Bacteria isolated from the lower respiratory tract of sheep and their relationship to clinical signs of sheep respiratory disease

Bactérias detectadas no trato respiratório inferior de ovinos e a relação com as manifestações clínicas da doença respiratória ovina

Mariane F. Franco Natália C. Gaeta Mario A.R. Alemán Priscilla A. Mellville Jorge Timenetsky Mário F.A. Balaro Lilian Gregory About the authors

ABSTRACT:

Respiratory diseases are among the most important diseases in sheep flocks. Herein was studied the bacterial etiology of respiratory disease and the clinical signs of 99 female and male sheep breed in the states of São Paulo (SP) and Rio de Janeiro (RJ), Brazil. After physical examination of animals, tracheobronchial flushing samples were obtained. The usual bacteria and Mycoplasma spp. were searched, as well as their association with the clinical status and clinical signs of sheep with respiratory disease. The main observed signs were: tachypnea (75%), increase of rectal temperature (09.4%), mucopurulent/purulent nasal discharge (21.9%), cough (25%), dyspnea (31.2%), changes of lung sounds at auscultation (87.5%) and chest percussion (28.1%) in pneumonic sheep. Non-fermenting gram-negative bacteria and Bacillus sp. were the most isolated bacteria. Microorganisms of the Mollicutes class were molecularly (PCR) detected in 33.3% of the animals. In addition, the specific detection of M. mycoides subsp. capri was described for the first time in sheep from the state of São Paulo, Brazil.

INDEX TERMS:
Bacteria; lower respiratory tract; sheep; clinics; sheep respiratory disease; Mycoplasma spp.; pneumonia; respiratory disease; bacterioses.

RESUMO:

A doença respiratória é uma das doenças mais importantes em rebanhos ovinos. Esta pesquisa teve como objetivo determinar a etiologia bacteriana da doença respiratória e sua relação com sinais clínicos em ovinos criados nos estados de São Paulo e Rio de Janeiro, Brasil. Noventa e nove ovelhas machos e fêmeas dos Estados de São Paulo (SP) e Rio de Janeiro (RJ) foram estudadas. Após o exame físico, amostras de lavagem traqueobrônquica foram obtidas. A presença de bactérias aeróbias e Mycoplasmaspp. foram estudados, assim como a associação entre os microrganismos e estado clínico e sinais clínicos de doença respiratória em ovinos. As principais manifestações clínicas observadas foram: taquipneia (75%), alta temperatura retal (09,4%), secreção nasal mucopurulenta/purulenta (21,9%), tosse (25%), dispneia (31,2%), sons pulmonares alterados na ausculta (87,5%) e na percussão torácica (28,1%) em ovelhas pneumônicas. Bactérias gram-negativas não fermentadoras e Bacillus sp. foram as bactérias mais isoladas. Microrganismos da classe Mollicutes foram detectados molecularmente (PCR) em 33,3% dos ovinos. Além disso, descreve-se pela primeira vez no estado de São Paulo, Brasil, a detecção do M. mycoides subsp. capri na espécie ovina utilizando a reação de polimerase em cadeia.

TERMOS DE INDEXAÇÃO:
Bactérias; trato respiratório inferior; ovinos; ovelhas; clínica; doença respiratória ovina; Mycoplasma spp.; pneumonia; bacterioses.

Introduction

Sheep respiratory disease is one of the most important diseases in sheep flocks (Marcondes et al. 2011Marcondes J.S., Martins M.T.A., Silva A.A., Rodrigues M.M.P., Ferreira D.O.L., Amorim R.L., Dias A. & Gonçalves R.C. 2011. Lavado traqueobrônquico por via nasotraqueal como metodologia de colheita de células do trato respiratório de ovinos sadios e portadores de afecções pulmonares. Pesq. Vet. Bras. 31(4):281-286. <http://dx.doi.org/10.1590/S0100-736X2011000400002>
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, Radostits et al. 2017Radostits O.M., Gay C., Blood D.C. & Hinchcliff K.W. 2017. Clinica Veterinária: um tratado de doenças dos bovinos, ovinos, suínos, caprinos e equinos. 10ª ed. Guanabara Koogan, Rio de Janeiro. 1737p.) due to its economic losses. In New Zealand, pneumonia in lambs cost NZ$28.1 million in 2008 (Goodwin-Ray et al. 2008Goodwin-Ray K.A., Stevenson M.A., Heuer C. & Cogger N. 2008. Economic effect of pneumonia and pleurisy in lambs in New Zealand. N. Z. Vet. J. 56(3):107-114. <http://dx.doi.org/10.1080/00480169.2008.36818> <PMid:18536769>
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). From 1996 to 2010, 10% of diagnosed diseases in lambs in the “Universidade Federal do Mato Grosso do Sul” were pneumonic related (Almeida et al. 2013Almeida T.L., Brum K.B., Lemos R.A.A., Leal C.R.B. & Borges F.A. 2013. Doenças de ovinos diagnosticadas no laboratório de anatomia patológica animal da Universidade Federal de Mato Grosso do Sul, Brasil (1996-2010). Pesq. Vet. Bras. 33(1):21-29. <http://dx.doi.org/10.1590/S0100-736X2013000100005>
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). The incidence of pneumonia in sheep and other respiratory diseases in Argentina was 57.9% (Suárez & Busetti 2009Suárez V.H. & Busetti M.R. 2009. Práticas de manejo sanitário e frequência de doenças em ovinos leiteiros na Argentina. Pesq. Vet. Bras. 29(11):931-937.). In Tanzania, 31.4% of lungs had pneumonic lesions in slaughter (Mellau et al. 2010Mellau L.S., Nonga H.E. & Karimuribo E.D. 2010. A slaughterhouse survey of lung lesions in slaughtered stocks at Arusha, Tanzania. Prev. Vet. Med. 97(2):77-82. <http://dx.doi.org/10.1016/j.prevetmed.2010.08.008> <PMid:20875689>
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). Recently, McRae et al. (2016)McRae K.M., Baird H.J., Dodds K.G., Bixley M.J. & Clarke S.M. 2016. Incidence and heritability of ovine pneumonia, and the relationship with production traits in New Zealand sheep. Small Rum. Res. 145:136-141. detected pneumonic lesions in 28% of lambs at slaughter in New Zealand.

