Bacterial pathogens of the lower respiratory tract of calves from Brazilian rural settlement herds and their association with clinical signs of bovine respiratory disease

Patógenos bacterianos do trato respiratório inferior de bezerros criados em rebanhos de assentamentos brasileiros e sua associação com os sinais clínicos da doença respiratória dos bovinos

Natália C. Gaeta Bruno L.M. Ribeiro Mario A.R. Alemán Eidi Yoshihara Alessandra F.C. Nassar Lucas M. Marques Jorge Timenetsky Lilian Gregory About the authors

ABSTRACT:

Bovine respiratory disease (BRD) is considered the major cause of economic losses in dairy and beef cattle production. The study aimed to detect the most important bacteria related to respiratory disease in tracheobronchial fluid samples of healthy and dairy calves with clinical signs of BRD in Brazilian rural settlements. Hundred and forty-one mongrel dairy calves were randomly selected from 42 family farm dairy herds from Brazilian settlements. Physical examination was performed and calves were classified as healthy (n=100) and BRD (n=41). Tracheobronchial fluid samples were collected. Isolation and molecular detection of Mycoplasma dispar, M. bovis and M. mycoides subsp. mycoides SC besides isolation of other aerobic bacteria were performed. Abnormal lung sounds (crackle/snoring/whistle), mucopurulent/purulent nasal discharge, body temperature >39.5°C and respiratory rate >40 breaths/min were higher in BRD calves compared to healthy calves (P<0.05). Bacillus sp., Staphylococcus intermedius and non-fermentative Gram-negative were the most prevalent bacteria isolated. Non-identified species from Enterobacteriaceae family was higher in BRD calves compared to healthy calves (P<0.05). Mollicutes were isolated in 7.4% of samples and only M. dispar was detected. Mollicutes was associated with purulent/mucopurulent nasal discharge (P=0.017). Pantoea agglomerans was associated to tachypnea (P=0.020), and Streptococcus spp. was associated with hyperthermia. Statistical tendencies were observed to M. dispar and tachypnea (P=0.066), and P. agglomerans and tachycardia (P=0.066). The obtained results describe the microorganisms found in tracheobronchial fluid of calves with BRD in some herds of Brazilian family farming and their relation to clinical signs of BRD.

INDEX TERMS:
Bacterial pathogens; respiratory tract; calves; Brazil; rural settlement; bovine respiratory disease; BRD; Mycoplasma spp.; aerobic bacteria; cattle; clinics

RESUMO:

A doença respiratória dos bovinos (DRB) é considerada a principal causa de perdas econômicas nas produções de leite e carne. O objetivo deste estudo foi detectar as mais importantes bactérias relacionadas a doença respiratória presentes em amostras de lavado traqueobrônquico de bezerros sadios e com sinais clínicos da DRB de assentamentos brasileiros. Cento e quarenta e um bezerros leiteiros sem raça definida foram randomicamente selecionados de 42 rebanhos leiteiros de assentamentos brasileiros. Exame físico foi realizado e os animais foram classificados em sadios (n=100) e com DRB (n=41). Amostras de lavado traqueobrônquico foram coletadas. Foram realizados o isolamento e a detecção molecular de Mycoplasma dispar, M. bovis e M. mycoides subsp. mycoides SC além de isolamento de outras bactérias aeróbias. Ruídos pulmonares anormais (crepitação/ ronco/sibilo), secreção nasal mucopurulenta/purulenta, temperatura corporal >39.5°C e frequência respiratória >40 movimentos respiratórios/min foram observados com maior frequência em bezerros com DRB comparado aos animais sadios (P<0.05). Bacillus sp, Staphylococcus intermedius e bactérias Gram-negativas não fermentadoras foram as bactérias mais prevalentes. Bactérias da família Enterobacteriaceae cuja espécie não fora identificada foram mais frequentes em bezerros com DRB comparado aos bezerros sadios (P<0.05). Mollicutes foram isolados em 7,4% das amostras e somente M. dispar foi detectado. Mollicutes foi associado à secreção nasal purulenta/mucopurulenta (P=0.017). Pantoea agglomerans foi associada a taquipneia (P=0.020), e Streptococcus spp. Foi associado a hipertermia. Tendência estatística foi observada para M. dispar e taquipneia (P=0.066), e P. agglomerans e taquicardia (P=0.066). Os resultados obtidos descrevem os micro-organismos encontrados no lavado traqueobrônquico de bezerros com DRB em rebanhos de agricultura familiar brasileira e sua relação com as manifestações clínicas da DRB.

TERMOS DE INDEXAÇÃO:
Patógenos bacterianos; trato respiratório inferior; bezerros; assentamento; doença respiratória dos bovinos; Mycoplasma spp.; bactéria aeróbica; bovinos; clínica

Introduction

Brazilian rural settlements are composed of small milk producers, which supply local dairies. They are important to local milk industry and social development, generating jobs in Brazilian rural area. However, this production is usually characterized as extensive, with mongrel dairy cattle, inadequate environment and sanitary management (Lima 2010Lima J.B.M. & Alves F.V. 2011. Diagnóstico das propriedades leiteiras do Assentamento São Manoel, Anastácio, MS. Cad. Agroec. 5(1):1-10.).

Bovine respiratory disease (BRD) is considered the major cause of economic losses in dairy and beef cattle production due to its high morbidity and mortality rates (Griffin 1997Griffin D. 1997. Economic impact associated with respiratory disease in beef cattle. Vet. Clin. N. Am., Food Anim. Pract. 13(3):367-377. http://dx.doi.org/10.1016/S0749-0720(15)30302-9. PMid:9368983.
https://doi.org/10.1016/S0749-0720(15)30...
, Miles 2009Miles D.G. 2009. Overview of the North American beef cattle industry and the incidence of bovine respiratory disease (BRD). Anim. Health Res. Rev. 10(2):101-103. http://dx.doi.org/10.1017/S1466252309990090. PMid:20003641.
https://doi.org/10.1017/S146625230999009...
, Hilton 2014Hilton W.M. 2014. BRD in 2014: where have we been, where are we now, and where do we want to go? Anim. Health Res. Rev. 15(2):120-122. http://dx.doi.org/10.1017/S1466252314000115. PMid:25358813.
https://doi.org/10.1017/S146625231400011...
), especially in less technology farms, such as family farms. BRD is the second major cause of losses in calf raising (Panciera & Confer 2010Panciera R.J. & Confer A.W. 2010. Pathogenesis and pathology of bovine pneumonia. Vet. Clin. N. Am., Food Anim. Pract. 26(2):191-214. http://dx.doi.org/10.1016/j.cvfa.2010.04.001. PMid:20619179.
https://doi.org/10.1016/j.cvfa.2010.04.0...
). Opportunistic bacteria are factors for the development of BRD (Caswell & Archambault 2007Caswell J.L. & Archambault M. 2007. Mycoplasma bovis pneumonia in cattle. Anim. Health Res. Rev. 8(2):161-186. http://dx.doi.org/10.1017/S1466252307001351. PMid:18218159.
https://doi.org/10.1017/S146625230700135...
, Angen et al. 2009Angen O., Thomsen J., Larsen L.E., Larsen J., Kokotovic B., Heegaard P.M.H. & Enemark J.M.D. 2009. Respiratory disease in calves: microbiological investigations on trans-tracheally aspirated bronchoalveolar fluid and acute phase protein response. Vet. Microbiol. 137(1/2):165-171. http://dx.doi.org/10.1016/j.vetmic.2008.12.024. PMid:19186010.
https://doi.org/10.1016/j.vetmic.2008.12...
, Griffin et al. 2010Griffin D., Chengappa M.M., Kuszak J. & McVey D.S. 2010. Bacterial pathogens of the bovine respiratory disease complex. Vet. Clin. N. Am., Food Anim. Pract. 26(2):381-394. http://dx.doi.org/10.1016/j.cvfa.2010.04.004. PMid:20619191.
https://doi.org/10.1016/j.cvfa.2010.04.0...
, Holman et al. 2015Holman D.B., McAllister T., Topp E., Wright A.-D.G. & Alexander T.W. 2015. The nasopharyngeal microbiota of feedlot cattle that develop bovine respiratory disease. Vet. Microbiol. 180(1/2):90-95. http://dx.doi.org/10.1016/j.vetmic.2015.07.031. PMid:26249828.
https://doi.org/10.1016/j.vetmic.2015.07...
). Stress conditions favor the immune response decay and the development of some bacteria in respiratory tract may cause a respiratory infection. (Panciera & Confer 2010Panciera R.J. & Confer A.W. 2010. Pathogenesis and pathology of bovine pneumonia. Vet. Clin. N. Am., Food Anim. Pract. 26(2):191-214. http://dx.doi.org/10.1016/j.cvfa.2010.04.001. PMid:20619179.
https://doi.org/10.1016/j.cvfa.2010.04.0...
).

