. hyodysenteriae, B. suanatina and B. hampsonii. Possible associations of epidemiologic data, such as multilocus sequence types (STs) to virulence gene profiles and antimicrobial susceptibility are rather scarce, particularly for B. hyodysenteriae isolates from Germany. In this study, B. hyodysenteriae (n = 116) isolated from diarrheic pigs between 1990 and 2016 in Germany were investigated for their STs, susceptibility to the major drugs used for treatment of SD (tiamulin and valnemulin) and genes that were previously linked with virulence and encode for hemolysins (tlyA, tlyB, tlyC, hlyA, BHWA1_RS02885, BHWA1_RS09085, BHWA1_RS04705, and BHWA1_RS02195), outer membrane proteins (OMPs) (bhlp16, bhlp17.6, bhlp29.7, bhmp39f, and bhmp39h) as well as iron acquisition factors (ftnA and bitC). Multilocus sequence typing (MLST) revealed that 79.4% of the isolates belonged to only three STs, namely ST52 (41.4%), ST8 (12.1%), and ST112 (25.9%) which have been observed in other European countries before. Another 24 isolates belonged to twelve new STs (ST113-118, ST120-123, ST131, and ST193). The temporal distribution of STs revealed the presence of new STs as well as the regular presence of ST52 over three decades (1990s–2000s). The proportion of strains that showed resistance to both tiamulin und valnemulin (39.1%) varied considerably among the most frequent STs ranging from 0% (0/14 isolates resistant) in ST8 isolates to 46.7% (14/30), 52.1% (25/48), and 85.7% (6/7) in isolates belonging to ST112, ST52, and ST114, respectively. All hemolysin genes as well as the iron-related gene ftnA and the OMP gene bhlp29.7 were regularly present in the isolates, while the OMP genes bhlp17.6 and bhmp39h could not be detected. Sequence analysis of hemolysin genes of selected isolates revealed co-evolution of tlyB, BHWA1_RS02885, BHWA1_RS09085, and BHWA1_RS02195 with the core genome and suggested independent evolution of tlyA, tlyC, and hlyA. Our data indicate that in Germany, swine dysentery might be caused by a limited number of B. hyodysenteriae clonal groups. Major STs (ST8, ST52, and ST112) are shared with other countries in Europe suggesting a possible role of the European intra-Community trade of pigs in the dissemination of certain clones. The identification of several novel STs, some of which are single or double locus variants of ST52, may on the other hand hint towards an ongoing diversification of the pathogen in the studied area. The linkage of pleuromutilin susceptibility and sequence type of an isolate might reflect a clonal expansion of the underlying resistance mechanism, namely mutations in the ribosomal RNA genes. A linkage between single virulence-associated genes (VAGs) or even VAG patterns and the phylogenetic background of the isolates could not be established, since almost all VAGs were regularly present in the isolates

Genome-Wide Associations for Microscopic Differential Somatic Cell Count and Specific Mastitis Pathogens in Holstein Cows in Compost-Bedded Pack and Cubicle Farming Systems

The aim of the present study was to detect significant SNP (single-nucleotide polymorphism) effects and to annotate potential candidate genes for novel udder health traits in two different farming systems. We focused on specific mastitis pathogens and differential somatic cell fractions from 2198 udder quarters of 537 genotyped Holstein Friesian cows. The farming systems comprised compost-bedded pack and conventional cubicle barns. We developed a computer algorithm for genome-wide association studies allowing the estimation of main SNP effects plus consideration of SNPs by farming system interactions. With regard to the main effect, 35 significant SNPs were detected on 14 different chromosomes for the cell fractions and the pathogens. Six SNPs were significant for the interaction effect with the farming system for most of the udder health traits. We inferred two possible candidate genes based on significant SNP interactions. HEMK1 plays a role in the development of the immune system, depending on environmental stressors. CHL1 is regulated in relation to stress level and influences immune system mechanisms. The significant interactions indicate that gene activity can fluctuate depending on environmental stressors. Phenotypically, the prevalence of mastitis indicators differed between systems, with a notably lower prevalence of minor bacterial indicators in compost systems

