Mycoplasma Bovis-pneumonia and Polyarthritis in Feedlot Calves in Argentina: First Local Isolation

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Infectious pneumonia usually has complex causes, involving several microorganisms. Mycoplasma is frequently detected in BRD, especially Mycoplasma bovis . This work reports the first local isolation of M. bovis from feedlot calves with pneumonia and polyarthritis in Argentina. Twenty four out of 545 calves showed progressive, subacute to chronic respiratory distress, coughing, and fever. Thirty percent of the affected calves also showed lameness and swelling of elbow or carpal, and knee or tarsal joints. Five necropsies were performed and severe multifocal to coalescent pulmonary nodules, containing white-yellowish caseous exudate encircled by fibrous tissue, and fibrinonecrotic arthritis and tenosynovitis were detected. Mycoplasma was isolated from lung and joint samples. The 16S-23S rRNA ITS consensus sequence obtained from these isolates showed 100 % similarity with the same region of M. bovis strains. This work should alert practitioners about the presence of mycoplasma infections as the cause of BRD in the region. Since there are no commercially available vaccines in the region for the prevention and control of M. bovis pneumonia and arthritis, surveillance is a priority to reduce the source of disease to naïve animals. Veterinary Epidemiology Mycoplasma bovis pneumonia polyarthritis feedlot cattle Figures Figure 1 Figure 2 Introduction Bovine respiratory disease (BRD) is one the most frequently occurring clinical problem in weaned calves after their arrival at the feedlot (O'Connor et al. 2001), causing important economic losses in the beef industry (Schneider et al. 2009). Infectious pneumonia usually has complex causes, involving two or more microorganisms and is commonly predisposed by environmental factors (Taylor et al. 2010). Viruses most frequently associated with BRD are bovine herpesvirus type 1 (BoHV1), bovine parainfluenza virus type 3 (BPIV-3), bovine respiratory syncytial virus (BRSV), and bovine viral diarrhea virus (BVDV). Secondary bacterial infections are usually associated with Mannheimia haemolytica , Pasteurella multocida and Histophilus somni . Other bacteria frequently detected in BRD are mycoplasmas, especially Mycoplasma bovis (Nicholas and Ayling 2003; Gagea et al. 2006; Booker et al. 2008; Fulton et al. 2009). M. bovis is most commonly recognized as a cause of pneumonia and arthritis in calves and mastitis in dairy cattle in North America and Europe (Gagea et al. 2006; Caswell et al. 2010; Murray et al. 2017). Margineda et al. (2017) reported for the first time the presence of M. bovis as a cause of pneumonia in feedlot cattle and dairy calves in Argentina. Nevertheless, the prevalence, morbidity, mortality, and economic relevance of M. bovis pneumonia in the BRD complex in Argentina is still unknown. Although M. bovis has been previously isolated from other clinical presentations of dairy cattle (Cerdá et al. 2000) and M. bovis -pneumonia has been previously diagnosed in Argentina (Margineda et al. 2017), this work reports the first local isolation of M. bovis from feedlot calves with pneumonia and polyarthritis in Buenos Aires province, Argentina. Materials and methods Clinical history of the herd The outbreak occurred in a feedlot in Carlos Tejedor department (35°11'01"S 62°36'16"W), Buenos Aires province, Argentina. During December 2018 until January 2019, 545 early-weaned calves of 45-55 kg arrived at the feedlot, from three farms. Upon arrival, calves were twice tilmicosin-treated (metaphylaxis, days 0 and 21 post-arrival) and immunized using a commercial polyvalent vaccine against BoHV1, P. multocida , Moraxella bovis , Clostridium chauvoei and Clostridium perfringens (days 0, 21 and 42 post-arrival). The diet consisted of cracked corn, soybean expeller, wheat bran and a commercial vitamins-minerals premix. Calves were allocated in three different lots: 200 in lot A, 143 in lot B and 202 in lot C. Post mortem examination and tissue sampling After clinical examination the five most clinically affected calves were euthanized according to the regulations of the Animal Ethics Committee of INTA and necropsied accordingly. Samples from central nervous system, heart, liver, spleen, kidney, muscle, lung, mediastinal lymph nodes and synovial capsules were collected and fixed in 10% neutral buffered formalin for histopathological and immunohistochemistry (IHC) examination. Also, lung, synovial fluid and capsules samples were collected for microbiological examination. Histopathology and Mycoplasma bovis immunohistochemistry Formalin fixed tissues were paraffin embedded, sectioned at 4-5 μm and stained with hematoxylin and eosin (HE) for histologic examination. Formalin fixed and paraffin embedded lung and synovial capsules were examined using IHC for the detection of M. bovis as described previously (Haines et al. 2004) using mouse anti- M. bovis monoclonal antibody (Millipore MAB970) at 1:100 dilution. Positive and negative controls were included (Margineda et al. 2017). No other Mycoplasma spp. were tested by IHC in this study. Microbiology Lung and synovial fluid samples were inoculated onto Mycoplasma Base Medium with Selective Mycoplasma supplement -MM- (Oxoid Ltd., Wad Road, Basingstoke, UK) and Columbia Blood Agar -CBA- (Oxoid Ltd., Wad Road, Basingstoke, UK) with 7% bovine blood and MacConkey agar -MC- (Oxoid Ltd., Wad Road, Basingstoke, UK). All plates were incubated at 37ºC, MM under 5% CO 2 , CBA under 10% CO 2 and MC under aerobiosis, and examined at 96, 48 and 24 h, respectively. Genera were classified according to the Bergey’s Manual of Determinative Bacteriology (Bergey and Holt 1994). Lung smears were heat-fixed and stained using Ziehl – Neelsen (ZN) methods to detect acid-fast bacteria (AFB). PCR and sequencing Molecular detection of Mycoplasma was performed for both, clinical samples and to confirm the presence of the agent after culture. Briefly, DNA was extracted using the commercial kit Puri-Prep S (Inbio Highway, Argentina) according to the manufacturer´s instructions. For mycoplasma detection, a nested PCR targeting 16S-23S rRNA intergenic spacer region (ITS) was performed under the conditions reported by Tang et al. (2000) using primers previously reported (Nakagawa et al. 1992; Harasawa et al. 1993). To identify the mycoplasma species, the obtained PCR products were purified (Puriprep-GP Kit, Inbio Highway), quantified and sequenced (ABI 3130xl; Applied Biosystems) using the inner primers described by Harasawa et al. (1993). The sequences were curated using the BioEdit software and aligned using Clustal