Biofilm characterization of Mycoplasma bovis co-cultured with Trueperella pyogenes

Biofilm characterization of Mycoplasma bovis co-cultured with Trueperella pyogenes | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Biofilm characterization of Mycoplasma bovis co-cultured with Trueperella pyogenes Koji Nishi, Satoshi Gondaira, Yuki Hirano, Masahide Ohashi, Ayano Sato, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4523720/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 30 Jan, 2025 Read the published version in Veterinary Research → Version 1 posted You are reading this latest preprint version Abstract Mycoplasma pneumonia caused by Mycoplasma bovis ( M. bovis ) is associated with severe inflammatory reactions in the trachea and lungs and can be difficult to treat with antibiotics. Biofilms play a significant role in the persistence of bacteria and contribute to chronic lesions. A recent study showed that polymicrobial interactions of species are an important factor in biofilm formation, but the detailed mechanism of biofilm formation of M. bovis remains unknown. Assuming multiple pathogen infections in bovine respiratory disease complex, this study examined the characterization of the polymicrobial relationship between M. bovis and Trueperella pyogenes ( T. pyogenes ) during biofilm formation. Bacterium-like aggregation structures (> 10 µm), which were assumed to be biofilms of M. bovis in vivo , were observed adhering to the cilia in calves with Mycoplasma pneumonia. M. bovis released extracellular matrix to connect with neighboring bacteria and form a mature biofilm on the plate. Biofilm formation in co-culture of M. bovis and T. pyogenes tended to increase compared to that in single culture of these bacteria. Additionally, some large aggregates (> 40 µm) composed of M. bovis and T. pyogenes were observed. The morphological characteristics of this biofilm were similar to those observed in vivo compared to a single culture. In conclusion, the polymicrobial interaction between M. bovis and T. pyogenes induced biofilm formation, which was associated with increased resistance to antimicrobial agents, thereby exacerbating the progression of chronic Mycoplasma pneumonia. antibiotics antimicrobial agents bovine respiratory disease extracellular matrix trachea Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Mycoplasma bovis ( M. bovis ) is a pathogen that causes mastitis ( 1 ), pneumonia ( 2 ), arthritis ( 3 ), and otitis media ( 4 ) in dairy cattle. Mycoplasma pneumonia caused by M. bovis is associated with severe inflammatory reactions in the trachea and lungs, can be difficult to treat with antibiotics, and may result in substantial economic losses in dairy and beef farms ( 5 ). M. bovis is an important etiological agent of the bovine respiratory disease complex (BRDC). BRDC is caused by an interaction between viral and bacterial pathogens such as bovine herpesvirus, bovine viral diarrhea virus, Pasteurella multocida , Mannheimia haemolytica , and Trueperella pyogenes ( T. pyogenes ) ( 6 ). Each pathogen has unique features, such as the suppression of the protective barrier function of the respiratory epithelium or the immune response in leukocytes ( 6 , 7 ), resulting in chronic pneumonia. T. pyogenes is part of the biota of the skin and mucous membranes of the upper respiratory, gastrointestinal, and urogenital tracts of animals and is also an opportunistic pathogen ( 8 ). T. pyogenes is involved in polymicrobial diseases, including mastitis, uterine infections, and pneumonia ( 8 ). Therefore, this bacterium is often isolated from a mixed infection of various bacterial species. The expression levels of virulence genes, including plo, fimA, nanH, and cbpA, in T. pyogenes isolates in co-culture with Fusobacterium necrophorum ( F. necrophorum ) and Escherichia coli ( E. coli ) were increased ( 9 ). It means that polymicrobial infection intensifies the virulence of T. pyogenes. A previous study reported that not only M. bovis but also T. pyogenes were isolated from lesions of chronic caseous pneumonia in cattle ( 10 ). It is speculated that co-infection with M. bovis and T. pyogenes induces a more severe inflammatory reaction in respiratory tissues than a single infection, but the detailed mechanism is unknown. Biofilms are communities of microorganisms that are attached to biotic or abiotic surfaces ( 11 ). Biofilms play a significant role in the persistence of bacteria and contribute to chronic lesions because it is difficult for antibiotics and immune responses to reach bacteria within biofilms ( 12 ). The structure of the extracellular polymeric substance (EPS) matrix of the biofilm consists of extracellular polysaccharides, DNA, and proteins ( 11 ). A recent study showed that EPS material, shared by multiple pathogens in co-culture, facilitates interspecies interactions through the formation of compact microcolony structures during biofilm formation ( 13 ). The interaction between Staphylococcus aureus ( S. aureus ) and Candida albicans ( C. albicans ) demonstrates synergistic activity, significantly enhancing biofilm formation and contributing to an increase in antimicrobial resistance in S. aureus ( 14 ). Therefore, the polymicrobial interaction between species are an important factor for biofilm formation. M. bovis is known to form biofilms, even though it has very limited genes ( 15 ). A previous study showed that the morphological characterization of M. bovis biofilm on the plate and its formation potential may be associated with the expression of an adhesion factor ( 15 ). However, M. bovis biofilms have never been observed in vivo , and the effect of polymicrobial relationships on biofilm formation is unknown. In this study, we analyzed the morphological characteristics of M. bovis biofilm in spontaneous Mycoplasma pneumonia in calves. Additionally, we examined the characterization of the polymicrobial relationship between M. bovis and T. pyogenes during biofilm formation to clarify its relevance to the development of pneumonia. Material and methods Animal Two Holstein calves, aged 2 months (Calf 1 and Calf 2), with chronic Mycoplasma pneumonia, and two control Holstein calves without clinical respiratory symptoms, aged 1 and 2 months, in Hokkaido, Japan, underwent pathological autopsies in accordance with the Guide for the Care and Use of Laboratory Animals of the School of Veterinary Medicine at Rakuno Gakuen University in 2021. Two calves with chronic Mycoplasma pneumonia also had arthritis and showed lameness. Tracheal and caseous necrotic foci were obtained from the lobe and subjected to PCR analyses using M. bovis –specific primers, as previously described ( 16 ). Additionally, swabs from these samples were cultured on blood agar plates (Eiken Kagaku, Tokyo, Japan) and incubated for 48 hours at 37°C. The bacterial colony obtained was identified by its 16S ribosomal RNA gene sequence as previously described ( 17 ). The sequence of the 16S 27F forward primer was 5′-AGAGTTTGATCCTGGCTCAG-3′, and the 1492R reverse primer was 5′-GGTTACCTTGTTACGACTT-3′. PCR products were extracted using the FastGene Gel/PCR Extraction Kit (Nippon Genetics, Tokyo, Japan). The product sequence was analyzed by Hokkaido System Science Co., Ltd. The sequence data was confirmed through database analysis using the Basic Local Alignment Search Tool. Bacterial strains The sixteen M. bovis strains and two T. pyogenes strains used in this study are listed in Table 1 . Before use, M. bovis and T. pyogenes strains were cultured in modified PPLO medium (Kanto Kagaku, Tokyo, Japan) and brain heart infusion supplemented with 5% fetal bovine serum (FBS) and then stored at − 80°C. Table 1 Information regarding the bacterial strains. Species Strain Origin Disease M. bovis PG45 ATCC 25523 - Strain M1 Nosal cavity Pneumonia Strain M2 Nosal cavity Pneumonia Strain M3 Nosal cavity Pneumonia Strain M4 Nosal cavity Pneumonia Strain M5 Nosal cavity Pneumonia Strain M6 Nosal cavity Pneumonia Strain M7 Synovial fluids Arthritis Strain M8 Synovial fluids Arthritis Strain M9 Milk Mastitis Strain M10 Milk Mastitis Strain M11 Lung Pneumonia Strain M12 Heart Endcarditis Strain M13 Heart Endcarditis Strain M14 Heart Endcarditis Strain M15 Heart Endcarditis Strain M16 Lung ( Mycoplasma pneumonia Calf1) Pneumonia T. pyogenes Strain T1 Lung ( Mycoplasma pneumonia Calf1) Pneumonia Strain T2 Lung ( Mycoplasma pneumonia Calf2) Pneumonia Light microscopy and immunohistochemical analysis Tracheal tissues were fixed in a 4% paraformaldehyde solution, dehydrated through an ethanol gradient from 70–100%, and then embedded in paraffin. After deparaffinization using xylene, the tissue sections were stained with hematoxylin and eosin, and then observed under a light microscope. Immunohistochemical staining was performed using indirect immunofluorescence analysis. The sections were heated in a microwave oven in the presence of 0.01 M sodium citrate buffer (pH 6.0) for 15 minutes and then immersed in a 3% hydrogen peroxide solution at room temperature for 10 minutes. After pretreatment, the sections were incubated with 5% normal goat serum at room temperature for 20 minutes as a blocking step. Subsequently, the sections were incubated with anti-cytokeratin18 (Proteintech, Chicago, IL, USA) and anti- M. bovis (Millipore, Billerica, MA, USA) antibodies at room temperature for 2 hours, and then incubated with rhodamine-conjugated goat anti-rabbit IgG antibody (Thermo Fisher Scientific, Waltham, MA, USA) and Alexa Fluor 488-conjugated goat anti-mouse IgG antibody (Thermo Fisher Scientific) at room temperature for 1 hour. The sections were stained with DAPI solution (DOJINDO, Kumamoto, Japan) at room temperature for 10 minutes and visualized using a Nikon C2 laser confocal microscope (Nikon, Tokyo, Japan). The obtained images were analyzed using NIS Elements AR Analysis and Fiji (a distribution of ImageJ software from the US National Institutes of Health, Bethesda, Maryland, USA) according to the following methods ( 18 ). Biofilm formation of M. bovis single culture Biofilm formation was performed with modifications as previously described ( 15 ). Store five strains of M. bovis (PG45, Strains M1–M4) were cultured in modified PPLO medium at 37°C for 24 hours without aeration. After culturing, 10 µL of planktonic M. bovis was inoculated into a non-coated 96-well cell culture plate (NIPPON Genetics) in triplicate. The culture medium used for biofilm formation was modified PPLO medium (PPLO rich), not modified PPLO broth medium (not adding horse serum and yeast extract, et al; PPLO broth: Kanto Kagaku), mueller hinton (MH: Beckton Dickinson, Franklin Lakes, NJ, USA), lysogeny broth (LB: Beckton Dickinson), brain heart infusion (BHI: Beckton Dickinson), trypticase soy broth (TSB: Beckton Dickinson), todd hewitt broth (THB: Kanto kagaku), Dulbecco’s modified Eagle’s medium (DMEM: Fujifilm Wako (Osaka, Japan), DMEM supplemented with 5% FBS, and Roswell Park Memorial Institute 1640 medium (RPMI: Fujifilm Wako), and RPMI supplemented with 5% FBS to determine the appropriate medium. These media were added at 190 µL per well in a 96-well plate. The planktonic M. bovis culture was then diluted 1:20 and incubated at 37°C with 5% CO 2 for 24 hours without aeration. Biofilm formation in co-culture with M. bovis and T. pyogenes Stored M. bovis was cultured in modified PPLO medium at 37°C for 24 hours, and then 10 µL of this cultured planktonic M. bovis was inoculated into a non-coated 96-well plate (NIPPON Genetics). Stored T. pyogenes was centrifuged (7000 rpm, 5 min, 4°C) and suspended in PPLO broth medium. T. pyogenes was inoculated at concentrations of 1 × 10 4 , 10 5 , 10 6 , and 10 7 colony-forming units (CFU) per 10 µL into a 96-well plate. PPLO broth medium was added at 180 µL per well in a 96-well plate and incubated at 37°C with 5% CO 2 for 24 hours without aeration. Crystal violet staining The biofilm in the 96-well plate was washed three times with phosphate-buffered saline (PBS) to remove planktonic cells and then fixed with 99.5% methanol for 15 minutes. The biofilm was stained with a 2% crystal violet solution for 20 minutes and rinsed three times with distilled water. The plate was dried and then decolorized with 200 µL of 99.5% ethanol to release the crystal violet. The released crystal violet was quantified using a microplate reader (BIO-RAD, Hercules, CA, USA) by measuring the absorbance at 595 nm. Scanning electron microscope Cultured planktonic M. bovis was prepared as previously described and inoculated with 300 µL into a 35 mm non-coated single culture dish (IWAKI, Chiba, Japan) with 2700 µL of PPLO broth medium. T. pyogenes was