Pasteurella multocida, Mannheimia haemolytica and Mycoplasma bovis, are the major bacterial pathogens of BRD (Caswell & Archambault 2007Caswell J.L. & Archambault M. 2007. Mycoplasma bovis pneumonia in cattle. Anim. Health Res. Rev. 8(2):161-186. http://dx.doi.org/10.1017/S1466252307001351. PMid:18218159.
https://doi.org/10.1017/S146625230700135...
, Dabo et al. 2007Dabo S.M., Taylor J.D. & Confer A.W. 2007. Pasteurella multocida and bovine respiratory disease. Anim. Health Res. Rev. 8(2):129-150. http://dx.doi.org/10.1017/S1466252307001399. PMid:18218157.
https://doi.org/10.1017/S146625230700139...
, Rice et al. 2007Rice J.A., Carrasco-Medina L., Hodgins D.C. & Shewen P.E. 2007. Mannheimia haemolytica and bovine respiratory disease. Anim. Health Res. Rev. 8(2):117-128. http://dx.doi.org/10.1017/S1466252307001375. PMid:18218156.
https://doi.org/10.1017/S146625230700137...
, Griffin et al. 2010Griffin D., Chengappa M.M., Kuszak J. & McVey D.S. 2010. Bacterial pathogens of the bovine respiratory disease complex. Vet. Clin. N. Am., Food Anim. Pract. 26(2):381-394. http://dx.doi.org/10.1016/j.cvfa.2010.04.004. PMid:20619191.
https://doi.org/10.1016/j.cvfa.2010.04.0...
). Mycoplasma mycoides subsp. mycoides SC is also an important microbe due to its role in Contagious Bovine Pleuropneumonia (OIE 2014OIE 2014. Contagious bovine pleuropneumonia: terrestrial manual. Available in <Available in http://www.oie.int/fileadmin/Home/eng/Health_standards/tahm/2.04.08_CBPP.pdf >. Accessed on Mar. 17, 2018.
http://www.oie.int/fileadmin/Home/eng/He...
), as well as M. dispar which is standing out as an important pathogen of BRD (Thomas et al. 2002Thomas 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(16):472-476. http://dx.doi.org/10.1136/vr.151.16.472. PMid:12418530.
https://doi.org/10.1136/vr.151.16.472...
, Marques et al. 2007Marques L.M., Buzinhani M., Oliveira R.C., Yamaguti M., Ferreira J.B., Neto R.L. & Timenetsky J. 2007. Prevalence of mycoplasmas in the respiratory tracts of calves in Brazil. Vet. Rec. 161(20):699-700. http://dx.doi.org/10.1136/vr.161.20.699. PMid:18024928.
https://doi.org/10.1136/vr.161.20.699...
, Angen et al. 2009Angen O., Thomsen J., Larsen L.E., Larsen J., Kokotovic B., Heegaard P.M.H. & Enemark J.M.D. 2009. Respiratory disease in calves: microbiological investigations on trans-tracheally aspirated bronchoalveolar fluid and acute phase protein response. Vet. Microbiol. 137(1/2):165-171. http://dx.doi.org/10.1016/j.vetmic.2008.12.024. PMid:19186010.
https://doi.org/10.1016/j.vetmic.2008.12...
, Šiugždaitė et al. 2015, Oliveira et al. 2016Oliveira B.A.F.D., Carrillo Gaeta N., Mendonça Ribeiro B.L., Reyes Alemán M.A., Miranda Marques L., Timenetsky J., Melville P.A., Avansi Marques J., Marvulle V. & Gregory L. 2016. Determination of bacterial aetiologic factor on tracheobronchial lavage in relation to clinical signs of bovine respiratory disease. J. Med. Microbiol. 65(10):1137-1142. http://dx.doi.org/10.1099/jmm.0.000345. PMid:27582268.
https://doi.org/10.1099/jmm.0.000345...
). In Brazil, there are few studies for the bacterial components of respiratory tract of healthy and BRD cattle (Gonçalves 1987Gonçalves R.C. 1987. Estudo da flora traqueobrônquica em bezerros clinicamente sadios e portadores de pneumonia, na região de Botucatu, SP. Tese de Doutorado, Universidade Estadual Paulista, Botucatu, SP. 44p., Barros et al. 1994Barros M.S.R.M., Castro R.S., Tabosa J.H.C., Brito M.F. & Amaral B. 1994. Colheita do fluido brônquio-alveolar de bezerros através da traqueocentese transcutânea. Arq. Bras. Med. Vet. Zootec. 46(1):41-49., Benesi et al. 2013Benesi F.J., Bertagnon H.G., Wachholz L., Leal M.L.R., Fernandes W.R., Benites N.R. & Melville P.A. 2013. Microbiota bacteriana e citologia da região traqueobrônquica de bezerros no período neonatal. Pesq. Vet. Bras. 33(6):700-704. http://dx.doi.org/10.1590/S0100-736X2013000600002.
https://doi.org/10.1590/S0100-736X201300...
, Oliveira et al. 2016Oliveira B.A.F.D., Carrillo Gaeta N., Mendonça Ribeiro B.L., Reyes Alemán M.A., Miranda Marques L., Timenetsky J., Melville P.A., Avansi Marques J., Marvulle V. & Gregory L. 2016. Determination of bacterial aetiologic factor on tracheobronchial lavage in relation to clinical signs of bovine respiratory disease. J. Med. Microbiol. 65(10):1137-1142. http://dx.doi.org/10.1099/jmm.0.000345. PMid:27582268.
https://doi.org/10.1099/jmm.0.000345...
). In addition, there is a lack of studies of microbes in the respiratory tract of calves of small producers such as those from family farms.

Animals diagnosed with BRD often show depression signals, weight loss, cough, mucopurulent or purulent nasal discharge, fever, increased respiratory rate, and abnormal pulmonary sound in auscultation (Radostits 2002Radostits C., Blood O.M. & Gay D.C. 2002. Clínica Veterinária: um tratado de doenças dos bovinos, ovinos, suínos, caprinos e equinos. Guanabara Koogan: Rio de Janeiro. 1737p., Dabo et al. 2007Dabo S.M., Taylor J.D. & Confer A.W. 2007. Pasteurella multocida and bovine respiratory disease. Anim. Health Res. Rev. 8(2):129-150. http://dx.doi.org/10.1017/S1466252307001399. PMid:18218157.
https://doi.org/10.1017/S146625230700139...
, Griffin et al. 2010Griffin D., Chengappa M.M., Kuszak J. & McVey D.S. 2010. Bacterial pathogens of the bovine respiratory disease complex. Vet. Clin. N. Am., Food Anim. Pract. 26(2):381-394. http://dx.doi.org/10.1016/j.cvfa.2010.04.004. PMid:20619191.
https://doi.org/10.1016/j.cvfa.2010.04.0...
). Because the similarities of clinical signs and variation of possible bacteria the presumptive diagnosis after physical examination remains difficult.

Because of the low knowledge of bacterial agents and microbial diagnosis for bovine respiratory disease in Brazilian rural settlements, the physical examination is the only way to help this activity. Thus, the aim of this study was to detect the most important bacteria related to respiratory disease in tracheobronchial fluid samples of healthy and dairy calves with clinical signs of BRD in Brazilian rural settlements.

Materials and Methods

Ethical statement. The present study was conducted at the “Laboratory of General Bacteriology” from Biological Institute, and at the Laboratory of Mycoplasmas and at the School of Veterinary Medicine and Animal Science, from University of São Paulo, Brazil. Samples were collected from August 2014 until March 2015. All procedures were carried out in agreement with the guidelines of the Committee of Ethics on Animal Use (Protocol number: 7973040214).

Area characterization and case definition. Pontal do Paranapanema is located at the extreme west region of the state of São Paulo, Brazil. The study was carried out at Caiuá, Presidente Epitácio and Mirante do Paranapanema, important cities from Pontal do Paranapanema. Hundred and forty-one bovine males and females were studied. The animals aged from one to twelve months, were mongrel dairy calves and randomly selected from 42 rural settlement dairy herds. Calves received colostrum directly from their mothers as confirmed by the owners. After weaning, calves received a diet based on pasture and mineral salt.

