Multilocus sequence typing schemes for the emerging swine pathogen Mycoplasma hyosynoviae.

Mycoplasma (M.) hyosynoviae is a commensal of the upper respiratory tract in swine, which has the potential to spread systemically, usually resulting in arthritis in fattening pigs and gilts. To date, very little is known about the epidemiology of M. hyosynoviae, mainly due to a lack of suitable typing methods. Therefore, this study aimed to develop both a conventional multi locus sequence typing (MLST) and a core genome (cg) MLST scheme. The development of the cgMLST was based on whole genome sequences of 64 strains isolated from pigs and wild boars during routine diagnostics as well as nine publicly available genomes. A cgMLST scheme containing 390 target genes was established using the Ridom© SeqSphere+ software. Using this scheme as a foundation, seven housekeeping genes were selected for conventional MLST based on their capability to reflect genome wide relatedness and subsequently, all 73 strains were typed by applying both methods. Core genome MLST results revealed a high diversity of the studied strain population and less than 100 allele differences between epidemiologically unrelated strains were only detected for four isolates from the US. On the other hand, seven clonal clusters (≤ 12 allele differences) comprising 20 isolates were identified. Comparison of the two typing methods resulted in highly congruent phylogenetic trees and an Adjusted Rand Coefficient of 0.893, while cgMLST showed marginally higher resolution when comparing closely related isolates, indicated by a slightly higher Simpson's ID (0.992) than conventional MLST (Simpson's ID = 0.990). Overall, both methods seem well suited for epidemiological analyses for scientific as well as diagnostic purposes. While MLST is faster and cheaper, cgMLST can be used to further differentiate closely related isolates.

Detection and disease diagnosis trends (2017-2022) for Streptococcus suis, Glaesserella parasuis, Mycoplasma hyorhinis, Actinobacillus suis and Mycoplasma hyosynoviae at Iowa State University Veterinary Diagnostic Laboratory.

BACKGROUND: Accurate measurement of disease associated with endemic bacterial agents in pig populations is challenging due to their commensal ecology, the lack of disease-specific antemortem diagnostic tests, and the polymicrobial nature of swine diagnostic cases. The main objective of this retrospective study was to estimate temporal patterns of agent detection and disease diagnosis for five endemic bacteria that can cause systemic disease in porcine tissue specimens submitted to the Iowa State University Veterinary Diagnostic Laboratory (ISU VDL) from 2017 to 2022. The study also explored the diagnostic value of specific tissue specimens for disease diagnosis, estimated the frequency of polymicrobial diagnosis, and evaluated the association between phase of pig production and disease diagnosis. RESULTS: S. suis and G. parasuis bronchopneumonia increased on average 6 and 4.3%, while S. suis endocarditis increased by 23% per year, respectively. M. hyorhinis and A. suis associated serositis increased yearly by 4.2 and 12.8%, respectively. A significant upward trend in M. hyorhinis arthritis cases was also observed. In contrast, M. hyosynoviae arthritis cases decreased by 33% average/year. Investigation into the diagnostic value of tissues showed that lungs were the most frequently submitted sample, However, the use of lung for systemic disease diagnosis requires caution due to the commensal nature of these agents in the respiratory system, compared to systemic sites that diagnosticians typically target. This study also explored associations between phase of production and specific diseases caused by each agent, showcasing the role of S. suis arthritis in suckling pigs, meningitis in early nursery and endocarditis in growing pigs, and the role of G. parasuis, A. suis, M. hyorhinis and M. hyosynoviae disease mainly in post-weaning phases. Finally, this study highlighted the high frequency of co-detection and -disease diagnosis with other infectious etiologies, such as PRRSV and IAV, demonstrating that to minimize the health impact of these endemic bacterial agents it is imperative to establish effective viral control programs. CONCLUSIONS: Results from this retrospective study demonstrated significant increases in disease diagnosis for S. suis, G. parasuis, M. hyorhinis, and A. suis, and a significant decrease in detection and disease diagnosis of M. hyosynoviae. High frequencies of interactions between these endemic agents and with viral pathogens was also demonstrated. Consequently, improved control programs are needed to mitigate the adverse effect of these endemic bacterial agents on swine health and wellbeing. This includes improving diagnostic procedures, developing more effective vaccine products, fine-tuning antimicrobial approaches, and managing viral co-infections.