inoculated at 3 × 10 8 CFU/well and incubated at 37°C with 5% CO 2 for 24 hours without aeration. Cultured bacterial biofilms and tracheal tissues isolated from pneumonia-affected and control calves were fixed with half-strength Karnovsky’s solution at 4°C overnight. The tissue samples were post-fixed with 1% osmium tetroxide for 30 minutes. The tissues and biofilm samples were then washed with 0.1 M cacodylate buffer and dehydrated through an ethanol gradient ranging from 30–100% (with concentrations at 30%, 50%, 70%, 80%, 90%, 95%, and 100%), spending 10 minutes at each step. After being dried using the t-butyl alcohol freeze-drying method, the specimens were coated with Pt–Pd and observed under a HITACHI S-2460N electron microscope at 8 kV. Confocal microscope Cultured plankonic M. bovis was prepared as previously described above and inoculated with 60 µL into a 24-well non-coated culture dish (IWAKI, Chiba, Japan) containing a 12 mm round cover glass and 540 µL of PPLO broth medium. T. pyogenes was inoculated at 6 × 10 7 CFU/well and incubated at 37°C with 5% CO 2 for 24 hours without aeration. The bacterial biofilm on the round cover glass was stained with DAPI solution (DOJINDO) or the LIVE/DEAD® BacLight™ Bacterial Viability Kit (Thermo Fisher Scientific) and then washed three times with PBS. The samples were fixed with a 4% paraformaldehyde solution and observed using a Nikon C2 laser confocal microscope. The images obtained were analyzed using NIS Elements AR Analysis and Fiji. Statistical analysis Data are expressed as the means ± SE. The Steel method was used for comparisons between different groups using the statistical analysis program MEPHAS ( http://www.gen-info.osaka-u.ac.jp/MEPHAS/ ). In all cases, a probability ( p ) value < 0.05 was considered to indicate a statistically significant difference. Results Pathological findings and morphological analysis of Mycoplasma pneumonia in calves A pathological autopsy was performed on calves affected by Mycoplasma pneumonia to evaluate the formation of M. bovis biofilm in vivo . The calf with Mycoplasma pneumonia (Calf 1) showed coagulation and caseous necrotic foci in the right cranial part and middle lobe (Fig. 1 A and B). Caseous discharge was observed on the cut surface of the right cranial region (Fig. 1 C). Tracheal tissues were obtained from control calves with no clinical respiratory symptoms and those with Mycoplasma pneumonia. M. bovis and T. pyogenes were detected in tracheal mucosa swabs and caseous necrotic foci in the lungs by PCR. Tracheal tissues were stained with hematoxylin and eosin (Fig. 1 D and E). Cilia were aligned on the epithelial cells of the tracheal mucosa in control calves, while in calves with Mycoplasma pneumonia, they were not located. Tissues from the trachea of control calves and those with Mycoplasma pneumonia were stained with cytokeratin-18 antibody, M. bovis antibody, and DAPI solution, and analyzed using a fluorescence microscope (Fig. 1 F–M). Areas positive for M. bovis were detected in the epithelial cells of tracheal tissues of calves with Mycoplasma pneumonia (Fig. 1 I and M). Micromorphological characterization of tracheal mucosa of control calves and those with Mycoplasma pneumonia was analyzed by SEM (Fig. 1 N–Q). The surface of the tracheal mucosa of control calves was completely covered with cilia (Fig. 1 N and O). Mycoplasma pneumonia in calves showed loss of cilia on the tracheal mucosa (Fig. 1 P). Bacterium-like aggregation structures (> 10 µm) were observed adhering to cilia in a calves with Mycoplasma pneumonia (Fig. 1 P and Q, yellow arrowheads). Bacteria were detected on the cilia of the tracheal mucosa (Fig. 1 Q, indicated by red arrows). Additionally, the boundaries between bacteria were obscured, and bacterial aggregation structures were observed (Fig. 1 Q, indicated by green arrows). These characteristic structures were also observed in the control and the one with Mycoplasma pneumonia (Calf 2; Additional File 1). Quantitative analysis of M. bovis biofilm formation We examined the appropriate medium to evaluate biofilm formation of M. bovis . Five strains of M. bovis were cultured in 96-well microplates across eleven culture media for 24 hours and then stained with crystal violet (Fig. 2 A). Biofilm from five M. bovis strains was not detected in the PPLO broth, which is a major growth medium for Mycoplasmas. In contrast, cultures in PPLO broth medium showed the highest level of biofilm formation among eleven culture media. Biofilm formation of five strains of M. bovis in DMEM or RPMI was higher than in DMEM + FBS or RPMI + FBS, respectively. Therefore, in subsequent experiments, PPLO broth medium was used to evaluate biofilm formation of M. bovis . Sixteen M. bovis strains were cultured in PPLO broth medium and stained with crystal violet (Fig. 2 B). PG45 was shown to be a moderate biofilm producer (OD value was 0.26 ± 0.04). No significant difference was found between PG45 and fifteen wild strains. However, Strains M1, M6, M7, and M8 were shown to be strong biofilm producers (OD value > 0.3). On the other hand, Strains M5, M11, M12, and M14 were shown to be weak biofilm producers (OD value < 0.2). It is shown that the ability to form biofilms differed depending on the strains. Morphological characterization of M. bovis biofilm in single culture The biofilm of Strain M7, a strong biofilm producer, was stained with DAPI solution and observed using a confocal microscope (Fig. 3 A). M. bovis biofilms were observed as aggregations of various sizes and scattered formations. The biofilm was stained with LIVE/DEAD BacLight and visualized in orthogonal sections (Fig. 3 B). Biofilm was formed by live M. bovis and there were almost no dead bacteria. The micromorphology of the biofilm was evaluated by SEM (Fig. 3 C and D). A single bacterium adhering to the plate and approximately 10 µm bacterial aggregates were observed (Fig. 3 D, red arrowheads). M. bovis cells were connected by filamentous structures and arranged in a rosary-like formation (Fig. 3 D, indicated by yellow arrows). The observation of aggregation and concatenated bacterial structures in these strains, which showed moderate levels of biofilm formation, was lower than that in Strain M7 (Additional File 2). Quantitative analysis of biofilm formation in co-culture with M. bovis and T. pyogenes M. bovis strains PG45 and M16: isolated from Calf 1) and T. pyogenes (Strain T1: isolated from Calf 1) were co-cultured in a 96-well microplate using PPLO-based medium for 24 hours and then stained with crystal violet (Fig. 4 ). Biofilm formations of PG45, when co-cultured with 1 × 10 4 , 1 × 10 5 , or 1 × 10 6 CFU/well of T. pyogenes (Strain T1), tended to be higher (OD values were 0.21 ± 0.03, 0.21 ± 0.03, and 0.19 ± 0.03, respectively) than those of the single culture (0.17 ± 0.01). Biofilm formations of Strain M16, when co-cultured with 1 × 10 4 , 1 × 10 5 , or 1 × 10 6 CFU/well of T. pyogenes , tended to be higher (OD values were 0.42 ± 0.07, 0.42 ± 0.06, and 0.42 ± 0.03, respectively) than those of the single culture (0.27 ± 0.04). T. pyogenes in single culture was shown to be a weak biofilm producer (OD value ≦ 0.1). However, biofilm formation of PG45 and Strain M16, when co-cultured with 1 × 10 7 CFU of T. pyogenes (OD values were 0.12 ± 0.03 and 0.24 ± 0.04, respectively), tended to be lower than when co-cultured with 1 × 10 4 , 1 × 10 5 , or 1 × 10 6 CFU/well of T. pyogenes . Quantitative analysis of biofilm formation co-cultured with M. bovis (PG45 and Strain M16) and T. pyogenes (Strain T2, isolated from Calf 2) is presented in Additional File 3. Biofilm formations of PG45, when co-cultured with T. pyogenes (Strain T2) showed no difference, while those of Strain M16 co-cultured with 1 × 10 4 , 1 × 10 5 , or 1 × 10 6 CFU/well of T. pyogenes tended to be higher than in single culture. The biofilm formation of Strain M16, when co-cultured with 1 × 10 7 CFU of T. pyogenes (Strain T2), also tended to be lower than when co-cultured with 1 × 10 5 or 1 × 10 6 CFU/well of T. pyogenes . Morphological characterization of biofilm in co-culture with M. bovis and T. pyogenes The biofilm of M. bovis (Strain M16) was stained with LIVE/DEAD BacLight and observed using a confocal microscope (Fig. 5 A and B). A single culture of M. bovis showed aggregations of biofilm composed of live bacteria. On the other hand, the biofilm co-cultured with M. bovis and T. pyogenes were formed with both live and dead bacteria, with the surface of the biofilm especially covered in dead bacteria (Fig. 5 C and D). The biofilm formation of single M. bovis (Strain M16), T. pyogenes (Strain T1), and the co-culture of these bacteria was analyzed by SEM (Fig. 5 E–H). M. bovis biofilms were observed as aggregations of varying sizes, which were formed through adhesion between neighboring bacteria (Fig. 5 E), whereas no biofilm formation by T. pyogenes and only minor conjugation of bacterial cells were observed in single culture (Fig. 5 F). In co-culture, M. bovis and T. pyogenes adhered, and there was no bacterium with cell-wall damage or erosion (Fig. 5 G and H). Interestingly, some large aggregates of bacteria (> 40 µm), consisting of M. bovis and T. pyogenes , were observed (Fig. 5 I). This aggregation was not observed in single cultures of M. bovis and T. pyogenes . The boundary between aggregating bacteria was obscure (Fig. 5 J, indicated by green arrowheads). Discussion M. bovis causes chronic pneumonia in calves, which is difficult to treat with antibiotics. It is known that bacteria in biofilms can become 10 to 1000 times more resistant to antimicrobial agents compared to planktonic bacteria ( 19 ). A previous study reported that distortion of fimbriae and edema of attached cells were observed in the tracheal tissues of calves experimentally infected with M. bovis , and in some cases, M. bovis was found residing in both ciliated and non-ciliated epithelial cells ( 20 ). However, biofilm formation of M. bovis in vivo has not been observed. In this study, we performed pathological autopsies on calves naturally infected with chronic Mycoplasma pneumonia and evaluated the biofilm formation of M. bovis . Calf 1 and Calf 2 showed pathological characteristics, which included coagulation and caseous necrosis foci in the lungs. T. pyogenes is an important secondary pathogen and is most commonly isolated from lung lesions in BRDC ( 21 ). Co-infection with M. bovis and T. pyogenes has also been identified as a risk factor for severe BRDC ( 21 ). In this study, not only M. bovis but also T. pyogenes were isolated as dominant bacteria from the trachea and lungs, suggesting that these pathogens were major factors causing lesions. We conducted a morphological analysis of the trachea from calves with pneumonia. Cilia were not detected on the epithelial cells of the tracheal mucosa in calves with Mycoplasma pneumonia. Additionally, M. bovis antigen was detected in the epithelial cells of the trachea using a fluorescent microscope. It has been reported that the production of hydrogen peroxide and other reactive oxygen species (ROS) may be important factors in the pathogenicity of mycoplasmas, as it causes damage to cell membranes ( 22 , 23 ). The genes involved in the production of hydrogen peroxide and ROS were encoded in the genome of commensal mycoplasma ( 24 ). M. bovis produces hydrogen peroxide, causing damage to epithelial cells under both in vivo and in vitro conditions ( 25 , 26 ). Exposure to high concentrations of hydrogen peroxide inhibited tracheal ciliary movement and induced apoptosis in epithelial cells ( 27 , 28 ). This damage to the respiratory mucosa reduces the immune barrier and induces a local inflammatory response. We suggested that M. bovis removes cilia from the trachea by producing hydrogen peroxide, causing a decline in tracheal immunity. Interestingly, bacterial aggregation structures were observed adhering to cilia from calves with Mycoplasma pneumonia in this study. It has been reported that large, mature biofilm structures, where networks of bacteria were embedded within structured matrices, were observed in the nasopharyngeal tissue of mice experimentally infected with Streptococcus pneumoniae ( S. pneumoniae ) ( 29 ). This large biofilm structure of S. pneumoniae was associated with increased resistance to antibiotics. In this study, it was assumed that the bacterial aggregation structures were biofilms of M. bovis in vivo and associated with resistance to antibiotics in Mycoplasma pneumonia in calves. T. pyogenes was also isolated from the trachea in calves with pneumonia, and it was thought to be involved in the biofilm formation of M. bovis . Thus, we examined biofilm formation during co-cultivation of M. bovis and T. pyogenes in vitro . It was previously reported that the methods for evaluating M. bovis biofilms were unstable ( 15 ), and we were unable to reproduce them. Initially, we identified the most suitable medium to assess the biofilm formation of M. bovis. Biofilm formation of M. bovis cultured in PPLO broth medium was higher than that in other media. Additionally, DMEM and RPMI promoted biofilm formation more than DMEM FBS and RPMI FBS, respectively. A previous study showed that M. bovis biofilm was evaluated using the “PPLO rich” growth medium ( 15 ). However, in our study, biofilm formation of M. bovis was the lowest in PPLO-rich medium, while the PPLO broth medium, which did not have added nutrients for growth such as horse serum and yeast extract, showed the highest biofilm formation. Generally, biofilm production increases in environments where bacterial growth is difficult ( 11 ). Since bovine serum is rich in nutritional factors essential for the growth of M. bovis , it is suggested that adding serum to DMEM or RPMI reduces biofilm formation. Thus, our data suggested that a PPLO broth without added nutrients for growth was suitable for evaluating the biofilm formation of M. bovis . We evaluated the biofilm formation of sixteen species, including M. bovis in PPLO broth medium. Strains M1, M6, M7, and M8 were strong biofilm producers, while Strains M5, M11, M12, and M14 were weak. Biofilm formation is characterized by three stages: attachment, maturation, and dispersion ( 11 ). The attachment is a crucial initial step in biofilm formation. Pseudomonas aeruginosa ( P. aeruginosa ) uses flagella for movement ( 30 ), and S. aureus adheres to host-derived EPS for attachment ( 31 ). Previous studies have indicated that the variable surface proteins B and O type, which are among the adhesion factors expressed by the wild strain M. bovis , form prolific biofilms ( 15 ). The difference in biofilm production ability among M. bovis strains was thought to be due to the expression levels of various adhesion factors. In this study, M. bovis biofilms were observed as aggregations of varying sizes and scattered formations, consisting of live bacteria, using a confocal microscope. Additionally, M. bovis was observed to have a moniliform structure bound together by filamentous material, using a electron microscope. It has been observed that a thick filamentous matrix connects bacterial cells in Clostridium difficile ( C. difficile ) and S. aureus biofilms ( 32 , 33 ). This matrix was assumed to be ECM released by bacteria ( 33 ). The filamentous matrix in biofilms connects neighboring groups of bacteria, which is an initial step in aggregation ( 32 , 33 ). Nuclease treatment inhibited Mycoplasma hyopneumoniae ( M. hyopneumoniae ) biofilm formation, suggesting that extracellular DNA released outside the cell is a crucial step for biofilm formation ( 34 ). Although it has been reported that the M. bovis biofilm consists of viable bacteria, a detailed morphological analysis has yet to be conducted ( 15 ). We observed aggregations of bacteria and moniliform structures between M. bovis cells, suggesting that M. bovis also released ECM or DNA to connect to neighboring bacteria, and then M. bovis formed compact microcolonies and biofilm. M. bovis and T. pyogenes are each a pathogen that causes pneumonia in cattle, and it is not uncommon for both bacteria to be detected from the same lesion, as in this study ( 10 , 35 ). The expression levels of multiple virulence genes in T. pyogenes were increased upon co-infection with E. coli and F. necrophorum in mice ( 9 ). Co-infection is assumed to affect biofilm formation in pathogens, and elucidating this process is important for understanding the pathology of pneumonia. Thus, we examined the polymicrobial relationship between M. bovis and T. pyogenes during biofilm formation. Biofilm formation of M. bovis (PG45 and Strain M16) co-cultured with 1 × 10 4 , 1 × 10 5 , and 1 × 10 6 CFU/well of T. pyogenes (Strain T1 and T2) was synergistically increased, while that with 1 × 10 7 CFU/well of T. pyogenes was lower than with 1 × 10 5 and 1 × 10 6 CFU/well of T. pyogenes. The biofilm resulting from co-cultivation with M. bovis (Strain M16) and T. pyogenes was composed of both live and dead bacteria, with a higher proportion of dead bacteria compared to single cultures. S. aureus -induced apoptosis in C. albicans was characterized by features such as intracytoplasmic disorganization, cell membrane discontinuity, vacuole formation, and chromatin condensation. ( 36 ). The induction of apoptosis was not due to cell-to-cell contact but rather to the presence of the supernatant of S. aureus . Our study showed that a co-cultivation condition induced bacterial death in M. bovis , T. pyogenes , or both bacteria, which was notably observed when the number of T. pyogenes was high. However, adherence of M. bovis (Strain M16) and T. pyogenes to each other and the formation of large bacterial aggregates were observed by SEM. S. aureus caused the death of Aspergillus fumigatus , but in co-cultivation, the morphological characteristics of the biofilm produced by these bacteria were similar to those observed in vivo ( 33 ). Additionally, eDNA acted as a crucial EPS component shared by S. aureus and P. aeruginosa in co-culture biofilms, facilitating interspecies interactions by promoting the formation of compact microcolony structures during biofilm development ( 13 ). This cell-to-cell communication is called quorum sensing, which is a major coordination factor in biofilm formation ( 37 , 38 ). A previous study indicated that M. bovis has a very limited genome but can produce biofilms that are helpful for surviving environmental stress ( 15 ). Our study showed that the combination of M. bovis and T. pyogenes led to the formation of a large microcolony, which is assumed to be associated with quorum sensing. This data indicated that M. bovis collaborated with other bacteria to form a mature biofilm, leading to increased antimicrobial resistance in the tracheal mucosa. Although the morphological characteristics of polymicrobial colonization were somewhat similar to those observed in the trachea of calves with Mycoplasma pneumonia in vivo compared to single cultures in vitro , they were not completely identical. It has been reported that the biofilm grown on the epithelial cells exhibited phenotypes similar to those observed during in vivo colonization ( 29 ). Epithelial cells play a crucial role in the biofilm formation process by potentially facilitating optimal interbacterial signaling and the expression of colonization-associated factors necessary for biofilm formation through adherence to in vivo ligands. Our study is the first to elucidate the interactions between M. bovis and T. pyogenes during biofilm formation, and these mechanisms may be involved in the progression of pathology in Mycoplasma pneumonia. Further analysis using bovine epithelial cells is required to elucidate the mechanism of biofilm formation of M. bovis . Conclusion In conclusion, we observed mature biofilms of M. bovis on the tracheae of calves naturally infected with pneumonia and established a method for evaluating the biofilm formation of M. bovis in vitro. M. bovis biofilms were observed as aggregations of various sizes and filamentous matrices connecting neighboring groups, suggesting that M. bovis releases ECM or DNA to connect to neighboring bacteria, and then forms compact microcolonies. Additionally, co-cultivation with M. bovis and T. pyogenes caused significant biofilm formation. This study indicated that the interaction between M. bovis and T. pyogenes led to increased resistance to antimicrobial agents, thereby exacerbating the progression of chronic Mycoplasma pneumonia. Declarations Funding This study was supported by JSPS KAKENHI [grant number 23K05574] and the Rakuno Gakuen University Fund [grant number 2020-04]. Acknowledgements The authors would like to thank Enago (www.enago.jp) for the English language review. Ethics approval and consent to participate Four calves were undergoing pathological autopsies in accordance with the Guide for the Care and Use of Laboratory Animals of the School of Veterinary Medicine at Rakuno Gakuen University in 2021. Competing interest The authors declare no conflicts of interest. Author Contribution Conception and design of the work: KN, SG, HH; data acquisition and analysis: KN, YH, MO, AS, KM, TI, TK, RU; interpretation of data: KN, SG, HH; first draft of the manuscript: KN; manuscript revision: SG, HH. All authors read and approved the final manuscript. References Fox LK (2012) Mycoplasma mastitis: causes, transmission, and control. Vet. Clin. North Am Food Anim Pract 28:225-237. Maunsell FP, Woolums AR, Francoz D, Rosenbusch RF, Step DL, Wilson DJ, Janzen ED (2011) Mycoplasma bovis infections in cattle. J Vet Intern Med 25:772-783. Desrochers A, Francoz D (2014) Clinical management of septic arthritis in cattle. 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Ramírez Granillo A, Canales MG, Espíndola ME, Martínez Rivera MA, de Lucio VM, Tovar AV (2015) Antibiosis interaction of Staphylococcus aureus on Aspergillus fumigatus assessed in vitro by mixed biofilm formation. BMC Microbiol 15:33. Raymond BBA, Jenkins C, Turnbull L, Whitchurch CB, Djordjevic SP (2018) Extracellular DNA release from the genome-reduced pathogen Mycoplasma hyopneumoniae is essential for biofilm formation on abiotic surfaces. Sci Rep 8:10373. Margineda CA, Zielinski GO, Jurado S, Alejandra F, Mozgovoj M, Alcaraz AC, López A (2017) Mycoplasma bovis pneumonia in feedlot cattle and dairy calves in Argentina. Braz J Vet Pathol 10(2):79-86. Camarillo-Márquez O, Córdova-Alcántara IM, Hernández-Rodríguez CH, García-Pérez BE, Martínez-Rivera MA, Rodríguez-Tovar AV (2018) Antagonistic interaction of Staphylococcus aureus toward Candida glabrata during in vitro biofilm formation is caused by an apoptotic mechanism. Front Microbiol 9:2031. Kong KF, Vuong C, Otto M (2006) Staphylococcus quorum sensing in biofilm formation and infection. Int J Med Microbiol 296:133-139. Solano C, Echeverz M, Lasa I (2014) Biofilm dispersion and quorum sensing. Curr Opin Microbiol 18:96-104. Supplementary Files Additionalfile1.docx Cite Share Download PDF Status: Published Journal Publication published 30 Jan, 2025 Read the published version in Veterinary Research → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4523720","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":312892388,"identity":"ef7f7e53-625c-41b5-8882-18307ffe85f5","order_by":0,"name":"Koji 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Daigaku","correspondingAuthor":false,"prefix":"","firstName":"Takuya","middleName":"","lastName":"Kanda","suffix":""},{"id":312892396,"identity":"50c73ffc-927a-451f-a750-b378035901d7","order_by":8,"name":"Ryoko Uemura","email":"","orcid":"","institution":"Miyazaki Daigaku","correspondingAuthor":false,"prefix":"","firstName":"Ryoko","middleName":"","lastName":"Uemura","suffix":""},{"id":312892397,"identity":"3c30901d-825e-4c2e-98e9-bf889d48a6e8","order_by":9,"name":"Hidetoshi Higuchi","email":"","orcid":"","institution":"Rakuno Gakuen University: Rakuno Gakuen Daigaku","correspondingAuthor":false,"prefix":"","firstName":"Hidetoshi","middleName":"","lastName":"Higuchi","suffix":""}],"badges":[],"createdAt":"2024-06-03 19:03:49","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4523720/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4523720/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s13567-025-01468-1","type":"published","date":"2025-01-30T15:57:25+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":59141087,"identity":"fde85c3f-2f1d-4e20-b097-227f638ef9db","added_by":"auto","created_at":"2024-06-26 20:25:02","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":3135622,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePathological findings and morphological analysis of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eMycoplasma\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e pneumonia in calves\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A and B) Calf infected with \u003cem\u003eMycoplasma bovis\u003c/em\u003e (\u003cem\u003eM. bovis\u003c/em\u003e) and \u003cem\u003eTrueperella pyogenes\u003c/em\u003e showed coagulation and caseous necrotic foci in the right cranial part and middle lobe (asterisk). (C) Caseous discharge was observed on the cut surface of the right cranial part. (D and E) Tracheal tissues from calves with no clinical respiratory symptoms (controls) and those with \u003cem\u003eMycoplasma\u003c/em\u003e pneumonia were stained with hematoxylin and eosin. Cilia were observed on the epithelial cells of the control calves (block arrows). Representative images are shown. Scale bar: 32 μm. (F–M) Tracheal tissues from control calves and those with \u003cem\u003eMycoplasma\u003c/em\u003e pneumonia were stained with cytokeratin-18 antibody, \u003cem\u003eM. bovis\u003c/em\u003e antibody, and DAPI solution, and analyzed using a fluorescence microscope. Cytokeratin-18 is stained red (F and G), \u003cem\u003eM. bovis\u003c/em\u003e is stained green (H and I), and nucleus is stained blue (J and K). These images are overlayed (L and M). Representative images are shown. Areas positive for \u003cem\u003eM. bovis \u003c/em\u003ewere detected in the epithelial cells of tracheal tissues from calves with \u003cem\u003eMycoplasma\u003c/em\u003e pneumonia (white arrows). Scale bar: 100 μm. (N–Q) The tracheal mucosa from control calves and those with \u003cem\u003eMycoplasma\u003c/em\u003e pneumonia were analyzed by SEM. Representative images are shown. (N and O) The tracheal mucosa surface of control calves was completely covered with cilia. Scale bar: 10 μm. (P) Bacterium-like aggregation structures were observed on the tracheal mucosa from calves with \u003cem\u003eMycoplasma\u003c/em\u003e pneumonia (yellow arrowheads). Scale bar: 10 μm. The yellow square was magnified in (Q) to provide a better view. (Q) Bacteria were detected on the cilia (red arrows). The boundary between bacterial cells was obscured (yellow arrows). Bacteria were detected on the bacterium-like aggregation structures (green arrows). Scale bar: 5 μm.