Omega software. Since all the sequences were identical, a unique consensus sequence was obtained and then aligned against the database using nucleotide BLAST ( http://www.ncbi.nlm.nih.gov/blast ) excluding uncultured and environmental sample sequences. Virology Lung samples were homogenized in Eagle´s minimum essential medium (MEM) (Gibco, 4150034; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum, and inoculated on Madin-Darby Bovine Kidney (MDBK) cells. Cell cultures were incubated at 37ºC and 5% CO 2 for 5 days and examined daily for cytopathic effect. After four consecutive passages, cultures were tested for BoHV-1, BPIV-3, BRSV and BVDV by direct fluorescent antibody tests. Results Clinical cases were initially reported in lots A (9 cases) and B (13 cases). The first two cases occurred during December 2018, 1 on January, 9 during February and 10 during March 2019. All the affected calves (24 out of 545; morbidity = 4.4%) showed progressive, subacute to chronic respiratory distress, coughing, hyperpnea, poor body condition, lethargy, dehydration and fever (39.5 to 41.3ºC). Thirty percent of the affected calves also showed lameness, grinding noise when they walk, and pain in one or more joints associated with visible swelling of the affected front (Fig. 1a) and hind leg joints (elbow or carpal, and knee or tarsal joints, respectively). Post mortem examinations were performed in five affected calves. Fibrinonecrotic arthritis and tenosynovitis were detected in the affected joints (Fig. 1b). The lungs of the five calves showed cranioventral consolidation affecting 20 to 50% of the pulmonary parenchyma. All the examined lungs showed multifocal to coalescent white nodules, some of them protruding above the pleural surface (Fig. 1c). On section, white nodules ranged 0.3 to 2.5 cm and contained white-yellowish caseous exudate, encircled by fibrous tissue (Fig. 1d). Pleural fibrosis was observed in one of the examined animals (Fig. 1e) and chronic pleural adhesions in two calves. No other gross lesions were observed in the affected necropsied calves. Histologically, multifocal necrotic areas in the pulmonary parenchyma were observed. These foci contained many necrotic inflammatory cells that retained their cellular outlines but had intensely eosinophilic cytoplasm and lysis of the nuclei (Fig. 2a and 2b). These foci were delineated by a band of neutrophils and macrophages, encircled by a layer of fibroblasts macrophages, lymphocytes, and plasma cells. The smaller bronchioles contained an accumulation of necrotic leukocytes in the bronchiolar lumen and the epithelium was discontinuous. The bronchiolar walls were thickened by edema and infiltrate of lymphocytes with fewer neutrophils and macrophages. Similar foci of caseous necrosis that contained recognizable necrotic leukocytes were occasionally present in the alveoli. Macrophages and scarce plasmocytes were infiltrating the interlobular septa. Follicular hyperplasia was observed in mediastinal lymph nodes. In joint capsule, fibrin admixed with many neutrophils was adherent to the synovium, with areas of hyperplasia alternating with areas of necrosis or denudation of synoviocytes. The subsynovial stroma had severe infiltration of many neutrophils, macrophages, lymphocytes, and plasma cells; and prominent fibroblast hyperplasia (Fig. 2c). No other microscopical lesions were detected in the affected calves. Immunohistochemistry showed abundant M. bovis antigen in the lungs and joint capsule of calves. In lungs, the positive staining was observed mainly at the margin of the necrotic lesions, and to a lesser extent in the center of the necrotic foci. In bronchioles containing caseous debris, antigen was present within the debris and adjacent to bronchiolar epithelial cells (Fig. 2d). Antigen of M. bovis was identified in synovium and stroma subsynovial within the debris and adjacent neutrophils and macrophages. Staining for M. bovis antigen was not visible in the sections of negative controls. Typical Mycoplasma fried-egg-shaped colonies were observed in all the lung samples from the five calves (Fig. 2e). In none of the synovial fluid samples compatible- Mycoplasma colonies were observed. Lung and synovial fluid sampled during the necropsies of calves #1, #2, #3 and #4 resulted negatives for the isolation of aerobic and microaerophilic bacteria using the routine diagnostic procedures. Trueperella pyogenes and H. somni were isolated from calf #5 lung sample. No AFB were observed in the ZN staining of lung smears. The lung samples from all calves and synovial samples from calves #1, #3, and #4 rendered PCR positive results. The presence of the agent was also confirmed in the lung cultures. The 16S-23S rRNA ITS consensus sequence obtained showed 100 % similarity with the same region of M. bovis strains NADC59 (CP042939.1), MJ1 (CP042938.1), KG4397 (AP019558.1), NADC61 (CP022599.1), NADC67 (CP022596.1), NADC62 (CP022595.1), NADC58 (CP022594.1), NADC57 (CP022593.1), NADC56 (CP022592.1), NADC55 (CP022591.1), NADC54 (CP022590.1), NADC18 (CP022589.1), MJ4 (CP022588.1), MJ3 (CP022587.1), MJ2 (CP022586.1), JF4278 (LT578453.1), Ningxia-1 (CP023663.1), 08M (CP019639.1), 72242 (KX687011.1), 393B08 (KX687010.1), 268B07 (KX687009.1), HB0801-P115 (CP007589.1), NM2012 (CP011348.1), 1982-M6152 (CP058969.1), 2019-043682 (CP058968.1), PG45 (CP002188.1), 70-213 (AY779747.1) and ATCC 25523 (AY729934.1). BoHV-1, BPIV-3, BRSV and BVDV isolation resulted negative in the lung samples tested. Discussion The bovine respiratory disease causes important economic losses in the beef industry (O'Connor et al. 2001) and is described as multifactorial with different etiological agents involved (Gagea et al. 2006; Fulton et al. 2009). Although M. bovis is frequently detected in association with BRD worldwide (Gagea et al. 2006; Fulton et al. 2009; Murray et al. 2017), only on description of the disease is available in Argentina (Margineda et al. 2017). This report describes an outbreak and the first isolation of M. bovis in feedlot calves with chronic pneumonia and polyarthritis in Argentina. Clinical signs observed in the animals are similar to the previous reports: subacute to chronic respiratory distress with fever and severe lameness resulting from polyarthritis (affecting carpal and tarsal joints, mainly), also known as “pneumonia-arthritis syndrome” (Adegboye et al. 1996; Gagea et al. 2006). Failure of antibiotic treatment (Ayling et al. 2000) and retarded growth are other characteristics of the disease (Shahriar et al. 2002), as it was observed in this outbreak. According to the information recorded during the occurrence of the outbreak, 4.4% of the exposed calves were affected. However, the