This thesis is an investigation of the objective assessment of pain through the use of pressure algometers and rumination tags and the effects of pain management therapy for experimentally-induced mastitis on behaviour and physiological measures in dairy cattle. Twenty-four lactating Holstein cows were enrolled in a lipopolysaccharide (LPS) endotoxin challenge study, where one mammary quarter was infused with 25 µg of Escherichia coli (E. coli) LPS endotoxin. Subsequently, a subcutaneous injection of either a non-steroidal anti-inflammatory drug (NSAID) (meloxicam; n=12) or placebo (n=12) was randomly allocated and administered using double-blind methods. Several behavioural, physiological and performance parameters were monitored throughout the study period. Beneficial effects of meloxicam administration on pain sensitivity, edema scores and dry matter intake were shown. For a subset of animals receiving placebo treatment, the algometer and rumination tags accurately detected changes in both pain sensitivity and rumination time after endotoxin challenge. / Canadian Bovine Mastitis Research Network and Boehringer-Ingelheim, Canada

Controlling mastitis in dairy herds relies on good prevention and detection methods. This dissertation describes two areas of research relating to mastitis control. In the first objective, the efficacy of 2 vaccines against Escherichia coli mastitis in mid-lactation dairy cows was evaluated. Secondly, in a series of 3 studies, milk and activity sensor data were used to derive models for clinical mastitis (CM) detection, and models were tested for their ability to indicate the causative pathogen type. Primiparous and multiparous animals were vaccinated with 1 of 2 commercially available J5 vaccines (V1 or V2) or served as unvaccinated controls (CTL). Intramammary challenge with E. coli approximately 84 d later resulted in few treatment differences in the clinical and behavioral responses, except that vaccinated cows exhibited fever (≥ 39.4 °C) 3 h earlier and laid down for longer periods than CTL. Although vaccinated cows had similar severity and duration of CM, V1 cows produced more serum IgG1 and IgG2 than V2 cows. Our results indicated that the effects of vaccination were diminished in mid-lactation, and that antibodies are not the limiting factor in defending against induced E. coli mastitis. Multiple regression models, incorporating the slope changes in relevant milk and activity sensor data, were developed to indicate all CM cases (ACM), or specifically, CM due to Gram-negative (GN) or Gram-positive (GP) bacteria. Gram-specific models had greater detection accuracy (> 80%) than the ACM model (75%) when evaluated using the model training dataset, but independent evaluation demonstrated reduced sensitivity (Se) of detecting CM by all models (GN, 62%, ACM, 56%, and GP, 32% Se). Data in the 3 d prior to CM were more important in detecting GN pathogens, whereas the best GP models incorporated changes more than 1 week prior to CM detection. Still, model performance was imperfect. Next, models were rederived from a dataset that better reflected the infection distribution of the herds its use was intended for. However, the Se of detecting CM in real-time, across 2 farms, was < 21% for all models, and categorization by Gram-status had no benefit. An insufficient number of CM cases was considered to contribute to the poor detection performance of models and limited repeatability across farms. Consequently, models derived in this study were inadequate for implementation as mastitis detection tools. In the future, development of new sensors and application of more sophisticated algorithms to the field of mastitis detection may improve the accuracy of models using sensor data. / Doctor of Philosophy / Mastitis is an important disease of dairy cattle that adversely affects animal welfare, productivity, and milk quality. Controlling mastitis in dairy herds relies on good prevention and detection methods. In this dissertation, we investigated two elements of mastitis control: 1) the effects of vaccination in protecting against mastitis, and 2) the ability of on-farm sensor data to detect clinical mastitis (CM) and indicate the causative pathogen type. Coliform bacteria commonly cause CM, and vaccination against these bacteria can reduce the severity of the disease. We evaluated the effect of 2 different vaccines on the clinical, behavioral, and immune response in cows with experimental mastitis caused by Escherichia coli. Our findings indicated that the effects of vaccination had diminished at the time of experimental mastitis, as vaccinated cows had no improvement in clinical recovery compared with unvaccinated controls. Although no clinical or behavioral differences were observed between the 2 different vaccines, the antibody response differed, suggesting that antibodies are not the key player underpinning the mechanisms of vaccination against induced coliform mastitis in mid-lactation. Rapid detection and diagnosis of mastitis is important to reduce effects on the cow, and to support decision making for the appropriate intervention. We aimed to develop and test mastitis detection models that utilized data collected by on-farm sensor technologies. Milk and activity parameters, which may be differentially affected by mastitis depending on the pathogen causing infection, were used in multiple regression models for detecting any CM case, or specifically CM caused by Gram-positive or Gram-negative bacteria. Gram-specific models were initially estimated to have > 80% accuracy in classifying cows with and without mastitis, but further validation demonstrated that the models were not repeatable when tested independently. Subsequently, models that were more suited to the farms they were to be implemented on were developed, and tested, revealing limited performance in detecting any case of CM, or CM due to the Gram-specific pathogens. Model derivation was limited by an insufficient number of CM cases to represent the variation in different cases of CM within the Gram-positive and Gram-positive classifications. Although our models did not show promise as a mastitis detection tool, milk and activity data may be incorporated with other sensor data for improved detection and diagnosis of mastitis.