Physical examination was performed in all randomly selected calves. Heart and respiratory rates, hydration level, color of mucous and specific physical examination to evaluate respiratory tract were included. Calves that showed at least two of the following parameters were considered unhealthy: mucopurulent or purulent nasal discharge, cough, crackle, snoring, respiratory rate above 40 breaths per minute and rectal temperature above 39,5oC (Benesi et al. 2013Benesi F.J., Bertagnon H.G., Wachholz L., Leal M.L.R., Fernandes W.R., Benites N.R. & Melville P.A. 2013. Microbiota bacteriana e citologia da região traqueobrônquica de bezerros no período neonatal. Pesq. Vet. Bras. 33(6):700-704. http://dx.doi.org/10.1590/S0100-736X2013000600002.
https://doi.org/10.1590/S0100-736X201300...
, Lima et al. 2016Lima S.F., Teixeira A.G.V., Higgins C.H., Lima F.S. & Bicalho R.C. 2016. The upper respiratory tract microbiome and its potential role in bovine respiratory disease and otitis media. Sci. Rep. 6(1):29050. http://dx.doi.org/10.1038/srep29050. PMid:27363739.
https://doi.org/10.1038/srep29050...
, Gaeta et al. 2017Gaeta N.C., Lima S.F., Teixeira A.G., Ganda E.K., Oikonomou G., Gregory L. & Bicalho R.C. 2017. Deciphering upper respiratory tract microbiota complexity in healthy calves and calves that develop respiratory disease using shotgun metagenomics. J. Dairy Sci. 100(2):1445-1458. http://dx.doi.org/10.3168/jds.2016-11522. PMid:27988122.
https://doi.org/10.3168/jds.2016-11522...
). Two experienced veterinarians performed the physical examination in all calves, that were classified as healthy (n=100) and calves showing clinical signs of bovine respiratory disease (n=41).

Clinical sample collection and microbiology identification. Tracheobronchial fluid samples were collected after antisepsis of the trachea. An Intracath® (BD, New Jersey, USA) was introduced by traqueocentesis, and 20mL of sterile saline 0.9% were instilled, recovering 1mL-5mL. An aliquote was added to a cryogenic tube with a transport solution for Mycoplasma spp. and glycerol, and stored in liquid nitrogen. Another aliquote was added to Brain Heart Infusion medium and stored at -4°C until further analysis.

Mycoplasma spp. culture and isolation was performed in SP-4 broth and agar (Tully 1995Tully J. & Razin J.G. 1995. Culture medium formulation for primary isolation and maintenance of mollicutes. p.33-40. In: Ibid. (Eds), Molecular and diagnostic procedures in mycoplasmology. Academic Press Inc. http://dx.doi.org/10.1016/B978-012583805-4/50005-4.
https://doi.org/10.1016/B978-012583805-4...
). Plates were incubated in aerobiosis at 37°C for fifteen days. The agar plates were daily observed for the production of “fried-egg” colonies. In broth, the glucose fermentation or arginine hydrolysis and the lack of turbidity, were confirmed. Molecular detection of Mycoplasma spp. was performed using a sub-culture in broth and the clinical samples. The DNA extraction followed the method described by Fan et al. (1995Fan H.H., Kleven S. & Jackwood M.W. 1995. Application of polymerase chain reaction with arbitrary primers to strain identification of mycoplasma gallisepticum. Avian Dis. 39(4):729-735. http://dx.doi.org/10.2307/1592409. PMid:8719206.
https://doi.org/10.2307/1592409...
). Polymerase chain reaction (PCR) was initially performed to detect Mollicutes (Van Kuppeveld et al. 1992Van Kuppeveld F.J., Van der Logt J.T., Angulo A.F., Van Zoest M.J., Quint W.G., Niesters H.G., Galama J.M. & Melchers W.J. 1992. Genus- and species-specific identification of mycoplasmas by 16S rRNA amplification. Appl. Environ. Microbiol. 58(8):2606-2615. PMid:1381174.). Then the positive samples were used to detect M. bovis (Chávez González et al. 1995Chávez González Y.R., Ros Bascuñana C., Bölske G., Mattsson J.G., Fernández Molina C. & Johansson K.E. 1995. In vitro amplification of the 16S rRNA genes from Mycoplasma bovis and Mycoplasma agalactiae by PCR. Vet. Microbiol. 47(1/2):183-190. http://dx.doi.org/10.1016/0378-1135(95)00058-I. PMid:8604550.
https://doi.org/10.1016/0378-1135(95)000...
), M. dispar (Marques et al. 2007Marques L.M., Buzinhani M., Oliveira R.C., Yamaguti M., Ferreira J.B., Neto R.L. & Timenetsky J. 2007. Prevalence of mycoplasmas in the respiratory tracts of calves in Brazil. Vet. Rec. 161(20):699-700. http://dx.doi.org/10.1136/vr.161.20.699. PMid:18024928.
https://doi.org/10.1136/vr.161.20.699...
), M. mycoides subsp. mycoides SC (Dedieu et al. 1994Dedieu L., Mady V. & Lefevre P.C. 1994. Development of a selective polymerase chain reaction assay for the detection of Mycoplasma mycoides subsp. Mycoides S.C. (Contagious bovine pleuropneumonia agent). Vet. Microbiol. 42(4):327-339. http://dx.doi.org/10.1016/0378-1135(94)90064-7. PMid:9133058.
https://doi.org/10.1016/0378-1135(94)900...
) and Ureaplasma diversum (Cardoso et al. 2002Cardoso M.V., Sforsin A.J., Scarcelli E., Teixeira S.R., Miyashiro S., Campos F.R. & Genovez M.R. 2002. Importância do diagnóstico diferencial em um surto de pneumonia enzoótica bovina. Arq. Inst. Biológico, São Paulo, 69(3):111-113.).

Regarding to samples in BHI medium, 10µL of this suspension were seeded on 5% sheep blood agar (Muller Hinton) and incubated for 48h at 37°C. The obtained colonies were gram stained and observed for hemolysis production. The colonies identification was performed for biochemical tests (Winn Jr et al. 2005Winn Jr W., Allen S., Janda W., Koneman E., Procop G., Schrsckenberger P. & Woods G. 2005. Koneman’s color atlas and textbook of diagnostic mycrobiology. 6th ed. LWW, Philadelphia, 1736p.).

Statistical analysis. All the statistical results were obtained on the Statistical Package of Social Science 16.0 (Chicago, NY) and a 95% confidence interval (CI). Descriptive analysis was performed to determine absolute and relative frequencies. Associations between categorical variables of health status (BRD and health), microorganisms (sheep blood agar bacteria and Mollicutes) and clinical signs (behavior, ocular mucosa, heart rate, respiratory rate, nasal discharge, cough, breathing pattern, percussion and auscultation) were analyzed by Pearson’s chi-square test or Fisher’s exact test in the form of univariate analysis (Hosmer & Lemeshow 1989Hosmer D. & Lemeshow S. 1989. Applied Logistic Regression. Wiley, New York. 307p.). Microbiological findings were considered the independent variables. Healthy status (healthy and BRD calves) and clinical signs were considered dependent variables. Odds Ratio was also calculated. Variables with P<0.05 were considered significant. Variables with 0.05<P<0.10 were considered statistical tendencies.

Results

Physical examination

Physical examination results are presented in Table 1. All clinical signs were detected in healthy calves, except tachycardia (heart rate >100bpm) and cough. In BRD calves, lethargy was not detected. Abnormal lung sounds (crackle/snoring/whistle) (P<0.001), mucopurulent/purulent nasal discharge (P=0.002), body temperature >39.5°C (P<0.001) and respiratory rate >40 breaths/min (P<0.001) were higher in BRD calves compared to healthy calves.

Table 1.
Clinical signs of healthy and bovine respiratory disease calves observed during the physical examination

Bacterial cultures in blood agar

Hundred and seventy-six bacterial isolates were obtained (77% Gram positive and 23% Gram negative). Bacillus sp. (56%), Staphylococcus intermedius (32.6%) and non-fermentative Gram-negative (9.2%) were the most prevalent bacteria isolated in samples of tracheobronchial fluid. Regarding to non-fermentative Gram-negative bacteria, coccobacilli positive for catalase and oxidase tests (38%; 05/13), bacilli positive for catalase and oxidase tests (31%; 04/13) and bacilli negative for catalase and oxidase tests (31%; 04/13) were detected. In lower prevalence, P. agglomerans, Staphylococcus aureus, Streptococcus sp., Serratia rubidae, Proteus spp., Pseudomonas spp., Escherichia coli, Enterobacter gergoviae, Enterobacter aerogenes, Stenotrophomonas maltophilia, Enterobacter cloacae and non-identified species from Enterobacteriaceae Family were detected (Table 1). Pasteurella multocida and Mannheimia haemolytica were not isolated.