Important risk factors related to respiratory disease of ruminants are the environmental high temperature and humidity, increased animal density, stress, dust, poor ventilation, and parasites (Goodwin-Ray et al. 2008Goodwin-Ray K.A., Stevenson M.A., Heuer C. & Cogger N. 2008. Economic effect of pneumonia and pleurisy in lambs in New Zealand. N. Z. Vet. J. 56(3):107-114. <http://dx.doi.org/10.1080/00480169.2008.36818> <PMid:18536769>
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, Taylor et al. 2010Taylor J.D., Fulton R.W., Lehenbauer T.W., Step D.L. & Confer A.W. 2010. The epidemiology of bovine respiratory disease: what is the evidence for predisposing factors? Can Vet J. 51(10):1095-1102. <PMid:21197200>, Scott 2011Scott P.R. 2011. Treatment and control of respiratory disease in sheep. Vet Clin. N. Am., Food Anim. Pract. 27(1):175-186. <http://dx.doi.org/10.1016/j.cvfa.2010.10.016> <PMid:21215901>
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). Some viruses are considered primary pathogens, changing the epithelial cells, and interfering in the immune response (Bosch et al. 2013Bosch A.A.T.M., Biesbroek G., Trzcinski K., Sanders E.A.M. & Bogaert D. 2013. Viral and bacterial interactions in the upper respiratory tract. PLoS Pathog. 9(1):e1003057. <http://dx.doi.org/10.1371/journal.ppat.1003057> <PMid:23326226>
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), leading to secondary bacterial infections. According to Sharp & Nettleton (2007)Sharp J.M. & Nettleton P.F. 2007. Acute respiratory virus infections, p.207-211. In: Aitken I. (Ed), Diseases of Sheep. Blackwell Publishing, New Jersey. http://dx.doi.org/10.1002/9780470753316.ch29.
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acute respiratory disease is not caused by virus infections alone.

Mannheimia (M.) haemolytica and Pasteurella (P.) multocida are important respiratory pathogens in sheep flocks (Radostits et al. 2017Radostits O.M., Gay C., Blood D.C. & Hinchcliff K.W. 2017. Clinica Veterinária: um tratado de doenças dos bovinos, ovinos, suínos, caprinos e equinos. 10ª ed. Guanabara Koogan, Rio de Janeiro. 1737p.). Both bacteria belong to family Pasteurellaceae, order Pasteurellales (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...
). These bacteria are part of the respiratory tract microbiota in sheep (Viana et al. 2007Viana L., Gonçalves R.C., Oliveira Filho J.P., Paes A.C. & Amorim R.M. 2007. Ocorrência de Mannheimia haemolytica e de Pasteurella multocida em ovinos sadios e com enfermidade respiratória. Arq. Bras. Med. Vet. Zootec. 59:1579-1582.) and as stated before, immunosuppression and viral infections predispose to respiratory disease caused by both species (Bosch et al. 2013Bosch A.A.T.M., Biesbroek G., Trzcinski K., Sanders E.A.M. & Bogaert D. 2013. Viral and bacterial interactions in the upper respiratory tract. PLoS Pathog. 9(1):e1003057. <http://dx.doi.org/10.1371/journal.ppat.1003057> <PMid:23326226>
https://doi.org/10.1371/journal.ppat.100...
, Viana et al. 2007Viana L., Gonçalves R.C., Oliveira Filho J.P., Paes A.C. & Amorim R.M. 2007. Ocorrência de Mannheimia haemolytica e de Pasteurella multocida em ovinos sadios e com enfermidade respiratória. Arq. Bras. Med. Vet. Zootec. 59:1579-1582.). Almost 10% of pneumonic sheep were positive for P. multocida in lungs in Nigeria (Odugbo et al. 2006Odugbo M.O., Odama L.E., Umoh J.U. & Lamorde A.G. 2006. Pasteurella multocida pneumonic infection in sheep: prevalence, clinical and pathological studies. Small Rum. Res. 66(1/3):273-277. <http://dx.doi.org/10.1016/j.smallrumres.2005.09.003>
https://doi.org/10.1016/j.smallrumres.20...
). Ten sheep died during an outbreak of M. haemolytica associated pneumonia in the State of Minas Gerais in 2009 (Araujo et al. 2009Araújo M.R., Costa M.C. & Ecco R. 2009. Ocorrência de pneumonia associada à infecção por Mannheimia haemolytica em ovinos de Minas Gerais. Pesq. Vet. Bras. 29(9):719-724. http://dx.doi.org/10.1590/S0100-736X2009000900007.
https://doi.org/10.1590/S0100-736X200900...
).

