Genome-Wide Association Study Reveals Genetic Markers for Antimicrobial Resistance in Mycoplasma bovis.

Mycoplasma bovis causes many health and welfare problems in cattle. Due to the absence of clear insights regarding transmission dynamics and the lack of a registered vaccine in Europe, control of an outbreak depends mainly on antimicrobial therapy. Unfortunately, antimicrobial susceptibility testing (AST) is usually not performed, because it is time-consuming and no standard protocol or clinical breakpoints are available. Fast identification of genetic markers associated with acquired resistance may at least partly resolve former issues. Therefore, the aims of this study were to implement a first genome-wide association study (GWAS) approach to identify genetic markers linked to antimicrobial resistance (AMR) in M. bovis using rapid long-read sequencing and to evaluate different epidemiological cutoff (ECOFF) thresholds. High-quality genomes of 100 M. bovis isolates were generated by Nanopore sequencing, and isolates were categorized as wild-type or non-wild-type isolates based on MIC testing results. Subsequently, a k-mer-based GWAS analysis was performed to link genotypes with phenotypes based on different ECOFF thresholds. This resulted in potential genetic markers for macrolides (gamithromycin and tylosin) (23S rRNA gene and 50S ribosomal unit) and enrofloxacin (GyrA and ParC). Also, for tilmicosin and the tetracyclines, previously described mutations in both 23S rRNA alleles and in one or both 16S rRNA alleles were observed. In addition, two new 16S rRNA mutations were possibly associated with gentamicin resistance. In conclusion, this study shows the potential of quick high-quality Nanopore sequencing and GWAS analysis in the evaluation of phenotypic ECOFF thresholds and the rapid identification of M. bovis strains with acquired resistance. IMPORTANCE Mycoplasma bovis is a leading cause of pneumonia but also causes other clinical signs in cattle. Since no effective vaccine is available, current M. bovis outbreak treatment relies primarily on the use of antimicrobials. However, M. bovis is naturally resistant to different antimicrobials, and acquired resistance against macrolides and fluoroquinolones is frequently described. Therefore, AST is important to provide appropriate and rapid antimicrobial treatment in the framework of AMR and to prevent the disease from spreading and/or becoming chronic. Unfortunately, phenotypic AST is time-consuming and, due to the lack of clinical breakpoints, the interpretation of AST in M. bovis is limited to the use of ECOFF values. Therefore, the objective of this study was to identify known and potentially new genetic markers linked to AMR phenotypes of M. bovis isolates, exploiting the power of a GWAS approach. For this, we used high-quality and complete Nanopore-sequenced M. bovis genomes of 100 isolates.

Effects of omega-3 fatty acids on immune, health and growth variables in veal calves.

Under the present intensive rearing conditions, calves face a series of stressors and multiple pathogens often necessitating antimicrobial use. Multiple feed additives are currently explored for their ability to prevent disease and limit the use of antimicrobials. Supplementation of the polyunsaturated long chain n-3 fatty acids eicosapentaenoic (EPA) and docohexaenoic (DHA) from marine origin has been proposed as a strategy to improve immune function and prevent excessive inflammation reactions. The aim of this randomized clinical trial was to explore the effects of n-3 fatty acids (PUFAs) used as feed supplement on health, production and immune variables in a veal calf setting. One hundred-seventy calves were randomly assigned to 3 treatment groups: microalgae (MA, n = 57, 2.5 g DHA/animal/day), fish oil (FO, n = 57, 2.5 g EPA + DHA/animal/day)] and a control group (CON, n = 56). Average daily gain (ADG), bodyweight at 12 weeks on feed and slaughter weight were determined. Health monitoring consisted of recording of clinical signs and repeated thoracic ultrasonography. After 5, 8 and 11 weeks of supplementation, the function of neutrophils, monocytes and peripheral blood mononuclear cells (PBMCs) was evaluated ex vivo by measuring reactive oxygen species (ROS) production by neutrophils and monocytes and proliferation of and cytokine release by PBMCs. Under the field conditions of this study, dietary supplementation of MA and FO showed very limited immunomodulatory effects. Feeding MA led to increased ROS production by neutrophils, Estimate (E) = 0.38, Standard Error (SE) = 0.14; P < 0.05, compared to the control calves after 5 weeks of in-feed supplementation. FO reduced IL-6 secretion E= -0.29, SE= 0.11; P < 0.05 compared to MA treated animals after 11 weeks on feed. Health and production variables were unaffected by treatments. The doses of EPA and DHA used in this study did not cause immunomodulatory changes in highly stressed calves to such an extent that this led to better health or growth of animals.