Comparisons by the health status showed that the frequency of non-identified species from Enterobacteriaceae family was higher in BRD calves compared to healthy calves (P=0.028). Bacillus sp. was numerically higher in BRD calves (59.5%) compared to healthy (54.5%) calves (P=0.586). The frequency of Staphylococcus intermedius was numerically higher in BRD (38.1%) calves compared to healthy (30.3%) ones (P=0.367). Non-fermentative Gram-negative bacteria showed a numerical higher frequency in BRD (11.9%) compared to healthy calves (9.1%) (P=0.473). As the fourth most prevalent bacteria, the finding of Pantoea agglomerans was numerically higher in healthy calves (9.1%) compared to BRD calves (2.4%) (P=0.281) (Table 2). Fungal colonies were detected in healthy calves only (8.1%).

Table 2.
Cultures in sheep blood agar of samples from tracheobronchial fluid samples of healthy and bovine respiratory disease calves according health status

Pure cultures were observed in 77 samples. Bacillus spp. was the most prevalent (31.2%), followed by S. intermedius (6.4%), P. agglomerans (5.0%) and non-fermentative Gram-negative bacteria (4.3%) in BRD calves. Non-identified species from Enterobacteriaceae family was only, that were classified detected in BRD calves. On the other hand, Serratia rubidae, Proteus spp., Pseudomonas sp, E. coli, E. aerogenes and S. maltophilia were detected in healthy calves only (Table 3).

Table 3.
Pure cultures in sheep blood agar plates from tracheobronchial fluid samples of healthy and bovine respiratory disease dairy calves according health status

Cutures to Mollicutes

“Fried-egg” colonies were obtained in healthy (7.1%; 07/99) and BRD calves (7.1%; 03/42) calves. All colonies in SP4 agar were identified as Mollicutes. After specific PCR, mostly colonies did not have the species determined by the primers used. M. dispar was isolated in 1.4% of samples. M. bovis and MmmSC were not isolated.

Regarding to the detection of Mollicutes in the direct material (tracheobronchial fluid samples) twenty-nine samples (20.6%) were positive for Mollicutes in healthy (20.2%; 20/99) and BRD (21.4%; 09/42) calves. Mostly samples of non-targeted molicutes were higher in BRD calves than in healthy calves (P = 0.013). Only M. dispar was detected (2.1%) by the primers used in this study.

Detected or isolated microorganisms and clinical signs of BRD

Comparison between the searched microorganisms and clinical signs of BRD revealed Mollicutes associated with purulent/mucopurulent nasal discharge (P=0.017) (Table 4). Regarding to the bacteria obtained in sheep blood agar plates, the absence of P. agglomerans was associated to tachypnea (P=0.020). On the other hand, the presence of Streptococcus spp. was associated with hyperthermia (P=0.025) (Table 4). Statistical tendencies were observed to M. dispar and tachypnea (P=0.066), and P. agglomerans and tachycardia (P=0.066).

Table 4.
Microorganisms detected in the lower respiratory tract of calves associated with clinical signs of bovine respiratory disease

Discussion

The association between the studied bacteria and clinical signs of BRD in family farms with limited care was evaluated. The finding of Mycoplasma dispar confirms in part its regular presence as microbiota in respiratory tract. However the detection of this molicute in BRD may also confirm the opportunist role of some Mollicutes to cause a disease (Tegtmeier et al. 1999Tegtmeier C., Uttenthal A., Friis N.F., Jensen N.E. & Jensen H.E. 1999. Pathological and microbiological studies on pneumonic lungs from Danish calves. Zentralbl Veterinarmed B 46(10):693-700. http://dx.doi.org/10.1046/j.1439-0450.1999.00301.x. PMid:10676147.
https://doi.org/10.1046/j.1439-0450.1999...
). In addition, our results suggest the potential role of other mycoplasma besides M. bovis, MmmSC and M. dispar in the development of BRD. Bacillus spp., Staphylococcus intermedius, non-fermentative Gram-negative bacteria, and Pantoea agglomerans were the most isolated regular bacteria. It was also detected a relation between of some clinical signs of BRD and the finding of Mollicutes, M. dispar, P. agglomerans and Streptococcus spp.

Tachypnea, hyperthermia, mucopurulent/purulent nasal discharge and auscultation were more intense in BRD calves (P<0.05). These observations are in agreement with the mention of clinical diagnosis of BRD (Radostits 2002Radostits C., Blood O.M. & Gay D.C. 2002. Clínica Veterinária: um tratado de doenças dos bovinos, ovinos, suínos, caprinos e equinos. Guanabara Koogan: Rio de Janeiro. 1737p., Dabo et al. 2007Dabo S.M., Taylor J.D. & Confer A.W. 2007. Pasteurella multocida and bovine respiratory disease. Anim. Health Res. Rev. 8(2):129-150. http://dx.doi.org/10.1017/S1466252307001399. PMid:18218157.
https://doi.org/10.1017/S146625230700139...
, Griffin et al. 2010Griffin D., Chengappa M.M., Kuszak J. & McVey D.S. 2010. Bacterial pathogens of the bovine respiratory disease complex. Vet. Clin. N. Am., Food Anim. Pract. 26(2):381-394. http://dx.doi.org/10.1016/j.cvfa.2010.04.004. PMid:20619191.
https://doi.org/10.1016/j.cvfa.2010.04.0...
) and show again the importance of this procedure.