Mycoplasma spp. belongs to class Mollicutes, order Mycoplasmatales and family Mycoplasmataceae (Razin & Hayflick 2010Razin S. & Hayflick L. 2010. Highlights of mycoplasma research: an historical perspective. Biologicals 38(2):183-190. <http://dx.doi.org/10.1016/j.biologicals.2009.11.008> <PMid:20149687>
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). They are the smallest organisms with self-replication (Razin & Hayflick 2010Razin S. & Hayflick L. 2010. Highlights of mycoplasma research: an historical perspective. Biologicals 38(2):183-190. <http://dx.doi.org/10.1016/j.biologicals.2009.11.008> <PMid:20149687>
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) and some are detected in healthy and diseased ruminants (Ayling et al. 2004Ayling R.D., Bashiruddin S.E. & Nicholas R.A.J. 2004. Mycoplasma species and related organisms isolated from ruminants in Britain between 1990 and 2000. Vet. Rec. 155(14):413-416. <http://dx.doi.org/10.1136/vr.155.14.413> <PMid:15508840>
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, Kumar et al. 2011Kumar P., Roy A., Bhanderi B.B. & Pal B. 2011. Isolation, identification and molecular characterization of Mycoplasma isolates from goats of Gujarat State, India. Vet. Arh. 81:443-458., Oliveira et al. 2016Oliveira B.A., Gaeta N.C., Ribeiro B.L.M., Alemán M.A.R., Marques L.M., Timenetsky J., Marques J.A., Melville P.A., 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>
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, Tortorelli et al. 2017Tortorelli G., Gaeta N.C., Ribeiro B.L.M., Marques L.M., Timenetsky J. & Gregory L. 2017. Evaluation of Mollicutes microorganisms in respiratory disease of cattle and their relationship to clinical signs. J. Vet. Intern. Med. 31(4):1215-1220. <http://dx.doi.org/10.1111/jvim.14721> <PMid:28602021>
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, Gaeta et al. 2018Gaeta N.C., Ribeiro B.L.M., Alemán M.A.R., Yoshihara E., Nassar A.F.C., Marques E.C., Timenetsky J. & Gregory L. 2018. Bacterial pathogens of the lower respiratory tract of calves from Brazilian rural settlement herds and their association with clinical signs of bovine respiratory disease. Pesq. Vet. Bras. 38(3):374-381. <http://dx.doi.org/10.1590/1678-5150-pvb-5323>
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). M. mycoides subsp. capri (Srivastava et al. 2000Srivastava N.C., Singh V.P., Sunder J., Singh V.P. & Thiaucourt F. 2000. Isolation of Mycoplasma mycoides small colony type from contagious caprine pleuropneumonia in India. Vet. Rec. 147(18):520-521. <http://dx.doi.org/10.1136/vr.147.18.520> <PMid:11110496>
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, Al-Momani et al. 2011Al-Momani W., Abo-Shehada M.N. & Nicholas R.A. 2011. Seroprevalence of and risk factors for Mycoplasma mycoides subspecies capri infection in small ruminants in Northern Jordan. Trop. Anim. Health Prod. 43(2):463-469. <http://dx.doi.org/10.1007/s11250-010-9717-9> <PMid:20960059>
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, Kumar et al. 2013Kumar V., Rana R., Mehra S. & Rout P.K. 2013. Isolation and characterization of Mycoplasma mycoides subsp. capri from milk of natural goat astitis cases. ISRN Vet Sci. 593029:5p. <PMid:23762593>), M. agalactiae (Kumar et al. 2014Kumar A., Rahal A., Chakraborty S., Verma A.K. & Dhama K. 2014. Mycoplasma agalactiae, an etiological agent of contagious agalactia in small ruminants: a review. Vet. Med. Int. 2014:1-13. <http://dx.doi.org/10.1155/2014/286752> <PMid:25097796>
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), M. capricolum subsp. capricolum (Fischer et al. 2012Fischer A., Shapiro B., Muriuki C., Heller M., Schnee C., Bongcam-Rudloff E., Vilei E.M., Frey J. & Jores J. 2012. The origin of the «Mycoplasma mycoides cluster» coincides with domestication of ruminants. PLoS One 7(4):e36150. <http://dx.doi.org/10.1371/journal.pone.0036150> <PMid:22558362>
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), M. capripneumoniae (Igbal et al. 2019Igbal Y.M., Raffiq P.O., Tauseef B.S., Ahmed B.R., Gopalakrishnan A., Karthik K., Dhama K. & Vir S.S. 2019. Contagious caprine pleuropneumonia - a comprehensive review. Vet. Q. 39(1):1-25.) and M. ovipneumoniae (George & Carmichael 1975George T.D.S. & Carmichael L.E. 1975. Isolation of Mycoplasma ovipneumoniae from sheep with chronic pneumonia. Vet. Rec. 97(11):205-206. <http://dx.doi.org/10.1136/vr.97.11.205> <PMid:1162872>
https://doi.org/10.1136/vr.97.11.205...
, Ayling et al. 2004Ayling R.D., Bashiruddin S.E. & Nicholas R.A.J. 2004. Mycoplasma species and related organisms isolated from ruminants in Britain between 1990 and 2000. Vet. Rec. 155(14):413-416. <http://dx.doi.org/10.1136/vr.155.14.413> <PMid:15508840>
https://doi.org/10.1136/vr.155.14.413...
, Dassanayake et al. 2010Dassanayake R.P., Shanthalingam S., Herndon C.N., Subramaniam R., Lawrence P.K., Bavananthasivam J., Cassirer E.F., Haldorson G.J., Foreyt W.J., Rurangirwa F.R., Knowles D.P., Besser T.E. & Srikumaran S. 2010. Mycoplasma ovipneumoniae can predispose bighorn sheep to fatal Mannheimia haemolytica pneumonia. Vet. Microbiol. 145(3/4):354-359. <http://dx.doi.org/10.1016/j.vetmic.2010.04.011> <PMid:20466492>
https://doi.org/10.1016/j.vetmic.2010.04...
) are described as important respiratory mycoplasmas for small ruminants.

