Ocorrência, caracterização fenotípica e genotípica de linhagens de Corynebacterium pseudotuberculosis isoladas de linfonodos de ovinos com e sem linfadenite
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Data
2013-07-29
Autores
Zamprogna, Thiago de Oliveira
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Universidade Estadual Paulista (Unesp)
Resumo
A linfadenite caseosa ovina se caracteriza pela complexidade etiológica, embora Corynebacterium pseudotuberculosis seja considerado o micro-organismo mais frequentemente isolado de ovinos e caprinos em vários países. O exame microbiológico ainda é considerado o método mais prático na rotina de diagnóstico da doença, porém a citologia aspirativa com agulha fina (CAAF) vem sendo utilizada nos últimos anos no diagnóstico à admissão de animais em exposições com sinais clínicos da doença. Recentemente, o diagnóstico molecular tem-se mostrado promissor na identificação do micro-organismo e biovares. O presente estudo investigou a ocorrência de micro-organismos, a caracterização fenotípica e genotípica de linhagens de Corynebacterium pseudotuberculosis isoladas de linfonodos de ovinos com e sem linfadenite. Foram citoaspirados 197 linfonodos de ovinos apresentando linfadenite (submandibulares, parotídeos, pré-femorais, poplíteos, pré-escapulares) sugestivos, clinicamente, de linfadenite caseosa, visando o diagnóstico microbiológico e citológico. Os animais eram provenientes de 14 rebanhos da região centro oeste e sudoeste do estado de São Paulo. Paralelamente, foram colhidos 202 linfonodos viscerais (mesentéricos e/ou mediastínicos), sem sinais aparentes de linfadenite, obtidos na linha de abate de frigorífico. O material colhido de todos os animais com e sem linfadenite foi submetido ao cultivo microbiológico e exame citológico. Os isolados de C. pseudotuberculosis foram submetidos ao teste de sensibilidade microbiana “in vitro” frente à ampicilina, azitromicina, ceftiofur, enrofloxacino, eritromicina, florfenicol, penicilina G, rifampicina e sulfadiazina/trimetoprim, além da confirmação diagnóstico pela PCR-multiplex para detecção dos genes 16S rRNA, rpoB e pld. Adicionalmente, foi realizado inquérito epidemiológico dos principais fatores de risco associados a linfenite caseosa nos criatórios. Foram isoladas 86 linhagens de C. pseudotuberculosis, das quais 64 (74,0%) foram confirmadas como C. pseudotuberculosis pela PCR-multiplex No diagnóstico citológico foram consideradas positivas 97 (49,2%) amostras que possuíam micro-organismos pleomórficos ou de aspecto “corineforme”. Das 202 amostras colhidas de linfonodos mediastínicos e mesentéricos de ovinos de abatedouro foi observado predomínio de isolamento de estafilococos, estreptococos e enterobactérias. Em nenhuma destas amostras foi isolado C. pseudotuberculosis. Foi observado alto “odds ratio” e associação estatística (P<0,05) para a presença da doença em ovinos em idade reprodutiva, em propriedades com histórico de comercialização de animais. Infere-se no presente estudo o predomínio de C. pseudotuberculosis na casuística da linfadenite caseosa ovina na região estudada. Apesar da alta sensibilidade “in vitro” de C. pseudotuberculosis aos diferentes antimicrobianos testados, o tratamento da doença geralmente é ineficaz, indicando a necessidade de adoção de medidas rigorosas de profilaxia e controle da doença nos criatórios, particularmente quanto à movimentação de animais. Foi observada também boa concordância entre a CAAF e o cultivo microbiológico, e entre o cultivo microbiológico e a PCR, reforçando que a associação de métodos aumenta a eficácia do diagnóstico de C. pseudotuberculosis na linfadenite caseosa ovina.
