RESSALVA Atendendo solicitação do(a) autor(a), o texto completo desta dissertação será disponibilizado somente a partir de 06/03/2023. UNIVERSIDADE ESTADUAL PAULISTA FACULDADE DE MEDICINA VETERINÁRIA E ZOOTECNIA ACURÁCIA DIAGNÓSTICA DE DIFERENTES ESTRATÉGIAS DE AMOSTRAGEM DE LEITE PARA DETECÇÃO DE INFECÇÕES INTRAMAMÁRIAS NA SECAGEM DE VACAS LEITEIRAS FÁBIO HENRIQUE HINGST FABRI Botucatu – SP 2022 UNIVERSIDADE ESTADUAL PAULISTA FACULDADE DE MEDICINA VETERINÁRIA E ZOOTECNIA ACURÁCIA DIAGNÓSTICA DE DIFERENTES ESTRATÉGIAS DE AMOSTRAGEM DE LEITE PARA DETECÇÃO DE INFECÇÕES INTRAMAMÁRIAS NA SECAGEM DE VACAS LEITEIRAS FÁBIO HENRIQUE HINGST FABRI Dissertação apresentada junto ao Programa de Pós-Graduação em Medicina Veterinária para obtenção do título de Mestre. Orientador: Prof. Dr. José Carlos de Figueiredo Pantoja Palavras-chave: Acurácia diagnóstica; Bovinocultura leiteira; Cultura microbiológica; Mastite; Vaca seca. Fabri, Fábio Henrique Hingst. Acurácia diagnóstica de diferentes estratégias de amostragem de leite para detecção de infecções intramamárias na secagem de vacas leiteiras / Fábio Henrique Hingst Fabri. - Botucatu, 2022 Dissertação (mestrado) - Universidade Estadual Paulista "Júlio de Mesquita Filho", Faculdade de Medicina Veterinária e Zootecnia Orientador: José Carlos de Figueiredo Pantoja Capes: 50502000 1. Bovino - Criação. 2. Bovino de leite. 3. Mastite. 4. Cultura e meios de cultura (Biologia). 5. Confiabilidade dos dados. DIVISÃO TÉCNICA DE BIBLIOTECA E DOCUMENTAÇÃO - CÂMPUS DE BOTUCATU - UNESP BIBLIOTECÁRIA RESPONSÁVEL: ROSANGELA APARECIDA LOBO-CRB 8/7500 FICHA CATALOGRÁFICA ELABORADA PELA SEÇÃO TÉC. AQUIS. TRATAMENTO DA INFORM. Nome do Autor: Fábio Henrique Hingst Fabri Título: ACURÁCIA DIAGNÓSTICA DE DIFERENTES ESTRATÉGIAS DE CULTIVO DE LEITE PARA DETECÇÃO DE INFECÇÕES INTRAMAMÁRIAS NA SECAGEM DE VACAS LEITEIRAS COMISSÃO EXAMINADORA Prof. Dr. José Carlos de Figueiredo Pantoja - Professor Orientador Departamento de Produção Animal e Medicina Veterinária Preventiva FMVZ – UNESP – Botucatu Prof. Dr. Juliano Gonçalves Pereira – Professor Titular Departamento de Produção Animal e Medicina Veterinária Preventiva FMVZ – UNESP – Botucatu Dr. Leonardo Leite Cardozo – Membro Titular Ordemilk Ltda. Data da Defesa: 06 de Setembro de 2022. DEDICATÓRIA Dedico esta dissertação aos meus pais, Fábio e Neusa, por todo o apoio e encorajamento ao longo do mestrado. AGRADECIMENTOS Agradeço, primeiramente, aos meus queridos pais, Neusa e Fábio Hingst Fabri, à minha irmã, Amanda Paola Hingst Fabri, à minha avó, Eli Benedette Hingst Arruda, e à minha namorada, Giovana Banin Mazzali, por todo o amor, apoio, carinho e incentivo ao longo dessa árdua jornada acadêmica, me dando toda a base e força que eu precisava para persistir. Ao meu digníssimo Professor Orientador, José Carlos de Figueiredo Pantoja, pela oportunidade, por acreditar em mim desde o início, pela orientação, ensinamentos, conselhos e por vivenciar as valiosas experiências que levarei para o resto da minha vida profissional e pessoal. Aos meus queridos colegas de pós-graduação, Lára Cristina Bastos Juliano, Fausto Ribeiro Fonseca e Eduardo Pires Macêdo, e, também, ao técnico laboratorial Paulo Capela, por toda a ajuda nas fases do projeto, pelo convívio na rotina do laboratório e por todo o aprendizado e amizade que me proporcionaram. Aos meus irmãos da Rep.Ública, por me acolherem mais uma vez, pela consideração, união, amizade e por sempre torcerem por mim. Aos veterinários, produtores e profissionais envolvidos com o grande ensaio clínico a campo no qual meu trabalho foi inserido. E, por fim, à instituição FMVZ-UNESP Botucatu pelo crescimento profissional e aos professores, residentes e colegas pós- graduandos do Departamento de Produção Animal e Medicina Veterinária Preventiva que contribuíram com este estudo. E, por fim, o presente trabalho foi realizado com apoio da Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Código de Financiamento 001. LISTA DE TABELAS Tabelas da Revisão de Literatura: Tabela 1 - Especificidade (ES) e sensibilidade (SE) de testes diagnósticos utilizados para detectar mastite subclínica na secagem e início da lactação......................................................................................17 Tabelas do Trabalho Científico: Table 1 - Microbiological results from quarter milk samples collected on three consecutive days before drying-off (D-2 = 2nd day prior to drying- off); D-1 = day before drying-off, and D0 = day of drying-off). ....................................................................................................45 Table 2 - Diagnostic accuracy of a single quarter milk sample collected on three consecutive days before drying-off (D-2 = 2nd day prior to drying- off; D-1 = day before drying-off, and D0 = day of drying-off), by pathogen category ....................................................................................................46 Table 3 - Diagnostic accuracy of a single