P. multocida and M. haemolytica are important pathogens of BRD (Griffin 2010Griffin D. 2010. Bovine pasteurellosis and other bacterial infections of the respiratory tract. Vet. Clin. N. Am., Food Anim. Pract. 26(1):57-71. http://dx.doi.org/10.1016/j.cvfa.2009.10.010. PMid:20117542.
https://doi.org/10.1016/j.cvfa.2009.10.0...
, Griffin et al. 2010Griffin D., Chengappa M.M., Kuszak J. & McVey D.S. 2010. Bacterial pathogens of the bovine respiratory disease complex. Vet. Clin. N. Am., Food Anim. Pract. 26(2):381-394. http://dx.doi.org/10.1016/j.cvfa.2010.04.004. PMid:20619191.
https://doi.org/10.1016/j.cvfa.2010.04.0...
), but in the present study, these microorganisms were not isolated. Similar results were obtained by Benesi et al. (2013)Benesi F.J., Bertagnon H.G., Wachholz L., Leal M.L.R., Fernandes W.R., Benites N.R. & Melville P.A. 2013. Microbiota bacteriana e citologia da região traqueobrônquica de bezerros no período neonatal. Pesq. Vet. Bras. 33(6):700-704. http://dx.doi.org/10.1590/S0100-736X2013000600002.
https://doi.org/10.1590/S0100-736X201300...
. However these species were isolated in other studies (Härtel et al. 2004Härtel H., Nikunen S., Neuvonen E., Tanskanen R., Kivelä S.L., Aho R., Soveri T. & Saloniemi H. 2004. Viral and bacterial pathogens in bovine respiratory disease in Finland. Acta Vet. Scand. 45(3/4):193-200. http://dx.doi.org/10.1186/1751-0147-45-193. PMid:15663079.
https://doi.org/10.1186/1751-0147-45-193...
, Autio et al. 2007Autio T., Pohjanvirta T., Holopainen R., Rikula U., Pentikäinen J., Huovilainen A., Rusanen H., Soveri T., Sihvonen L. & Pelkonen S. 2007. Etiology of respiratory disease in non-vaccinated, non-medicated calves in rearing herds. Vet. Microbiol. 119(2-4):256-265. http://dx.doi.org/10.1016/j.vetmic.2006.10.001. PMid:17084565.
https://doi.org/10.1016/j.vetmic.2006.10...
, Angen et al. 2009Angen O., Thomsen J., Larsen L.E., Larsen J., Kokotovic B., Heegaard P.M.H. & Enemark J.M.D. 2009. Respiratory disease in calves: microbiological investigations on trans-tracheally aspirated bronchoalveolar fluid and acute phase protein response. Vet. Microbiol. 137(1/2):165-171. http://dx.doi.org/10.1016/j.vetmic.2008.12.024. PMid:19186010.
https://doi.org/10.1016/j.vetmic.2008.12...
, Oliveira et al. 2016Oliveira B.A.F.D., Carrillo Gaeta N., Mendonça Ribeiro B.L., Reyes Alemán M.A., Miranda Marques L., Timenetsky J., Melville P.A., Avansi Marques J., Marvulle V. & Gregory L. 2016. Determination of bacterial aetiologic factor on tracheobronchial lavage in relation to clinical signs of bovine respiratory disease. J. Med. Microbiol. 65(10):1137-1142. http://dx.doi.org/10.1099/jmm.0.000345. PMid:27582268.
https://doi.org/10.1099/jmm.0.000345...
). The high occurrence of Bacillus spp. in the studied samples may justify their inhibition of other bacteria due to the secretion of bacteriocins (Cherif et al. 2001Cherif A., Ouzari H., Daffonchio D., Cherif H., Ben Slama K., Hassen A., Jaoua S. & Boudabous A. 2001. Thuricin 7: a novel bacteriocin produced by Bacillus thuringiensis BMG1.7, a new strain isolated from soil. Lett. Appl. Microbiol. 32(4):243-247. http://dx.doi.org/10.1046/j.1472-765X.2001.00898.x. PMid:11298934.
https://doi.org/10.1046/j.1472-765X.2001...
, Shelburne et al. 2007Shelburne C.E., An F.Y., Dholpe V., Ramamoorthy A., Lopatin D.E. & Lantz M.S. 2007. The spectrum of antimicrobial activity of the bacteriocin subtilosin A. J. Antimicrob. Chemother. 59(2):297-300. http://dx.doi.org/10.1093/jac/dkl495. PMid:17213266.
https://doi.org/10.1093/jac/dkl495...
). In fact, Bacillus spp. inhibit the growth of P. multocida, M. haemolytica and H. somni (Xie et al. (2009)Xie J., Zhang R., Shang C. & Guo Y. 2009. Isolation and characterization of a bacteriocin produced by an isolated Bacillus subtilis LFB112 that exhibits antimicrobial activity against domestic animal. Afr. J. Biotechnol. 8(20):5611-5619.. Other hypothesis is the faster growth of opportunistic bacteria such as Bacillus sp., which prevent the growth of M. haemolytica and P. multocida. Bacillus sp., S. intermedius, and non-fermentative Gram-negative were highly isolated from BRD calves. The sheep blood agar also allowed the isolation of Streptococcus spp., E. coli, and Pseudomonas spp. from tracheobronchial fluid samples. Moreover, non-identified species from Enterobacteriaceae family isolates were recovered more frequently from BRD calves (P<0.05) compared to the healthy calves. Similar results were obtained in two Brazilian studies. Benesi et al. (2013)Benesi F.J., Bertagnon H.G., Wachholz L., Leal M.L.R., Fernandes W.R., Benites N.R. & Melville P.A. 2013. Microbiota bacteriana e citologia da região traqueobrônquica de bezerros no período neonatal. Pesq. Vet. Bras. 33(6):700-704. http://dx.doi.org/10.1590/S0100-736X2013000600002.
https://doi.org/10.1590/S0100-736X201300...
detected Staphylococcus spp., Bacillus spp., Streptococcus spp., P. aeruginosa and enterobacteria. Evaluating calves from an intensive production type, Oliveira et al. (2016)Oliveira B.A.F.D., Carrillo Gaeta N., Mendonça Ribeiro B.L., Reyes Alemán M.A., Miranda Marques L., Timenetsky J., Melville P.A., Avansi Marques J., Marvulle V. & Gregory L. 2016. Determination of bacterial aetiologic factor on tracheobronchial lavage in relation to clinical signs of bovine respiratory disease. J. Med. Microbiol. 65(10):1137-1142. http://dx.doi.org/10.1099/jmm.0.000345. PMid:27582268.
https://doi.org/10.1099/jmm.0.000345...
also detected Bacillus spp., Staphylococcus spp., P. aeruginosa and E. coli, but in lower prevalence. In other countries, Elshafee (2003)Elshafee S.I.M.A. 2003. Isolation and characterization of aerobic bacteria associated with pneumonic lungs of cattle in Singa slaughter house, Sinnar State. M.Sc. Dissertation, University of Khartoum. 80p. detected Staphylococcus spp., Bacillus spp., Enterobacter spp., Escherichia spp., Pseudomonas spp. and Serratia spp. in bovine pneumonic lungs. Many of mentioned microorganisms are present in the environment and could be inhaled by calves and detected in both upper and lower respiratory tracts. In particular situations, Bacillus cereus (Miller et al. 1997Miller J.M., Hair J., Hebert M., Hebert L., Roberts Jr F.J. & Weyant R. 1997. Fulminating bacteremia and pneumonia due to Bacillus cereus. J. Clin. Microbiol. 35(2):504-507. PMid:9003628.), and S. intermedius (Gerstadt et al. 1999Gerstadt K., Daly J.S., Mitchell M., Wessolossky M. & Cheeseman S.H. 1999. Methicillin-resistant Staphylococcus intermedius pneumonia following coronary artery bypass grafting. Clin. Infect. Dis. 29(1):218-219. http://dx.doi.org/10.1086/520168. PMid:10433599.
https://doi.org/10.1086/520168...
) were responsible for human pneumonia. Non-identified species from Enterobacteriaceae family obtained in pure cultures, herein, were recovered from BRD calves (P<0.05). In fact, the species are not considered important pathogens related to BRD (Loneragan et al. 2001Loneragan G.H., Gould D.H., Mason G.L., Garry F.B., Yost G.S., Miles D.G., Hoffman B.W. & Mills L.J. 2001. Involvement of microbial respiratory pathogens in acute interstitial pneumonia in feedlot cattle. Am. J. Vet. Res. 62(10):1519-1524. http://dx.doi.org/10.2460/ajvr.2001.62.1519. PMid:11592313.
https://doi.org/10.2460/ajvr.2001.62.151...
, Griffin et al. 2010Griffin D., Chengappa M.M., Kuszak J. & McVey D.S. 2010. Bacterial pathogens of the bovine respiratory disease complex. Vet. Clin. N. Am., Food Anim. Pract. 26(2):381-394. http://dx.doi.org/10.1016/j.cvfa.2010.04.004. PMid:20619191.
https://doi.org/10.1016/j.cvfa.2010.04.0...
), however their opportunistic role should be considered.

