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Additional file 2. Detailed results from the statistical models used to analyse the serology (ELISA) data.

Additional file 1. Detailed results from the statistical models used to analyse the infection status (PCR) data.

Additional file 3. Optical density values for M. hyopneumoniae-specific antibodies in serum of sows and 2-day-old piglets. Farm A: endemically infected with M. hyopneumoniae; Farm B: free of M. hyopneumoniae. Optical density (OD) investigated with a commercial blocking ELISA (IDEIA™ Mycoplasma hyopneumoniae EIA kit, Oxoid Limited, Hampshire, UK). On farm A, sows were vaccinated against M. hyopneumoniae at 6 and 3 weeks before farrowing and blood samples were taken before the first vaccination (pre vacc.) and at the time of farrowing (post vacc.), on farm B blood was sampled of the sows at the time of farrowing. On farm A 47 piglets were sampled (24 cross-fostered and 23 non-cross fostered) and on farm B 24 piglets were sampled. Samples were considered positive if the OD of the sample was lower than 50% of the average OD of the buffer control.

Additional file 4: Table S2. Sequencing summary of the M. bovis MiSeq (Illumina) sequencing run with resulting genome coverages after quality-filtering and adapter-clipping.

Additional file 1.

Additional file 1: Figure S1. In-depth analyses of de novo consensus genomes between the Canu and Flye assemblers, with or without four rounds of Racon. While both Canu and Flye result in a similar number of mismatches (per 100 kbp), significantly more indels (per 10 kbp) are observed when implementing the Flye assembler in the bioinformatics workflow. Decreased Flye performance is suggested to lie within decreased insertion accuracies as compared to Canu. Implementation of four rounds of Racon allows increased Flye insertion Q-scores when custom-pg45 basecalling is used. However, Canu without four rounds of Racon still results in an overall better performance.

Additional file 3: Table S1. Sequencing summary of multiplexed M. bovis and E. coli sequencing runs (48 h) on MinION R9.4.1 flow cells and resulting coverages after qcat and NanoFilt filtering and trimming..

Additional file 2: Table S3. Sequencing summary of single M. bovis sequencing runs (24h) on Flongle R9.4.1 flow cells and resulting coverages after qcat and NanoFilt filtering and trimming.

Table S1. Broncho-alveolar lavage fluid total nucleated cell count and differential cell counts according to ultrasonographic lesion score (ULS) based on 352 group-housed calves. (DOCX 26 kb)

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.

Table S2. Descriptives of broncho-alveolar fluid cellular characteristics from 352 group-housed calves. (DOCX 37 kb)

Pasteurella multocida is notorious for its role as an opportunistic pathogen in infectious bronchopneumonia, the economically most important disease facing cattle industry and leading indication for antimicrobial therapy. To rationalize antimicrobial use, avoiding imprudent use of highly and critically important antimicrobials for human medicine, availability of a rapid antimicrobial susceptibility test is crucial. The objective of the present study was to design a MALDI Biotyper antibiotic susceptibility test rapid assay (MBT-ASTRA) procedure for tetracycline resistance detection in P. multocida. This procedure was validated on 100 clinical isolates with MIC-gradient strip test, and a comparison with disk diffusion was made. Sensitivity and specificity of the MBT-ASTRA procedure were 95.7% (95% confidence interval (CI) = 89.8-101.5) and 100% (95% CI = 100-100), respectively, classifying 98% of the isolates correctly after only three hours of incubation. Sensitivity and specificity of disk diffusion were 91.3% (95% CI = 83.2-99.4) and 92.6% (95% CI = 85.6-99.6) respectively, classifying 91% of the isolates correctly. In conclusion, this MBT-ASTRA procedure has all the potential to fulfil the need for a rapid and highly accurate tetracycline susceptibility testing in P. multocida to rationalize antimicrobial use in outbreaks of bronchopneumonia in cattle or other clinical presentations across species.

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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.