Sheep with respiratory disease often show depression, inappetence, high rectal temperature, dyspnea, coughing, mucopurulent or purulent nasal discharge and increased cardiac and respiratory rates (Scott 2011Scott P.R. 2011. Treatment and control of respiratory disease in sheep. Vet Clin. N. Am., Food Anim. Pract. 27(1):175-186. <http://dx.doi.org/10.1016/j.cvfa.2010.10.016> <PMid:21215901>
https://doi.org/10.1016/j.cvfa.2010.10.0...
). Only a few studies aimed detect the bacterial etiology of pneumonic sheep in Brazil (Coutinho et al. 2009Coutinho A.S., Oliveira Filho J.P., Silva D.P.G., Oliveira A.P., Marcondes J.S., Chiacchio S.B., Paes A.C., Siqueira A.K., Amorim R.M. & Gonçalves R.C. 2009. Mannheimiose pulmonar experimental em bezerros: swab nasal e nasofaringeano como auxílio diagnóstico. Pesq. Vet. Bras. 29(1):83-88. <http://dx.doi.org/10.1590/S0100-736X2009000100013>
https://doi.org/10.1590/S0100-736X200900...
, Marcondes et al. 2011Marcondes J.S., Martins M.T.A., Silva A.A., Rodrigues M.M.P., Ferreira D.O.L., Amorim R.L., Dias A. & Gonçalves R.C. 2011. Lavado traqueobrônquico por via nasotraqueal como metodologia de colheita de células do trato respiratório de ovinos sadios e portadores de afecções pulmonares. Pesq. Vet. Bras. 31(4):281-286. <http://dx.doi.org/10.1590/S0100-736X2011000400002>
https://doi.org/10.1590/S0100-736X201100...
). Therefore, herein it was detected the bacterial etiology of respiratory disease and their relationship to clinical signs of sheep respiratory disease.

Materials and Methods

All procedures were carried out in agreement with the guidelines of the Committee of Ethics on Animal Use (Protocol no. 3498060716).

In this cross-sectional research, 99 male and female sheep from 12 flocks from the States of São Paulo (SP=6) and Rio de Janeiro (RJ=6) were enrolled in this study, according to a non-probabilistic sampling for convenience.

Animals were classified as healthy and unhealthy (showing clinical signs of respiratory disease) after physical exam (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...
, Gaeta et al. 2018Gaeta N.C., Ribeiro B.L.M., Alemán M.A.R., Yoshihara E., Nassar A.F.C., Marques E.C., Timenetsky J. & Gregory L. 2018. Bacterial pathogens of the lower respiratory tract of calves from Brazilian rural settlement herds and their association with clinical signs of bovine respiratory disease. Pesq. Vet. Bras. 38(3):374-381. <http://dx.doi.org/10.1590/1678-5150-pvb-5323>
https://doi.org/10.1590/1678-5150-pvb-53...
). Sheep were considered unhealthy if show at least two of the following parameters: respiratory rate more than 30 respiratory movements per minute, coughing, rectal temperature higher than 40°C and abnormal sound on thoracic auscultation.