Mollicutes were isolated in BRD and healthy calves. These microorganisms were also detected by PCR in 20.6% of the direct material (tracheobronchial fluid), and this result were lower in percentage compared to those reported by Oliveira et al. (2016)Oliveira B.A.F.D., Carrillo Gaeta N., Mendonça Ribeiro B.L., Reyes Alemán M.A., Miranda Marques L., Timenetsky J., Melville P.A., Avansi Marques J., Marvulle V. & Gregory L. 2016. Determination of bacterial aetiologic factor on tracheobronchial lavage in relation to clinical signs of bovine respiratory disease. J. Med. Microbiol. 65(10):1137-1142. http://dx.doi.org/10.1099/jmm.0.000345. PMid:27582268.
https://doi.org/10.1099/jmm.0.000345...
and Marques et al. (2007)Marques L.M., Buzinhani M., Oliveira R.C., Yamaguti M., Ferreira J.B., Neto R.L. & Timenetsky J. 2007. Prevalence of mycoplasmas in the respiratory tracts of calves in Brazil. Vet. Rec. 161(20):699-700. http://dx.doi.org/10.1136/vr.161.20.699. PMid:18024928.
https://doi.org/10.1136/vr.161.20.699...
. Only M. dispar was detected in samples. Oliveira et al. (2016)Oliveira B.A.F.D., Carrillo Gaeta N., Mendonça Ribeiro B.L., Reyes Alemán M.A., Miranda Marques L., Timenetsky J., Melville P.A., Avansi Marques J., Marvulle V. & Gregory L. 2016. Determination of bacterial aetiologic factor on tracheobronchial lavage in relation to clinical signs of bovine respiratory disease. J. Med. Microbiol. 65(10):1137-1142. http://dx.doi.org/10.1099/jmm.0.000345. PMid:27582268.
https://doi.org/10.1099/jmm.0.000345...
reported a higher frequency of M. dispar in trachoebronchial lavage samples, particularly in healthy calves. However, Autio et al. (2007)Autio T., Pohjanvirta T., Holopainen R., Rikula U., Pentikäinen J., Huovilainen A., Rusanen H., Soveri T., Sihvonen L. & Pelkonen S. 2007. Etiology of respiratory disease in non-vaccinated, non-medicated calves in rearing herds. Vet. Microbiol. 119(2-4):256-265. http://dx.doi.org/10.1016/j.vetmic.2006.10.001. PMid:17084565.
https://doi.org/10.1016/j.vetmic.2006.10...
, Marques et al. (2007)Marques L.M., Buzinhani M., Oliveira R.C., Yamaguti M., Ferreira J.B., Neto R.L. & Timenetsky J. 2007. Prevalence of mycoplasmas in the respiratory tracts of calves in Brazil. Vet. Rec. 161(20):699-700. http://dx.doi.org/10.1136/vr.161.20.699. PMid:18024928.
https://doi.org/10.1136/vr.161.20.699...
, and Angen et al. (2009)Angen O., Thomsen J., Larsen L.E., Larsen J., Kokotovic B., Heegaard P.M.H. & Enemark J.M.D. 2009. Respiratory disease in calves: microbiological investigations on trans-tracheally aspirated bronchoalveolar fluid and acute phase protein response. Vet. Microbiol. 137(1/2):165-171. http://dx.doi.org/10.1016/j.vetmic.2008.12.024. PMid:19186010.
https://doi.org/10.1016/j.vetmic.2008.12...
reported higher prevalence in BRD animals. M. bovis is a well-known pathogen related to BRD (Griffin et al. 2010Griffin D., Chengappa M.M., Kuszak J. & McVey D.S. 2010. Bacterial pathogens of the bovine respiratory disease complex. Vet. Clin. N. Am., Food Anim. Pract. 26(2):381-394. http://dx.doi.org/10.1016/j.cvfa.2010.04.004. PMid:20619191.
https://doi.org/10.1016/j.cvfa.2010.04.0...
) that was not detected in this study. Same results were observed by Nikunen et al. (2007)Nikunen S., Härtel H., Orro T., Neuvonen E., Tanskanen R., Kivelä S.L., Sankari S., Aho P., Pyörälä S., Saloniemi H. & Soveri T. 2007. Association of bovine respiratory disease with clinical status and acute phase proteins in calves. Comp. Immunol. Microbiol. Infect. Dis. 30(3):143-151. http://dx.doi.org/10.1016/j.cimid.2006.11.004. PMid:17258318.
https://doi.org/10.1016/j.cimid.2006.11....
and Angen et al. (2009)Angen O., Thomsen J., Larsen L.E., Larsen J., Kokotovic B., Heegaard P.M.H. & Enemark J.M.D. 2009. Respiratory disease in calves: microbiological investigations on trans-tracheally aspirated bronchoalveolar fluid and acute phase protein response. Vet. Microbiol. 137(1/2):165-171. http://dx.doi.org/10.1016/j.vetmic.2008.12.024. PMid:19186010.
https://doi.org/10.1016/j.vetmic.2008.12...
. M. mycoides subsp. mycoides SC were not detected in this study and is in agreement with other Brazilian researches (Marques et al. 2007Marques L.M., Buzinhani M., Oliveira R.C., Yamaguti M., Ferreira J.B., Neto R.L. & Timenetsky J. 2007. Prevalence of mycoplasmas in the respiratory tracts of calves in Brazil. Vet. Rec. 161(20):699-700. http://dx.doi.org/10.1136/vr.161.20.699. PMid:18024928.
https://doi.org/10.1136/vr.161.20.699...
, Oliveira et al. 2016Oliveira B.A.F.D., Carrillo Gaeta N., Mendonça Ribeiro B.L., Reyes Alemán M.A., Miranda Marques L., Timenetsky J., Melville P.A., Avansi Marques J., Marvulle V. & Gregory L. 2016. Determination of bacterial aetiologic factor on tracheobronchial lavage in relation to clinical signs of bovine respiratory disease. J. Med. Microbiol. 65(10):1137-1142. http://dx.doi.org/10.1099/jmm.0.000345. PMid:27582268.
https://doi.org/10.1099/jmm.0.000345...
). The targeted Mollicutes such as M. bovis and M. mycoides subsp. mycoides SC, herein, to BRD with used primers were not detected. This may suggest that other mycoplasmas may have a role to BRD. M. bovirhinis (Angen et al. 2009Angen O., Thomsen J., Larsen L.E., Larsen J., Kokotovic B., Heegaard P.M.H. & Enemark J.M.D. 2009. Respiratory disease in calves: microbiological investigations on trans-tracheally aspirated bronchoalveolar fluid and acute phase protein response. Vet. Microbiol. 137(1/2):165-171. http://dx.doi.org/10.1016/j.vetmic.2008.12.024. PMid:19186010.
https://doi.org/10.1016/j.vetmic.2008.12...
), Acholeplasma spp. (Zinka & Maid 2012Zinka M. & Maid R. 2012. Mycoplasmas isolated from the respiratory tract of cattle in Bosnia and Herzegovina. Anim. Vet. 28:79-83), M. alkalensis and M. arginini (Thomas et al. 1986Thomas L.H., Howard C.J., Stott E. & Parsons K.R. 1986. Mycoplasma bovis infection in gnotobiotic calves and combined infection with respiratory syncytial virus. Vet. Pathol. 23(5):571-578. http://dx.doi.org/10.1177/030098588602300505. PMid:3535220.
https://doi.org/10.1177/0300985886023005...
) were species that have been described in both upper and lower respiratory tract of cattle

The present data revealed relations between microorganisms and clinical signs of BRD. Mollicutes microorganisms were associated with mucopurulent/purulent nasal discharge. Maeda et al. (2003)Maeda T., Shibahara T., Kimura K., Wada Y., Sato K., Imada Y., Ishikawa Y. & Kadota K. 2003. Mycoplasma bovis associated suppurative otitis media and pneumonia in bull calves. J. Comp. Pathol. 129(2/3):100-110. http://dx.doi.org/10.1016/S0021-9975(03)00009-4. PMid:12921715.
https://doi.org/10.1016/S0021-9975(03)00...
reported an association between M. bovis and a non-characterized nasal discharge. Griffin et al. (2010)Griffin D., Chengappa M.M., Kuszak J. & McVey D.S. 2010. Bacterial pathogens of the bovine respiratory disease complex. Vet. Clin. N. Am., Food Anim. Pract. 26(2):381-394. http://dx.doi.org/10.1016/j.cvfa.2010.04.004. PMid:20619191.
https://doi.org/10.1016/j.cvfa.2010.04.0...
referred M. bovis pneumonia with or without nasal discharge. Oliveira et al. (2016)Oliveira B.A.F.D., Carrillo Gaeta N., Mendonça Ribeiro B.L., Reyes Alemán M.A., Miranda Marques L., Timenetsky J., Melville P.A., Avansi Marques J., Marvulle V. & Gregory L. 2016. Determination of bacterial aetiologic factor on tracheobronchial lavage in relation to clinical signs of bovine respiratory disease. J. Med. Microbiol. 65(10):1137-1142. http://dx.doi.org/10.1099/jmm.0.000345. PMid:27582268.
https://doi.org/10.1099/jmm.0.000345...
reported the association between submassive sound on acoustic percussion of the thorax and the absence of Mollicutes. In the present research, important clinical signs of BRD, such as tachypnea and hyperthermia were associated with P. agglomerans and Streptococcus spp., while both microorganisms were described as the etiologic agent of human pneumonia (Kays et al. 2002Kays M.B., Smith D.W., Wack M.F. & Denys G.A. 2002. Levofloxacin treatment failure in a patient with fluoroquinolone-resistant streptococcus pneumoniae pneumonia. Pharmacotherapy 22(3):395-399. http://dx.doi.org/10.1592/phco.22.5.395.33185. PMid:11898897.
https://doi.org/10.1592/phco.22.5.395.33...
, Shubov et al. 2011Shubov A., Jagannathan P. & Chin-Hong P.V. 2011. Pantoea agglomerans pneumonia in a heart-lung transplant recipient: case report and a review of an emerging pathogen in immunocompromised hosts. Transpl. Infect. Dis. 13(5):536-539. http://dx.doi.org/10.1111/j.1399-3062.2011.00630.x. PMid:21504526.
https://doi.org/10.1111/j.1399-3062.2011...
). Statistical tendency was observed between M. dispar and tachypnea. In an experimental infection of M. dispar in calves, Ribeiro (1979)Ribeiro O.C. 1979. Experimental infection of calves with Mycoplasma dispar. Ames: Iowa State University. 162p. reported that only one calf showed clinical signs of BRD and that M. dispar pneumonia might be a mild infection.

Conclusions

The obtained results described the microorganisms detected in tracheobronchial fluid of healthy and calve with BRD in Brazilian rural settlements. Bacillus sp., Staphylococcus intermedius and non-fermentative Gram-negative bacteria were the most prevalent.

Besides, important bacteria such as Pasteurella multocida, Mannheimia haemolytica and Mycoplasma bovis were not detected. Mycoplasma dispar was found out, but mollicutes that did not have the species confirmed were more prevalent, suggesting the potential role of other species in BRD.

