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Master's Dissertation
DOI
https://doi.org/10.11606/D.10.2020.tde-19012021-100518
Document
Author
Full name
Brunna de Mattos Granja
E-mail
Institute/School/College
Knowledge Area
Date of Defense
Published
Pirassununga, 2020
Supervisor
Committee
Santos, Marcos Veiga dos (President)
Cerqueira, Mônica Maria Oliveira Pinho
Pantoja, José Carlos de Figueiredo
Title in Portuguese
Avaliação de meios de cultura cromogênicos para identificação rápida de microrganismos causadores de mastite bovina
Keywords in Portuguese
Cultura na fazenda
Mastite
Meios cromogênicos
Vaca leiteira
Abstract in Portuguese
Este estudo objetivou avaliar o desempenho diagnóstico (especificidade, Sp; sensibilidade, Se; acurácia, Ac; valor preditivo positivo, VPP; valor preditivo negativo, VPN; e coeficiente de concordância kappa de Cohen, k) de meios de cultura cromogênicos para identificação rápida de microrganismos causadores de mastite clínica (MC) e subclínica (MSC). Para tanto, dois experimentos foram realizados: 1) Avaliação de biplaca de meios de cultura cromogênicos para identificação rápida de microrganismos causadores de mastite; 2) Avaliação de triplaca de meios de cultura cromogênicos para identificação rápida de microrganismos causadores de mastite. Em ambos estudos (1 e 2), a identificação de microrganismos com o uso de meios cromogênicos baseou-se na leitura visual da coloração das colônias microbianas após 18 a 24 h de incubação. Para avaliação da Ac, Se, Sp, VPP, VPN e k dos meios cromogênicos a identificação dos microrganismos por espectrometria de massas (MALDI-TOF) foi considerada a metodologia padrão. No experimento 1, foram avaliadas 476 amostras de leite coletadas de vacas com MC e 660 de MSC, as quais foram inoculadas em dois meios de cultura cromogênicos seletivos para bactérias Gram-positivas (GP) e outro para Gram-negativas (GN) (CHROMagar, França). No experimento 2 foram avaliadas 476 amostras de leite de vacas com MC e 500 de vacas com MSC, as quais foram inoculadas em triplaca de meios de cultura cromogênicos (Smartcolor2, Onfarm, Piracicaba, Brasil), seletivos para Streptococcus, Staphylococcus e bactérias Gram-negativas. No experimento 1, do total de amostras de MC houve crescimento de colônias com morfologia única em 53,6% das amostras (255/476) e em 2,7% (13/476) com duas morfologias. O uso de biplacas com os meios de cultura GP/GN apresentou Ac que variou de 95% (Strep. agalactiae/dysgalactiae) a 100% (Pseudomonas spp.). Com relação às amostras de MSC, houve crescimento de colônias com uma morfologia em 31,1% (205/660) das amostras e com duas morfologias em 4,5% (30/660). A acurácia essas variou de 95% (Strep. uberis) a 100% (Klebsiella/Enterobacter/Citrobacter; Pseudomonas spp.) em amostras de MSC. No experimento 2, do total de amostras de MC, houve crescimento de colônias com morfologia única em 53,2% (253/476) das amostras e com duas morfologias em 4,2% (20/476). A Ac da triplaca de meios cromogênicos em amostras de MC variou de 93% (Staph. não-aureus) a 99% (Pseudomonas spp.). Para as amostras de MSC, houve crescimento de colônias com uma morfologia em 60,2% (301/500) das amostras e com duas morfologias em 7,4% (37/500). O uso da triplaca com os meios de cultura cromogênicos em amostras de MSC, apresentou Ac que variou de 87% (Staphylococcus não-aureus) a 100% (Pseudomonas spp.). O uso dos métodos de biplaca e da triplaca de meios de cultura cromogênicos apresentou alta acurácia (Ac >92%) para os diagnósticos dos principais microrganismos causadores de mastite, o que indica que podem ser utilizados em sistemas de cultura na fazenda, com base na visualização de coloração de colônias dos principais patógenos causadores de mastite.
Title in English
Evaluation of chromogenic culture media for rapid identification of bovine mastitis-causing microorganisms
Keywords in English
Chromogenic media
Mastitis
Milky cow
On farm culture
Abstract in English
This study aimed to evaluate the diagnostic performance (specificity, Sp; sensitivity; Se; accuracy, Ac; positive predictive value, PPV; negative predictive value, NPV; and Cohen's kappa coefficient of agreement, k) for chromogenic culture media for rapid identification of microorganisms that cause clinical (CM) and subclinical (SCM) mastitis. For this, two experiments were carried out: 1) Evaluation of biplate of chromogenic culture media for rapid identification of mastitis-causing microorganisms; 2) Evaluation of tri-plate of chromogenic culture media for rapid identification of mastitis-causing microorganisms. In both studies (1 and 2), the identification of microorganisms using chromogenic media was based on the visual reading of the color of the microbial colonies after 18 to 24 h of incubation. For the evaluation of Ac, Se, Sp, PPV, NPV and kappa coefficient of chromogenic media, the identification of microorganisms by mass spectrometry (MALDI-TOF) was considered the standard methodology. In experiment 1, 476 milk samples collected from cows with CM and 667 from SCM were inoculated in two culture media selective for Gram-positive (GP) bacteria and another for Gram-negative (GN) (CHROMagar, France). In experiment 2, 476 milk samples from cows with CM and 500 from cows with SCM were inoculated in three chromogenic culture media, selective for Streptococcus, Staphylococcus and Gram-negative bacteria (Smartcolor2, Onfarm, Piracicaba, Brazil). In experiment 1, of the total number of CM samples, 53.6% (255/476) had growth of colonies with one morphology and 2.7% (13/476) with two morphologies. The use of biplates with GP / GN culture media showed Ac that ranged from 95% (Strep. agalactiae / dysgalactiae) to 100% (Pseudomonas spp.). Regarding the samples of SCM, 31.1% (205/660) had growth of colonies with one morphology and 4.5% (30/660) with two morphologies. The accuracy for SCM samples ranged from 95% (Strep. uberis) to 100% (Klebsiella / Enterobacter / Citrobacter; Pseudomonas spp.). In experiment 2, of the total number of CM samples, 53.2% (253/476) had growth of colonies with one morphology and 4.2% (20/476) with two morphologies. The Ac of the triple plate with chromogenic media in CM samples ranged from 93% (Staph. Non-aureus) to 99% (Pseudomonas spp.). For SMC samples, 60.2% (301/500) had growth of colonies with one morphology and 7.4% (37/500) with two morphologies. The Ac use of the triple plate with chromogenic culture media in SCM samples varied from 87% (Staphylococcus non-aureus) to 100% (Pseudomonas spp.). The use of the bi-plate and tri-plate of chromogenic culture media showed high accuracy (Ac> 92%) for the diagnosis of the main mastitis causing microorganisms, which indicates that they can be used in on farm culture systems, based on the visualization of colony color of the main pathogens causing mastitis.
 
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Publishing Date
2021-07-27
 
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