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.

Bacterial polyarthritis in post-weaning pigs in a high-density swine breeding area in Italy.

We assessed the bacterial agents found in 8-12-wk-old post-weaning pigs with arthritis. The bodies of 178 post-weaning pigs from 90 farms (average of 2 pigs/farm) with recurrent problems of lameness and swollen joints in a high-density breeding area were submitted for autopsy and sampled for further bacterial investigation. The most common articular gross lesions and histopathologic findings were serofibrinous (95 of 178; 53%) or serous (65 of 178; 37%) arthritis; suppurative lesions were less frequent (18 of 178; 10%). In 133 of 178 (74.7%) cases, a bacterial agent was detected in joints. Mycoplasma hyorhinis was the most common bacterium detected (82 of 133; 61.6%). Haemophilus parasuis and Streptococcus spp. were observed in 27 of 133 (20.3%) and 24 of 133 (18.0%) cases, respectively. Other bacteria in the 113 cases, considered less important, in order of their low frequency, were Mycoplasma spp. (13; 9.8%), Trueperella pyogenes (11; 8.2%), Mycoplasma hyosynoviae (4; 3.0%), Staphylococcus spp. (3; 2.2%), Escherichia coli (2; 1.5%), and Actinobacillus spp. (2; 1.5%). Our results highlight the primary role of M. hyorhinis compared to other microorganisms involved in young pigs with arthritis.

Antimicrobial susceptibility profiles of porcine mycoplasmas isolated from samples collected in southern Europe.

BACKGROUND: Mycoplasma (M.) hyopneumoniae, M. hyorhinis and M. hyosynoviae are significant pathogens for the porcine industry worldwide. The aim of the present study was to determine the antimicrobial susceptibility of six key antimicrobials (tylosin, tilmicosin, tylvalosin, lincomycin, tiamulin and valnemulin) routinely used for treating infections caused by these pathogens. Twenty-seven M. hyopneumoniae, 48 M. hyorhinis and 40 M. hyosynoviae field strains isolated from clinical samples from different Southern European countries between 2013 and 2018 using broth microdilution method were evaluated. RESULTS: Tylvalosin exhibited the highest in vitro activity among the macrolides assayed, with MIC90 values 4 to 5 two-fold dilutions lower than those of tylosin and tilmicosin. The pleuromutilin valnemulin showed one of the highest in vitro activities against the three mycoplasma species. On the contrary, lincomycin exhibited the highest MIC values of the antimicrobials tested. CONCLUSIONS: The data obtained in the present study supports the use of pleuromutilins and macrolides for the control of infections caused by porcine mycoplasmas. The use of lincomycin for the treatment of porcine mycoplasma infections should be carefully evaluated due to the presence of circulating field isolates with decreased susceptibility to this antimicrobial.

Detection of Mycoplasma hyorhinis and Mycoplasma hyosynoviae in oral fluids and correlation with pig lameness scores.

This study was designed to detect Mycoplasma hyorhinis and M. hyosynoviae in oral fluids and determine their correlation with lameness scores in pigs. Thirty-seven nursery and/or finisher herds were included in this study. Oral fluids were collected by pen. Using species specific real-time PCR M. hyorhinis was detected in 97% of sampled herds, whereas 70% were positive for M. hyosynoviae. Lameness scores were determined for all pigs in each pen where oral fluids were collected. Lameness was identified in 3.9% of pigs across all sampled pens. No correlation was observed between lameness in pigs in a pen and detection of M. hyorhinis in oral fluid samples (p > 0.05), whereas a significant correlation was observed between M. hyosynoviae detection in oral fluids and lameness (p < 0.05). A negative correlation was observed between the proportion of lame pigs in the pen and Ct values for M. hyosynoviae in oral fluids (p < 0.05; r = -0.27). An age-related effect was observed with M. hyosynoviae detection in oral fluids, indicating an increased prevalence of the bacterium in finishers compared to nursery pigs. Under the conditions of this study, M. hyorhinis was frequently detected in oral fluids from nursery and finisher pigs regardless of the clinical presentation of lameness, whereas the detection of M. hyosynoviae varied depending on the age of sample pigs. Our results suggest that oral fluids may not be an informative diagnostic sample for M. hyorhinis associated lameness. However, the association of lameness and M. hyosynoviae detection in oral fluids warrants prospective population-based diagnostic studies.