Evaluation of different diagnostic methods for the detection of Mycobacterium avium subsp. paratuberculosis in boot swabs and liquid manure samples

Abstract Background Environmental sampling based on boot swabs and/or liquid manure samples is an upcoming strategy for the identification of paratuberculosis (paraTB) positive herds, but only limited data are available regarding the diagnostic performance of molecular detection methods (qPCR) versus faecal culture (FC) for this purpose. In the present study, the test characteristics of two different qPCR protocols (A and B) and a standardized FC protocol, for the detection of Mycobacterium avium subsp. paratuberculosis in boot swabs and liquid manure samples were evaluated. Results In 19 paraTB unsuspicious and 58 paraTB positive herds boot swabs and liquid manure were sampled simultaneously and analyzed in three different diagnostic laboratories. Using boot swabs and liquid manure, a substantial to excellent accordance was found between both qPCRs, for boot swabs also with culture, while for liquid manure the detection rate of culture was decreased after prolonged storage at −20 °C. The quantitative results of both qPCR methods correlated well for the same sample and also for boot swabs and liquid manure from the same herd. When cut-off threshold cycle (CT-)-values were applied as recommended by the manufacturers, herd level specificity (Sp) of qPCR B was below 100% for boot swabs and for both qPCRs for liquid manure. A decreased herd level sensitivity was encountered after adjustment of Sp to 100% and re-calculation of the cut-off CT-values. Conclusions qPCR is equally suitable as bacterial culture for the detection of Mycobacterium avium subsp. paratuberculosis in boot swabs and liquid manure samples. Both matrices represent easily accessible composite environmental samples which can be tested with reliable results. The data encourage qPCR testing of composite environmental samples for paraTB herd diagnosis

Patterns of virulence-associated genes (VAGs) in 116 <i>B</i>. <i>hyodysenteriae</i> field isolates from pigs in Germany.

<p>Patterns of virulence-associated genes (VAGs) in 116 <i>B</i>. <i>hyodysenteriae</i> field isolates from pigs in Germany.</p

MIC₅₀^* values of pleuromutilins for <i>B</i>. <i>hyodysenteriae</i> isolates of major multilocus sequence types according to the year of isolation.

<p>MIC₅₀^{<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0190928#t004fn001" target="_blank">*</a>} values of pleuromutilins for <i>B</i>. <i>hyodysenteriae</i> isolates of major multilocus sequence types according to the year of isolation.</p

Single nucleotide polymorphism in the <i>B</i>. <i>hyodysenteriae</i> ribosomal protein L3 gene/protein in relation to sequence type and pleuromutilin susceptibility.

<p>Single nucleotide polymorphism in the <i>B</i>. <i>hyodysenteriae</i> ribosomal protein L3 gene/protein in relation to sequence type and pleuromutilin susceptibility.</p

Fig 1A and 1B. MSTs of 746 <i>B</i>. <i>hyodysenteriae</i> isolates (n = 741 from pigs; n = 5 from birds and mice) from the MLST database and from this study.

<p>MST A mirrors the spatial distribution of isolates. Each circle represents a different sequence type (n = 186), the color indicates the country of origin and the size reflects the number of isolates deposited. MST B depicts the temporal distribution of isolates. Each circle represents a different year of isolation and the size reflects the number of isolates from the year in question. Clonal complexes (CC) of STs sharing at least six common loci are indicated by grey shading. The predicted founder of a CC is marked with an asterisk (*); # = CCs with unclear founders.</p

Minimum inhibitory concentration of pleuromutilins used in this study and classification of isolates as being resistant, intermediate or susceptible.

<p>Minimum inhibitory concentration of pleuromutilins used in this study and classification of isolates as being resistant, intermediate or susceptible.</p