Thesis (MScAgric)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: Mastitis is the most prevalent and costly production disease of dairy cattle; hence mastitis incidence is a distinctly important trait in dairy cattle. The primary objective of the study was to determine the economic value, and develop a model for genetic prediction of clinical mastitis in South African Holstein cattle. These procedures are a prerequisite to including this trait in the breeding objective. The cost of clinical mastitis per incident was calculated as the sum of revenue loss due to discarded milk during the infection period and the associated treatment costs. Economic value (ZAR/incident) was calculated as the change in profit (increase in costs) resulting from a simulated marginal increase in mastitis incidence in an average herd. Average economic losses due to clinical mastitis were estimated at ZAR919.96/cow/year and the average incidence was 0.9cases/cow/year. The economic value of clinical mastitis was ‐ZAR1079.51/incident. A model for predicting estimated breeding values (EBVs) for clinical mastitis using somatic cell score (SCS), fore teat length (FTL), udder depth (UD) and rear udder height (RUH) was developed, using genetic (co)variances among these traits. Since EBVs for SCS, FTL, UD and RUH are routinely estimated under the national genetic evaluation programme, EBVs for clinical mastitis can be predicted from the model developed in the current study. Thus, the results of the study provide the basis for including clinical mastitis in the breeding objective for South African Holstein cattle. / AFRIKAANSE OPSOMMING: Mastitis is die mees algemeenste en duursteproduksie siekte wat voorkom by melkbeeste, daarom is die voorkoms van mastitis 'n belangrike eienskap in melkbeeste. Die primêre doel van die studie was om die ekonomiese waarde te bepaal, asook die ontwikkeling van 'n model vir genetiese voorspelling van kliniese mastitis in Suid‐Afrikaanse Holstein beeste. Hierdie prosedures is 'n voorvereiste vir insluiting van hierdie eienskap as ‘n teeldoelwit in seleksie programme. Die koste van kliniese mastitis per voorval is bereken as die som van die inkomste verlies weens melk weggegooi tydens die infeksie periode en die gepaardgaande koste vir die behandeling. Ekonomiese waarde (ZAR / voorval) is bereken as die verandering in wins (toename in koste) wat voortspruit uit 'n gesimuleerde marginale toename in mastitis voorkoms in 'n gemiddelde kudde. Gemiddelde ekonomiese verliese as gevolg van kliniese mastitis was beraam op ZAR919.96/koei/jaar en die gemiddelde voorkoms was 0.9gevalle/koei/jaar. Die ekonomiese waarde van kliniese mastitis was ‐ ZAR1079.51/geval. 'n Model vir die voorspelling van beraamde teelwaardes (EBV’s) vir kliniese mastitis is ontwikkel deur gebruik te maak van die ko‐variansies tussen die onderskeie eienskappe: somatiese sel telling (SST), voorspeen lengte (VSL), uier diepte (UD) en agter uier hoogte (AUH). Aangesien teelwaardes vir SST, VSL, UD en AUH gereeld beraam word onder die Nasionale genetiese evaluasie program, kan teelwaardes vir kliniese mastitis voorspel word vanuit die model wat ontwikkel is in die huidige studie. Dus verskaf die resultate van hierdie studie ‘n basis vir die insluiting van kliniese mastitis as ‘n teeldoelwit in seleksie programme van die Suid‐Afrikaanse Holstein beeste.