Salmonellose bij de mens wordt vaak veroorzaakt door Salmonella enterica subspecies enterica serovar Typhimurium (Salmonella Typhimurium). De ziekte veroorzaakt door deze bacterie is voornamelijk geassocieerd met het eten van varkensvlees. Varkens die geïnfecteerd zijn met Salmonella Typhimurium zijn meestal symptoomloze dragers. De mechanismen die Salmonella Typhimurium gebruikt om deze dragerdieren blijvend te infecteren zijn niet gekend. Het doel van deze thesis was om inzicht te verkrijgen in de mechanismen die Salmonella Typhimurium gebruikt om varkens te kolonizeren en er in te persisteren. In het eerste hoofdstuk van deze thesis werd een Salmonella Typhimurium stam geselecteerd die een persisterende infectie bij varkens kan veroorzaken. Hiervoor werd een veldstam, geïsoleerd uit een persistent geïnfecteerd varken, vergeleken met een standaard laboratorium stam die vaak wordt gebruikt voor onderzoek in muizen. De laboratorium stam was virulenter in muizen, maar de varkensstam was efficiënter in het veroorzaken van een persisterende infectie bij varkens. Deze varkensstam werd gekozen om te gebruiken in verdere experimenten. In het tweede hoofdstuk werden verschillende in vitro en in vivo modellen op punt gesteld om de pathogenese van Salmonella Typhimurium infecties bij varkens te onderzoeken. In het derde hoofdstuk werd de rol van verschillende virulentiegenen van Salmonella in de pathogenese van Salmonella Typhimurium infecties bij het varken onderzocht. In een eerste reeks experimenten werd de rol nagegaan van de genen die gelegen zijn op het Salmonella Pathogeniciteitseiland 1 (SPI-1). Deze genen waren essentieel voor Salmonella Typhimurium om varkensdarmcellen en macrofagen te kunnen binnen dringen. Alle SPI-1 mutanten waren sterk verzwakt in hun vermogen om diarree te veroorzaken. Zowel vroege als late celdood werd gezien in de macrofagen, maar enkel de vroege celdood bleek SPI-1 afhankelijk. Wanneer varkens peroraal geïnfecteerd werden met een combinatie van de Salmonella Typhimurium veldstam enerzijds en een SPI-1 deletiemutant anderzijds, bleek dat de mutant stam sterk verzwakt was in het invaderen en kolonizeren van de darmen, maar niet in het kolonizeren van de tonsillen. In een tweede reeks experimenten werd de rol nagegaan van de genen die gelegen zijn op het Salmonella Pathogeniciteitseiland 2 (SPI-2). Hierbij werd gebruik gemaakt van een mutant in het ssrA gen. Deze mutant was minder goed in staat om in vitro in macrofagen te vermeerderen en om de organen te kolonizeren van varkens die intraveneus geïnfecteerd werden. Bij biggen die oraal geïnoculeerd werden met de ssrA mutant stam verliep de infectie evenwel gelijkaardig in vergelijking met biggen die geïnoculeerd waren met de wild type stam. In een derde reeks experimenten, werd het belang van het fibronectine bindend eiwit ShdA in het ontstaan van een persisterende infectie onderzocht. Alhoewel dit gen bij muizen recent geïdentificeerd is als een belangrijke factor in de intestinale kolonizatie en persistentie in muizen, kon dit in deze experimenten niet worden bevestigd voor Salmonella Typhimurium infecties bij varkens. De resultaten van deze thesis tonen aan dat zowel de gastheermodellen als de Salmonella stammen met zorg gekozen moeten worden om relevant onderzoek te kunnen doen naar de pathogenese van Salmonella infecties. Tevens kan geconcludeerd worden dat SPI-1 afhankelijke invasie cruciaal is voor de kolonizatie van de darmen, maar niet van de tonsillen. De bijdrage van SPI-2 en shdA tot de persistentie van Salmonella Typhimurium in varkens is veel kleiner dan beschreven voor muizen.

New vaccine formulations that include novel strains of Mycoplasma hyopneumoniae and innovative adjuvants designed to induce cellular immunity could improve vaccine efficacy against this pathogen. The aim of this experimental study was to assess the efficacy of three experimental bacterin formulations based on M. hyopneumoniae field strain F7.2C which were able to induce cellular immunity. The formulations included a cationic liposome formulation with the Mincle receptor ligand trehalose 6,6-dibehenate (Lipo_DDA:TDB), a squalene-in-water emulsion with Toll-like receptor (TLR) ligands targeting TLR1/2, TLR7/8 and TLR9 (SWE_TLR), and a poly(lactic-co-glycolic acid) micro-particle formulation with the same TLR ligands (PLGA_TLR). Four groups of 12 M. hyopneumoniae-free piglets were primo- (day (D) 0; 39 days of age) and booster vaccinated (D14) intramuscularly with either one of the three experimental bacterin formulations or PBS. The pigs were endotracheally inoculated with a highly and low virulent M. hyopneumoniae strain on D28 and D29, respectively, and euthanized on D56. The main efficacy parameters were: respiratory disease score (RDS; daily), macroscopic lung lesion score (D56) and log copies M. hyopneumoniae DNA determined with qPCR on bronchoalveolar lavage (BAL) fluid (D42, D56). All formulations were able to reduce clinical symptoms, lung lesions and the M. hyopneumoniae DNA load in the lung, with formulation SWE_TLR being the most effective (RDSD28-D56 -61.90%, macroscopic lung lesions -88.38%, M. hyopneumoniae DNA load in BAL fluid (D42) -67.28%). Further experiments raised under field conditions are needed to confirm these results and to assess the effect of the vaccines on performance parameters.