composite sample collected on the day of drying-off (D0), by pathogen category. The gold standard was based on quarter milk samples collected at D0. A cow was considered infected when at least one mammary quarter was infected......................................................................................47 Table 4 - Diagnostic accuracy of a single composite sample collected on the day of drying-off (D0), by pathogen category. The gold standard9 was based on quarter milk samples collected on three consecutive days before drying-off (D-2 = 2nd day prior to drying-off; D-1 = day before drying-off, and D0 = day of drying- off).............................................................................................48 LISTA DE ABREVIAÇÕES IIM – Infecção intramamária SDCT – Selective dry-cow therapy (terapia seletiva de vacas secas) BDCT – Blanket dry-cow therapy (terapia convencional de vacas secas) DCT – Dry-cow therapy SE – Sensibilidade ES – Especificidade VPP – Valor preditivo positivo VPN – Valor preditivo negativo FP – Falsos-positivos FN – Falsos-negativos SCN – Staphylococcus coagulase-negativa CCN – Cocos catalase-negativa CMT – California Mastitis Test CCS – Contagem de células somáticas MC – Mastite Clínica SUMÁRIO Resumo ......................................................................................................................... 9 Abstract ...................................................................................................................... 10 CAPÍTULO 1 .............................................................................................................. 1 1. Introdução ................................................................................................................ 1 2. Revisão de Literatura ............................................................................................... 3 CAPÍTULO 2 – Trabalho científico ....................................................................... 22 Introduction ................................................................................................................ 26 Material and Methods ................................................................................................ 27 Results ........................................................................................................................ 31 Discussion .................................................................................................................. 34 Conclusions ................................................................................................................ 37 References .................................................................................................................. 39 REFERÊNCIAS ....................................................................................................... 51 FABRI, F. H. H. Acurácia diagnóstica de diferentes estratégias de amostragem de leite para detecção de infecções intramamárias na secagem de vacas leiteiras. Botucatu, 2022. 77p. Dissertação (Mestrado) – Faculdade de Medicina Veterinária e Zootecnia, Campus de Botucatu, Universidade Estadual Paulista “Júlio de Mesquita Filho” – UNESP. RESUMO O objetivo principal foi avaliar a acurácia de abordagens de cultivo de leite para detectar infecções intramamárias (IIM) na secagem. Uma amostra de 184 vacas de 28 fazendas foi utilizada para tanto. As fazendas foram visitadas em três dias consecutivos antes da secagem (D-2, D-1 e D0) para coleta de amostras de leite para cultivo microbiológico. Amostras compostas foram preparadas em laboratório. A sensibilidade (SE), especificidade (ES) e valores preditivos positivo (VPP) e negativo (VPN) foram estimados para: 1) uma amostra dos quartos coletada em D-2, D-1 ou D0, em relação ao padrão ouro em nível de quarto (isolamento do mesmo patógeno em ≥ 2 das três amostras consecutivas); 2) uma amostra composta, em relação ao padrão-ouro em nível de vaca (≥ 1 quarto infectado por vaca). A prevalência de IIM na secagem foi de 47,36 (quartos) e 60,44% (compostas). Considerando todos os patógenos, a SE das amostras de quarto variou entre 93,62-95,35%. A ES variou entre 86,91-91,24%. A acurácia variou entre os patógenos. A SE para detectar Streptococcus agalactiae e outros cocos catalase-negativos foi alta (> 92,11%), enquanto para detectar Staphylococcus aureus a SE variou entre 88,24-94,44%. Em geral, a ES e o VPN foram elevados. Para amostras compostas, houve perda de acurácia. A SE variou de 50,00% (Corynebacterium bovis) a 76,32% (S. aureus). Amostras de leite (quarto ou compostas) colhidas no D-2 podem ser adequadas para detectar IIM na secagem, mas diferenças na acurácia entre os patógenos devem ser consideradas ao tomar decisões no tratamento da vaca seca. Palavras-chave: mastite; vaca seca; bovinocultura leiteira; acurácia diagnóstica; cultura microbiológica. FABRI, F. H. H. Diagnostic accuracy of different milk sampling strategies for detection of intramammary infections at drying-off of dairy cows. Botucatu, 2022. 