The distribution of yeast fungi has been ascertained as their influence on SCM etiology. Our developed preparation for treatment for yeast and microbe caused mastitis “Gentafung” is composition of wide spectrum antibiotics and antifungal medications. An evaluation was performed of commercially available homeopathic preparations in treating SCM. A new recommended preparation “OrbeScal” was evaluated for drying cows. A comparison of Candida genus commercial identification systems diagnostic accuracy was via classical methods.

No description available.

Peu d’études ont exploré l’utilisation du traitement prolongé pour les mammites cliniques et aucune ne portait sur l’utilisation du traitement prolongé à base de ceftiofur pour le traitement des mammites cliniques légères à modérées. L’objectif de cette étude était d’évaluer l’efficacité du traitement intra-mammaire prolongé à base de ceftiofur pour les mammites cliniques légères à modérées, en considérant toutes les bactéries responsables ou seulement Staphylococcus aureus (S. aureus) et les streptocoques. Des vaches laitières (n = 241) de 22 élevages du Québec et de l’Ontario ont été inclues. L’étude a été conçue comme un essai clinique à allocation aléatoire. Pour chaque cas de mammite clinique légère à modérée, 125 mg d’hydrochloride de ceftiofur (Spectramast® LC) a été administré par voie intra-mammaire une fois par jour pour 2 ou 8 jours. Le pourcentage de guérison clinique 21 jours après la fin du traitement était de 89% (n = 98/110 pour chaque groupe, p = 0,95). Les pourcentages de guérison bactériologique 21 jours après la fin du traitement pour les groupes 2 jours et 8 jours étaient 32% (n = 15/47) et 61% (n = 25/41) respectivement pour toutes les bactéries (p < 0,01); 64% (n = 9/14) et 82% (n = 9/11) respectivement pour les streptocoques (p = 0,50); et 0% (n = 0/20) et 47% (n = 9/19) pour S. aureus (p < 0,01). Il n'y avait pas de différence entre les 2 groupes pour les nouvelles infections intra-mammaires (p = 0,30). Le traitement prolongé à base de ceftiofur est un choix raisonnable pour le traitement des mammites cliniques légères à modérées, en particulier causées par S. aureus. / Little research has focused on extended therapy in lactating cows with clinical mastitis and none were with ceftiofur. The objective of the present study was to evaluate the efficacy of an intramammary extended ceftiofur treatment for mild to moderate clinical mastitis, and to determine whether it would increase cure rates when considering any bacteria or more particularly Staphylococcus aureus (S. aureus) and streptococci. Holstein dairy cows (n = 241) from 22 herds located in Quebec and Ontario, Canada, were included. The study was designed as a randomized clinical trial. For each case of mild to moderate clinical mastitis diagnosed, 125 mg of ceftiofur hydrochloride (Spectramast® LC) was administered intramammary once a day for 2 or 8 consecutive days. Clinical cure rate 21 days after the last treatment was identical in each group (89%; n = 98/110; p = 0.95). Bacteriological cure rates 21 days after the last treatment for the 2- and 8-day regimens were 32% (n = 15/47) and 61% (n = 25/41), respectively, for all bacteria (p < 0.01); 64% (n = 9/14) and 82% (n = 9/11), respectively, for streptococci (p = 0.50); and 0% (n = 0/20) and 47% (n = 9/19), respectively, for S. aureus (p < 0.01). There were no statistical differences between experimental groups for new intramammary infections. Extended therapy appears to be a reasonable choice for the treatment of mild to moderate clinical mastitis, particularly in cases where S. aureus is the pathogen involved.

