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Different mycobacterial species are encountered in bovine medicine. The fastidiously growing mycobacteria (Mycobacterium bovis as the cause of bovine tuberculosis, and Mycobacterium avium ssp. paratuberculosis, MAP, as the cause of paratuberculosis) are well known and targeted in eradication/control or monitoring programs in different countries, whereas the rapidly growing species is only rarely identified from bovine disease. The latter have occasionally been reported as the cause of bovine clinical mastitis, but recent reports are scarce. In this study, Mycolicibacterium smegmatis (basonym Mycobacterium smegmatis) was identified as cause of granulomatous, relapsing clinical mastitis in 2 cows from one Belgian dairy herd. Milk, blood, and fecal samples were collected, as well as tissue samples after the cows were culled. Serological analysis conducted on milk and serum samples resulted in positive reactions for MAP, but negative for Mycobacterium bovis. Production of IFN-gamma showed sensitization with mycobacteria or similar organisms, other than M. bovis, in one cow. Detection of MAP by bacteriological culture and IS900-based quantitative PCR on milk and feces remained negative. In conclusion, this paper describes M. smegmatis as a cause of bovine clinical mastitis in Belgium and suggests cross-reactivity of the intramammary M. smegmatis infection with routinely used serological tests for MAP.

The antibacterial potential of organic acids and essential oil components against Brachyspira hyodysenteriae, the causative pathogen of swine dysentery, was evaluated Minimum inhibitory concentrations (MIC) of 15 compounds were determined at pH 7.2 and pH 6.0, using a broth microdilution assay. In addition, possible synergism was determined. MIC values for the three tested strains were similar. For organic acids, MIC values at pH 6.0 were lower than at pH 7.2. B. hyodysenteriae was most sensitive to cinnamaldehyde and lauric acid, with MIC values

The aim of this study was to determine the link between the characteristics of antimicrobial therapy and occurrence of antimicrobial resistance in Escherichia coli of clinically healthy pigs exposed to antimicrobial treatments. A total of 918 Escherichia coli isolates were obtained from faecal samples, collected from 50 pig herds at the end of the fattening period and susceptibility was tested towards 15 different antimicrobial agents, using the disk diffusion method. The Antimicrobial Resistance Index (ARI) of each isolate was calculated, as the number of antimicrobials to which resistance was found divided by the number of drugs tested. The antimicrobial resistance percentage per class (ARclass) was defined as the percentage of E. coli strains clinically resistant to that specific class. Data on group level antimicrobial use in the sampled herds was collected and quantified as treatment incidences (TI) based on the used daily dose pig (UDDpig) and the animal daily dose pig (ADDpig) (number of pigs treated with one ADDpig or UDDpig/1000 pigs at risk/day). The UDDpig/ADDpig ratio gives an indication of the correctness of dosing. The TIADDpig for group level use was 235.7 per 1000 pigs at risk per day, whereas the TIUDDpig equaled 200.7. This means that in reality, fewer pigs were treated with the same amount of antimicrobials than theoretically expected and thus antimicrobials were generally overdosed. Generalized linear regression analysis showed a significant relation between the TIADDpig and the ARI (p< 0.01), whereas there were no significant links for the TIUDDpig (p> 0.05). Analysis of the antimicrobial resistance for β-lactam antimicrobials and tetracyclines suggests that the effect of correct or incorrect dosing on resistance development was different for the different antimicrobial classes tested. Besides the amount of administered antimicrobial agents, the frequency of drug administration may play a role in the selection of antimicrobial resistance in commensal E. coli.