In addition, the association between clinical signs and microorganisms, such as Mollicutes X purulent/mucopurulent nasal discharge, could help clinicians during the diagnosis of the etiological agent of BRD

Acknowledgements

Authors are grateful to ITESP for personal support, Aricelma França Pinheiro for the technical support, farmers that allowed our presence in their farms and São Paulo Research Foundation (FAPESP) (Protocol number: 2014/03188-3) for financial support

References

  • Angen O., Thomsen J., Larsen L.E., Larsen J., Kokotovic B., Heegaard P.M.H. & Enemark J.M.D. 2009. Respiratory disease in calves: microbiological investigations on trans-tracheally aspirated bronchoalveolar fluid and acute phase protein response. Vet. Microbiol. 137(1/2):165-171. http://dx.doi.org/10.1016/j.vetmic.2008.12.024. PMid:19186010.
    » https://doi.org/10.1016/j.vetmic.2008.12.024
  • Autio T., Pohjanvirta T., Holopainen R., Rikula U., Pentikäinen J., Huovilainen A., Rusanen H., Soveri T., Sihvonen L. & Pelkonen S. 2007. Etiology of respiratory disease in non-vaccinated, non-medicated calves in rearing herds. Vet. Microbiol. 119(2-4):256-265. http://dx.doi.org/10.1016/j.vetmic.2006.10.001. PMid:17084565.
    » https://doi.org/10.1016/j.vetmic.2006.10.001
  • Barros M.S.R.M., Castro R.S., Tabosa J.H.C., Brito M.F. & Amaral B. 1994. Colheita do fluido brônquio-alveolar de bezerros através da traqueocentese transcutânea. Arq. Bras. Med. Vet. Zootec. 46(1):41-49.
  • Benesi F.J., Bertagnon H.G., Wachholz L., Leal M.L.R., Fernandes W.R., Benites N.R. & Melville P.A. 2013. Microbiota bacteriana e citologia da região traqueobrônquica de bezerros no período neonatal. Pesq. Vet. Bras. 33(6):700-704. http://dx.doi.org/10.1590/S0100-736X2013000600002.
    » https://doi.org/10.1590/S0100-736X2013000600002
  • Cardoso M.V., Sforsin A.J., Scarcelli E., Teixeira S.R., Miyashiro S., Campos F.R. & Genovez M.R. 2002. Importância do diagnóstico diferencial em um surto de pneumonia enzoótica bovina. Arq. Inst. Biológico, São Paulo, 69(3):111-113.
  • Caswell J.L. & Archambault M. 2007. Mycoplasma bovis pneumonia in cattle. Anim. Health Res. Rev. 8(2):161-186. http://dx.doi.org/10.1017/S1466252307001351. PMid:18218159.
    » https://doi.org/10.1017/S1466252307001351
  • Chávez González Y.R., Ros Bascuñana C., Bölske G., Mattsson J.G., Fernández Molina C. & Johansson K.E. 1995. In vitro amplification of the 16S rRNA genes from Mycoplasma bovis and Mycoplasma agalactiae by PCR. Vet. Microbiol. 47(1/2):183-190. http://dx.doi.org/10.1016/0378-1135(95)00058-I. PMid:8604550.
    » https://doi.org/10.1016/0378-1135(95)00058-I
  • Cherif A., Ouzari H., Daffonchio D., Cherif H., Ben Slama K., Hassen A., Jaoua S. & Boudabous A. 2001. Thuricin 7: a novel bacteriocin produced by Bacillus thuringiensis BMG1.7, a new strain isolated from soil. Lett. Appl. Microbiol. 32(4):243-247. http://dx.doi.org/10.1046/j.1472-765X.2001.00898.x. PMid:11298934.
    » https://doi.org/10.1046/j.1472-765X.2001.00898.x
  • Dabo S.M., Taylor J.D. & Confer A.W. 2007. Pasteurella multocida and bovine respiratory disease. Anim. Health Res. Rev. 8(2):129-150. http://dx.doi.org/10.1017/S1466252307001399. PMid:18218157.
    » https://doi.org/10.1017/S1466252307001399
  • Dedieu L., Mady V. & Lefevre P.C. 1994. Development of a selective polymerase chain reaction assay for the detection of Mycoplasma mycoides subsp. Mycoides S.C. (Contagious bovine pleuropneumonia agent). Vet. Microbiol. 42(4):327-339. http://dx.doi.org/10.1016/0378-1135(94)90064-7. PMid:9133058.
    » https://doi.org/10.1016/0378-1135(94)90064-7
  • Elshafee S.I.M.A. 2003. Isolation and characterization of aerobic bacteria associated with pneumonic lungs of cattle in Singa slaughter house, Sinnar State. M.Sc. Dissertation, University of Khartoum. 80p.
  • Fan H.H., Kleven S. & Jackwood M.W. 1995. Application of polymerase chain reaction with arbitrary primers to strain identification of mycoplasma gallisepticum. Avian Dis. 39(4):729-735. http://dx.doi.org/10.2307/1592409. PMid:8719206.
    » https://doi.org/10.2307/1592409
  • Gaeta N.C., Lima S.F., Teixeira A.G., Ganda E.K., Oikonomou G., Gregory L. & Bicalho R.C. 2017. Deciphering upper respiratory tract microbiota complexity in healthy calves and calves that develop respiratory disease using shotgun metagenomics. J. Dairy Sci. 100(2):1445-1458. http://dx.doi.org/10.3168/jds.2016-11522. PMid:27988122.
    » https://doi.org/10.3168/jds.2016-11522
  • Gerstadt K., Daly J.S., Mitchell M., Wessolossky M. & Cheeseman S.H. 1999. Methicillin-resistant Staphylococcus intermedius pneumonia following coronary artery bypass grafting. Clin. Infect. Dis. 29(1):218-219. http://dx.doi.org/10.1086/520168. PMid:10433599.
    » https://doi.org/10.1086/520168
  • Gonçalves R.C. 1987. Estudo da flora traqueobrônquica em bezerros clinicamente sadios e portadores de pneumonia, na região de Botucatu, SP. Tese de Doutorado, Universidade Estadual Paulista, Botucatu, SP. 44p.
  • Griffin D. 1997. Economic impact associated with respiratory disease in beef cattle. Vet. Clin. N. Am., Food Anim. Pract. 13(3):367-377. http://dx.doi.org/10.1016/S0749-0720(15)30302-9. PMid:9368983.
    » https://doi.org/10.1016/S0749-0720(15)30302-9
  • Griffin D. 2010. Bovine pasteurellosis and other bacterial infections of the respiratory tract. Vet. Clin. N. Am., Food Anim. Pract. 26(1):57-71. http://dx.doi.org/10.1016/j.cvfa.2009.10.010. PMid:20117542.
    » https://doi.org/10.1016/j.cvfa.2009.10.010
  • Griffin D., Chengappa M.M., Kuszak J. & McVey D.S. 2010. Bacterial pathogens of the bovine respiratory disease complex. Vet. Clin. N. Am., Food Anim. Pract. 26(2):381-394. http://dx.doi.org/10.1016/j.cvfa.2010.04.004. PMid:20619191.
    » https://doi.org/10.1016/j.cvfa.2010.04.004
  • Härtel H., Nikunen S., Neuvonen E., Tanskanen R., Kivelä S.L., Aho R., Soveri T. & Saloniemi H. 2004. Viral and bacterial pathogens in bovine respiratory disease in Finland. Acta Vet. Scand. 45(3/4):193-200. http://dx.doi.org/10.1186/1751-0147-45-193. PMid:15663079.
    » https://doi.org/10.1186/1751-0147-45-193
  • Hilton W.M. 2014. BRD in 2014: where have we been, where are we now, and where do we want to go? Anim. Health Res. Rev. 15(2):120-122. http://dx.doi.org/10.1017/S1466252314000115. PMid:25358813.
    » https://doi.org/10.1017/S1466252314000115
  • Holman D.B., McAllister T., Topp E., Wright A.-D.G. & Alexander T.W. 2015. The nasopharyngeal microbiota of feedlot cattle that develop bovine respiratory disease. Vet. Microbiol. 180(1/2):90-95. http://dx.doi.org/10.1016/j.vetmic.2015.07.031. PMid:26249828.
    » https://doi.org/10.1016/j.vetmic.2015.07.031
  • Hosmer D. & Lemeshow S. 1989. Applied Logistic Regression. Wiley, New York. 307p.
  • Kays M.B., Smith D.W., Wack M.F. & Denys G.A. 2002. Levofloxacin treatment failure in a patient with fluoroquinolone-resistant streptococcus pneumoniae pneumonia. Pharmacotherapy 22(3):395-399. http://dx.doi.org/10.1592/phco.22.5.395.33185. PMid:11898897.