Additional file 1. Results of the M. hyopneumoniae-specific antibodies measured at different time points in serum and in BAL fluid. Pigs were prime-boost vaccinated on D0 and D14 with three different experimental M. hyopneumoniae bacterins (Lipo_DDA:TDB, PLGA_TLR, SWE_TLR), challenge infected on D28–29 and euthanized on D56. M. hyopneumoniae-specific antibodies were determined by the IDEIA™ Mycoplasma hyopneumoniae EIA kit (Oxoid Limited, Hampshire, UK) and by indirect in-house ELISAs. For the in-house ELISAs, NetOD-values were calculated by subtracting the OD-value of the blank from the OD-value of the sample. BAL, bronchoalveolar lavage; NCG, non-challenge control group (PBS-injected, non-challenge infected); PCG, PBS-injected control group (PBS-injected, challenge infected); OD, optical density.

Additional file 1. Results of the M. hyopneumoniae-specific antibodies measured at different time points in serum and in BAL fluid. Pigs were prime-boost vaccinated on D0 and D14 with three different experimental M. hyopneumoniae bacterins (Lipo_DDA:TDB, PLGA_TLR, SWE_TLR), challenge infected on D28–29 and euthanized on D56. M. hyopneumoniae-specific antibodies were determined by the IDEIA™ Mycoplasma hyopneumoniae EIA kit (Oxoid Limited, Hampshire, UK) and by indirect in-house ELISAs. For the in-house ELISAs, NetOD-values were calculated by subtracting the OD-value of the blank from the OD-value of the sample. BAL, bronchoalveolar lavage; NCG, non-challenge control group (PBS-injected, non-challenge infected); PCG, PBS-injected control group (PBS-injected, challenge infected); OD, optical density.

We characterized five different vaccine candidates and a commercial vaccine in terms of safety, immunogenicity and using a systems vaccinology approach, with the aim to select novel vaccine candidates against Mycoplasma hyopneumoniae. Seven groups of six M. hyopneumoniae-free piglets were primo- and booster vaccinated with the different experimental bacterin formulations, the commercial vaccine Hyogen® as a positive control or PBS as a negative control. The experimental bacterin was formulated with cationic liposomes + c-di-AMP (Lipo_AMP), cationic liposomes + Toll-like receptor (TLR) 2/1, TLR7, and TLR9 ligands (TLR ligands; Lipo_TLR), micro-particles + TLR ligands (PLGA_TLR), squalene-in-water emulsion + TLR ligands (SWE_TLR), or DDA:TDB liposomes (Lipo_DDA:TDB). Lipo_DDA:TDB and Lipo_AMP were the most potent in terms of serum antibody induction, and Lipo_DDA:TDB, Lipo_AMP, and SWE_TLR significantly induced Th1 cytokine-secreting T-cells. Only PLGA_TLR appeared to induce Th17 cells, but was unable to induce serum antibodies. The transcriptomic analyses demonstrated that the induction of inflammatory and myeloid cell blood transcriptional modules (BTM) in the first 24 h after vaccination correlated well with serum antibodies, while negative correlations with the same modules were found 7 days post-vaccination. Furthermore, many cell cycle and T-cell BTM upregulated at day seven correlated positively with adaptive immune responses. When comparing the delivery of the identical TLR ligands with the three formulations, we found SWE_TLR to be more potent in the induction of an early innate immune response, while the liposomal formulation more strongly promoted late cell cycle and T-cell BTM. For the PLGA formulation we found signs of a delayed and weak perturbation of these BTM. Lipo_AMP was found to be the most potent vaccine at inducing a BTM profile similar to that correlating with adaptive immune response in this and other studies. Taken together, we identified four promising vaccine candidates able to induce M. hyopneumoniae-specific antibody and T-cell responses. In addition, we have adapted a systems vaccinology approach developed for human to pigs and demonstrated its capacity in identifying early immune signatures in the blood relating to adaptive immune responses. This approach represents an important step in a more rational design of efficacious vaccines for pigs.

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.