Peu d’études ont exploré l’utilisation du traitement prolongé pour les mammites cliniques et aucune ne portait sur l’utilisation du traitement prolongé à base de ceftiofur pour le traitement des mammites cliniques légères à modérées. L’objectif de cette étude était d’évaluer l’efficacité du traitement intra-mammaire prolongé à base de ceftiofur pour les mammites cliniques légères à modérées, en considérant toutes les bactéries responsables ou seulement Staphylococcus aureus (S. aureus) et les streptocoques. Des vaches laitières (n = 241) de 22 élevages du Québec et de l’Ontario ont été inclues. L’étude a été conçue comme un essai clinique à allocation aléatoire. Pour chaque cas de mammite clinique légère à modérée, 125 mg d’hydrochloride de ceftiofur (Spectramast® LC) a été administré par voie intra-mammaire une fois par jour pour 2 ou 8 jours. Le pourcentage de guérison clinique 21 jours après la fin du traitement était de 89% (n = 98/110 pour chaque groupe, p = 0,95). Les pourcentages de guérison bactériologique 21 jours après la fin du traitement pour les groupes 2 jours et 8 jours étaient 32% (n = 15/47) et 61% (n = 25/41) respectivement pour toutes les bactéries (p < 0,01); 64% (n = 9/14) et 82% (n = 9/11) respectivement pour les streptocoques (p = 0,50); et 0% (n = 0/20) et 47% (n = 9/19) pour S. aureus (p < 0,01). Il n'y avait pas de différence entre les 2 groupes pour les nouvelles infections intra-mammaires (p = 0,30). Le traitement prolongé à base de ceftiofur est un choix raisonnable pour le traitement des mammites cliniques légères à modérées, en particulier causées par S. aureus. / Little research has focused on extended therapy in lactating cows with clinical mastitis and none were with ceftiofur. The objective of the present study was to evaluate the efficacy of an intramammary extended ceftiofur treatment for mild to moderate clinical mastitis, and to determine whether it would increase cure rates when considering any bacteria or more particularly Staphylococcus aureus (S. aureus) and streptococci. Holstein dairy cows (n = 241) from 22 herds located in Quebec and Ontario, Canada, were included. The study was designed as a randomized clinical trial. For each case of mild to moderate clinical mastitis diagnosed, 125 mg of ceftiofur hydrochloride (Spectramast® LC) was administered intramammary once a day for 2 or 8 consecutive days. Clinical cure rate 21 days after the last treatment was identical in each group (89%; n = 98/110; p = 0.95). Bacteriological cure rates 21 days after the last treatment for the 2- and 8-day regimens were 32% (n = 15/47) and 61% (n = 25/41), respectively, for all bacteria (p < 0.01); 64% (n = 9/14) and 82% (n = 9/11), respectively, for streptococci (p = 0.50); and 0% (n = 0/20) and 47% (n = 9/19), respectively, for S. aureus (p < 0.01). There were no statistical differences between experimental groups for new intramammary infections. Extended therapy appears to be a reasonable choice for the treatment of mild to moderate clinical mastitis, particularly in cases where S. aureus is the pathogen involved.

Orientador: José Carlos de Figueiredo Pantoja / Resumo: Objetivou-se avaliar a eficácia do cetoprofeno em casos de mastite clínica (MC) com ausência de isolamento microbiológico nas amostras de leite cultivadas no sistema de cultura na fazenda (SCF). Vacas de três rebanhos foram alocadas aleatoriamente em dois grupos experimentais. Grupo cetoprofeno (N = 65 quartos) foi tratado com cetoprofeno (3 mg/kg, via intramuscular, dose única) e grupo controle (N = 64 quartos) não recebeu terapia. Amostras de leite foram coletadas no momento do diagnóstico da MC e 14 (D14) e 21 (D21) dias após o caso. Os desfechos avaliados foram risco de cura clínica, recidiva e recorrência de MC, risco de nova infecção intramamária (IIM) em D14 e D21 e contagem de células somáticas em D14 e D21. A proporção de cura clínica, recidiva e recorrência de MC nos grupos cetoprofeno e controle foi de 83,08% e 85,94%, 18,52% e 18,18% e 16,67% e 16,36%, respectivamente. O risco relativo de todos os desfechos mencionados não foi diferente entre os grupos experimentais. Em ambas as análises as chances de nova IIM não foram diferentes entre os grupos experimentais. Não houve diferença na CCS entre os grupos entre os dias de amostragem ou grupos experimentais. Os resultados sugerem que o uso de cetoprofeno em casos de MC com cultura negativa