incidence of mycoplasma pneumonia can be as high as 100% (Pfützner and Sachse 1996). Nevertheless, before the disease was confirmed in this feedlot, some of the calves in the affected lots (A and B) were moved into different lots with other animals. Then, Mycoplasma -like disease was observed in these animals (veterinary practitioner personal communication), showing how easy transmission occurred (Pfützner 1990). Therefore, the exact epidemiological rates of this outbreak are actually unknown. Certain animals may act as reservoirs of Mycoplasma in the respiratory tract without developing the clinical disease (Thomas et al. 2002) and probably, reservoir calves may have been introduced in December or January, providing the source for infection to in-contact calves, as it was previously reported (Allen et al. 1992). No previous history of these calves was available to explain this issue. Tilmicosin-treatment of these calves was probably not efficient in order to reduce their reservoir status, since this is not recommended as effective for M. bovis therapy. On the other hand, enrofloxacin, florfenicol and spectinomycin would be better options as metaphylactic antibiotic treatment (Caswell et al. 2010). Post mortem diagnostic during BRD should be carried out in untreated animals in the initial stages of the clinical disease. Therefore, diagnosis of BRD due to M. bovis sometimes have some difficulties, since chronically affected calves probably have been already treated with a variety of antimicrobials (Cooper and Brodersen 2010). Nevertheless, pathological changes associated with Mycoplasma pneumonia are characteristic and can provide useful information. Mycoplasma pneumonia is characterized as subacute or chronic suppurative bronchopneumonia with multiple foci of caseous necrosis (Adegboye et al. 1995; Pfützner and Sachse 1996; Gagea et al. 2006; Caswell et al. 2010), as they were observed during the five necropsied calves in this outbreak. Histopathologically, foci of acute coagulative necrosis surrounded by a densely basophilic border of necrotic leukocytes (“oat cells”) are also morphologically distinctive from other bacterial etiologies of BRD (Pfützner and Sachse 1996; Gagea et al. 2006). Bovine respiratory disease is usually caused by multiple microorganisms and their identification in tissue samples from an affected calf should be carefully interpreted (Hodgins et al. 2002; Nicholas and Ayling 2003). In one of the sampled lungs, T. pyogenes and H. somni were isolated. These bacteria could be responsible for BRD. However, the clinical history and the pathological findings resemble “pneumonia-arthritis syndrome” previously associated with M. bovis (Adegboye et al. 1996; Gagea et al. 2006). Molecular diagnostics have substituted classic diagnostic procedures such as culture for Mycoplasma spp and other fastidious microorganisms, providing very specific, sensible and rapid tests (Cooper and Brodersen 2010). During this work, PCR was applied as a screening test, and then, the tissue samples were cultured and Mycoplasma was isolated. Although previous reports mentioned similar isolation success on lung and synovial samples (Adegboye et al. 1996), in this work, only lung samples resulted positive for Mycoplasma isolation. Previous reports DNA amplicons were then sequenced and 100% nucleotide identity was observed with M. bovis reference strains, confirming the etiological agent involved during this outbreak. Nevertheless, bacteriological results should be interpreted in conjunction with the presence of pathological changes associated with this infection, since M. bovis can be part of the microbiota of healthy bovine upper respiratory tract. In accordance with Margineda et al. (2017), this work should alert practitioners about the presence of Mycoplasma infections as the cause of BRD in Argentina, moreover considering that other species of Mollicutes causing arthritis and pneumonia as U. diversum , M. bovigenitalum , M. bovirhinis , M. alkalescens and M. leachii has been reported in Argentina (Seitz et al. 2018; Sosa et al. 2018; Neder et al. 2019). Since there are no commercially available vaccines in the region for the prevention and control of M. bovis pneumonia and arthritis, and the disease caused by M. bovis is refractory to delayed antimicrobial therapy, surveillance is a priority to reduce the source of disease to naïve animals. Removal of clinically affected animals and quarantine of the affected lots is useful to reduce the dissemination of mycoplasmosis to unaffected lots. Conclusions This work reports the first local isolation of M. bovis from feedlot calves with pneumonia and polyarthritis in Argentina. Further work should be done in order to broaden the regional information about the clinical prevalence of this pathogen. Declarations Funding This study was financially supported by Red Nacional de Laboratorios de Diagnóstico Veterinario (RIST.I111; INTA, Argentina); Ministerio de Ciencia, Tecnología e Innovación, Argentina (PICT 0442/2015 and PICT 02148/2018); Universidad Nacional de Rio Cuarto, Argentina (PPI 2016-2018, 188/2016) and Innovaciones Tecnológicas Agropecuarias S.A. Competing Interests None of the co-authors have any financial or personal relationships that could inappropriately influence or bias the content of the paper. Availability of data and materials All data and materials are available for publication. Ethical approval The manuscript in part or in full has not been submitted or published anywhere. The manuscript will not be submitted elsewhere until the editorial process is completed. Consent to participate and for publication All the co-authors have agreed for authorship, read and approved the manuscript, and given consent for submission and subsequent publication of the manuscript. Authors' contributions Germán Cantón conceived of the presented manuscript. Germán Cantón, Ignacio Llada, Facundo Urtizbiría and Sofía Fanti performed the post mortem examination and sampling of the animals. Germán Cantón, Ignacio Llada, Carlos Margineda, Valeria Scioli and Eleonora Morrell carried out the histopathological analysis. Carlos Margineda performed the inmmunohistochemical analysis of the tissue samples. María Andrea Fiorentino, Enrique Louge Uriarte, Erika Sticotti and Pablo Tamiozzo carried out the microbiological examination of the specimens. All authors discussed the results and contributed to the final manuscript. Acknowledgements We acknowledge Dr. Fernando Ibañez, Susana Pereyra, Jorgelina Lomónaco and Paula Nievas for technical assistance. 