Tracheobronchial wash samples were obtained from both health and pneumonic sheep (Anton & Mayayo 2007Anton J.J.R. & Mayayo L.M.F. 2007. La exploracion clinica del ganado ovino y su entorno. Servet, Zaragoza. 448p.). Briefly, trichotomy and antisepsis using 70% alcohol, chlorhexidine and iodopovidone were performed in the third part of the trachea. An Intracath® (BD, USA) was introduced by tracheocentesis and 20 to 50mL of sterile saline 0.9% was instilled, recovering up to 5mL. An aliquot (500μL) was added to Stuart transport media and stored at 4°C until the isolation of bacteria. Another aliquot was added to transport media for Mycoplasma spp. containing glycerol and stored in liquid nitrogen.

Tracheal suspensions were inoculated in Brain Heart Infusion broth and the cultures in agar plates were performed by plating 10μL of these samples in MacConkey and Blood agar (5%). The cultures were incubated for 24 to 72 hours at 37°C in aerobic atmosphere. The obtained colonies were noted for their morphology, Gram stained and screened for biochemical tests (Staphylococcus sp.: catalase test, sensitivity to bacitracin and furazolidone and oxidase test; Streptococcus sp.: catalase test, sensitivity to bacitracin, growth at 65% NaCl, bile-esculin test and growth at 45°C; Bacillus sp.: catalase test; Klebsiella sp. and non-fermenting bacteria: triple sugar iron growth, production of indole, urease and fenilalanine desaminase, oxidation-fermentation test, motility test, lysine descarboxylation test, use of citrate and malonate, lactose and glucose fermentation, nitrate reduction, Voges-Proskauer test and Methyl-red test) (Murray et al. 1999Murray P.R., Baron E.J., Pfaller M., Tenover F.C. & Yolken R. 1999. Manual of Clinical Microbiology. 7th ed. American Society for Microbiology, Washington, DC.).

Mycoplasma spp. isolation was performed using solid and liquid SP-4 media (Tully 1995Tully J. 1995. Culture medium formulation for primary isolation and maintenance of mollicutes, p.33-39. In: Razin S. & Tully J. (Eds), Molecular and Diagnostic Procedures in Mycoplasmology. Academic Press, Massachusetts. http://dx.doi.org/10.1016/B978-012583805-4/50005-4.
https://doi.org/10.1016/B978-012583805-4...
). Agar plates and broths were incubated in aerobiosis at 37°C for 15 days. Mycoplasma spp. were screened for producing “fried-egg”-like colonies and differentiated for glucose fermentation and/or arginine hydrolysis without turbiding the broths. Polymerase chain reaction (PCR) was performed to confirm the DNA of Mollicutes in samples (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 PCR positive samples were tested again for M. bovis and M. agalactiae (Chávez González et al. 1995Chávez González Y.R., Bascuñana C.R., Bölske G., Mattsson J.G., Molina C.F. & 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...
) and M. mycoides subsp. capri (Monnerat et al. 1999Monnerat M.P., Thiaucourt F., Poveda J.B., Nicolet J. & Frey J. 1999. Genetic and serological analysis of lipoprotein LppA in Mycoplasma mycoides subsp. mycoides LC and Mycoplasma mycoides subsp. capri. Clin. Diagn. Lab. Immunol. 6(2):224-230. <PMid:10066658>).

The association between health status and clinical signs and microbiologic findings were calculated using Chi-square test or Fisher’s Exact test. Variables with a P-value <0,05 were considered significant. All calculations were performed using Software Statistical Package for Social Sciences 16.0 (SPSS, IBM).

Results

After clinical examination, the sheep were classified as healthy (67.7%, 67/99) and pneumonic (32.32%, 32/99). Clinical signs such as tachypnea, high rectal temperature, mucopurulent/purulent nasal discharge, coughing, dyspnea, abnormal pulmonary sounds on auscultation and thoracic percussion were increased in pneumonic sheep compared to health sheep (Table 1).

Table 1.
Clinical signs detected in healthy and pneumonic sheep from the States of São Paulo and Rio de Janeiro, Brazil

Forty-five bacterial isolates were obtained. Bacillus sp., non-fermentative gram-negative bacteria, Streptococcus sp. and Staphylococcus sp. were the main species. P. multocida and M. haemolytica were not isolated (Table 2). Fried-egg-like colonies were observed in 23.3% (17/73) of samples. The PCR methodology with generic primers for Mollicutes spp. confirmed all isolates of Mollicutes. In addition, PCR was also performed directly in tracheobronchial wash samples, and the DNA of Mollicutes spp. was detected in 45.2% (33/73) of samples (38% in pneumonic sheep and 49% in healthy sheep). M. mycoides subsp. capri (01/33) was detected by PCR. The used specific primers in this study did not allow detect other Mollicute species. No association between species detected and health status were detected.