Early detection and differential serodiagnosis of Mycoplasma hyorhinis and Mycoplasma hyosynoviae infections under experimental conditions.

Mycoplasma hyorhinis (MHR) and Mycoplasma hyosynoviae (MHS) are common opportunistic pathogens in the upper respiratory tract and tonsils of swine. The identification of the specific species involved in clinical cases using conventional diagnostic methods is challenging. Therefore, a recombinant chimeric polypeptide based on the seven known variable lipoproteins (A-G) specific of MHR and a cocktail of surface proteins detergent-extracted from MHS cultures were generated and their suitability as antemortem biomarkers for serodiagnosis of MHR- and MHS-infection were evaluated by ELISA. M. hyorhinis and MHS ELISA performance, evaluated using serum samples collected over a 56-day observation period from pigs inoculated with MHR, MHS, M. hyopneumoniae, M. flocculare, or Friis medium, varied by assay, targeted antibody isotype, and cutoffs. The progressions of MHR and MHS clinical diseases were evaluated in relation to the kinetics of the isotype-specific antibody response in serum and bacterial shedding in oral fluids during the observation period. In pigs inoculated with MHR, bacterial DNA was detected in one or more of the 5 pens at all sampling points throughout the study, IgA was first detected at DPI 7, one week before the first clinical signs, with both IgA and IgG detected in all samples collected after DPI 14. The peak of MHS shedding (DPI 8) coincided with the onset of the clinical signs, with both IgA and IgG detected in all serum samples collected ≥ DPI 14. This study demonstrated, under experimental conditions, that both ELISAs were suitable for early detection of specific antibodies against MHR or MHS. The diagnostic performance of the MHR and MHS ELISAs varied depending on the selected cutoff and the antibody isotype evaluated. The high diagnostic and analytical specificity of the ELISAs was particularly remarkable. This study also provides insights into the infection dynamics of MHR-associated disease and MHS-associated arthritis not previously described.

Mycoplasma hyorhinis and Mycoplasma hyosynoviae dual detection patterns in dams and piglets.

Mycoplasma hyorhinis and M. hyosynoviae are agents associated with arthritis in pigs. This study investigated the tonsillar detection patterns of M. hyorhinis and M. hyosynoviae in a swine population with a history of lameness. The plausibility of dual PCR detection of these agents in dams at one and three weeks post-farrowing and their offspring at the same time was determined. The association between M. hyorhinis and M. hyosynoviae detection in piglets and potential development of lameness in wean-to-finish stages was evaluated by correlating individual piglet lameness scores and PCR detection in tonsils. Approximately 40% of dams were detected positive for M. hyorhinis and M. hyosynoviae at both one and three weeks post-farrowing. In first parity dams, M. hyorhinis was detected in higher proportions (57.1% and 73.7%) at both weeks of sampling compared to multi-parity dams. A lower proportion of first parity dams (37.5%) were detected positive at week one with M. hyosynoviae and an increase in this proportion to 50% was identified in week three. Only 8.3% of piglets were detected positive for M. hyorhinis in week one compared to week three (50%; p<0.05). The detection of M. hyosynoviae was minimal in piglets at both weeks of sampling (0% and 0.9%). Lameness was scored in pigs 5-22 weeks of age, with the highest score observed at week 5. The correlation between PCR detection and lameness scores revealed that the relative risk of developing lameness post-weaning was significantly associated with detection of M. hyorhinis in piglets at three weeks of age (r = 0.44; p<0.05).The detection pattern of M. hyorhinis and M. hyosynoviae in dams did not reflect the detection pattern in piglets. Results of this study suggest that positive detection of M. hyorhinis in piglets pre-weaning could act as a predictor for lameness development at later production stages.