não foi associado ao risco de cura clínica, recidiva e recorrência de MC, novas IIM e CCS. / Abstract: The objective of this study was to evaluate the efficacy of ketoprofen in culture-negative clinical mastitis (CM) cases. Cows from three commercial herds were randomly allocated into two experimental groups. The ketoprofen group (N = 65 quarters) received a single intramuscular injection of 3 mg/kg of ketoprofen. The control group (N = 64 quarters). Milk samples were collected at the time of CM diagnosis and at 14 (D14) and 21 (D21) days after the case. The outcomes evaluated were clinical cure, relapse and recurrence of CM, risk of new intramammary infection (IIM) at D14 and D21, and somatic cell count at D14 and D21. The proportion of clinical cure, relapse and recurrence of CM in the ketoprofen and control groups was 83.08%, 85.94%, 18.52% and 18,18%, 16,67% and 16,36%, respectively. The relative risk of all mentioned outcomes was not different between the experimental groups. For both analyses, the odds of new IIM were not different between the experimental groups. Mean SCC was not different between sampling days or experimental groups. Results suggest that the use of ketoprofen for culture-negative CM was not associated with the risk of clinical cure, relapse and recurrence of CM, new IIM and SCC. / Mestre

The general objectives of this thesis were: (i) to determine the etiological and molecular profile of clinical mastitis (CM) in 20 dairy herds of Southeast, Brazil; and (ii) to quantify antimicrobial used for treatment of CM in the study population. To achieve this goals, four studies were performed. In the Study 1, we characterized the pathogen frequency and severity of CM in dairy herds. In addition, we determined the incidence rate of clinical mastitis (IRCM) and its association with the following herd-level descriptors: bulk milk somatic cell count (BMSCC), bulk milk total bacterial count (BMTBC), herd size (number of lactating cows), milk yield, housing system and season. The association between herd-level descriptors and IRCM were determined by two groups of mixed regression models: one based on the overall IRCM, and five based on the following specific-pathogen groups: contagious, other Gram-positive, Gram-negative, other (composed of yeast and Prototheca spp), and negative culture. A total of 5,957 quarter-cases of CM were recorded and the most frequently isolated pathogens were Escherichia coli (6.6% of total cultures), Streptococcus uberis (6.1%), and Streptococcus agalactiae (5.9%). The majority of CM cases were mild (60.3%), while 34.1% were moderate and 5.6% severe. Overall, the IRCM was 9.7 quarter-cases per 10,000 quarter-days at risk (QDAR), and the only herd-level parameter associated with overall IRCM was BMSCC, in which the highest IRCM was observed for herds with BMSCC &gt;600.000 × 103 cells/mL. In the models evaluating the specific-pathogen groups, IRCM with isolation of major contagious pathogens was associated with BMSCC, milk yield and housing system. For the evaluation of other Gram-positive pathogens, the IRCM was higher in the rainy season of 2015 in comparison with the other seasonal categories. In addition, for the model evaluating the Gram-negative group, the IRCM was highest in herds with BMTBC &gt;30 × 103 cfu/mL. The Study 2 aimed to characterize the treatment profile and quantify the antimicrobial consumption for treatment of CM in dairy herds; and to determine the association of antimicrobial use (AMU) and the same herd-level descriptors as described in the Study 1. Data on treatment practices and AMU were obtained from 19 dairy herds for a period of 12 months per herd. The AMU for treatment of CM was quantified monthly in units of defined daily dose (DDD) and expressed as antimicrobial treatment incidence (ATI; number of DDD per 1,000 lactating cows-day). The overall monthly mean ATI was 17.7 DDD per 1,000 lactating cow-days (15.4 for intramammary compounds, and 2.2 for systematically administered antimicrobials). Among intramammary drugs, aminoglycosides had the highest ATI (11.7 DDD per 1,000 lactating cow-days), while for systematically administrated antimicrobials, fluoroquinolones (4.2 DDD per 1,000 lactating cow-days) were the most frequently used antimicrobials. Herd size and BMSCC were positively associated with ATI. In addition, herd-level ATI was higher in freestall herds than in compost