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4523720/v1/a34a60ac7563637f1b00ca99.jpg"},{"id":59141085,"identity":"6e6cbc4c-ac96-4d04-aa0f-49f3aec0eb17","added_by":"auto","created_at":"2024-06-26 20:25:02","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1051194,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eQuantitative analysis of biofilm formation of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eM. bovis\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e(A) Five strains of \u003cem\u003eM. bovis\u003c/em\u003e (PG45, Strains M1–M4) were cultured in eleven culture media on a 96-well microplate. \u003cem\u003eM. bovis\u003c/em\u003e biofilm was quantified using crystal violet staining. PBS: phosphate buffer saline; PPLO rich: modified PPLO medium; PPLO broth: PPLO broth medium; MH: mueller hinton; LB: lysogeny broth; BHI: brain heart infusion; TSB: trypticase soy broth; THB: todd hewitt broth; DMEM: Dulbecco’s modified Eagle’s medium; DMEM+FBS: 5% fetal bovine serum in DMEM; RPMI: Roswell Park Memorial Institute 1640 medium; RPMI+FBS: 5% fetal bovine serum in RPMI. (B) Biofilm formation of sixteen M. bovis strains was quantified using crystal violet staining in PPLO broth medium for 24 hours. Biofilm biomass was shown as mean OD values ± SE.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4523720/v1/034892f3fd90e724fb7c4076.jpg"},{"id":59141086,"identity":"de38544e-0a97-48ef-a125-fd6f896e9f98","added_by":"auto","created_at":"2024-06-26 20:25:02","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":3277968,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMorphological characterization of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eM. bovis\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e biofilm\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Topographical image of M. bovis biofilm was analyzed using a confocal microscope. \u003cem\u003eM. bovis\u003c/em\u003e (Strain M7) was cultured in PPLO broth for 24 hours and then stained with DAPI solution. A representative image is shown. (B) Orthogonal sections showing horizontal (z) and side views (x and y) of three-dimensional biofilm images reconstructed by confocal microscopy. \u003cem\u003eM. bovis\u003c/em\u003e (Strain M7) was stained with LIVE/DEAD BacLight, resulting in live bacteria appearing green and dead bacteria appearing red. A representative image is shown. Scale bar: 50 μm. (C) \u003cem\u003eM. bovis\u003c/em\u003e biofilm (Strain M7) was analyzed by SEM. Scale bar: 20 μm. The red square was magnified in (D) to provide a better view. (D) \u003cem\u003eM. bovis \u003c/em\u003ewas connected by filamentous structures and arranged in a rosary-like formation (yellow arrows). \u003cem\u003eM. bovis\u003c/em\u003e aggregation was observed (red arrowheads). A representative image is shown. Scale bar: 5 μm.\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4523720/v1/3f7cf498bdbc55f2cf54fdb3.jpg"},{"id":59141090,"identity":"36488c96-0287-4489-b424-4cad14ab06db","added_by":"auto","created_at":"2024-06-26 20:25:02","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":615399,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eQuantitative analysis of biofilm formation in co-culture with M. bovis and T. pyogenes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eM. bovis\u003c/em\u003e strains PG45 and M16 were co-cultured with 1 × 10\u003csup\u003e4\u003c/sup\u003e, 1 × 10\u003csup\u003e5\u003c/sup\u003e, 1 × 10\u003csup\u003e6\u003c/sup\u003e, or 1 × 10\u003csup\u003e7\u003c/sup\u003e CFU of \u003cem\u003eT. pyogenes\u003c/em\u003e per well in PPLO broth medium for 24 hours. Biofilms were quantified using crystal violet staining. Gray bars represent \u003cem\u003eM. bovis\u003c/em\u003e in single culture; black bars represent \u003cem\u003eM. bovis \u003c/em\u003eand \u003cem\u003eT. pyogenes\u003c/em\u003e in co-culture; white bars represent \u003cem\u003eT. pyogenes\u003c/em\u003e in single culture. Biofilm biomass was shown as mean OD values ± SE.\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4523720/v1/dc1569081d05d51e1ef8e33f.jpg"},{"id":59141578,"identity":"48786a04-c955-453c-b975-2654183d0c3b","added_by":"auto","created_at":"2024-06-26 20:41:02","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":4596332,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMorphological characterization of biofilm co-cultured with \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eM. bovis\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eT. pyogenes\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e(A and B) \u003cem\u003eM. bovis\u003c/em\u003e (Strain M16) was cultured in PPLO broth medium for 24 hours and then stained with LIVE/DEAD BacLight, which made the live bacteria appear green and the dead bacteria red. Images of orthogonal sections (A) and topographical images (B) were observed using a confocal microscope. A representative image is shown. Scale bar: 50 μm (C and D) \u003cem\u003eM. bovis \u003c/em\u003e(Strain M16) and \u003cem\u003eT. pyogenes \u003c/em\u003e(Strain T1) were co-cultured and stained with the LIVE/DEAD BacLight. Orthogonal sections (C) and topographical images (D) of biofilms were observed using a confocal microscope. A representative image is shown. Scale bar: 50 μm. (E–J) Biofilms of single \u003cem\u003eM. bovis\u003c/em\u003e (E: Strain M16), \u003cem\u003eT. pyogenes\u003c/em\u003e (F), and those co-cultured with these bacteria (G–J) were observed by SEM. Representative images are shown. (G) The red square was magnified in (H) to provide a better view. (H) Under the co-culture condition, \u003cem\u003eM. bovis\u003c/em\u003e (yellow arrowheads) and \u003cem\u003eT. pyogenes \u003c/em\u003e(red arrowheads) were connected. (I) Biofilm of \u003cem\u003eM. bovis\u003c/em\u003e and \u003cem\u003eT. pyogenes \u003c/em\u003eformed a large aggregation. The red square was magnified in (J) to provide a better view. (J) The boundary between the bacteria was obscured (green arrowheads). Scale bar of E–J: 5 μm.\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4523720/v1/96b128963701cdbd6dfe710d.jpg"},{"id":75351248,"identity":"cf55c56f-9674-48ca-8b83-014c55323ae6","added_by":"auto","created_at":"2025-02-03 16:08:26","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":13842934,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4523720/v1/0e529b1f-9b30-41f1-9419-83e653b5dded.pdf"},{"id":59141229,"identity":"14f482dd-43ed-46c7-9976-eb12638062d4","added_by":"auto","created_at":"2024-06-26 20:33:02","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":6764270,"visible":true,"origin":"","legend":"","description":"","filename":"Additionalfile1.docx","url":"https://assets-eu.researchsquare.com/files/rs-4523720/v1/c014cee1907e2c5a17891134.docx"}],"financialInterests":"","formattedTitle":"Biofilm characterization of Mycoplasma bovis co-cultured with Trueperella pyogenes","fulltext":[{"header":"Introduction","content":"\u003cp\u003e \u003cem\u003eMycoplasma bovis\u003c/em\u003e (\u003cem\u003eM. bovis\u003c/em\u003e) is a pathogen that causes mastitis (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e), pneumonia (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e), arthritis (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e), and otitis media (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e) in dairy cattle. \u003cem\u003eMycoplasma\u003c/em\u003e pneumonia caused by \u003cem\u003eM. bovis\u003c/em\u003e is associated with severe inflammatory reactions in the trachea and lungs, can be difficult to treat with antibiotics, and may result in substantial economic losses in dairy and beef farms (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). \u003cem\u003eM. bovis\u003c/em\u003e is an important etiological agent of the bovine respiratory disease complex (BRDC). BRDC is caused by an interaction between viral and bacterial pathogens such as bovine herpesvirus, bovine viral diarrhea virus, \u003cem\u003ePasteurella multocida\u003c/em\u003e, \u003cem\u003eMannheimia haemolytica\u003c/em\u003e, and \u003cem\u003eTrueperella pyogenes\u003c/em\u003e (\u003cem\u003eT. pyogenes\u003c/em\u003e) (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). Each pathogen has unique features, such as the suppression of the protective barrier function of the respiratory epithelium or the immune response in leukocytes (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e), resulting in chronic pneumonia.\u003c/p\u003e \u003cp\u003e \u003cem\u003eT. pyogenes\u003c/em\u003e is part of the biota of the skin and mucous membranes of the upper respiratory, gastrointestinal, and urogenital tracts of animals and is also an opportunistic pathogen (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). \u003cem\u003eT. pyogenes\u003c/em\u003e is involved in polymicrobial diseases, including mastitis, uterine infections, and pneumonia (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). Therefore, this bacterium is often isolated from a mixed infection of various bacterial species. The expression levels of virulence genes, including plo, fimA, nanH, and cbpA, in \u003cem\u003eT. pyogenes\u003c/em\u003e isolates in co-culture with \u003cem\u003eFusobacterium necrophorum\u003c/em\u003e (\u003cem\u003eF. necrophorum\u003c/em\u003e) and \u003cem\u003eEscherichia coli\u003c/em\u003e (\u003cem\u003eE. coli\u003c/em\u003e) were increased (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). It means that polymicrobial infection intensifies the virulence of \u003cem\u003eT. pyogenes.\u003c/em\u003e A previous study reported that not only \u003cem\u003eM. bovis\u003c/em\u003e but also T. \u003cem\u003epyogenes\u003c/em\u003e were isolated from lesions of chronic caseous pneumonia in cattle (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). It is speculated that co-infection with \u003cem\u003eM. bovis\u003c/em\u003e and \u003cem\u003eT. pyogenes\u003c/em\u003e induces a more severe inflammatory reaction in respiratory tissues than a single infection, but the detailed mechanism is unknown.\u003c/p\u003e \u003cp\u003eBiofilms are communities of microorganisms that are attached to biotic or abiotic surfaces (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). Biofilms play a significant role in the persistence of bacteria and contribute to chronic lesions because it is difficult for antibiotics and immune responses to reach bacteria within biofilms (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). The structure of the extracellular polymeric substance (EPS) matrix of the biofilm consists of extracellular polysaccharides, DNA, and proteins (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). A recent study showed that EPS material, shared by multiple pathogens in co-culture, facilitates interspecies interactions through the formation of compact microcolony structures during biofilm formation (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). The interaction between \u003cem\u003eStaphylococcus aureus\u003c/em\u003e (\u003cem\u003eS. aureus\u003c/em\u003e) and \u003cem\u003eCandida albicans\u003c/em\u003e (\u003cem\u003eC. albicans\u003c/em\u003e) demonstrates synergistic activity, significantly enhancing biofilm formation and contributing to an increase in antimicrobial resistance in \u003cem\u003eS. aureus\u003c/em\u003e (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). Therefore, the polymicrobial interaction between species are an important factor for biofilm formation.