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J Microbiol Methods 39: 121-126. https://dx.doi.org/10.1016/s0167-7012(99)00107-4 Taylor JD, Fulton RW, Lehenbauer TW, Step DL, Confer AW (2010) The epidemiology of bovine respiratory disease: What is the evidence for predisposing factors? Can Vet J 51: 1095-1102. Thomas A, Dizier I, Trolin A, Mainil J, Linden A, Ball H, Bell C (2002) Isolation of mycoplasma species from the lower respiratory tract of healthy cattle and cattle with respiratory disease in Belgium. Vet Rec 151: 472-476. http://dx.doi.org/10.1136/vr.151.16.472 Cite Share Download PDF Status: Posted 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. 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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-215610","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":11489769,"identity":"bd0f55b7-7221-4cb1-b2bb-97dea7ab9688","order_by":0,"name":"Germán Cantón","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABCElEQVRIiWNgGAWjYPCDCgkIzdiAVxmy9BkJBh7StDC2MRDWwt9+9viDjzsY7PqlTyd+LpxnkWfPfoD5BVBE3hyHNokzeYmNM88wJM/sy90sPXObRDEPTwKbJVDEcM4B7FoMGHIMm3nbGJINzvBukObdJpHYw5DAZgwUYZyBw2EG/G8gWuzP8G7+zTsHqIX/AViLPU4tEhBb7Ax4eLdJ8zYAtUgkMD8GiiTi0iJx443hzJltEgkSZ3i3WfMcA2q58bCNceYZiWRcWvj7cww+fGyzsefv4d18m6emLrG9P/nwh487bGxxaYFZltiA4DC2AdOABH4NQGCPzGH+QFD9KBgFo2AUjCQAAKA2VNmH3G8nAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0003-3494-8193","institution":"Instituto Nacional de Tecnologia Agropecuaria","correspondingAuthor":true,"prefix":"","firstName":"Germán","middleName":"","lastName":"Cantón","suffix":""},{"id":11489770,"identity":"898026e4-25c1-4c55-82bf-a263700eb66b","order_by":1,"name":"Ignacio Llada","email":"","orcid":"","institution":"INTA: Instituto Nacional de Tecnologia Agropecuaria","correspondingAuthor":false,"prefix":"","firstName":"Ignacio","middleName":"","lastName":"Llada","suffix":""},{"id":11489771,"identity":"9a0ddd5e-078c-477b-9635-43c76f6cc67a","order_by":2,"name":"Carlos Margineda","email":"","orcid":"","institution":"INTA: Instituto Nacional de Tecnologia Agropecuaria","correspondingAuthor":false,"prefix":"","firstName":"Carlos","middleName":"","lastName":"Margineda","suffix":""},{"id":11489772,"identity":"179135b1-360f-4746-84be-cff768799e4a","order_by":3,"name":"Facundo Urtizbiria","email":"","orcid":"","institution":"INTA: Instituto Nacional de Tecnologia Agropecuaria","correspondingAuthor":false,"prefix":"","firstName":"Facundo","middleName":"","lastName":"Urtizbiria","suffix":""},{"id":11489773,"identity":"bcf81303-5bc6-4232-98ee-3bafe406f848","order_by":4,"name":"Sofia Fanti","email":"","orcid":"","institution":"INTA: Instituto Nacional de Tecnologia Agropecuaria","correspondingAuthor":false,"prefix":"","firstName":"Sofia","middleName":"","lastName":"Fanti","suffix":""},{"id":11489774,"identity":"899932b7-45f0-4566-ac1f-80f988b842b0","order_by":5,"name":"Valeria Scioli","email":"","orcid":"","institution":"INTA: Instituto Nacional de Tecnologia Agropecuaria","correspondingAuthor":false,"prefix":"","firstName":"Valeria","middleName":"","lastName":"Scioli","suffix":""},{"id":11489775,"identity":"1c586cb9-6ede-4086-9224-4c8b90279ca2","order_by":6,"name":"Maria Andrea Fiorentino","email":"","orcid":"","institution":"INTA: Instituto Nacional de Tecnologia Agropecuaria","correspondingAuthor":false,"prefix":"","firstName":"Maria","middleName":"Andrea","lastName":"Fiorentino","suffix":""},{"id":11489776,"identity":"17d32095-a03a-419c-a73b-982db2d923e6","order_by":7,"name":"Enrique Louge Uriarte","email":"","orcid":"","institution":"INTA: Instituto Nacional de Tecnologia Agropecuaria","correspondingAuthor":false,"prefix":"","firstName":"Enrique","middleName":"Louge","lastName":"Uriarte","suffix":""},{"id":11489777,"identity":"3696b009-82cd-43fb-8182-9b369a018f8b","order_by":8,"name":"Eleonora Morrell","email":"","orcid":"","institution":"INTA: Instituto Nacional de Tecnologia Agropecuaria","correspondingAuthor":false,"prefix":"","firstName":"Eleonora","middleName":"","lastName":"Morrell","suffix":""},{"id":11489778,"identity":"d444e4f1-28e9-4e9f-9b7e-b1be88b33c76","order_by":9,"name":"Erika Sticotti","email":"","orcid":"","institution":"UNRC: Universidad Nacional de Rio Cuarto","correspondingAuthor":false,"prefix":"","firstName":"Erika","middleName":"","lastName":"Sticotti","suffix":""},{"id":11489779,"identity":"e65bb3b9-7601-4da2-9c73-96d40c5889fd","order_by":10,"name":"Pablo Tamiozzo","email":"","orcid":"","institution":"UNRC: Universidad Nacional de Rio Cuarto","correspondingAuthor":false,"prefix":"","firstName":"Pablo","middleName":"","lastName":"Tamiozzo","suffix":""}],"badges":[],"createdAt":"2021-02-07 03:09:32","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-215610/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-215610/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":6230804,"identity":"229440ba-3d09-4a75-b1d0-ce6fdc13b1a3","added_by":"auto","created_at":"2021-02-23 00:06:04","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":5713,"visible":true,"origin":"","legend":"Post mortem findings in necropsied calves. (a) Bilateral swollen carpal joints of a calf with mycoplasma arthritis. (b) Necropsy #3. Fibrinonecrotic carpal arthritis and tenosynovitis in calf with Mycoplasmosis. (c) Necropsy #4. Focally extensive subacute to chronic pneumonia affecting approximately 90% of the right lung (apical, cranial and caudal lobes) of a calf with mycoplasma pneumonia. Multifocal caseonecrotic nodules are present in the cranial lung lobe (blue arrows). (d) Necropsy #1. Multifocal caseonecrotic nodules in the lung parenchyma. Formalin fixed tissue. (e) Necropsy #1. Focally extensive subacute to chronic pneumonia mainly affecting the whole right apical and cranial lobes and the cranial region (20% approximately of parenchyma) of the right caudal lobe of a calf with mycoplasma pneumonia (*).","description":"","filename":"fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-215610/v1/76734041fa994d188234343c.png"},{"id":6231344,"identity":"21405667-6eed-40b0-b02f-441e45a2d884","added_by":"auto","created_at":"2021-02-23 00:09:04","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":5713,"visible":true,"origin":"","legend":"Laboratory findings. (a) Necropsy #4. Lung. Necrotic focus delineated by a band of neutrophils and macrophages, encircled by a layer of fibroblasts, macrophages, lymphocytes, and plasma cells. Hematoxylin and eosin, 100×. (b) Necropsy #3. Lung. Large area of caseous necrosis with mineralization surrounded by inflammatory infiltrate characterized by fibroblast, macrophages and lymphocytes, mainly, typical of Mycoplasma pneumonia. Macrophages and scarce plasmocytes were infiltrating the interlobular septa. Hematoxylin and eosin, 40×. (c) Necropsy #4. Carpal joint. Necrotic foci or denudation of synoviocytes