bedded-pack barns. In the Study 3, we determined the phylogeny of E. coli strains isolated from CM in dairy cows and the association of most frequent phylogroups with antimicrobial susceptibility. A total of 100 E. coli isolates recovered from CM cases described in the Study 1 were categorized according to their phylogenetic group using a quadruplex PCR method; antimicrobial susceptibility pattern was also evaluated. Most isolates were assigned to phylogenetic group A (52%), followed by B1 (38%), B2 (2%), C (4%), D (3%), and E (1%). Resistant isolates were observed for all evaluated antimicrobials. Overall, more than 96% of E. coli isolates were resistant to ampicillin, and more than 23% were resistant to cephalothin, sulphadimethoxine or tetracycline. High levels of resistance (&gt;70%) were also found to erythromycin, oxacillin, penicillin, penicillin associated with novobiocin, and pirlimycin. In contrary, high susceptibility was observed to ceftiofur (96.8%) among E. coli isolates. Difference in the antimicrobial susceptibility among phylogenetic groups was observed only for cephalothin, in which E. coli strains belonging to the phylogroup A were inhibited at lower antimicrobial concentrations than strains assigned to the phylogroup B1. In Study 4, we evaluated the genotypic diversity among Strep. agalactiae and Strep. uberis isolates recovered from CM in dairy cows; in addition, the study evaluated the association of genotypes clustered by genetic similarity with antimicrobial susceptibility pattern. Isolates were subtyped using randomly amplified polymorphic DNA (RAPD) analysis. A great genotypic diversity was found for both Strep. agalactiae (45 subtypes out of 89 isolates) and Strep. uberis (56 subtypes out of 88 isolates). For evaluation of antimicrobial susceptibility, subtypes of Strep. agalactiae were clustered into three groups (Ia, Ib and II), while Strep. uberis subtypes were clustered into two groups (I and II) according to their genetic similarity. Overall, Strep. agalactiae isolates showed high susceptibility to most antimicrobials, except to tetracycline and erythromycin. Differences in the antimicrobial susceptibility among clusters of Strep. agalactiae were observed for ampicillin, ceftiofur, erythromycin, pirlimycin, sulphadimethoxine and tetracycline. In contrary, Strep. uberis isolates were categorized as resistant to most antimicrobials, except to cephalothin and penicillin+novobiocin. No differences were observed among clusters for all antimicrobials in the analysis of Strep. uberis. In conclusion, the results of this thesis indicated a high IRCM in the evaluated herds, and although environmental pathogens were the most common cause of CM in these herds, contagious pathogens such as Strep. agalactiae and Staph. aureus, are still a concern in some dairy herds of Brazil. Furthermore, high frequencies of AMU and off-label protocols were observed among the evaluated herds. The non-judicious use of antimicrobials can become a risk factor for the development of antimicrobial resistance, which was even observed for isolates belonging to the three most prevalent bacterial species identified from CM cases in our study (E. coli, Strep. agalactiae and Strep. uberis). Finally, because there were some herd-level descriptors associated with the IRCM and AMU in our study, there may be opportunity for management strategies aiming to improve the control of CM in dairy herds of southeastern Brazil. / Os objetivos gerais desta tese foram: (i) determinar o perfil etiológico e molecular da mastite clínica (MC) em 20 rebanhos leiteiros do Sudeste do Brasil; e, (ii) quantificar os antimicrobianos usados para tratamento da MC na população estudada. Para alcançar esses objetivos, quatro estudos foram realizados. No Estudo 1, foi caracterizada a frequência de patógenos causadores de MC e a gravidade das infecções nos rebanhos leiteiros. Além disso, foi determinada a taxa de incidência de mastite clínica (TIMC) e sua associação com as seguintes variáveis em nível de rebanho: contagem de células somáticas em leite de tanque (CCSLT), contagem bacteriana total em leite de tanque (CBTLT), tamanho (número de vacas em lactação), produção de leite, sistema de alojamento e estação do ano. A associação entre as variáveis em nível de rebanho e a TIMC foi determinada por dois grupos de modelos de regressão logística multivariada: um baseado na TIMC