\u003c/p\u003e \u003cp\u003e \u003cem\u003eM. bovis\u003c/em\u003e is known to form biofilms, even though it has very limited genes (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). A previous study showed that the morphological characterization of \u003cem\u003eM. bovis\u003c/em\u003e biofilm on the plate and its formation potential may be associated with the expression of an adhesion factor (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). However, \u003cem\u003eM. bovis\u003c/em\u003e biofilms have never been observed \u003cem\u003ein vivo\u003c/em\u003e, and the effect of polymicrobial relationships on biofilm formation is unknown. In this study, we analyzed the morphological characteristics of \u003cem\u003eM. bovis\u003c/em\u003e biofilm in spontaneous \u003cem\u003eMycoplasma\u003c/em\u003e pneumonia in calves. Additionally, we examined the characterization of the polymicrobial relationship between \u003cem\u003eM. bovis\u003c/em\u003e and \u003cem\u003eT. pyogenes\u003c/em\u003e during biofilm formation to clarify its relevance to the development of pneumonia.\u003c/p\u003e"},{"header":"Material and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003eAnimal\u003c/h2\u003e\n \u003cp\u003eTwo Holstein calves, aged 2 months (Calf 1 and Calf 2), with chronic \u003cem\u003eMycoplasma\u003c/em\u003e pneumonia, and two control Holstein calves without clinical respiratory symptoms, aged 1 and 2 months, in Hokkaido, Japan, underwent pathological autopsies in accordance with the Guide for the Care and Use of Laboratory Animals of the School of Veterinary Medicine at Rakuno Gakuen University in 2021. Two calves with chronic \u003cem\u003eMycoplasma\u003c/em\u003e pneumonia also had arthritis and showed lameness. Tracheal and caseous necrotic foci were obtained from the lobe and subjected to PCR analyses using \u003cem\u003eM. bovis\u003c/em\u003e\u0026ndash;specific primers, as previously described (\u003cspan class=\"CitationRef\"\u003e16\u003c/span\u003e). Additionally, swabs from these samples were cultured on blood agar plates (Eiken Kagaku, Tokyo, Japan) and incubated for 48 hours at 37\u0026deg;C. The bacterial colony obtained was identified by its 16S ribosomal RNA gene sequence as previously described (\u003cspan class=\"CitationRef\"\u003e17\u003c/span\u003e). The sequence of the 16S 27F forward primer was 5\u0026prime;-AGAGTTTGATCCTGGCTCAG-3\u0026prime;, and the 1492R reverse primer was 5\u0026prime;-GGTTACCTTGTTACGACTT-3\u0026prime;. PCR products were extracted using the FastGene Gel/PCR Extraction Kit (Nippon Genetics, Tokyo, Japan). The product sequence was analyzed by Hokkaido System Science Co., Ltd. The sequence data was confirmed through database analysis using the Basic Local Alignment Search Tool.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\n \u003ch2\u003eBacterial strains\u003c/h2\u003e\n \u003cp\u003eThe sixteen \u003cem\u003eM. bovis\u003c/em\u003e strains and two \u003cem\u003eT. pyogenes\u003c/em\u003e strains used in this study are listed in Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e. Before use, \u003cem\u003eM. bovis\u003c/em\u003e and \u003cem\u003eT. pyogenes\u003c/em\u003e strains were cultured in modified PPLO medium (Kanto Kagaku, Tokyo, Japan) and brain heart infusion supplemented with 5% fetal bovine serum (FBS) and then stored at \u0026minus;\u0026thinsp;80\u0026deg;C.\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003e\u003cstrong\u003eInformation regarding the bacterial strains.\u003c/strong\u003e\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"4\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSpecies\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eStrain\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eOrigin\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eDisease\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eM. bovis\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePG45\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eATCC 25523\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eStrain M1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNosal cavity\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePneumonia\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eStrain M2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNosal cavity\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePneumonia\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eStrain M3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNosal cavity\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePneumonia\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eStrain M4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNosal cavity\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePneumonia\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eStrain M5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNosal cavity\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePneumonia\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eStrain M6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNosal cavity\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePneumonia\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eStrain M7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSynovial fluids\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eArthritis\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eStrain M8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSynovial fluids\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eArthritis\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eStrain M9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMilk\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMastitis\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eStrain M10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMilk\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMastitis\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eStrain M11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLung\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePneumonia\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eStrain M12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHeart\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eEndcarditis\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eStrain M13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHeart\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eEndcarditis\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eStrain M14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHeart\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eEndcarditis\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eStrain M15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHeart\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eEndcarditis\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eStrain M16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLung (\u003cem\u003eMycoplasma\u003c/em\u003e pneumonia Calf1)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePneumonia\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eT. pyogenes\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eStrain T1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLung (\u003cem\u003eMycoplasma\u003c/em\u003e pneumonia Calf1)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePneumonia\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eStrain T2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLung (\u003cem\u003eMycoplasma\u003c/em\u003e pneumonia Calf2)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePneumonia\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\n \u003ch2\u003eLight microscopy and immunohistochemical analysis\u003c/h2\u003e\n \u003cp\u003eTracheal tissues were fixed in a 4% paraformaldehyde solution, dehydrated through an ethanol gradient from 70\u0026ndash;100%, and then embedded in paraffin. After deparaffinization using xylene, the tissue sections were stained with hematoxylin and eosin, and then observed under a light microscope. Immunohistochemical staining was performed using indirect immunofluorescence analysis. The sections were heated in a microwave oven in the presence of 0.01 M sodium citrate buffer (pH 6.0) for 15 minutes and then immersed in a 3% hydrogen peroxide solution at room temperature for 10 minutes. After pretreatment, the sections were incubated with 5% normal goat serum at room temperature for 20 minutes as a blocking step. Subsequently, the sections were incubated with anti-cytokeratin18 (Proteintech, Chicago, IL, USA) and anti-\u003cem\u003eM. bovis\u003c/em\u003e (Millipore, Billerica, MA, USA) antibodies at room temperature for 2 hours, and then incubated with rhodamine-conjugated goat anti-rabbit IgG antibody (Thermo Fisher Scientific, Waltham, MA, USA) and Alexa Fluor 488-conjugated goat anti-mouse IgG antibody (Thermo Fisher Scientific) at room temperature for 1 hour. The sections were stained with DAPI solution (DOJINDO, Kumamoto, Japan) at room temperature for 10 minutes and visualized using a Nikon C2 laser confocal microscope (Nikon, Tokyo, Japan). The obtained images were analyzed using NIS Elements AR Analysis and Fiji (a distribution of ImageJ software from the US National Institutes of Health, Bethesda, Maryland, USA) according to the following methods (\u003cspan class=\"CitationRef\"\u003e18\u003c/span\u003e).\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eBiofilm formation of\u003c/strong\u003e \u003cstrong\u003eM. bovis\u003c/strong\u003e \u003cstrong\u003esingle culture\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eBiofilm formation was performed with modifications as previously described (\u003cspan class=\"CitationRef\"\u003e15\u003c/span\u003e). Store five strains of \u003cem\u003eM. bovis\u003c/em\u003e (PG45, Strains M1\u0026ndash;M4) were cultured in modified PPLO medium at 37\u0026deg;C for 24 hours without aeration. After culturing, 10 \u0026micro;L of planktonic \u003cem\u003eM. bovis\u003c/em\u003e was inoculated into a non-coated 96-well cell culture plate (NIPPON Genetics) in triplicate. The culture medium used for biofilm formation was modified PPLO medium (PPLO rich), not modified PPLO broth medium (not adding horse serum and yeast extract, et al; PPLO broth: Kanto Kagaku), mueller hinton (MH: Beckton Dickinson, Franklin Lakes, NJ, USA), lysogeny broth (LB: Beckton Dickinson), brain heart infusion (BHI: Beckton Dickinson), trypticase soy broth (TSB: Beckton Dickinson), todd hewitt broth (THB: Kanto kagaku), Dulbecco\u0026rsquo;s modified Eagle\u0026rsquo;s medium (DMEM: Fujifilm Wako (Osaka, Japan), DMEM supplemented with 5% FBS, and Roswell Park Memorial Institute 1640 medium (RPMI: Fujifilm Wako), and RPMI supplemented with 5% FBS to determine the appropriate medium. These media were added at 190 \u0026micro;L per well in a 96-well plate. The planktonic \u003cem\u003eM. bovis\u003c/em\u003e culture was then diluted 1:20 and incubated at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e for 24 hours without aeration.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eBiofilm formation in co-culture with\u003c/strong\u003e \u003cstrong\u003eM. bovis\u003c/strong\u003e \u003cstrong\u003eand\u003c/strong\u003e \u003cstrong\u003eT. pyogenes\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eStored \u003cem\u003eM. bovis\u003c/em\u003e was cultured in modified PPLO medium at 37\u0026deg;C for 24 hours, and then 10 \u0026micro;L of this cultured planktonic \u003cem\u003eM. bovis\u003c/em\u003e was inoculated into a non-coated 96-well plate (NIPPON Genetics). Stored \u003cem\u003eT. pyogenes\u003c/em\u003e was centrifuged (7000 rpm, 5 min, 4\u0026deg;C) and suspended in PPLO broth medium. \u003cem\u003eT. pyogenes\u003c/em\u003e was inoculated at concentrations of 1 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e, 10\u003csup\u003e5\u003c/sup\u003e, 10\u003csup\u003e6\u003c/sup\u003e, and 10\u003csup\u003e7\u003c/sup\u003e colony-forming units (CFU) per 10 \u0026micro;L into a 96-well plate. PPLO broth medium was added at 180 \u0026micro;L per well in a 96-well plate and incubated at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e for 24 hours without aeration.