Foodborne salmonellosis is one of the most important bacterial zoonotic diseases worldwide. Salmonella Typhimurium is the serovar most frequently isolated from slaughter pigs in Europe. Circumvention of the host’s immune system by Salmonella might contribute to persistent infection of pigs. We found that Salmonella Typhimurium strain 112910a, which is able to persist in pigs, was capable of downregulating the expression of major histocompatibility class II (MHC II) molecules on porcine alveolar macrophages (PAM) in a Salmonella pathogenicity island 2 (SPI-2) dependent way and that MHC II downregulation was Salmonella strain dependent. The MHC II downregulation capacity was abolished when bacteria were opsonized with Salmonella-specific antibodies. Furthermore, intracellular proliferation of Salmonella Typhimurium opsonized with Salmonella positive pig serum was significantly impaired compared to that of the bacteria opsonized with negative pig serum. In a subsequent in vivo experiment, Salmonella Typhimurium strain MB2216 that did not induce MHC II downregulation in vitro, was shed less and persisted less but induced earlier seroconversion in pigs than strain 112910a. From the in vitro data, it is proposed that Salmonella Typhimurium downregulates the humoral immune response to promote intracellular survival inside porcine macrophages, contributing to long-term Salmonella persistence in pigs. The fact that the less persistent strain MB2216 induced earlier seroconversion than strain 112910a is of major interest for Salmonella-monitoring programs primarily based on serology, since this indicates that more persisting strains are more likely to escape serological detection.

Salmonellosis is one of the most important bacterial zoonotic diseases and represents a considerable burden to humans in both developing and developed countries1. The consumption of contaminated pork is a major source of Salmonella Typhimurium infections in humans. Porcine carcass contamination is correlated with long-term Salmonella persistence in pigs, resulting in so called asymptomatic ‘carrier pigs’. Since interference of several pathogens with the host’s immune system has already been shown to be of major importance for successful long-term infection2, Salmonella Typhimurium persistence in pigs could be the result of the bacterium being able to circumvent the host’s cellular and/or humoral immune responses. It was the aim of the present study to examine if, how and why Salmonella Typhimurium escapes the porcine humoral immune system.

Persistent Salmonella Typhimurium infections in pigs often result in asymptomatic carrier pigs and are a major concern for food safety and human health. Tonsils and lymph nodes play a key role in the persistence of Salmonella Typhimurium in pigs, but very little is known about the underlying mechanisms. After bacterial invasion in pigs, the porcine immune system will respond to clear the Salmonella infection and bacterial survival strategies for (long-)term persistence will become important. For the identification of Salmonella Typhimurium genes specifically induced in tonsils and lymph nodes at 3 weeks post inoculation, a genome-wide screening method was performed using in vivo expression technology (IVET). Using IVET we were able to identify different Salmonella Typhimurium genes that play a role in long-term persistence in pigs. Furthermore, we were able to show a role for Salmonella Typhimurium genes htpG (encoding a heat-shock protein) and STM4067 (encoding a protein with an unknown function) in long-term persistence in the porcine intestines and lymph nodes, although their exact role remains to be clarified.

Mycoplasma hyopneumoniae is het primaire agens van enzoötische pneumonie en speelt een belangrijke rol in het porcien respiratoir ziektecomplex. In de meeste landen is de kiem endemisch aanwezig en varkens van alle leeftijden kunnen besmet worden. De meeste economische schade door een infectie met M. hyopneumoniae vindt plaats bij de vleesvarkens, maar fokdieren spelen een belangrijke rol in de epidemiologie van de kiem. Ze fungeren namelijk als een permanente bron van M. hyopneumoniae circulatie op het bedrijf en kunnen de nakomelingen reeds op jonge leeftijd besmetten. Om de ziekte en de economische verliezen te controleren, wordt vaccinatie tegen M. hyopneumoniae vaak toegepast op varkensbedrijven. Hierbij worden voornamelijk de fokgelten en de biggen gevaccineerd. Na vaccinatie moet er in het lichaam van het varken een M. hyopneumoniae-specifieke humorale en celgemedieerde immuniteit aanwezig zijn waardoor het dier snel kan reageren wanneer het in contact komt met de kiem. Bovendien wordt de M. hyopneumoniae-specifieke immuniteit overgedragen van zeug naar big via colostrum. Voor de bescherming tegen een M. hyopneumoniae infectie zou de celgemedieerde immuniteit een belangrijkere rol spelen dan de antistoffen in het serum. Echter, de duur van de humorale en celgemedieerde immuniteit opgewerkt door M. hyopneumoniae vaccinatie is onvoldoende onderzocht. Mycoplasma hyopneumoniae-specifieke celgemedieerde immuniteit wordt overgedragen van zeug naar big, maar tot dusver werden de verschillende immuuncellen niet verder gekarakteriseerd.