Transfer of maternal immunity to piglets is involved in early protection against Mycoplasma hyosynoviae infection.

Mycoplasma hyosynoviae causes arthritis in pigs older than 12 weeks. The role of colostrum in protection of piglets against M. hyosynoviae infection is not clear. Our objective was therefore to investigate whether transfer of maternal immunity to piglets was involved in early protection against the infection. Experimental infections were carried out in three groups of weaners receiving different levels of M. hyosynoviae-specific colostrum components; Group NC derived from Mycoplasma free sows and possessed no specific immunity to M. hyosynoviae. Group CAb pigs, siblings of the NC group, received colostrum with M. hyosynoviae-specific antibodies immediately after birth. Group CCE pigs were born and raised by infected sows and presumably had the full set of colostrally transferred factors, including specific antibodies. When 4½ weeks old, all pigs were inoculated intranasally with M. hyosynoviae. The course of infection was measured through clinical observations of lameness, cultivation of M. hyosynoviae from tonsils, blood and synovial fluid and observation for gross pathological lesions in selected joints. Specific immune status in the pigs was evaluated through detection of antibodies by immunoblotting and measurement of M. hyosynoviae-specific T-cell proliferation. The latter analysis may possibly indicate that M. hyosynoviae infection induces a T-cell response. The CCE piglets were significantly protected against development of lameness and pathology, as well as infection with M. hyosynoviae in tonsils, blood and joints, when compared to the two other groups. Raising the CCE pigs in an infected environment until weaning, with carrier sows as mothers, apparently made them resistant to M. hyosynoviae-arthritis when challenge-infected at 4½ weeks of age. More pigs in group NC had M. hyosynoviae related pathological lesions than in group CAb, a difference that was significant for cubital joints when analysed on joint type level. This finding indicates a partially protective effect of passively transferred M. hyosynoviae-specific colostral antibodies upon development of M. hyosynoviae related pathology. Thus, the level of passive immunity transferred from sow to piglet seems to provide, at least partial, protection against development of arthritis. It cannot be ruled out that the CCE pigs, by growing up in an infected environment, have had the chance to establish an active anti-M. hyosynoviae immune response that complements the maternally transferred immune factors. Evident from this study is that the general absence of M. hyosynoviae arthritis in piglets can be ascribed mainly to their immunological status.

Two clinical isolates of Mycoplasma hyosynoviae showed differing pattern of lameness and pathogen detection in experimentally challenged pigs.

Mycoplasma (M.) hyosynoviae is known to colonize and cause disease in growing-finishing pigs. In this study, two clinical isolates of M. hyosynoviae were compared by inoculating cesarean-derived colostrum-deprived and specific-pathogen-free growing pigs. After intranasal or intravenous inoculation, the proportion and distribution pattern of clinical cases was compared in addition to the severity of lameness. Tonsils were found to be the primary site of colonization, while bacteremia was rarely detected prior to the observation of clinical signs. Regardless of the clinical isolate, route of inoculation, or volume of inocula, histopathological alterations and tissue invasion were detected in multiple joints, indicating an apparent lack of specific joint tropism. Acute disease was primarily observed 7 to 10 days post-inoculation. The variability in the severity of synovial microscopic lesions and pathogen detection in joint cavities suggests that the duration of joint infection may influence the diagnostic accuracy. In summary, these findings demonstrate that diagnosis of M. hyosynoviae-associated arthritis can be influenced by the clinical isolate, and provides a study platform to investigate the colonization and virulence potential of field isolates. This approach can be particularly relevant to auxiliate in surveillance and testing of therapeutic and/or vaccine candidates.

Quantitative real-time polymerase chain reaction for detecting Mycoplasma hyosynoviae and Mycoplasma hyorhinis in pen-based oral, tonsillar, and nasal fluids.