Caseous lymphadenitis is an infectious disease which etiology is considered very complex, even with frequently isolation of Corynebacterium pseudotuberculosis from lesions in both sheep and goats. The main sources of infection are infected animals in the herd and lymphadenitis is the most important clinical sign, from which the disease is known. Microbiological culture is still the most suitable test for routine diagnostic, however fine needle aspiration cytology (FNAC) is being widely applied in recent years as triage to assess possibly infected animals. Further, molecular diagnostic has been promising in identification of the microorganism and its respective biotypes In the present study, 14 sheep herds were evaluated. From the total of animals,197 sheeps presented enlarged lymph nodes (submandibular, superficial parotid, prefemoral, popliteal, prescapular) suggesting presence of existing disease. Fine needle aspiration citology was performed in animals with lymphadenitis in order to collect samples for microbiological and cytological exams. Therefore, 202 visceral lymph nodes (mesenteric and mediastinic) were collected from slaughtered sheep without signs of lymphadenitis. All lymph nodes were submitted to cytological evaluation, microbiological culture and, in samples where the pathogen was obtained, traditional polymerase chain reaction (PCR) and antimicrobial resistance test were also performed. In culture, 86 samples presented the isolation of C. pseudotuberculosis, from which, 64 (74%) were confirmed as C. pseudotuberculosis by PCR and 22 (26%) as negative. The cytology presented caracteristics of pleomorphism in 97 (49.2%) of the samples whilst 100 (50.8%) lymph nodes did not present any cytological modification. Among the lymph nodes obtained from carcasses, no C. pseudotuberculosis isolation was observed. Moreover, the isolates submitted to antimicrobial resistance test presented high susceptibility to all antimicrobials tested (ampicillin, azithromycin, ceftiofur, enrofloxacin, erythromycin, florfenicol, penicillin G, rifampicin and sulfadiazine-trimethoprim. Additionally, we conducted an epidemiological survey of the main risk factors associated with caseous lymphadenitis in farms. We isolated 86 strains of C. pseudotuberculosis, of which 64 (74.0%) were confirmed as C. pseudotuberculosis by PCR-multiplex in cytological diagnosis were considered positive 97 (49.2%) samples that had micro-organisms or aspect pleomorphic. Of the 202 samples taken from mediastinal and mesenteric lymph nodes of sheep slaughterhouse was observed prevalence of isolation of staphylococci, streptococci and enterobacteria. In none of these samples was isolated C. pseudotuberculosis. We observed a high "odds ratio" and statistical association (P <0.05) in the presence of the disease in sheep reproductive age in herds with marketing of animals. Despite the high sensitivity "in vitro" of C. pseudotuberculosis to different antimicrobials, treatment of disease is usually ineffective, indicating the need to adopt strict measures to prevent and control the disease in farms, particularly as to the movement of animals. It was also observed good agreement between FNAC and microbiological culture and between microbiological culture and PCR, reinforcing the association of methods increases the diagnostic efficacy of C. pseudotuberculosis in ovine caseous lymphadenitis.
Caseous lymphadenitis is an infectious disease which etiology is considered very complex, even with frequently isolation of Corynebacterium pseudotuberculosis from lesions in both sheep and goats. The main sources of infection are infected animals in the herd and lymphadenitis is the most important clinical sign, from which the disease is known. Microbiological culture is still the most suitable test for routine diagnostic, however fine needle aspiration cytology (FNAC) is being widely applied in recent years as triage to assess possibly infected animals. Further, molecular diagnostic has been promising in identification of the microorganism and its respective biotypes In the present study, 14 sheep herds were evaluated. From the total of animals,197 sheeps presented enlarged lymph nodes (submandibular, superficial parotid, prefemoral, popliteal, prescapular) suggesting presence of existing disease. Fine needle aspiration citology was performed in animals with lymphadenitis in order to collect samples for microbiological and cytological exams. Therefore, 202 visceral lymph nodes (mesenteric and mediastinic) were collected from slaughtered sheep without signs of lymphadenitis. All lymph nodes were submitted to cytological evaluation, microbiological culture and, in samples where the pathogen was obtained, traditional polymerase chain reaction (PCR) and antimicrobial resistance test were also performed. In culture, 86 samples presented the isolation of C. pseudotuberculosis, from which, 64 (74%) were confirmed as C. pseudotuberculosis by PCR and 22 (26%) as negative. The cytology presented caracteristics of pleomorphism in 97 (49.2%) of the samples whilst 100 (50.8%) lymph nodes did not present any cytological modification. Among the lymph nodes obtained from carcasses, no C. pseudotuberculosis isolation was observed. Moreover, the isolates submitted to antimicrobial resistance test presented high susceptibility to all antimicrobials tested (ampicillin, azithromycin, ceftiofur, enrofloxacin, erythromycin, florfenicol, penicillin G, rifampicin and sulfadiazine-trimethoprim. Additionally, we conducted an epidemiological survey of the main risk factors associated with caseous lymphadenitis in farms. We isolated 86 strains of C. pseudotuberculosis, of which 64 (74.0%) were confirmed as C. pseudotuberculosis by PCR-multiplex in cytological diagnosis were considered positive 97 (49.2%) samples that had micro-organisms or aspect pleomorphic. Of the 202 samples taken from mediastinal and mesenteric lymph nodes of sheep slaughterhouse was observed prevalence of isolation of staphylococci, streptococci and enterobacteria. In none of these samples was isolated C. pseudotuberculosis. We observed a high "odds ratio" and statistical association (P <0.05) in the presence of the disease in sheep reproductive age in herds with marketing of animals. Despite the high sensitivity "in vitro" of C. pseudotuberculosis to different antimicrobials, treatment of disease is usually ineffective, indicating the need to adopt strict measures to prevent and control the disease in farms, particularly as to the movement of animals. It was also observed good agreement between FNAC and microbiological culture and between microbiological culture and PCR, reinforcing the association of methods increases the diagnostic efficacy of C. pseudotuberculosis in ovine caseous lymphadenitis.
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Palavras-chave
Linfadenite caseosa, Corynebacterium pseudotuberculosis, Microbiologia, Caseous lymphadenitis, Microbiology