Mycoplasma bovis is a leading, primary cause of pneumonia, arthritis, otitis and mastitis in cattle, resulting in impaired animal welfare and huge economic losses in all cattle sectors worldwide. This small bacterium lost its cell wall and several physiological mechanisms through evolution, whereupon it acquired inherent resistance against many conventional antimicrobials (e.g. penicillines, cephalosporines, sulfonamides, ..). Next to this natural resistance, M. bovis may acquire resistance against other antimicrobials as well. Currently, isolation and identification of M. bovis by culture takes 1-2 weeks, and subsequent antimicrobial susceptibility testing is currently not performed in routine diagnostics. No standard protocol is available and the lack of clinical breakpoints limits the translation of in vitro results to clinical outcome predictions of antimicrobial treatment. At a higher price, faster identification is possible with PCR (2 days). Although diagnostic accuracy of PCR is expected to be higher than culture, scientific information on this matter is limited. To be able to control M. bovis and start appropriate antimicrobial treatment immediately, there is a great need for rapid and reliable diagnostic tools for this pathogen. However, next to control, prevention of M. bovis spreading into/within the herd is also very important. How M. bovis is exactly transmitted, and whether there are specific M. bovis strains associated with antimicrobial resistance or sectors, has not been elucidated yet. Key factors for successful control and prevention are the formulation of specific biosecurity protocols and guidelines targeted to M. bovis. To achieve this, a rapid diagnosis of infected or carrier animals and better insights into the spread of M. bovis between herds, sectors, and countries are needed. Therefore, the general aim of this thesis was to develop new Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) and nanopore sequencing based diagnostic methods for rapid identification, strain typing, and antimicrobial susceptibility testing of M. bovis, and to apply those methods on Belgian field samples, gaining better insight into the epidemiology of M. bovis. In the general introduction (Chapter 1) a literature overview is provided, presenting current state-of-the-art on disease course, risk factors and treatment of M. bovis. Subsequently, the many different diagnostic techniques available for identification, strain typing, and susceptibility testing are described. Next to the existing techniques, more innovative techniques, such as MALDI-TOF MS and nanopore sequencing and their potential as rapid diagnostic methods are explained. In the first experimental study different methods were explored to identify M. bovis cultures grown on solid medium with MALDI-TOF MS (Chapter 3.1). The most straight-forward method, being the direct transfer method, is broadly applied for most bacteria, but faced several problems for M. bovis identification. In this study, these problems were better identified and it was shown that medium-related peaks (mostly obtained from horse serum and colistin) can result in false positive Mycoplasma alkalescens and Mycoplasma arginini identification. Unfortunately, it was not possible to obtain a more reliable direct transfer protocol. Therefore, in Chapter 3.2 the identification of M. bovis with MALDI-TOF MS from liquid medium was further explored and optimized. Here it was shown that identification was possible within 24 hours after inoculation of one colony from a solid medium into liquid medium. Supplementing pleuropneumonia-like organism broth (PPLO-broth) with pyruvate prolonged the possibility of M. bovis identification to at least 120 hours after inoculation. Also, supplementation with antimicrobials prevented overgrowth with other bacteria, and did not influence the identification score. Although with the previous two methods, a step towards more rapid identification of M. bovis was set, prior isolation of M. bovis from any sample is still necessary and could easily take 5-10 days. Therefore, methods to identify M. bovis directly from bronchoalveolar lavage fluid (BALf) with MALDI-TOF MS (Chapter 3.3) and nanopore sequencing (Chapter 3.4) were developed and validated in a Bayesian latent class model on 104 and 100 BALf from calves, respectively. It was possible to identify M. bovis with MALDI-TOF MS within 2-3 days with a sensitivity and specificity of 86.6% (CI95%: 69.4-97.6%) and 86.4% (76.1-93.8%), respectively. While sensitivity and specificity of nanopore sequencing were 77.4% (58.6-92.3%) and 97.3% (91.1-99.7%), respectively. Also when 5 BALf were pooled, both methods were still reliable, and therefore very cost-effective possibilities. In addition, the in Belgium widely used selective-indicative agar method based on lipase-activity, which was never validated before using a large number of field samples, showed a sensitivity of 70.5% (52.1-87.1%) and specificity of 93.9% (85.9-98.4). All three methods are useful in routine laboratories, depending on the diagnostic needs of the