Table 2.
Aerobic bacteria detected in tracheobronchial wash samples of sheep in relation to respiratory health status

Regarding to clinical manifestation, only Bacillus sp. was associated to respiratory rate (P=0.005) and submassive/massive sound on thoracic percussion (P=0.024).

Discussion

The present study helps to better understand the main bacteria involved in the respiratory disease of Brazilian sheep from the States of São Paulo and Rio de Janeiro, Brazil. Bronchopneumonia was detected in 32.3% of sheep as detected in other studies in Brazil (Viana et al. 2007Viana L., Gonçalves R.C., Oliveira Filho J.P., Paes A.C. & Amorim R.M. 2007. Ocorrência de Mannheimia haemolytica e de Pasteurella multocida em ovinos sadios e com enfermidade respiratória. Arq. Bras. Med. Vet. Zootec. 59:1579-1582.) and Ethiopia (Garedew et al. 2010Garedew L., Gelagay A., Yilma R., Zelek A. & Gelaye E. 2010. Isolation of diverse bacterial species associated with Maedi-Visna infection of sheep in Ethiopia. African J. Microbiol. Res. 4:14-21.).

Respiratory rate, rectal temperature, nasal discharge, cough, dyspnea, percussion and altered sounds in pulmonary auscultation were increased in pneumonic sheep (P<0.05). These findings confirm the importance of the observed clinical signs of sheep to determine the respiratory health during physical examination (Viana et al. 2007Viana L., Gonçalves R.C., Oliveira Filho J.P., Paes A.C. & Amorim R.M. 2007. Ocorrência de Mannheimia haemolytica e de Pasteurella multocida em ovinos sadios e com enfermidade respiratória. Arq. Bras. Med. Vet. Zootec. 59:1579-1582., Legesse et al. 2018Legesse A., Abayneh T., Mamo G., Gelaye E., Tesfaw L., Yami M. & Belay A. 2018. Molecular characterization of Mannheimia haemilytica isolates associated with pneumonic cases of sheep in selected areas of central Ethiopia. BMC Microbiol. 18(1):205. <http://dx.doi.org/10.1186/s12866-018-1338-x> <PMid:30518323>
https://doi.org/10.1186/s12866-018-1338-...
).