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\n \u003ch2\u003eCrystal violet staining\u003c/h2\u003e\n \u003cp\u003eThe biofilm in the 96-well plate was washed three times with phosphate-buffered saline (PBS) to remove planktonic cells and then fixed with 99.5% methanol for 15 minutes. The biofilm was stained with a 2% crystal violet solution for 20 minutes and rinsed three times with distilled water. The plate was dried and then decolorized with 200 \u0026micro;L of 99.5% ethanol to release the crystal violet. The released crystal violet was quantified using a microplate reader (BIO-RAD, Hercules, CA, USA) by measuring the absorbance at 595 nm.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\n \u003ch2\u003eScanning electron microscope\u003c/h2\u003e\n \u003cp\u003eCultured planktonic \u003cem\u003eM. bovis\u003c/em\u003e was prepared as previously described and inoculated with 300 \u0026micro;L into a 35 mm non-coated single culture dish (IWAKI, Chiba, Japan) with 2700 \u0026micro;L of PPLO broth medium. \u003cem\u003eT. pyogenes\u003c/em\u003e was inoculated at 3 \u0026times; 10\u003csup\u003e8\u003c/sup\u003e CFU/well and incubated at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e for 24 hours without aeration. Cultured bacterial biofilms and tracheal tissues isolated from pneumonia-affected and control calves were fixed with half-strength Karnovsky\u0026rsquo;s solution at 4\u0026deg;C overnight. The tissue samples were post-fixed with 1% osmium tetroxide for 30 minutes. The tissues and biofilm samples were then washed with 0.1 M cacodylate buffer and dehydrated through an ethanol gradient ranging from 30\u0026ndash;100% (with concentrations at 30%, 50%, 70%, 80%, 90%, 95%, and 100%), spending 10 minutes at each step. After being dried using the t-butyl alcohol freeze-drying method, the specimens were coated with Pt\u0026ndash;Pd and observed under a HITACHI S-2460N electron microscope at 8 kV.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n \u003ch2\u003eConfocal microscope\u003c/h2\u003e\n \u003cp\u003eCultured plankonic \u003cem\u003eM. bovis\u003c/em\u003e was prepared as previously described above and inoculated with 60 \u0026micro;L into a 24-well non-coated culture dish (IWAKI, Chiba, Japan) containing a 12 mm round cover glass and 540 \u0026micro;L of PPLO broth medium. \u003cem\u003eT. pyogenes\u003c/em\u003e was inoculated at 6 \u0026times; 10\u003csup\u003e7\u003c/sup\u003e CFU/well and incubated at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e for 24 hours without aeration. The bacterial biofilm on the round cover glass was stained with DAPI solution (DOJINDO) or the LIVE/DEAD\u0026reg; BacLight\u0026trade; Bacterial Viability Kit (Thermo Fisher Scientific) and then washed three times with PBS. The samples were fixed with a 4% paraformaldehyde solution and observed using a Nikon C2 laser confocal microscope. The images obtained were analyzed using NIS Elements AR Analysis and Fiji.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\n \u003ch2\u003eStatistical analysis\u003c/h2\u003e\n \u003cp\u003eData are expressed as the means\u0026thinsp;\u0026plusmn;\u0026thinsp;SE. The Steel method was used for comparisons between different groups using the statistical analysis program MEPHAS (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.gen-info.osaka-u.ac.jp/MEPHAS/\u003c/span\u003e\u003c/span\u003e). In all cases, a probability (\u003cem\u003ep\u003c/em\u003e) value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered to indicate a statistically significant difference.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003ePathological findings and morphological analysis of\u003c/b\u003e \u003cb\u003eMycoplasma\u003c/b\u003e \u003cb\u003epneumonia in calves\u003c/b\u003e\u003c/p\u003e \u003cp\u003eA pathological autopsy was performed on calves affected by \u003cem\u003eMycoplasma\u003c/em\u003e pneumonia to evaluate the formation of \u003cem\u003eM. bovis\u003c/em\u003e biofilm \u003cem\u003ein vivo\u003c/em\u003e. The calf with \u003cem\u003eMycoplasma\u003c/em\u003e pneumonia (Calf 1) showed coagulation and caseous necrotic foci in the right cranial part and middle lobe (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA and B). Caseous discharge was observed on the cut surface of the right cranial region (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Tracheal tissues were obtained from control calves with no clinical respiratory symptoms and those with \u003cem\u003eMycoplasma\u003c/em\u003e pneumonia. \u003cem\u003eM. bovis\u003c/em\u003e and \u003cem\u003eT. pyogenes\u003c/em\u003e were detected in tracheal mucosa swabs and caseous necrotic foci in the lungs by PCR. Tracheal tissues were stained with hematoxylin and eosin (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD and E). Cilia were aligned on the epithelial cells of the tracheal mucosa in control calves, while in calves with \u003cem\u003eMycoplasma\u003c/em\u003e pneumonia, they were not located. Tissues from the trachea of control calves and those with \u003cem\u003eMycoplasma\u003c/em\u003e pneumonia were stained with cytokeratin-18 antibody, \u003cem\u003eM. bovis\u003c/em\u003e antibody, and DAPI solution, and analyzed using a fluorescence microscope (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF\u0026ndash;M). Areas positive for \u003cem\u003eM. bovis\u003c/em\u003e were detected in the epithelial cells of tracheal tissues of calves with \u003cem\u003eMycoplasma\u003c/em\u003e pneumonia (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eI and M). Micromorphological characterization of tracheal mucosa of control calves and those with \u003cem\u003eMycoplasma\u003c/em\u003e pneumonia was analyzed by SEM (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eN\u0026ndash;Q). The surface of the tracheal mucosa of control calves was completely covered with cilia (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eN and O). \u003cem\u003eMycoplasma\u003c/em\u003e pneumonia in calves showed loss of cilia on the tracheal mucosa (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eP). Bacterium-like aggregation structures (\u0026gt;\u0026thinsp;10 \u0026micro;m) were observed adhering to cilia in a calves with \u003cem\u003eMycoplasma\u003c/em\u003e pneumonia (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eP and Q, yellow arrowheads). Bacteria were detected on the cilia of the tracheal mucosa (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eQ, indicated by red arrows). Additionally, the boundaries between bacteria were obscured, and bacterial aggregation structures were observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eQ, indicated by green arrows). These characteristic structures were also observed in the control and the one with \u003cem\u003eMycoplasma\u003c/em\u003e pneumonia (Calf 2; Additional File 1).\u003c/p\u003e \u003cp\u003e \u003cb\u003eQuantitative analysis of\u003c/b\u003e \u003cb\u003eM. bovis\u003c/b\u003e \u003cb\u003ebiofilm formation\u003c/b\u003e\u003c/p\u003e \u003cp\u003eWe examined the appropriate medium to evaluate biofilm formation of \u003cem\u003eM. bovis\u003c/em\u003e. Five strains of \u003cem\u003eM. bovis\u003c/em\u003e were cultured in 96-well microplates across eleven culture media for 24 hours and then stained with crystal violet (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Biofilm from five \u003cem\u003eM. bovis\u003c/em\u003e strains was not detected in the PPLO broth, which is a major growth medium for Mycoplasmas. In contrast, cultures in PPLO broth medium showed the highest level of biofilm formation among eleven culture media. Biofilm formation of five strains of \u003cem\u003eM. bovis\u003c/em\u003e in DMEM or RPMI was higher than in DMEM\u0026thinsp;+\u0026thinsp;FBS or RPMI\u0026thinsp;+\u0026thinsp;FBS, respectively. Therefore, in subsequent experiments, PPLO broth medium was used to evaluate biofilm formation of \u003cem\u003eM. bovis\u003c/em\u003e. Sixteen \u003cem\u003eM. bovis\u003c/em\u003e strains were cultured in PPLO broth medium and stained with crystal violet (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). PG45 was shown to be a moderate biofilm producer (OD value was 0.26\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04). No significant difference was found between PG45 and fifteen wild strains. However, Strains M1, M6, M7, and M8 were shown to be strong biofilm producers (OD value\u0026thinsp;\u0026gt;\u0026thinsp;0.3). On the other hand, Strains M5, M11, M12, and M14 were shown to be weak biofilm producers (OD value\u0026thinsp;\u0026lt;\u0026thinsp;0.2). It is shown that the ability to form biofilms differed depending on the strains.\u003c/p\u003e \u003cp\u003e \u003cb\u003eMorphological characterization of\u003c/b\u003e \u003cb\u003eM. bovis\u003c/b\u003e \u003cb\u003ebiofilm in single culture\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe biofilm of Strain M7, a strong biofilm producer, was stained with DAPI solution and observed using a confocal microscope (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). \u003cem\u003eM. bovis\u003c/em\u003e biofilms were observed as aggregations of various sizes and scattered formations. The biofilm was stained with LIVE/DEAD BacLight and visualized in orthogonal sections (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Biofilm was formed by live \u003cem\u003eM. bovis\u003c/em\u003e and there were almost no dead bacteria. The micromorphology of the biofilm was evaluated by SEM (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC and D). A single bacterium adhering to the plate and approximately 10 \u0026micro;m bacterial aggregates were observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD, red arrowheads). \u003cem\u003eM. bovis\u003c/em\u003e cells were connected by filamentous structures and arranged in a rosary-like formation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD, indicated by yellow arrows). The observation of aggregation and concatenated bacterial structures in these strains, which showed moderate levels of biofilm formation, was lower than that in Strain M7 (Additional File 2).\u003c/p\u003e \u003cp\u003e \u003cb\u003eQuantitative analysis of biofilm formation in co-culture with\u003c/b\u003e \u003cb\u003eM. bovis\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003eT. pyogenes\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003cem\u003eM. bovis\u003c/em\u003e strains PG45 and M16: isolated from Calf 1) and \u003cem\u003eT. pyogenes\u003c/em\u003e (Strain T1: isolated from Calf 1) were co-cultured in a 96-well microplate using PPLO-based medium for 24 hours and then stained with crystal violet (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Biofilm formations of PG45, when co-cultured with 1 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e, 1 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e, or 1 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e CFU/well of \u003cem\u003eT. pyogenes\u003c/em\u003e (Strain T1), tended to be higher (OD values were 0.21\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03, 0.21\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03, and 0.19\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03, respectively) than those of the single culture (0.