with severe infiltration of neutrophils, macrophages, lymphocytes, and plasma cells; and prominent fibroblast hyperplasia. Hematoxylin and eosin, 40×. (d) Necropsy #3. Lung. Immunohistochemistry labelling of M. bovis in the lung observed mainly in bronchioles containing caseous debris. (e) Typical Mycoplasma fried-egg-shaped colonies were observed in the lung culture collected during necropsy #1.","description":"","filename":"placeholderimage.png","url":"https://assets-eu.researchsquare.com/files/rs-215610/v1/40c48f2198f833f089388b5f.png"},{"id":13669316,"identity":"b588b933-42c5-4945-bc63-a850e5eb4378","added_by":"auto","created_at":"2021-09-17 11:00:17","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":265363,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-215610/v1/07003e43-03d7-4ab0-94f4-1cf72efcd682.pdf"}],"financialInterests":"","formattedTitle":"\u003cp\u003eMycoplasma Bovis-pneumonia and Polyarthritis in Feedlot Calves in Argentina: First Local Isolation\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eBovine respiratory disease (BRD) is one the most frequently occurring clinical problem in weaned calves after their arrival at the feedlot (O'Connor et al. 2001), causing important economic losses in the beef industry (Schneider et al. 2009). Infectious pneumonia usually has complex causes, involving two or more microorganisms and is commonly predisposed by environmental factors (Taylor et al. 2010). Viruses most frequently associated with BRD are bovine herpesvirus type 1 (BoHV1), bovine parainfluenza virus type 3 (BPIV-3), bovine respiratory syncytial virus (BRSV), and bovine viral diarrhea virus (BVDV). Secondary bacterial infections are usually associated with \u003cem\u003eMannheimia haemolytica\u003c/em\u003e, \u003cem\u003ePasteurella multocida\u003c/em\u003e and \u003cem\u003eHistophilus somni\u003c/em\u003e. Other bacteria frequently detected in BRD are mycoplasmas, especially \u003cem\u003eMycoplasma bovis\u003c/em\u003e (Nicholas and Ayling 2003; Gagea et al. 2006; Booker et al. 2008; Fulton et al. 2009).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eM. bovis\u003c/em\u003e is most commonly recognized as a cause of pneumonia and arthritis in calves and mastitis in dairy cattle in North America and Europe (Gagea et al. 2006; Caswell et al. 2010; Murray et al. 2017). Margineda et al. (2017) reported for the first time the presence of \u003cem\u003eM. bovis\u003c/em\u003e as a cause of pneumonia in feedlot cattle and dairy calves in Argentina. Nevertheless, the prevalence, morbidity, mortality, and economic relevance of \u003cem\u003eM. bovis\u003c/em\u003e pneumonia in the BRD complex in Argentina is still unknown.\u003c/p\u003e\n\u003cp\u003eAlthough \u003cem\u003eM. bovis\u003c/em\u003e has been previously isolated from other clinical presentations of dairy cattle (Cerd\u0026aacute; et al. 2000) and \u003cem\u003eM. bovis\u003c/em\u003e-pneumonia has been previously diagnosed in Argentina (Margineda et al. 2017), this work reports the first local isolation of \u003cem\u003eM. bovis\u003c/em\u003e from feedlot calves with pneumonia and polyarthritis in Buenos Aires province, Argentina.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e\u003cem\u003eClinical history of the herd\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe outbreak occurred in a feedlot in Carlos Tejedor department (35\u0026deg;11'01\"S 62\u0026deg;36'16\"W), Buenos Aires province, Argentina. During December 2018 until January 2019, 545 early-weaned calves of 45-55 kg arrived at the feedlot, from three farms. Upon arrival, calves were twice tilmicosin-treated (metaphylaxis, days 0 and 21 post-arrival) and immunized using a commercial polyvalent vaccine against BoHV1, \u003cem\u003eP. multocida\u003c/em\u003e, \u003cem\u003eMoraxella bovis\u003c/em\u003e, \u003cem\u003eClostridium chauvoei\u003c/em\u003e and \u003cem\u003eClostridium perfringens\u003c/em\u003e (days 0, 21 and 42 post-arrival). The diet consisted of cracked corn, soybean expeller, wheat bran and a commercial vitamins-minerals premix. Calves were allocated in three different lots: 200 in lot A, 143 in lot B and 202 in lot C.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003ePost mortem examination and tissue sampling\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eAfter clinical examination the five most clinically affected calves were euthanized according to the regulations of the Animal Ethics Committee of INTA and necropsied accordingly. Samples from central nervous system, heart, liver, spleen, kidney, muscle, lung, mediastinal lymph nodes and synovial capsules were collected and fixed in 10% neutral buffered formalin for histopathological and immunohistochemistry (IHC) examination. Also, lung, synovial fluid and capsules samples were collected for microbiological examination.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eHistopathology and Mycoplasma bovis immunohistochemistry\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eFormalin fixed tissues were paraffin embedded, sectioned at 4-5 \u0026mu;m and stained with hematoxylin and eosin (HE) for histologic examination. Formalin fixed and paraffin embedded lung and synovial capsules were examined using IHC for the detection of \u003cem\u003eM. bovis\u003c/em\u003e as described previously (Haines et al. 2004) using mouse anti-\u003cem\u003eM. bovis\u003c/em\u003e monoclonal antibody (Millipore MAB970) at 1:100 dilution. Positive and negative controls were included (Margineda et al. 2017). No other \u003cem\u003eMycoplasma\u003c/em\u003e spp. were tested by IHC in this study.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eMicrobiology\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eLung and synovial fluid samples were inoculated onto Mycoplasma Base Medium with Selective Mycoplasma supplement -MM- (Oxoid Ltd., Wad Road, Basingstoke, UK) and Columbia Blood Agar -CBA- (Oxoid Ltd., Wad Road, Basingstoke, UK) with 7% bovine blood and MacConkey agar -MC- (Oxoid Ltd., Wad Road, Basingstoke, UK). All plates were incubated at 37\u0026ordm;C, MM under 5% CO\u003csup\u003e2\u003c/sup\u003e, CBA under 10% CO\u003csup\u003e2\u003c/sup\u003e and MC under aerobiosis, and examined at 96, 48 and 24 h, respectively. Genera were classified according to the Bergey\u0026rsquo;s Manual of Determinative Bacteriology (Bergey and Holt 1994). Lung smears were heat-fixed and stained using Ziehl \u0026ndash; Neelsen (ZN) methods to detect acid-fast bacteria (AFB).