Opportunistic agents may have a controversy importance due their detection in healthy hosts. Bacillus sp. was the most isolated bacteria, especially in pneumonic sheep. This microbial genera was also detected in other studies with the same propose (Asaye et al. 2015Asaye M.B. & Habtamu B.M. 2015. Isolation and characterization of respiratory tract bacterial species from domestic animals with pneumonic lungs from Elphora abattoir, Ethiopia. Int. J. Microbiol. Res. 6:13-19., Megra et al. 2006Megra T., Sisay T. & Asseged B. 2006. The aerobic bacterial flora of the respiratory passageways of healthy goats in Dire Dawa Abattoir, Eastern Ethiopia. Rev. Med. Vet. 157:84-87., Gebremeskel et al. 2017Gebremeskel A., Tesema T., Yegoraw A. & Mekuria B. 2017. Isolation and characterization of bacterial species from respiratory tracts of cattle slaughtered in Addis Abada City, Central Ethiopia. World’s Vet. J. 1(1):14-20. <http://dx.doi.org/10.5455/wvj.20170289>
https://doi.org/10.5455/wvj.20170289...
, Yegoraw et al. 2017Yegoraw A.A., Gebremeskel A.K., Tesema T.S. & Birhanu B.T. 2017. Aerobic and anaerobica bacterial isolates from the respiratory tract of sheep slaughteres at Addis Abada Abattoirs Enterprises, Central Ethiopia. J. Vet. Med. Anim. Health 10:284-289., Yimer & Asseged 2007Yimer N. & Asseged B. 2007. Aerobic bacterial flora of respiratory tract of healthy sheep slaughtered in Dessie municipal abattoir, northeastern Ethiopia. Rev. Med. Vet. 158:473-478.). In fact, Bacillus sp. is an opportunistic infectious agent that belongs to the ovine respiratory tract microbiota (Rajivkumar & Ghaar 2000Rajivkumar K.R.C. & Ghaar P. 2000. Bacteriological studies on pneumonic Gaddi sheep of Himachal Predsh. Indian Vet. J. 77:846-848., Garedew et al. 2010Garedew L., Gelagay A., Yilma R., Zelek A. & Gelaye E. 2010. Isolation of diverse bacterial species associated with Maedi-Visna infection of sheep in Ethiopia. African J. Microbiol. Res. 4:14-21.). The presence of non-anthracis Bacillus microorganism in clinical samples was considered a paradigm for a long time. As mentioned in literature, many infectious agents were previously considered not be pathogenic and became be accepted as pathogenic (Farrar Jr. 1963Farrar Junior W.E. 1963. Serious infections due to “non-pathogenic” organisms of the genus Bacillus. Review of their status as pathogens. Am. J. Med. 34(1):134-141. http://dx.doi.org/10.1016/0002-9343(63)90047-0. PMid:13944444.
https://doi.org/10.1016/0002-9343(63)900...
) as happened with Bacillus sp. considered a non-contaminant in clinical samples (Shimoyama et al. 2017Shimoyama Y., Umegaki O., Ooi Y., Agui T., Kadono N. & Minami T. 2017. Bacillus cereus pneumonia in an immunocompetent patient: a case report. JA Clin. Reports 3(1):25. <http://dx.doi.org/10.1186/s40981-017-0096-3> <PMid:29457069>
https://doi.org/10.1186/s40981-017-0096-...
). Otherwise Bacillus cereus is known to be responsible for nosocomial pneumonia in human, and in immunocompromised or immunocompetent human patients (Gray et al. 1999Gray J., George R.H., Durbin G.M., Ewer A.K., Hocking M.D. & Morgan M.E.I. 1999. An outbreak of Bacillus cereus respiratory tract infections on a neonatal unit due to contaminated ventilator circuits. J. Hosp. Infect. 41(1):19-22. <http://dx.doi.org/10.1016/S0195-6701(99)90032-4> <PMid:9949960>
https://doi.org/10.1016/S0195-6701(99)90...
, Miyata et al. 2013Miyata J., Tasaka S., Miyazaki M., Yoshida S., Naoki K., Sayama K., Asano K., Fujiwara H., Ohkusu K., Hasegawa N. & Betsuyaku T. 2013. Bacillus cereus necrotizing pneumonia in a patient with nephrotic syndrome. Intern. Med. 52(1):101-104. <http://dx.doi.org/10.2169/internalmedicine.52.7282> <PMid:23291682>
https://doi.org/10.2169/internalmedicine...
, Shimoyama et al. 2017Shimoyama Y., Umegaki O., Ooi Y., Agui T., Kadono N. & Minami T. 2017. Bacillus cereus pneumonia in an immunocompetent patient: a case report. JA Clin. Reports 3(1):25. <http://dx.doi.org/10.1186/s40981-017-0096-3> <PMid:29457069>
https://doi.org/10.1186/s40981-017-0096-...
). Bacillus sp. has been isolated from lung tissue (Elshafee 2003Elshafee S. 2003. Isolation and characterization of aerobic bacteria associated with pneumonic lungs of cattle in Singa slaughter house, Sinnar State. Master’s Dissertation, University of Khartoum, Khartoum State, Sudan. 75p.) and tracheobronchial flushing samples of cattle (Oliveira et al. 2016Oliveira B.A., Gaeta N.C., Ribeiro B.L.M., Alemán M.A.R., Marques L.M., Timenetsky J., Marques J.A., Melville P.A., 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...
, Gaeta et al. 2018Gaeta N.C., Ribeiro B.L.M., Alemán M.A.R., Yoshihara E., Nassar A.F.C., Marques E.C., Timenetsky J. & Gregory L. 2018. Bacterial pathogens of the lower respiratory tract of calves from Brazilian rural settlement herds and their association with clinical signs of bovine respiratory disease. Pesq. Vet. Bras. 38(3):374-381. <http://dx.doi.org/10.1590/1678-5150-pvb-5323>
https://doi.org/10.1590/1678-5150-pvb-53...
). The present study stated the association of clinical signs of sheep, the respiratory disease and the isolation of Bacillus sp. (high respiratory rate and the presence of abnormal sound on thoracic percussion).

Klebsiella oxytoca and K. pneumoniae are virulent bacteria that are often associated with pneumonia in sheep (Ajuwape & Aregbesola 2002Ajuwape T.P. & Aregbesola E.A. 2002. The bacterial flora of the upper respiratory tract of normal rabbits. Isr. Vet. Med. Assoc. 57:1-5., Patel et al. 2017Patel S.S., Chauhan H.C., Patel A.C., Shrimali M.D., Patel K.B., Prajapati B.I., Kala J.K., Patel M.G., Rajgor M. & Patel M.A. 2017. Isolation and identification of Klebsiella pneumoniae from sheep. Int. J. Curr. Microbiol. Appl. Sci. 6(5):331-334. <http://dx.doi.org/10.20546/ijcmas.2017.605.037>
https://doi.org/10.20546/ijcmas.2017.605...
). Herein, both species were detected, as well was in other studies in Ethiopia et al. (2010) and Iraqi (Al-Sultan 1995Al-Sultan I.I. 1995. Bacterial isolation from pneumonic lungs in sheep. Iraq J. Vet. Sci. 8:213-215.).