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01). Biofilm formations of Strain M16, when co-cultured with 1 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e, 1 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e, or 1 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e CFU/well of \u003cem\u003eT. pyogenes\u003c/em\u003e, tended to be higher (OD values were 0.42\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07, 0.42\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06, and 0.42\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03, respectively) than those of the single culture (0.27\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04). \u003cem\u003eT. pyogenes\u003c/em\u003e in single culture was shown to be a weak biofilm producer (OD value\u0026thinsp;≦\u0026thinsp;0.1). However, biofilm formation of PG45 and Strain M16, when co-cultured with 1 \u0026times; 10\u003csup\u003e7\u003c/sup\u003e CFU of \u003cem\u003eT. pyogenes\u003c/em\u003e (OD values were 0.12\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03 and 0.24\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04, respectively), tended to be lower than when co-cultured with 1 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e, 1 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e, or 1 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e CFU/well of \u003cem\u003eT. pyogenes\u003c/em\u003e. Quantitative analysis of biofilm formation co-cultured with \u003cem\u003eM. bovis\u003c/em\u003e (PG45 and Strain M16) and \u003cem\u003eT. pyogenes\u003c/em\u003e (Strain T2, isolated from Calf 2) is presented in Additional File 3. Biofilm formations of PG45, when co-cultured with \u003cem\u003eT. pyogenes\u003c/em\u003e (Strain T2) showed no difference, while those of Strain M16 co-cultured with 1 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e, 1 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e, or 1 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e CFU/well of \u003cem\u003eT. pyogenes\u003c/em\u003e tended to be higher than in single culture. The biofilm formation of Strain M16, when co-cultured with 1 \u0026times; 10\u003csup\u003e7\u003c/sup\u003e CFU of \u003cem\u003eT. pyogenes\u003c/em\u003e (Strain T2), also tended to be lower than when co-cultured with 1 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e or 1 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e CFU/well of \u003cem\u003eT. pyogenes\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eMorphological characterization of biofilm in co-culture with\u003c/b\u003e \u003cb\u003eM. bovis\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003eT. pyogenes\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe biofilm of \u003cem\u003eM. bovis\u003c/em\u003e (Strain M16) was stained with LIVE/DEAD BacLight and observed using a confocal microscope (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA and B). A single culture of \u003cem\u003eM. bovis\u003c/em\u003e showed aggregations of biofilm composed of live bacteria. On the other hand, the biofilm co-cultured with \u003cem\u003eM. bovis\u003c/em\u003e and \u003cem\u003eT. pyogenes\u003c/em\u003e were formed with both live and dead bacteria, with the surface of the biofilm especially covered in dead bacteria (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC and D). The biofilm formation of single \u003cem\u003eM. bovis\u003c/em\u003e (Strain M16), \u003cem\u003eT. pyogenes\u003c/em\u003e (Strain T1), and the co-culture of these bacteria was analyzed by SEM (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE\u0026ndash;H). \u003cem\u003eM. bovis\u003c/em\u003e biofilms were observed as aggregations of varying sizes, which were formed through adhesion between neighboring bacteria (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE), whereas no biofilm formation by \u003cem\u003eT. pyogenes\u003c/em\u003e and only minor conjugation of bacterial cells were observed in single culture (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF). In co-culture, \u003cem\u003eM. bovis\u003c/em\u003e and \u003cem\u003eT. pyogenes\u003c/em\u003e adhered, and there was no bacterium with cell-wall damage or erosion (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG and H). Interestingly, some large aggregates of bacteria (\u0026gt;\u0026thinsp;40 \u0026micro;m), consisting of \u003cem\u003eM. bovis\u003c/em\u003e and \u003cem\u003eT. pyogenes\u003c/em\u003e, were observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eI). This aggregation was not observed in single cultures of \u003cem\u003eM. bovis\u003c/em\u003e and \u003cem\u003eT. pyogenes\u003c/em\u003e. The boundary between aggregating bacteria was obscure (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eJ, indicated by green arrowheads).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003e \u003cem\u003eM. bovis\u003c/em\u003e causes chronic pneumonia in calves, which is difficult to treat with antibiotics. It is known that bacteria in biofilms can become 10 to 1000 times more resistant to antimicrobial agents compared to planktonic bacteria (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). A previous study reported that distortion of fimbriae and edema of attached cells were observed in the tracheal tissues of calves experimentally infected with \u003cem\u003eM. bovis\u003c/em\u003e, and in some cases, \u003cem\u003eM. bovis\u003c/em\u003e was found residing in both ciliated and non-ciliated epithelial cells (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). However, biofilm formation of \u003cem\u003eM. bovis in vivo\u003c/em\u003e has not been observed. In this study, we performed pathological autopsies on calves naturally infected with chronic \u003cem\u003eMycoplasma\u003c/em\u003e pneumonia and evaluated the biofilm formation of \u003cem\u003eM. bovis\u003c/em\u003e. Calf 1 and Calf 2 showed pathological characteristics, which included coagulation and caseous necrosis foci in the lungs. \u003cem\u003eT. pyogenes\u003c/em\u003e is an important secondary pathogen and is most commonly isolated from lung lesions in BRDC (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). Co-infection with \u003cem\u003eM. bovis\u003c/em\u003e and \u003cem\u003eT. pyogenes\u003c/em\u003e has also been identified as a risk factor for severe BRDC (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). In this study, not only \u003cem\u003eM. bovis\u003c/em\u003e but also \u003cem\u003eT. pyogenes\u003c/em\u003e were isolated as dominant bacteria from the trachea and lungs, suggesting that these pathogens were major factors causing lesions. We conducted a morphological analysis of the trachea from calves with pneumonia. Cilia were not detected on the epithelial cells of the tracheal mucosa in calves with \u003cem\u003eMycoplasma\u003c/em\u003e pneumonia. Additionally, \u003cem\u003eM. bovis\u003c/em\u003e antigen was detected in the epithelial cells of the trachea using a fluorescent microscope. It has been reported that the production of hydrogen peroxide and other reactive oxygen species (ROS) may be important factors in the pathogenicity of mycoplasmas, as it causes damage to cell membranes (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). The genes involved in the production of hydrogen peroxide and ROS were encoded in the genome of commensal mycoplasma (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). \u003cem\u003eM. bovis\u003c/em\u003e produces hydrogen peroxide, causing damage to epithelial cells under both \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e conditions (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). Exposure to high concentrations of hydrogen peroxide inhibited tracheal ciliary movement and induced apoptosis in epithelial cells (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). This damage to the respiratory mucosa reduces the immune barrier and induces a local inflammatory response. We suggested that \u003cem\u003eM. bovis\u003c/em\u003e removes cilia from the trachea by producing hydrogen peroxide, causing a decline in tracheal immunity. Interestingly, bacterial aggregation structures were observed adhering to cilia from calves with \u003cem\u003eMycoplasma\u003c/em\u003e pneumonia in this study. It has been reported that large, mature biofilm structures, where networks of bacteria were embedded within structured matrices, were observed in the nasopharyngeal tissue of mice experimentally infected with \u003cem\u003eStreptococcus pneumoniae\u003c/em\u003e (\u003cem\u003eS. pneumoniae\u003c/em\u003e) (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). This large biofilm structure of \u003cem\u003eS. pneumoniae\u003c/em\u003e was associated with increased resistance to antibiotics. In this study, it was assumed that the bacterial aggregation structures were biofilms of \u003cem\u003eM. bovis in vivo\u003c/em\u003e and associated with resistance to antibiotics in \u003cem\u003eMycoplasma\u003c/em\u003e pneumonia in calves. \u003cem\u003eT. pyogenes\u003c/em\u003e was also isolated from the trachea in calves with pneumonia, and it was thought to be involved in the biofilm formation of \u003cem\u003eM. bovis\u003c/em\u003e. Thus, we examined biofilm formation during co-cultivation of \u003cem\u003eM. bovis\u003c/em\u003e and \u003cem\u003eT. pyogenes in vitro\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eIt was previously reported that the methods for evaluating \u003cem\u003eM. bovis\u003c/em\u003e biofilms were unstable (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e), and we were unable to reproduce them. Initially, we identified the most suitable medium to assess the biofilm formation of \u003cem\u003eM. bovis.\u003c/em\u003e Biofilm formation of \u003cem\u003eM. bovis\u003c/em\u003e cultured in PPLO broth medium was higher than that in other media. Additionally, DMEM and RPMI promoted biofilm formation more than DMEM FBS and RPMI FBS, respectively. A previous study showed that \u003cem\u003eM. bovis\u003c/em\u003e biofilm was evaluated using the \u0026ldquo;PPLO rich\u0026rdquo; growth medium (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). However, in our study, biofilm formation of \u003cem\u003eM. bovis\u003c/em\u003e was the lowest in PPLO-rich medium, while the PPLO broth medium, which did not have added nutrients for growth such as horse serum and yeast extract, showed the highest biofilm formation. Generally, biofilm production increases in environments where bacterial growth is difficult (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). Since bovine serum is rich in nutritional factors essential for the growth of \u003cem\u003eM. bovis\u003c/em\u003e, it is suggested that adding serum to DMEM or RPMI reduces biofilm formation. Thus, our data suggested that a PPLO broth without added nutrients for growth was suitable for evaluating the biofilm formation of \u003cem\u003eM. bovis\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eWe evaluated the biofilm formation of sixteen species, including \u003cem\u003eM. bovis\u003c/em\u003e in PPLO broth medium. Strains M1, M6, M7, and M8 were strong biofilm producers, while Strains M5, M11, M12, and M14 were weak. Biofilm formation is characterized by three stages: attachment, maturation, and dispersion (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). The attachment is a crucial initial step in biofilm formation. \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e (\u003cem\u003eP. aeruginosa\u003c/em\u003e) uses flagella for movement (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e), and \u003cem\u003eS. aureus\u003c/em\u003e adheres to host-derived EPS for attachment (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). Previous studies have indicated that the variable surface proteins B and O type, which are among the adhesion factors expressed by the wild strain \u003cem\u003eM. bovis\u003c/em\u003e, form prolific biofilms (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). The difference in biofilm production ability among \u003cem\u003eM. bovis\u003c/em\u003e strains was thought to be due to the expression levels of various adhesion factors. In this study, \u003cem\u003eM. bovis\u003c/em\u003e biofilms were observed as aggregations of varying sizes and scattered formations, consisting of live bacteria, using a confocal microscope. Additionally, \u003cem\u003eM. bovis\u003c/em\u003e was observed to have a moniliform structure bound together by filamentous material, using a electron microscope. It has been observed that a thick filamentous matrix connects bacterial cells in \u003cem\u003eClostridium difficile\u003c/em\u003e (\u003cem\u003eC. difficile\u003c/em\u003e) and \u003cem\u003eS. aureus\u003c/em\u003e biofilms (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e). This matrix was assumed to be ECM released by bacteria (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e). The filamentous matrix in biofilms connects neighboring groups of bacteria, which is an initial step in aggregation (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e). Nuclease treatment inhibited \u003cem\u003eMycoplasma hyopneumoniae\u003c/em\u003e (\u003cem\u003eM. hyopneumoniae\u003c/em\u003e) biofilm formation, suggesting that extracellular DNA released outside the cell is a crucial step for biofilm formation (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e). Although it has been reported that the \u003cem\u003eM. bovis\u003c/em\u003e biofilm consists of viable bacteria, a detailed morphological analysis has yet to be conducted (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). We observed aggregations of bacteria and moniliform structures between \u003cem\u003eM. bovis\u003c/em\u003e cells, suggesting that \u003cem\u003eM. bovis\u003c/em\u003e also released ECM or DNA to connect to neighboring bacteria, and then \u003cem\u003eM. bovis\u003c/em\u003e formed compact microcolonies and biofilm.