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003ePCR and sequencing\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eMolecular detection of \u003cem\u003eMycoplasma\u003c/em\u003e was performed for both, clinical samples and to confirm the presence of the agent after culture. Briefly, DNA was extracted using the commercial kit Puri-Prep S (Inbio Highway, Argentina) according to the manufacturer\u0026acute;s instructions. For mycoplasma detection, a nested PCR targeting 16S-23S rRNA intergenic spacer region (ITS) was performed under the conditions reported by Tang et al. (2000) using primers previously reported (Nakagawa et al. 1992; Harasawa et al. 1993). To identify the mycoplasma species, the obtained PCR products were purified (Puriprep-GP Kit, Inbio Highway), quantified and sequenced (ABI 3130xl; Applied Biosystems) using the inner primers described by Harasawa et al. (1993). The sequences were curated using the BioEdit software and aligned using Clustal Omega software. Since all the sequences were identical, a unique consensus sequence was obtained and then aligned against the database using nucleotide BLAST (\u003ca href=\"http://www.ncbi.nlm.nih.gov/blast\"\u003ehttp://www.ncbi.nlm.nih.gov/blast\u003c/a\u003e) excluding uncultured and environmental sample sequences.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eVirology\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eLung samples were homogenized in Eagle\u0026acute;s minimum essential medium (MEM) (Gibco, 4150034; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum, and inoculated on Madin-Darby Bovine Kidney (MDBK) cells. Cell cultures were incubated at 37\u0026ordm;C and 5% CO\u003csup\u003e2\u003c/sup\u003e for 5 days and examined daily for cytopathic effect. After four consecutive passages, cultures were tested for BoHV-1, BPIV-3, BRSV and BVDV by direct fluorescent antibody tests.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eClinical cases were initially reported in lots A (9 cases) and B (13 cases). The first two cases occurred during December 2018, 1 on January, 9 during February and 10 during March 2019. All the affected calves (24 out of 545; morbidity = 4.4%) showed progressive, subacute to chronic respiratory distress, coughing, hyperpnea, poor body condition, lethargy, dehydration and fever (39.5 to 41.3\u0026ordm;C). Thirty percent of the affected calves also showed lameness, grinding noise when they walk, and pain in one or more joints associated with visible swelling of the affected front (Fig. 1a) and hind leg joints (elbow or carpal, and knee or tarsal joints, respectively).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003ePost mortem\u003c/em\u003e examinations were performed in five affected calves. Fibrinonecrotic arthritis and tenosynovitis were detected in the affected joints (Fig. 1b). The lungs of the five calves showed cranioventral consolidation affecting 20 to 50% of the pulmonary parenchyma. All the examined lungs showed multifocal to coalescent white nodules, some of them protruding above the pleural surface (Fig. 1c). On section, white nodules ranged 0.3 to 2.5 cm and contained white-yellowish caseous exudate, encircled by fibrous tissue (Fig. 1d). Pleural fibrosis was observed in one of the examined animals (Fig. 1e) and chronic pleural adhesions in two calves. No other gross lesions were observed in the affected necropsied calves.\u003c/p\u003e\n\u003cp\u003eHistologically, multifocal necrotic areas in the pulmonary parenchyma were observed. These foci contained many necrotic inflammatory cells that retained their cellular outlines but had intensely eosinophilic cytoplasm and lysis of the nuclei (Fig. 2a and 2b). These foci were delineated by a band of neutrophils and macrophages, encircled by a layer of fibroblasts macrophages, lymphocytes, and plasma cells. The smaller bronchioles contained an accumulation of necrotic leukocytes in the bronchiolar lumen and the epithelium was discontinuous. The bronchiolar walls were thickened by edema and infiltrate of lymphocytes with fewer neutrophils and macrophages. Similar foci of caseous necrosis that contained recognizable necrotic leukocytes were occasionally present in the alveoli. Macrophages and scarce plasmocytes were infiltrating the interlobular septa. Follicular hyperplasia was observed in mediastinal lymph nodes. In joint capsule, fibrin admixed with many neutrophils was adherent to the synovium, with areas of hyperplasia alternating with areas of necrosis or denudation of synoviocytes. The subsynovial stroma had severe infiltration of many neutrophils, macrophages, lymphocytes, and plasma cells; and prominent fibroblast hyperplasia (Fig. 2c). No other microscopical lesions were detected in the affected calves.\u003c/p\u003e\n\u003cp\u003eImmunohistochemistry showed abundant \u003cem\u003eM. bovis\u003c/em\u003e antigen in the lungs and joint capsule of calves. In lungs, the positive staining was observed mainly at the margin of the necrotic lesions, and to a lesser extent in the center of the necrotic foci. In bronchioles containing caseous debris, antigen was present within the debris and adjacent to bronchiolar epithelial cells (Fig. 2d). Antigen of \u003cem\u003eM. bovis\u003c/em\u003e was identified in synovium and stroma subsynovial within the debris and adjacent neutrophils and macrophages. Staining for \u003cem\u003eM. bovis\u003c/em\u003e antigen was not visible in the sections of negative controls.\u003c/p\u003e\n\u003cp\u003eTypical \u003cem\u003eMycoplasma\u003c/em\u003e fried-egg-shaped colonies were observed in all the lung samples from the five calves (Fig. 2e). In none of the synovial fluid samples compatible-\u003cem\u003eMycoplasma\u003c/em\u003e colonies were observed. Lung and synovial fluid sampled during the necropsies of calves #1, #2, #3 and #4 resulted negatives for the isolation of aerobic and microaerophilic bacteria using the routine diagnostic procedures. \u003cem\u003eTrueperella pyogenes\u003c/em\u003e and \u003cem\u003eH. somni\u003c/em\u003e were isolated from calf #5 lung sample. No AFB were observed in the ZN staining of lung smears.\u003c/p\u003e\n\u003cp\u003eThe lung samples from all calves and synovial samples from calves #1, #3, and #4 rendered PCR positive results. The presence of the agent was also confirmed in the lung cultures. The 16S-23S rRNA ITS consensus sequence obtained showed 100 % similarity with the same region of \u003cem\u003eM. bovis\u003c/em\u003e strains NADC59 (CP042939.1), MJ1 (CP042938.1), KG4397 (AP019558.1), NADC61 (CP022599.1), NADC67 (CP022596.1), NADC62 (CP022595.1), NADC58 (CP022594.1), NADC57 (CP022593.1), NADC56 (CP022592.1), NADC55 (CP022591.1), NADC54 (CP022590.1), NADC18 (CP022589.1), MJ4 (CP022588.1), MJ3 (CP022587.1), MJ2 (CP022586.1), JF4278 (LT578453.1), Ningxia-1 (CP023663.1), 08M (CP019639.1), 72242 (KX687011.1), 393B08 (KX687010.1), 268B07 (KX687009.1), HB0801-P115 (CP007589.1), NM2012 (CP011348.1), 1982-M6152 (CP058969.1), 2019-043682 (CP058968.1), PG45 (CP002188.1), 70-213 (AY779747.1) and ATCC 25523 (AY729934.1).