Mycoplasmas are frequently related to respiratory disease in sheep, and are responsible for important economic losses (Kumar et al. 2012Kumar A., Verma A.K., Gangwar N.K. & Rahal A. 2012. Isolation, characterization and antibiogram of Mycoplasma bovis in sheep pneumonia. Asian J. Anim. Vet. Adv. 7(2):149-157. <http://dx.doi.org/10.3923/ajava.2012.149.157>
https://doi.org/10.3923/ajava.2012.149.1...
). Mollicutes were detected by PCR in 45.2% of studied sheep tracheal samples. Al-Momani et al. (2006)Al-Momani W., Halablab M.A., Abo-Shehada N., Miles K., Mcauliffe L. & Nicholas R.A.J. 2006. Isolation and molecular identification os small ruminant mycoplasmas in Jordan. Small Rumin. Res. 65(1/2):106-112. <http://dx.doi.org/10.1016/j.smallrumres.2005.05.022>
https://doi.org/10.1016/j.smallrumres.20...
obtained similar results in Jordan (35% samples). In addition, we detected Mollicutes in 38% of pneumonic and 49% in healthy sheep. It is well stablished that Mollicutes are part of the respiratory tract microbiota of ruminants, and after a stress, they may become pathogenic. These findings are also in agreement with other studies Swedish flocks (Tauni 2017Tauni F.A. 2017. Association of Mycoplasma ovipneumoniae infection with respiratory disease in Swedish sheep. Degree Project in Veterinary Medicine, Swedish University of Agricultural Sciences, Uppsala, Sweden. 33p.), and India (Kumar et al. 2012Kumar A., Verma A.K., Gangwar N.K. & Rahal A. 2012. Isolation, characterization and antibiogram of Mycoplasma bovis in sheep pneumonia. Asian J. Anim. Vet. Adv. 7(2):149-157. <http://dx.doi.org/10.3923/ajava.2012.149.157>
https://doi.org/10.3923/ajava.2012.149.1...
). Mollicutes have also been detected in the lower respiratory tract of calves (Oliveira et al. 2016Oliveira B.A., Gaeta N.C., Ribeiro B.L.M., Alemán M.A.R., Marques L.M., Timenetsky J., Marques J.A., Melville P.A., 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...
, Gaeta et al. 2018Gaeta N.C., Ribeiro B.L.M., Alemán M.A.R., Yoshihara E., Nassar A.F.C., Marques E.C., Timenetsky J. & Gregory L. 2018. Bacterial pathogens of the lower respiratory tract of calves from Brazilian rural settlement herds and their association with clinical signs of bovine respiratory disease. Pesq. Vet. Bras. 38(3):374-381. <http://dx.doi.org/10.1590/1678-5150-pvb-5323>
https://doi.org/10.1590/1678-5150-pvb-53...
) in Brazil. M. mycoides subsp. capri was detected by PCR in present study in a healthy sheep. Opportunistic bacteria are easily spread into the flocks, particularly in animals in close proximity. As stated before, any situation that compromises the immune system favors the disease development. During a respiratory outbreak by M. mycoides subsp. capri, Hernandez et al. (2006)Hernandez L., Lopez J., St-Jacques M., Ontiveros L., Acosta J. & Handel K. 2006. Mycoplasma mycoides subsp. capri associated with goat respiratory disease and high flock mortality. Can. Vet. J. 47(4):366-369. <PMid:16642877> described a 60% mortality in a Mexican goat flock. Animals showed abundant nasal discharge, fever, dyspnea, prostration, ear drop, and decrease in milk production. Depending on the number of affected animals, economic losses might be severe. Finally, most of Mollicutes detected by PCR in studied samples was not specifically identified. This questioned the role of other species of Mollicutes in respiratory disease of sheep, such as M. bovis (Kumar et al. 2012Kumar A., Verma A.K., Gangwar N.K. & Rahal A. 2012. Isolation, characterization and antibiogram of Mycoplasma bovis in sheep pneumonia. Asian J. Anim. Vet. Adv. 7(2):149-157. <http://dx.doi.org/10.3923/ajava.2012.149.157>
https://doi.org/10.3923/ajava.2012.149.1...
), Acholeplasma sp., and M. arginine (DaMassa et al. 1992DaMassa A.J., Wakenell P.S. & Brooks D.L. 1992. Mycoplasmas of gots and sheep. J. Vet. Diagn. Invest. 4(1):101-113. <http://dx.doi.org/10.1177/104063879200400126> <PMid:1554763>
https://doi.org/10.1177/1040638792004001...
).

Conclusions

In the present study, Bacillus sp. was the main bacteria detected in both healthy and pneumonic sheep. Important pathogens such as Klebsiella oxytoca and K. pneumoniae were both isolated in pneumonic sheep, confirming their importance in the etiology of the disease in the southeastern region of Brazil.

Mollicutes is part of the ruminant’s microbiota, but they are also important to ovine pneumonia.

This seems to be the first study that detected Mycoplasma mycoides subsp. capri in the State of São Paulo.

Acknowledgements

Authors are thankful for all participants and The Brazilian National Council for Scientific and Technological Development for the financial support.

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Publication Dates

  • Publication in this collection
    02 Dec 2019
  • Date of issue
    Oct 2019

History

  • Received
    06 May 2019
  • Accepted
    04 June 2019
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