\u003c/p\u003e \u003cp\u003e \u003cem\u003eM. bovis\u003c/em\u003e and \u003cem\u003eT. pyogenes\u003c/em\u003e are each a pathogen that causes pneumonia in cattle, and it is not uncommon for both bacteria to be detected from the same lesion, as in this study (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e). The expression levels of multiple virulence genes in \u003cem\u003eT. pyogenes\u003c/em\u003e were increased upon co-infection with \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eF. necrophorum\u003c/em\u003e in mice (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). Co-infection is assumed to affect biofilm formation in pathogens, and elucidating this process is important for understanding the pathology of pneumonia. Thus, we examined the polymicrobial relationship between \u003cem\u003eM. bovis\u003c/em\u003e and \u003cem\u003eT. pyogenes\u003c/em\u003e during biofilm formation. Biofilm formation of \u003cem\u003eM. bovis\u003c/em\u003e (PG45 and Strain M16) co-cultured with 1 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e, 1 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e, and 1 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e CFU/well of \u003cem\u003eT. pyogenes\u003c/em\u003e (Strain T1 and T2) was synergistically increased, while that with 1 \u0026times; 10\u003csup\u003e7\u003c/sup\u003e CFU/well of \u003cem\u003eT. pyogenes\u003c/em\u003e was lower than with 1 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e and 1 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e CFU/well of \u003cem\u003eT. pyogenes.\u003c/em\u003e The biofilm resulting from co-cultivation with \u003cem\u003eM. bovis\u003c/em\u003e (Strain M16) and \u003cem\u003eT. pyogenes\u003c/em\u003e was composed of both live and dead bacteria, with a higher proportion of dead bacteria compared to single cultures. \u003cem\u003eS. aureus\u003c/em\u003e-induced apoptosis in \u003cem\u003eC. albicans\u003c/em\u003e was characterized by features such as intracytoplasmic disorganization, cell membrane discontinuity, vacuole formation, and chromatin condensation. (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e). The induction of apoptosis was not due to cell-to-cell contact but rather to the presence of the supernatant of \u003cem\u003eS. aureus\u003c/em\u003e. Our study showed that a co-cultivation condition induced bacterial death in \u003cem\u003eM. bovis\u003c/em\u003e, \u003cem\u003eT. pyogenes\u003c/em\u003e, or both bacteria, which was notably observed when the number of \u003cem\u003eT. pyogenes\u003c/em\u003e was high. However, adherence of \u003cem\u003eM. bovis\u003c/em\u003e (Strain M16) and \u003cem\u003eT. pyogenes\u003c/em\u003e to each other and the formation of large bacterial aggregates were observed by SEM. \u003cem\u003eS. aureus\u003c/em\u003e caused the death of \u003cem\u003eAspergillus fumigatus\u003c/em\u003e, but in co-cultivation, the morphological characteristics of the biofilm produced by these bacteria were similar to those observed \u003cem\u003ein vivo\u003c/em\u003e (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e). Additionally, eDNA acted as a crucial EPS component shared by \u003cem\u003eS. aureus\u003c/em\u003e and \u003cem\u003eP. aeruginosa\u003c/em\u003e in co-culture biofilms, facilitating interspecies interactions by promoting the formation of compact microcolony structures during biofilm development (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). This cell-to-cell communication is called quorum sensing, which is a major coordination factor in biofilm formation (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e). A previous study indicated that \u003cem\u003eM. bovis\u003c/em\u003e has a very limited genome but can produce biofilms that are helpful for surviving environmental stress (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). Our study showed that the combination of \u003cem\u003eM. bovis\u003c/em\u003e and \u003cem\u003eT. pyogenes\u003c/em\u003e led to the formation of a large microcolony, which is assumed to be associated with quorum sensing. This data indicated that \u003cem\u003eM. bovis\u003c/em\u003e collaborated with other bacteria to form a mature biofilm, leading to increased antimicrobial resistance in the tracheal mucosa. Although the morphological characteristics of polymicrobial colonization were somewhat similar to those observed in the trachea of calves with \u003cem\u003eMycoplasma\u003c/em\u003e pneumonia \u003cem\u003ein vivo\u003c/em\u003e compared to single cultures \u003cem\u003ein vitro\u003c/em\u003e, they were not completely identical. It has been reported that the biofilm grown on the epithelial cells exhibited phenotypes similar to those observed during \u003cem\u003ein vivo\u003c/em\u003e colonization (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). Epithelial cells play a crucial role in the biofilm formation process by potentially facilitating optimal interbacterial signaling and the expression of colonization-associated factors necessary for biofilm formation through adherence to \u003cem\u003ein vivo\u003c/em\u003e ligands. Our study is the first to elucidate the interactions between \u003cem\u003eM. bovis\u003c/em\u003e and \u003cem\u003eT. pyogenes\u003c/em\u003e during biofilm formation, and these mechanisms may be involved in the progression of pathology in \u003cem\u003eMycoplasma\u003c/em\u003e pneumonia. Further analysis using bovine epithelial cells is required to elucidate the mechanism of biofilm formation of \u003cem\u003eM. bovis\u003c/em\u003e.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn conclusion, we observed mature biofilms of \u003cem\u003eM. bovis\u003c/em\u003e on the tracheae of calves naturally infected with pneumonia and established a method for evaluating the biofilm formation of \u003cem\u003eM. bovis\u003c/em\u003e in vitro. \u003cem\u003eM. bovis\u003c/em\u003e biofilms were observed as aggregations of various sizes and filamentous matrices connecting neighboring groups, suggesting that \u003cem\u003eM. bovis\u003c/em\u003e releases ECM or DNA to connect to neighboring bacteria, and then forms compact microcolonies. Additionally, co-cultivation with \u003cem\u003eM. bovis\u003c/em\u003e and \u003cem\u003eT. pyogenes\u003c/em\u003e caused significant biofilm formation. This study indicated that the interaction between \u003cem\u003eM. bovis\u003c/em\u003e and \u003cem\u003eT. pyogenes\u003c/em\u003e led to increased resistance to antimicrobial agents, thereby exacerbating the progression of chronic \u003cem\u003eMycoplasma\u003c/em\u003e pneumonia.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by JSPS KAKENHI\u0026nbsp;[grant\u0026nbsp;number\u0026nbsp;23K05574]\u0026nbsp;and the Rakuno Gakuen University Fund\u0026nbsp;[grant\u0026nbsp;number\u0026nbsp;2020-04].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors would like to thank Enago (www.enago.jp) for the English language review.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFour calves were undergoing pathological autopsies in accordance with the Guide for the Care and Use of Laboratory Animals of the School of Veterinary Medicine at Rakuno Gakuen University in 2021.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConception and design of the work: KN, SG, HH; data acquisition and analysis: KN, YH, MO, AS, KM, TI, TK, RU; interpretation of data: KN, SG, HH; first draft of the manuscript: KN; manuscript revision: SG, HH. All authors read and approved the final manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eFox LK (2012) Mycoplasma mastitis: causes, transmission, and control. Vet. Clin. North Am Food Anim Pract 28:225-237.\u003c/li\u003e\n\u003cli\u003eMaunsell FP, Woolums AR, Francoz D, Rosenbusch RF, Step DL, Wilson DJ, Janzen ED (2011) \u003cem\u003eMycoplasma bovis\u003c/em\u003e infections in cattle. J Vet Intern Med 25:772-783.\u003c/li\u003e\n\u003cli\u003eDesrochers A, Francoz D (2014) Clinical management of septic arthritis in cattle. Vet Clin North Am Food Anim Pract 30:177-203, vii.\u003c/li\u003e\n\u003cli\u003eLamm CG, Munson L, Thurmond MC, Barr BC, George LW (2004) Mycoplasma otitis in California calves. 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Braz J Vet Pathol 10(2):79-86.\u003c/li\u003e\n\u003cli\u003eCamarillo-M\u0026aacute;rquez O, C\u0026oacute;rdova-Alc\u0026aacute;ntara IM, Hern\u0026aacute;ndez-Rodr\u0026iacute;guez CH, Garc\u0026iacute;a-P\u0026eacute;rez BE, Mart\u0026iacute;nez-Rivera MA, Rodr\u0026iacute;guez-Tovar AV (2018) Antagonistic interaction of \u003cem\u003eStaphylococcus aureus\u003c/em\u003e toward \u003cem\u003eCandida glabrata \u003c/em\u003eduring \u003cem\u003ein vitro\u003c/em\u003e biofilm formation is caused by an apoptotic mechanism. Front Microbiol 9:2031.\u003c/li\u003e\n\u003cli\u003eKong KF, Vuong C, Otto M (2006) Staphylococcus quorum sensing in biofilm formation and infection. Int J Med Microbiol 296:133-139.\u003c/li\u003e\n\u003cli\u003eSolano C, Echeverz M, Lasa I (2014) Biofilm dispersion and quorum sensing. Curr Opin Microbiol 18:96-104.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"antibiotics, antimicrobial agents, bovine respiratory disease, extracellular matrix, trachea","lastPublishedDoi":"10.21203/rs.3.rs-4523720/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4523720/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e \u003cem\u003eMycoplasma\u003c/em\u003e pneumonia caused by \u003cem\u003eMycoplasma bovis\u003c/em\u003e (\u003cem\u003eM. bovis\u003c/em\u003e) is associated with severe inflammatory reactions in the trachea and lungs and can be difficult to treat with antibiotics. Biofilms play a significant role in the persistence of bacteria and contribute to chronic lesions. A recent study showed that polymicrobial interactions of species are an important factor in biofilm formation, but the detailed mechanism of biofilm formation of \u003cem\u003eM. bovis\u003c/em\u003e remains unknown. Assuming multiple pathogen infections in bovine respiratory disease complex, this study examined the characterization of the polymicrobial relationship between \u003cem\u003eM. bovis\u003c/em\u003e and \u003cem\u003eTrueperella pyogenes\u003c/em\u003e (\u003cem\u003eT. pyogenes\u003c/em\u003e) during biofilm formation. Bacterium-like aggregation structures (\u0026gt;\u0026thinsp;10 \u0026micro;m), which were assumed to be biofilms of \u003cem\u003eM. bovis in vivo\u003c/em\u003e, were observed adhering to the cilia in calves with \u003cem\u003eMycoplasma\u003c/em\u003e pneumonia. \u003cem\u003eM. bovis\u003c/em\u003e released extracellular matrix to connect with neighboring bacteria and form a mature biofilm on the plate. Biofilm formation in co-culture of \u003cem\u003eM. bovis\u003c/em\u003e and \u003cem\u003eT. pyogenes\u003c/em\u003e tended to increase compared to that in single culture of these bacteria. Additionally, some large aggregates (\u0026gt;\u0026thinsp;40 \u0026micro;m) composed of \u003cem\u003eM. bovis\u003c/em\u003e and \u003cem\u003eT. pyogenes\u003c/em\u003e were observed. The morphological characteristics of this biofilm were similar to those observed \u003cem\u003ein vivo\u003c/em\u003e compared to a single culture. In conclusion, the polymicrobial interaction between \u003cem\u003eM. bovis\u003c/em\u003e and \u003cem\u003eT. pyogenes\u003c/em\u003e induced biofilm formation, which was associated with increased resistance to antimicrobial agents, thereby exacerbating the progression of chronic \u003cem\u003eMycoplasma\u003c/em\u003e pneumonia.\u003c/p\u003e","manuscriptTitle":"Biofilm characterization of Mycoplasma bovis co-cultured with Trueperella pyogenes","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-06-26 20:24:57","doi":"10.21203/rs.3.rs-4523720/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"fdeb0224-ebea-46c9-a4c6-61b4d14ee2af","owner":[],"postedDate":"June 26th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-02-03T16:01:52+00:00","versionOfRecord":{"articleIdentity":"rs-4523720","link":"https://doi.org/10.1186/s13567-025-01468-1","journal":{"identity":"veterinary-research","isVorOnly":false,"title":"Veterinary Research"},"publishedOn":"2025-01-30 15:57:25","publishedOnDateReadable":"January 30th, 2025"},"versionCreatedAt":"2024-06-26 20:24:57","video":"","vorDoi":"10.1186/s13567-025-01468-1","vorDoiUrl":"https://doi.org/10.1186/s13567-025-01468-1","workflowStages":[]},"version":"v1","identity":"rs-4523720","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4523720","identity":"rs-4523720","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}