geral, e cinco baseados nos seguintes grupos específicos de patógenos: contagiosos, outros Gram-positivos, Gram-negativos, outros patógenos (composto de leveduras e Prototheca spp.), e cultura negativa. Um total de 5.957 casos de MC em nível de quarto mamário foi registrado e os patógenos mais prevalentes foram Escherichia coli (6,6% de todas as culturas), Streptococcus uberis (6,1%), e Streptococcus agalactiae (5,9%). A maioria dos casos de MC foi de gravidade leve (60,3%), enquanto 34,1% dos casos foram moderados e 5,6% foram graves. A TIMC geral foi de 9,7 casos por 10.000 quartos-dia em risco (QDR), e o único parâmetro em nível de rebanho associado com a TIMC geral foi a CCSLT, em que a TIMC mais alta foi observada em rebanhos com CCSLT &gt;600.000 × 103 células/mL. Nos modelos que avaliaram os grupos específicos de patógenos, a TIMC de patógenos contagiosos foi associada com a CCSLT, produção de leite e sistema de alojamento. Na avaliação de outros patógenos Gram-positivos, a TIMC foi maior na estação chuvosa de 2015 em comparação com as outras categorias referentes à estação do ano. Adicionalmente, para o modelo avaliando o grupo de patógenos Gram-negativos, a TIMC foi mais alta em rebanhos com CBTLT &gt;30.000 × 103 ufc/mL. O Estudo 2 teve como objetivo caracterizar o perfil de tratamento e o consumo de antimicrobianos em rebanhos leiteiros; e determinar a associação de uso de antimicrobianos (UAM) e as mesmas variáveis em nível de rebanho descritas no Estudo 1. Dados sobre as práticas terapêuticas e UAM foram obtidos de 19 rebanhos leiteiros durante um período de 12 meses por rebanho. A frequência de UAM para tratamento da MC foi quantificada mensalmente em unidades de doses definidas diárias (DDD) e expressa como incidência de tratamento antimicrobiano (ITA: número de DDD por 1.000 vacas em lactação-dia). A média de ITA mensal foi de 17,7 DDD por 1.000 vacas em lactação-dia (15,4 para compostos intramamários, e 2,2 para compostos sistêmicos). Entre os produtos intramamários, os aminoglicosídeos tiveram a ITA mais alta (11,7 DDD por 1.000 vacas em lactação-dia), enquanto que para os compostos administrados pela via sistêmica, as fluoroquinolonas (4,2 DDD por 1.000 vacas em lactação-dia) foram os antimicrobianos mais frequentemente usados. O tamanho do rebanho e CCSLT foram positivamente associados com a ITA. Além disso, a ITA foi mais alta em rebanhos com freestall do que em rebanhos com sistema tipo compost barn. No Estudo 3, determinou-se a filogenia de cepas de E. coli isoladas de casos de MC em vacas leiteiras, e a associação dos filogrupos mais frequentes com a susceptibilidade aos antimicrobianos. Um total de 100 isolados de E. coli identificados nos casos de MC descritos no Estudo 1 foram categorizados de acordo com os grupos filogenéticos por meio de um método de PCR quadruplex; o perfil de susceptibilidade aos antimicrobianos também foi avaliado. A maioria dos isolados pertenceram ao grupo A (52%), seguido dos grupos B1 (38%), B2 (2%), C (4%), D (3%), e E (1%). Foram encontrados isolados resistentes para todos os antimicrobianos avaliados. De forma geral, mais de 96% dos isolados de E. coli foram resistentes a ampicilina, e mais de 23% foram resistentes a cefalotina, sulfadimetoxina ou tetraciclina. Altos níveis de resistência (&gt;70%) foram encontrados também para eritromicina, oxacilina, penicilina e penicilina associada a novobiocina. Ao contrário, foi observado alta susceptibilidade ao ceftiofur (96.8%) entre os isolados de E. coli. Diferenças na susceptibilidade entre os grupos filogenéticos foi observada apenas para a cefalotina, em que os isolados de E. coli pertencentes ao filogrupo A foram inibidos em concentrações de antimicrobianas mais baixas que isolados pertencentes ao filogrupo B1. No Estudo 4, avaliou-se a diversidade genotípica entre isolados de Strep. agalactiae e Strep. uberis identificados em casos de MC em vacas leiteiras; adicionalmente, o estudo avaliou a associação dos genótipos agrupados de acordo com a similaridade genética com o perfil de susceptibilidade aos antimicrobianos. Os isolados foram genotipados por meio do método de amplificação randômica de DNA polimórfico (RAPD). Grande diversidade genotípica foi observada tanto para o Strep. agalactiae (45 subtipos de 89 isolados) quanto para Strep. uberis (56 subtipos de 89 isolados). Para a avaliação de susceptibilidade aos antimicrobianos, os subtipos