Mycoplasma (M.) hyorhinis and M. hyosynoviae are pathogens known to cause disease in pigs post-weaning. Due to their fastidious nature, there is increased need for culture-independent diagnostic platforms to detect these microorganisms. Therefore, this study was performed to develop and optimize quantitative real-time PCR (qPCR) assays to rapidly detect M. hyorhinis and M. hyosynoviae in pen-based oral fluids as well as nasal and tonsillar fluids as proxies for samples used in swine herd surveillance. Two methods of genomic DNA extraction, automated versus manual, were used to compare diagnostic test performance. A wean-to-finish longitudinal study was also carried out to demonstrate the reproducibility of using pen-based oral fluids. Overall, pen-based oral and tonsillar fluids were more likely to be positive for both types of bacteria whereas only M. hyorhinis was detected in nasal fluids. DNA extraction protocols were shown to significantly influence test result. Although the initial detection time somewhat differed, both organisms were repeatedly detected in the longitudinal study. Overall, this study evaluated two qPCR methods for rapid and specific detection of either mycoplasma. Results from the present investigation can serve as a foundation for future studies to determine the prevalence of the two microorganisms, environmental load, and effectiveness of veterinary interventions for infection control.

Optimization of an antibiotic sensitivity assay for Mycoplasma hyosynoviae and susceptibility profiles of field isolates from 1997 to 2011.

Mycoplasma hyosynoviae is a common agent responsible for polyarthritis leading to decreased production in swine herds worldwide. Antimicrobial agents are used to combat infections; however breakpoints for M. hyosynoviae have not yet been established. A number of methods have previously been utilized to analyze minimum inhibitory concentrations (MICs) for antibiotics against M. hyosynoviae; however these techniques as currently described are not easily standardized between laboratories. A dry microbroth dilution method was conducted to compare the minimum inhibitory concentrations (MICs) for 18 antibiotics, representative of different classes, against 24 recent isolates (23 field isolates and the type strain) of M. hyosynoviae. The MICs were determined using standard, commercially available 96-well Sensititre(®) plates containing various freeze-dried antibiotics at a range of concentrations appropriate to their potency. Clindamycin (CLI), a lincosamide antibiotic, showed the highest activity and most consistent inhibition for all isolates with an MIC(50) of ≤ 0.12 µg/ml. Tiamulin (TIA), a pleuromutilin derivative, exhibited an MIC(50) of ≤ 0.25 µg/ml. The isolates had similar levels of susceptibility to the quinolones, enrofloxacin (ENRO) and danofloxacin (DANO), exhibiting an MIC(50) of 0.25 µg/ml and 0.5 µg/ml, respectively. For the macrolides, the MIC(50) for tylosin (TYLT) and tilmicosin (TIL) was ≤ 0.25 µg/ml and ≤ 2 µg/ml respectively, but was ≤ 16 µg/ml for tulathromycin (TUL). For the aminoglycosides, the MIC(50) for gentamicin (GEN) was ≤ 0.5 µg/ml, while spectinomycin (SPE) and neomycin (NEO) had an MIC(50) of ≤ 4 µg/ml. The tetracyclines, oxytetracycline (OXY) and chlortetracycline (CTET) both had an MIC(50) of ≤ 2 µg/ml. Florfenicol (FFN) exhibited a MIC(50) of ≤ 1 µg/ml. All isolates were resistant to penicillin (PEN), ampicillin (AMP), ceftiofur (TIO), trimethoprim/sulfamethoxazole (SXT), and sulphadimethoxine (SDM) at all concentrations. Within the isolates tested, there was a range of sensitivity detected, with some isolates being overall more resistant while others appeared more susceptible. Further research is required to demonstrate how this MIC data correlates to clinical efficacy of the various antibiotics in the field.

Optimal combinations of acute phase proteins for detecting infectious disease in pigs.