\u003c/p\u003e\n\u003cp\u003eBoHV-1, BPIV-3, BRSV and BVDV isolation resulted negative in the lung samples tested.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe bovine respiratory disease causes important economic losses in the beef industry (O'Connor et al. 2001) and is described as multifactorial with different etiological agents involved (Gagea et al. 2006; Fulton et al. 2009). Although \u003cem\u003eM. bovis \u003c/em\u003eis frequently detected in association with BRD worldwide (Gagea et al. 2006; Fulton et al. 2009; Murray et al. 2017), only on description of the disease is available in Argentina (Margineda et al. 2017).\u003c/p\u003e\n\u003cp\u003eThis report describes an outbreak and the first isolation of \u003cem\u003eM. bovis\u003c/em\u003e in feedlot calves with chronic pneumonia and polyarthritis in Argentina. Clinical signs observed in the animals are similar to the previous reports: subacute to chronic respiratory distress with fever and severe lameness resulting from polyarthritis (affecting carpal and tarsal joints, mainly), also known as \u0026ldquo;pneumonia-arthritis syndrome\u0026rdquo; (Adegboye et al. 1996; Gagea et al. 2006). Failure of antibiotic treatment (Ayling et al. 2000) and retarded growth are other characteristics of the disease (Shahriar et al. 2002), as it was observed in this outbreak. According to the information recorded during the occurrence of the outbreak, 4.4% of the exposed calves were affected. However, the incidence of mycoplasma pneumonia can be as high as 100% (Pf\u0026uuml;tzner and Sachse 1996). Nevertheless, before the disease was confirmed in this feedlot, some of the calves in the affected lots (A and B) were moved into different lots with other animals. Then, \u003cem\u003eMycoplasma\u003c/em\u003e-like disease was observed in these animals (veterinary practitioner personal communication), showing how easy transmission occurred (Pf\u0026uuml;tzner 1990). Therefore, the exact epidemiological rates of this outbreak are actually unknown. Certain animals may act as reservoirs of \u003cem\u003eMycoplasma\u003c/em\u003e in the respiratory tract without developing the clinical disease (Thomas et al. 2002) and probably, reservoir calves may have been introduced in December or January, providing the source for infection to in-contact calves, as it was previously reported (Allen et al. 1992). No previous history of these calves was available to explain this issue. Tilmicosin-treatment of these calves was probably not efficient in order to reduce their reservoir status, since this is not recommended as effective for \u003cem\u003eM. bovis \u003c/em\u003etherapy. On the other hand, enrofloxacin, florfenicol and spectinomycin would be better options as metaphylactic antibiotic treatment (Caswell et al. 2010).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003ePost mortem\u003c/em\u003e diagnostic during BRD should be carried out in untreated animals in the initial stages of the clinical disease. Therefore, diagnosis of BRD due to \u003cem\u003eM. bovis\u003c/em\u003e sometimes have some difficulties, since chronically affected calves probably have been already treated with a variety of antimicrobials (Cooper and Brodersen 2010). Nevertheless, pathological changes associated with \u003cem\u003eMycoplasma\u003c/em\u003e pneumonia are characteristic and can provide useful information. \u003cem\u003eMycoplasma\u003c/em\u003e pneumonia is characterized as subacute or chronic suppurative bronchopneumonia with multiple foci of caseous necrosis (Adegboye et al. 1995; Pf\u0026uuml;tzner and Sachse 1996; Gagea et al. 2006; Caswell et al. 2010), as they were observed during the five necropsied calves in this outbreak. Histopathologically, foci of acute coagulative necrosis surrounded by a densely basophilic border of necrotic leukocytes (\u0026ldquo;oat cells\u0026rdquo;) are also morphologically distinctive from other bacterial etiologies of BRD (Pf\u0026uuml;tzner and Sachse 1996; Gagea et al. 2006).\u003c/p\u003e\n\u003cp\u003eBovine respiratory disease is usually caused by multiple microorganisms and their identification in tissue samples from an affected calf should be carefully interpreted (Hodgins et al. 2002; Nicholas and Ayling 2003). In one of the sampled lungs, \u003cem\u003eT. pyogenes\u003c/em\u003e and \u003cem\u003eH. somni \u003c/em\u003ewere isolated. These bacteria could be responsible for BRD. However, the clinical history and the pathological findings resemble \u0026ldquo;pneumonia-arthritis syndrome\u0026rdquo; previously associated with \u003cem\u003eM. bovis\u003c/em\u003e (Adegboye et al. 1996; Gagea et al. 2006).\u003c/p\u003e\n\u003cp\u003eMolecular diagnostics have substituted classic diagnostic procedures such as culture for \u003cem\u003eMycoplasma\u003c/em\u003e spp and other fastidious microorganisms, providing very specific, sensible and rapid tests (Cooper and Brodersen 2010). During this work, PCR was applied as a screening test, and then, the tissue samples were cultured and \u003cem\u003eMycoplasma\u003c/em\u003e was isolated. Although previous reports mentioned similar isolation success on lung and synovial samples (Adegboye et al. 1996), in this work, only lung samples resulted positive for \u003cem\u003eMycoplasma\u003c/em\u003e isolation. Previous reports DNA amplicons were then sequenced and 100% nucleotide identity was observed with \u003cem\u003eM. bovis\u003c/em\u003e reference strains, confirming the etiological agent involved during this outbreak. Nevertheless, bacteriological results should be interpreted in conjunction with the presence of pathological changes associated with this infection, since \u003cem\u003eM. bovis\u003c/em\u003e can be part of the microbiota of healthy bovine upper respiratory tract.\u003c/p\u003e\n\u003cp\u003eIn accordance with Margineda et al. (2017), this work should alert practitioners about the presence of \u003cem\u003eMycoplasma\u003c/em\u003e infections as the cause of BRD in Argentina, moreover considering that other species of Mollicutes causing arthritis and pneumonia as \u003cem\u003eU. diversum\u003c/em\u003e, \u003cem\u003eM. bovigenitalum\u003c/em\u003e, \u003cem\u003eM. bovirhinis\u003c/em\u003e,\u003cem\u003e M. alkalescens\u003c/em\u003e and \u003cem\u003eM. leachii\u003c/em\u003e has been reported in Argentina (Seitz et al. 2018; Sosa et al. 2018; Neder et al. 2019).