applicant. Currently the prevalence of M. bovis in the Belgian dairy and beef sector is estimated at 30%, whereas 100% of the veal calf herds tested positive. Together with the high antimicrobial use in the veal sector, the question has been raised whether there is a possible reservoir of multi-resistant and sector-specific M. bovis strains in this sector, as previously shown for other respiratory bacteria. To better understand the molecular epidemiology and genetic relatedness of different M. bovis isolates, the full genome of 100 Belgian M. bovis isolates collected from dairy, beef and veal herds was obtained using nanopore sequencing (Chapter 4). A single nucleotide polymorphism (SNP) analysis was performed and the phylogenetic tree showed five separate genomic clusters of M. bovis isolates and one outlier circulating in Belgium between 2014 and 2019. No sector-specific isolates and no association with spatial location in Belgium were identified. At world-scale, the Belgian M. bovis isolates clustered together with European, American and Israeli strains. These results contribute to emphasizing the importance of purchase protocols and biosecurity to prevent M. bovis from entering the country or herd. In Chapter 5.1, antimicrobial susceptibility testing of 141 M. bovis isolates retrieved from Belgian dairy, beef and veal calf herds was performed with broth microdilution. Minimum inhibitory concentration values were used to establish the epidemiological cut-off (ECOFF) with visual and statistical methods to distinguish the population in wild type M. bovis and those with acquired antimicrobial resistance (non-wild type). The results showed high percentages of acquired resistance for macrolides (tilmicosin, tylosin, and gamithromycin), but no acquired resistance for tetracyclines (oxytetracycline, doxycycline). Only little acquired resistance was observed for florfenicol, gentamicin, and tiamulin, while there was limited acquired resistance to enrofloxacin. Only M. bovis isolates from beef cattle or the third genomic cluster had a significantly higher change to have acquired resistance against gamithromycin than those collected from other sectors or genomic clusters. These results support the current national formulary for respiratory disease associated with M. bovis, recommending florfenicol as first choice, and oxytetracycline and macrolides as second choice. Possibly, a small remark for gamithromycin is needed, as higher risk for acquired resistance for this antimicrobial was seen in beef cattle. In vitro susceptibility testing results should be interpreted carefully, as the association with in vivo efficacy has not confirmed yet, due to the lack of clinical breakpoints. Finally, in Chapter 5.2, upgraded genomes derived from Chapter 4 and the susceptibility data from Chapter 5.1 were combined to compare genotype and phenotype antimicrobial susceptibility of M. bovis isolates. A genome wide association study to reveal genetic markers for antimicrobial resistance in M. bovis and verifying the ECOFF values obtained by the previously used different methods was executed. Many point mutations were associated with antimicrobial resistance against the critically important antibiotics of the macrolide (A2058G in the 23S rRNA gene, Gln83His in the L22 protein) and fluoroquinolone classes. For enrofloxacin the combination of different mutations in the GyrA and ParC gene showed the step-wise acquired resistance. Also previously described mutations for tilmicosin (G478A mutation in 23S rRNA alleles), and new markers for gentamicin (A1408G and G1488A in 16S rRNA) were identified. The visual estimation of de ECOFF showed to be a reliable method, although statistical methods can help when step-wise resistance results in difficult to interpret “tailing”. Even when phenotypical resistance is not yet obtained, in case of first-step mutations it should be discouraged to use fluoroquinolones as antimicrobial therapy, as selection pressure will eventually result in phenotypical resistance as well. In the general discussion (Chapter 6), the innovations in M. bovis diagnostics achieved with this thesis are discussed. In the second part, practical recommendations for diagnostics in M. bovis outbreak management, purchase policy, and eradication or herd status certificates are proposed. In this thesis new methods to identify, strain type, and access the antimicrobial susceptibility of M. bovis were developed. When rapid identification of M. bovis with MALDI-TOF MS (Chapter 3.3) is followed by the determination of antimicrobial resistance with nanopore sequencing (Chapter 5.2) it is now possible to obtain identification, strain typing and an antibiogram for critically important antibiotics within 3-5 days. This is a major step towards better control of M. bovis in clinical outbreaks and prevent herd infection when purchasing animals. Together with these new methods, also substantial epidemiological information came to light, showing the importance of a more national approach for the prevention of introducing M. bovis into the herd and country.