The acute phase protein (APP) response is an early systemic sign of disease, detected as substantial changes in APP serum concentrations and most disease states involving inflammatory reactions give rise to APP responses. To obtain a detailed picture of the general utility of porcine APPs to detect any disease with an inflammatory component seven porcine APPs were analysed in serum sampled at regular intervals in six different experimental challenge groups of pigs, including three bacterial (Actinobacillus pleuropneumoniae, Streptococcus suis, Mycoplasma hyosynoviae), one parasitic (Toxoplasma gondii) and one viral (porcine respiratory and reproductive syndrome virus) infection and one aseptic inflammation. Immunochemical analyses of seven APPs, four positive (C-reactive protein (CRP), haptoglobin (Hp), pig major acute phase protein (pigMAP) and serum amyloid A (SAA)) and three negative (albumin, transthyretin, and apolipoprotein A1 (apoA1)) were performed in the more than 400 serum samples constituting the serum panel. This was followed by advanced statistical treatment of the data using a multi-step procedure which included defining cut-off values and calculating detection probabilities for single APPs and for APP combinations. Combinations of APPs allowed the detection of disease more sensitively than any individual APP and the best three-protein combinations were CRP, apoA1, pigMAP and CRP, apoA1, Hp, respectively, closely followed by the two-protein combinations CRP, pigMAP and apoA1, pigMAP, respectively. For the practical use of such combinations, methodology is described for establishing individual APP threshold values, above which, for any APP in the combination, ongoing infection/inflammation is indicated.

Isolation of Mycoplasma hyosynoviae from pneumonic lung of swine.

The isolation of Mycoplasma hyosynoviae from a piglet with severe pneumonia is described. This is the first report of M. hyosynoviae isolation in the country. The lung sample where the isolation was made was severely consolidated, suppurative and pleurisy. The pathogenicity of the M. hyosynoviae isolated has yet to be determined.

Absence of strictly age-related resistance to Mycoplasma hyosynoviae infection in 6-week-old pigs.

Mycoplasma hyosynoviae has never been reported to cause arthritis in pigs younger than 10 weeks of age. In order to investigate whether a strict age-related resistance exists, four 6-week-old pigs and four 13-week-old pigs, all immunologically naïve with respect to M. hyosynoviae, were inoculated intranasally with the agent and autopsied at day 11 or 13 after infection. One uninoculated pig per age group was included as a negative control. Just as the 13-week-old pigs, the 6-week-old piglets were susceptible to blood, joint and tonsillar infection with M. hyosynoviae and developed clinical arthritis following inoculation with the agent. Thus, we found no evidence of an age-related resistance to the infection.

Use of a novel serum ELISA method and the tonsil-carrier state for evaluation of Mycoplasma hyosynoviae distributions in pig herds with or without clinical arthritis.

A novel indirect ELISA test using deoxycholate extracted antigens coated onto a hydrophobic polystyrene surface for measurement of serum antibodies specific for Mycoplasma hyosynoviae was developed. Sensitivity and specificity of the test were found to be superior to previous ELISAs as tested on porcine serum following experimental Mycoplasma infections as well as by analysis of samples from one Danish herd known to be free of M. hyosynoviae and samples from two Norwegian herds without clinical suspicion of M. hyosynoviae infections since their establishment. The epidemiology of M. hyosynoviae infection was then investigated in Danish pig herds with evidence of clinical M. hyosynoviae arthritis (MhA herds, n = 4) and in herds with M. hyosynoviae-carriers among slaughter pigs, but with limited clinical lameness (MhC herds, n = 4). M. hyosynoviae bacteriaemia and tonsil-carrier state were determined by culture of cross-sectional samples of whole-blood (n = 238) and tonsil scrapings (n = 322), respectively. Levels of serum antibodies (n = 396) were measured by the novel indirect ELISA test. There was no significant difference in the ELISA results between the MhA and the MhC herds. Pigs that were tonsil-carriers had a significantly higher response in the ELISA test (P < 0.001) than non-carriers. Slaughter pigs showed higher ELISA values (P < 0.001) and they were more prone to be tonsil-carriers (P < 0.001). The most critical period for spread of the infection seems to be the nursery period (4-12 weeks). The results indicate that M. hyosynoviae infection progresses similarly in herds irrespective of the presence of clinical arthritis. Thus, clinical arthritis due to M. hyosynoviae is probably triggered by other host or herd factors than low levels of serum antibodies or by differences in virulence factors between M. hyosynoviae strains.