    » https://doi.org/10.1592/phco.22.5.395.33185
  • Lima J.B.M. & Alves F.V. 2011. Diagnóstico das propriedades leiteiras do Assentamento São Manoel, Anastácio, MS. Cad. Agroec. 5(1):1-10.
  • Lima S.F., Teixeira A.G.V., Higgins C.H., Lima F.S. & Bicalho R.C. 2016. The upper respiratory tract microbiome and its potential role in bovine respiratory disease and otitis media. Sci. Rep. 6(1):29050. http://dx.doi.org/10.1038/srep29050. PMid:27363739.
    » https://doi.org/10.1038/srep29050
  • Loneragan G.H., Gould D.H., Mason G.L., Garry F.B., Yost G.S., Miles D.G., Hoffman B.W. & Mills L.J. 2001. Involvement of microbial respiratory pathogens in acute interstitial pneumonia in feedlot cattle. Am. J. Vet. Res. 62(10):1519-1524. http://dx.doi.org/10.2460/ajvr.2001.62.1519. PMid:11592313.
    » https://doi.org/10.2460/ajvr.2001.62.1519
  • Maeda T., Shibahara T., Kimura K., Wada Y., Sato K., Imada Y., Ishikawa Y. & Kadota K. 2003. Mycoplasma bovis associated suppurative otitis media and pneumonia in bull calves. J. Comp. Pathol. 129(2/3):100-110. http://dx.doi.org/10.1016/S0021-9975(03)00009-4. PMid:12921715.
    » https://doi.org/10.1016/S0021-9975(03)00009-4
  • Marques L.M., Buzinhani M., Oliveira R.C., Yamaguti M., Ferreira J.B., Neto R.L. & Timenetsky J. 2007. Prevalence of mycoplasmas in the respiratory tracts of calves in Brazil. Vet. Rec. 161(20):699-700. http://dx.doi.org/10.1136/vr.161.20.699. PMid:18024928.
    » https://doi.org/10.1136/vr.161.20.699
  • Miles D.G. 2009. Overview of the North American beef cattle industry and the incidence of bovine respiratory disease (BRD). Anim. Health Res. Rev. 10(2):101-103. http://dx.doi.org/10.1017/S1466252309990090. PMid:20003641.
    » https://doi.org/10.1017/S1466252309990090
  • Miller J.M., Hair J., Hebert M., Hebert L., Roberts Jr F.J. & Weyant R. 1997. Fulminating bacteremia and pneumonia due to Bacillus cereus J. Clin. Microbiol. 35(2):504-507. PMid:9003628.
  • Nikunen S., Härtel H., Orro T., Neuvonen E., Tanskanen R., Kivelä S.L., Sankari S., Aho P., Pyörälä S., Saloniemi H. & Soveri T. 2007. Association of bovine respiratory disease with clinical status and acute phase proteins in calves. Comp. Immunol. Microbiol. Infect. Dis. 30(3):143-151. http://dx.doi.org/10.1016/j.cimid.2006.11.004. PMid:17258318.
    » https://doi.org/10.1016/j.cimid.2006.11.004
  • OIE 2014. Contagious bovine pleuropneumonia: terrestrial manual. Available in <Available in http://www.oie.int/fileadmin/Home/eng/Health_standards/tahm/2.04.08_CBPP.pdf >. Accessed on Mar. 17, 2018.
    » http://www.oie.int/fileadmin/Home/eng/Health_standards/tahm/2.04.08_CBPP.pdf
  • Oliveira B.A.F.D., Carrillo Gaeta N., Mendonça Ribeiro B.L., Reyes Alemán M.A., Miranda Marques L., Timenetsky J., Melville P.A., Avansi Marques J., Marvulle V. & Gregory L. 2016. Determination of bacterial aetiologic factor on tracheobronchial lavage in relation to clinical signs of bovine respiratory disease. J. Med. Microbiol. 65(10):1137-1142. http://dx.doi.org/10.1099/jmm.0.000345. PMid:27582268.
    » https://doi.org/10.1099/jmm.0.000345
  • Panciera R.J. & Confer A.W. 2010. Pathogenesis and pathology of bovine pneumonia. Vet. Clin. N. Am., Food Anim. Pract. 26(2):191-214. http://dx.doi.org/10.1016/j.cvfa.2010.04.001. PMid:20619179.
    » https://doi.org/10.1016/j.cvfa.2010.04.001
  • Radostits C., Blood O.M. & Gay D.C. 2002. Clínica Veterinária: um tratado de doenças dos bovinos, ovinos, suínos, caprinos e equinos. Guanabara Koogan: Rio de Janeiro. 1737p.
  • Ribeiro O.C. 1979. Experimental infection of calves with Mycoplasma dispar Ames: Iowa State University. 162p.
  • Rice J.A., Carrasco-Medina L., Hodgins D.C. & Shewen P.E. 2007. Mannheimia haemolytica and bovine respiratory disease. Anim. Health Res. Rev. 8(2):117-128. http://dx.doi.org/10.1017/S1466252307001375. PMid:18218156.
    » https://doi.org/10.1017/S1466252307001375
  • Shelburne C.E., An F.Y., Dholpe V., Ramamoorthy A., Lopatin D.E. & Lantz M.S. 2007. The spectrum of antimicrobial activity of the bacteriocin subtilosin A. J. Antimicrob. Chemother. 59(2):297-300. http://dx.doi.org/10.1093/jac/dkl495. PMid:17213266.
    » https://doi.org/10.1093/jac/dkl495
  • Shubov A., Jagannathan P. & Chin-Hong P.V. 2011. Pantoea agglomerans pneumonia in a heart-lung transplant recipient: case report and a review of an emerging pathogen in immunocompromised hosts. Transpl. Infect. Dis. 13(5):536-539. http://dx.doi.org/10.1111/j.1399-3062.2011.00630.x. PMid:21504526.
    » https://doi.org/10.1111/j.1399-3062.2011.00630.x
  • Tegtmeier C., Uttenthal A., Friis N.F., Jensen N.E. & Jensen H.E. 1999. Pathological and microbiological studies on pneumonic lungs from Danish calves. Zentralbl Veterinarmed B 46(10):693-700. http://dx.doi.org/10.1046/j.1439-0450.1999.00301.x. PMid:10676147.
    » https://doi.org/10.1046/j.1439-0450.1999.00301.x
  • 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(16):472-476. http://dx.doi.org/10.1136/vr.151.16.472. PMid:12418530.
    » https://doi.org/10.1136/vr.151.16.472
  • Thomas L.H., Howard C.J., Stott E. & Parsons K.R. 1986. Mycoplasma bovis infection in gnotobiotic calves and combined infection with respiratory syncytial virus. Vet. Pathol. 23(5):571-578. http://dx.doi.org/10.1177/030098588602300505. PMid:3535220.
    » https://doi.org/10.1177/030098588602300505
  • Tully J. & Razin J.G. 1995. Culture medium formulation for primary isolation and maintenance of mollicutes. p.33-40. In: Ibid. (Eds), Molecular and diagnostic procedures in mycoplasmology. Academic Press Inc. http://dx.doi.org/10.1016/B978-012583805-4/50005-4.
    » https://doi.org/10.1016/B978-012583805-4/50005-4
  • Van Kuppeveld F.J., Van der Logt J.T., Angulo A.F., Van Zoest M.J., Quint W.G., Niesters H.G., Galama J.M. & Melchers W.J. 1992. Genus- and species-specific identification of mycoplasmas by 16S rRNA amplification. Appl. Environ. Microbiol. 58(8):2606-2615. PMid:1381174.
  • Winn Jr W., Allen S., Janda W., Koneman E., Procop G., Schrsckenberger P. & Woods G. 2005. Koneman’s color atlas and textbook of diagnostic mycrobiology. 6th ed. LWW, Philadelphia, 1736p.
  • Xie J., Zhang R., Shang C. & Guo Y. 2009. Isolation and characterization of a bacteriocin produced by an isolated Bacillus subtilis LFB112 that exhibits antimicrobial activity against domestic animal. Afr. J. Biotechnol. 8(20):5611-5619.
  • Zinka M. & Maid R. 2012. Mycoplasmas isolated from the respiratory tract of cattle in Bosnia and Herzegovina. Anim. Vet. 28:79-83

Publication Dates

  • Publication in this collection
    Mar 2018

History

  • Received
    09 Mar 2017
  • Accepted
    07 June 2017
Colégio Brasileiro de Patologia Animal - CBPA Pesquisa Veterinária Brasileira, Caixa Postal 74.591, 23890-000 Rio de Janeiro, RJ, Brasil, Tel./Fax: (55 21) 2682-1081 - Rio de Janeiro - RJ - Brazil
E-mail: pvb@pvb.com.br