77p. Dissertação (Mestrado) – Faculdade de Medicina Veterinária e Zootecnia, Campus de Botucatu, Universidade Estadual Paulista “Julio de Mesquita Filho” – UNESP. ABSTRACT The main objective was to evaluate the accuracy of milk culture approaches to detect intramammary infections (IMI) at drying-off. A sample of 184 cows from 28 farms was used for this purpose. Farms were visited on three consecutive days before drying-off (D- 2, D-1 and D0) to collect milk samples for microbiological culture. Composite samples were prepared in the laboratory. Sensitivity (SE), specificity (SP), and positive (PPV) and negative (NPV) predictive values were estimated for: 1) one quarter sample collected on D-2, D-1 or D0, relative to the gold standard in quarter-level (isolation of the same pathogen in ≥ 2 of the three consecutive samples); 2) one composite sample, relative to the gold standard at the cow-level (≥ 1 infected quarter per cow). The prevalence of IMI in drying-off was 47.36 (quarters) and 60.44% (composites). Considering all pathogens, the SE of the quarter samples varied between 93.62-95.35%. The SP ranged between 86.91-91.24%. The accuracy varied among the pathogens. The SE for detecting Streptococcus agalactiae and other catalase-negative cocci was high (> 92.11%), while for detecting Staphylococcus aureus the SE ranged between 88.24-94.44%. In general, SP and NPV were high. For composite samples, there was a loss of accuracy. The SE ranged from 50.00% (Corynebacterium bovis) to 76.32% (S. aureus). Milk samples (quarter or composite) collected at D-2 may be adequate to detect IMI at drying-off, but differences in accuracy between pathogens must be considered when making decisions on dry cow treatment. Keywords: mastitis; dry cow; dairy; diagnostic accuracy; microbiological culture. 1 CAPÍTULO 1 1. INTRODUÇÃO A mastite é considerada uma das enfermidades mais prevalentes nos rebanhos bovinos leiteiros do mundo (BRITO et al.et al.., 1997), capaz de oferecer riscos à saúde pública (RIBEIRO, 1996; BUENO et al.., 2002), gerar perdas financeiras aos produtores (AGHAMOHAMMADI et al., 2018), à indústria de laticínios (SCHÄELLIBAUM, 2000) e trazer impacto negativo na qualidade do leite (MÜLLER, 2002). O período seco é caracterizado por um alto risco de infeções intramamárias (IIM), podendo este ser maior do que o risco na lactação (HASSAN et al., 1999; ROBERT et al., 2006). A terapia convencional da vaca seca (Blanket Dry-cow Therapy, BDCT) consiste na infusão de antimicrobianos intramamários em todos os quartos de todas as vacas, no momento da secagem. A BDCT foi um dos cinco pontos originalmente propostos para o controle da mastite bovina (NEAVE et al., 1969) e tem sido utilizada mundialmente desde 1951 (HASSAN et al., 1999) para eliminar as IIM existentes na secagem e prevenir novas IIM durante o período seco (HALASA et al., 2009a e 2009b; VILAR et al., 2018). O uso da BDCT contribuiu historicamente para a redução da prevalência de mastite e da contagem de células somáticas (CCS) no leite a granel (PATEL et al., 2018). No entanto, devido ao uso indiscriminado e de caráter profilático de antimicrobianos, decorrente do tratamento de quartos mamários não infectados, a BDCT não tem sido permitida em regiões leiteiras desenvolvidas (SANTMAN-BERENDS et al., 2016). Alternativamente, a terapia seletiva de vacas secas (Selective Dry-cow Therapy, SDCT) tem sido recomendada (PATEL et al., 2018), na qual os quartos infectados recebem antimicrobiano, e os não infectados recebem apenas selante de teto à base de subnitrato de bismuto, visando a diminuição no risco de IIM durante o período seco (CAMERON et al., 2014). Assim, o uso da SDCT visa reduzir o uso desnecessário de antimicronianos na secagem sem aumentar o risco de IIM no período seco, o que resultaria em economia para os produtores e proteção da saúde pública (RINDSIG et al., 1978). Algorítimos têm sido utilizados para realizar a seleção de vacas a serem tratadas ou não no momento da secagem, dentre os quais os 2 mais comuns são baseados no histórico mensal da CCS e ocorrência de casos clínicos na lactação (TORRES et al., 2008; VASQUEZ et al., 2018; ROWE et al., 2020). No entanto, o método de amostragem de leite para realização da cultura microbiológica anteriormente à secagem não é padronizado, podendo-se utilizar amostras compostas (mistura do leite de todos os quartos mamários) ou de quartos mamários individuais, colhidas em um único dia ou de forma repetida. Amostras compostas possuem menor custo e maior facilidade de colheita, mas identificam apenas as vacas infectadas e sadias, sem especificar o quarto acometido, resultando no tratamento desnecessário de quartos não infectados (CAMERON et al., 2014). Amostras seriadas requerem maior uso de mão de obra, mas um possível ganho em acurácia diagnóstica ainda não é bem estabelecido. 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