The presence of Mycoplasma bovis in colostrum.

In herds with Mycoplasma bovis circulation, colostrum is often considered infectious. However, in contrast to milk, the presence of M. bovis in colostrum was not previously evidenced. In this survey, the presence of M. bovis DNA was determined with real-time PCR in 368 colostrum samples from 17 herds, recently infected with M. bovis. Only 1.9% of the samples tested positive, with 13 herds having no positive samples and an overall within-herd prevalence of 3.2% (SD: 4.9%; Range: 0-30.0%). These results show that in infected herds M. bovis DNA can be retrieved in colostrum. To what extend colostrum is infectious remains to be determined.

Effects of glycerol-esters of saturated short- and medium chain fatty acids on immune, health and growth variables in veal calves.

In veal and dairy beef production systems, Holstein bull calves experience many stressors and excessive pathogen exposure, necessitating the use of antimicrobials for welfare and production reasons. The aim of this randomized clinical trial was to explore the effects of esterified fatty acids used as feed supplement on health, production and immune variables in veal calves. Different glycerol-esters of fatty acids were used: short chain fatty acid (SCFA)-based glycerol-mono- (C4) and tributyrate (C4), and medium chain fatty acid (MCFA)-based glycerol-monocaprylate/monocaprinate (C8/C10) and glycerol-monolaurate (C12) in two different doses. One hundred sixty eight calves (2-to 4-week-old) were randomly assigned to 6 treatment groups; tributyrate (0.5 g/animal/day); monobutyrate (1 g/animal/day); low C8/C10 (7 g/animal/day) and high C8/C10 (10 g/animal/day); low C12 (4 g/animal/day) and high C12 (6 g/animal/day) and a control group (CON). Duration of in-feed supplementation was 14 weeks. Average daily gain, bodyweight at 14 weeks on feed and slaughter weight were determined. Health monitoring consisted of clinical signs and repeated thoracic ultrasonography. After 4, 8 and 12 weeks of supplementation, the function of neutrophils, monocytes and peripheral blood mononuclear cells (PBMCs) was evaluated ex vivo by measuring reactive oxygen species (ROS) production by neutrophils and monocytes, proliferation of and cytokine release by PBMCs. Study power was based upon ROS production by neutrophils and treatment groups were too limited to detect significant differences in growth and health variables. Glycerol-ester supplementation resulted in different effects on immune cell function, depending on the type and dose of the glycerol-ester as well as duration of supplementation. Our main findings were increased secretion of interleukin IL-17A by PBMCs at 4 weeks of feed supplementation in high C8/C10 (P< 0.01), low C12 (P < 0.01) and monobutyrate (P< 0.01) groups, combined with decreased ROS production in neutrophils (P < 0.001) and monocytes (P < 0.05) in the high C8/C10 and monocytes (P < 0.05) in low C12 groups compared to the control animals. After 12 weeks on feed, ROS production by neutrophils (P < 0.001) and monocytes (P < 0.01) of monobutyrate and by monocytes (P < 0.01) of tributyrate groups was decreased compared to control calves. In summary, supplementation of glycerol-esters of MCFAs resulted in immune-modulatory effects, which did not manifest themselves in improved health and growth of calves under the conditions and limitations of this study. Especially doses of high C8/C10 and low C12 show potential to promote an early, robust pro-inflammatory response with diminished ROS production. This might be beneficial for clearance of pathogens in young calves in periods of stress and high pathogen load.