\u003c/p\u003e\n\u003cp\u003eSince there are no commercially available vaccines in the region for the prevention and control of \u003cem\u003eM. bovis\u003c/em\u003e pneumonia and arthritis, and the disease caused by \u003cem\u003eM. bovis\u003c/em\u003e is refractory to delayed antimicrobial therapy, surveillance is a priority to reduce the source of disease to na\u0026iuml;ve animals. Removal of clinically affected animals and quarantine of the affected lots is useful to reduce the dissemination of mycoplasmosis to unaffected lots.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThis work reports the first local isolation of \u003cem\u003eM. bovis\u003c/em\u003e from feedlot calves with pneumonia and polyarthritis in Argentina. Further work should be done in order to broaden the regional information about the clinical prevalence of this pathogen.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was financially supported by Red Nacional de Laboratorios de Diagn\u0026oacute;stico Veterinario (RIST.I111; INTA, Argentina); Ministerio de Ciencia, Tecnolog\u0026iacute;a e Innovaci\u0026oacute;n, Argentina (PICT 0442/2015 and PICT 02148/2018); Universidad Nacional de Rio Cuarto, Argentina (PPI 2016-2018, 188/2016) and Innovaciones Tecnol\u0026oacute;gicas Agropecuarias S.A.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNone of the co-authors have any financial or personal relationships that could inappropriately influence or bias the content of the paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data and materials are available for publication.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe manuscript in part or in full has not been submitted or published anywhere. The manuscript will not be submitted elsewhere until the editorial process is completed.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate and for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll the co-authors have agreed for authorship, read and approved the manuscript, and given consent for submission and subsequent publication of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors' contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGerm\u0026aacute;n Cant\u0026oacute;n conceived of the presented manuscript. Germ\u0026aacute;n Cant\u0026oacute;n, Ignacio Llada, Facundo Urtizbir\u0026iacute;a and Sof\u0026iacute;a Fanti performed the post mortem examination and sampling of the animals. Germ\u0026aacute;n Cant\u0026oacute;n, Ignacio Llada, Carlos Margineda, Valeria Scioli and Eleonora Morrell carried out the histopathological analysis. Carlos Margineda performed the inmmunohistochemical analysis of the tissue samples. Mar\u0026iacute;a Andrea Fiorentino, Enrique Louge Uriarte, Erika Sticotti and Pablo Tamiozzo carried out the microbiological examination of the specimens. All authors discussed the results and contributed to the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe acknowledge Dr. Fernando Iba\u0026ntilde;ez, Susana Pereyra, Jorgelina Lom\u0026oacute;naco and Paula Nievas for technical assistance.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAdegboye DS, Halbur PG, Cavanaugh DL, Werdin RE, Chase CCL, Miskimins DW, Rosenbusch RF (1995) Immunohistochemical and pathological study of\u003cem\u003e Mycoplasma bovis\u003c/em\u003e-associated lung abscesses in calves. 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J Microbiol Methods 39: 121-126. \u003ca href=\"https://dx.doi.org/10.1016/s0167-7012(99)00107-4\"\u003ehttps://dx.doi.org/10.1016/s0167-7012(99)00107-4\u003c/a\u003e\u003c/li\u003e\n\u003cli\u003eTaylor JD, Fulton RW, Lehenbauer TW, Step DL, Confer AW (2010) The epidemiology of bovine respiratory disease: What is the evidence for predisposing factors? Can Vet J 51: 1095-1102.\u003c/li\u003e\n\u003cli\u003eThomas A, Dizier I, Trolin A, Mainil J, Linden A, Ball H, Bell C (2002) Isolation of mycoplasma species from the lower respiratory tract of healthy cattle and cattle with respiratory disease in Belgium. Vet Rec 151: 472-476. \u003ca href=\"http://dx.doi.org/10.1136/vr.151.16.472\"\u003ehttp://dx.doi.org/10.1136/vr.151.16.472\u003c/a\u003e\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"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":"Mycoplasma bovis, pneumonia, polyarthritis, feedlot, cattle","lastPublishedDoi":"10.21203/rs.3.rs-215610/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-215610/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBovine respiratory disease (BRD) is one the most frequently occurring clinical problem in weaned calves after their arrival at the feedlot. Infectious pneumonia usually has complex causes, involving several microorganisms. Mycoplasma is frequently detected in BRD, especially \u003cem\u003eMycoplasma bovis\u003c/em\u003e. This work reports the first local isolation of \u003cem\u003eM. bovis\u003c/em\u003e from feedlot calves with pneumonia and polyarthritis in Argentina. Twenty four out of 545 calves showed progressive, subacute to chronic respiratory distress, coughing, and fever. Thirty percent of the affected calves also showed lameness and swelling of elbow or carpal, and knee or tarsal joints. Five necropsies were performed and severe multifocal to coalescent pulmonary nodules, containing white-yellowish caseous exudate encircled by fibrous tissue, and fibrinonecrotic arthritis and tenosynovitis were detected. \u003cem\u003eMycoplasma\u003c/em\u003e was isolated from lung and joint samples. The 16S-23S rRNA ITS consensus sequence obtained from these isolates showed 100 % similarity with the same region of \u003cem\u003eM. bovis\u003c/em\u003e strains. This work should alert practitioners about the presence of mycoplasma infections as the cause of BRD in the region. Since there are no commercially available vaccines in the region for the prevention and control of \u003cem\u003eM. bovis\u003c/em\u003e pneumonia and arthritis, surveillance is a priority to reduce the source of disease to na\u0026iuml;ve animals.\u003c/p\u003e","manuscriptTitle":"Mycoplasma Bovis-pneumonia and Polyarthritis in Feedlot Calves in Argentina: First Local Isolation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2021-02-23 00:06:02","doi":"10.21203/rs.3.rs-215610/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":"eb65155c-bb5a-4bec-943f-e4da360940ef","owner":[],"postedDate":"February 23rd, 2021","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":2533520,"name":"Veterinary Epidemiology"}],"tags":[],"updatedAt":"2021-03-17T22:39:44+00:00","versionOfRecord":[],"versionCreatedAt":"2021-02-23 00:06:02","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-215610","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-215610","identity":"rs-215610","version":["v1"]},"buildId":"_2-kVJe1T_tPrBINL-cwx","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}