de Strep. agalactiae foram agrupados em três clusters (Ia, Ib e II), enquanto que os subtipos de Strep. uberis foram agrupados em dois clusters (I e II) de acordo com a similaridade genética. De forma geral, os isolados de Strep. agalactiae apresentaram alta susceptibilidade à maioria dos antimicrobianos, exceto para tetraciclina e eritromicina. Diferenças na susceptibilidade aos antimicrobianos entre os clusters de Strep. agalactiae foram observadas para ampicilina, ceftiofur, eritromicina, pirlimicina, sulfadimetoxina e tetraciclina. Por outro lado, os isolados de Strep. uberis foram resistentes à maioria dos antimicrobianos, exceto para cefalotina e penicilina + novobiocina. Não foram encontradas diferenças entre os clusters para todos os antimicrobianos na análise de Strep. uberis. Em conclusão, os resultados desta tese indicaram alta TIMC nos rebanhos avaliados, e apesar de os patógenos ambientais serem a causa mais comum de MC nestes rebanhos, patógenos contagiosos como Strep. agalactiae e Staph. aureus, ainda são uma preocupação em alguns rebanhos do Brasil. Além disso, observaram-se altas frequências de UAM e de terapias não recomendadas em bula entre os rebanhos avaliados. O uso não judicioso de antimicrobianos pode se tornar um fator de risco para o desenvolvimento da resistência bacteriana aos antimicrobianos, o que foi inclusive observado para isolados pertencentes as três espécies bacterianas mais prevalentes nos casos de MC no nosso estudo (E. coli, Strep. agalactiae e Strep. uberis). Finalmente, pelo fato de algumas variáveis em nível de rebanho terem sido associadas com a TIMC e com o UAM em nosso estudo, é possível que hajam oportunidades para implementação de estratégias de manejo com o objetivo de melhorar o controle da MC em rebanhos leiteiros do sudeste do Brasil.

The general objectives of this thesis were: (i) to determine the etiological and molecular profile of clinical mastitis (CM) in 20 dairy herds of Southeast, Brazil; and (ii) to quantify antimicrobial used for treatment of CM in the study population. To achieve this goals, four studies were performed. In the Study 1, we characterized the pathogen frequency and severity of CM in dairy herds. In addition, we determined the incidence rate of clinical mastitis (IRCM) and its association with the following herd-level descriptors: bulk milk somatic cell count (BMSCC), bulk milk total bacterial count (BMTBC), herd size (number of lactating cows), milk yield, housing system and season. The association between herd-level descriptors and IRCM were determined by two groups of mixed regression models: one based on the overall IRCM, and five based on the following specific-pathogen groups: contagious, other Gram-positive, Gram-negative, other (composed of yeast and Prototheca spp), and negative culture. A total of 5,957 quarter-cases of CM were recorded and the most frequently isolated pathogens were Escherichia coli (6.6% of total cultures), Streptococcus uberis (6.1%), and Streptococcus agalactiae (5.9%). The majority of CM cases were mild (60.3%), while 34.1% were moderate and 5.6% severe. Overall, the IRCM was 9.7 quarter-cases per 10,000 quarter-days at risk (QDAR), and the only herd-level parameter associated with overall IRCM was BMSCC, in which the highest IRCM was observed for herds with BMSCC &gt;600.000 × 103 cells/mL. In the models evaluating the specific-pathogen groups, IRCM with isolation of major contagious pathogens was associated with BMSCC, milk yield and housing system. For the evaluation of other Gram-positive pathogens, the IRCM was higher in the rainy season of 2015 in comparison with the other seasonal categories. In addition, for the model evaluating the Gram-negative group, the IRCM was highest in herds with BMTBC &gt;30 × 103 cfu/mL. The Study 2 aimed to characterize the treatment profile and quantify the antimicrobial consumption for treatment of CM in dairy herds; and to determine the association of antimicrobial use (AMU) and the same herd-level descriptors as described in the Study 1. Data on treatment practices and AMU were obtained from 19 dairy herds for a period of 12 months per herd. The AMU for treatment of CM was quantified monthly in units of defined daily dose (DDD) and expressed as antimicrobial treatment incidence (ATI; number of DDD per 1,000 lactating cows-day). The overall monthly mean ATI was 17.7 DDD per 1,000 lactating cow-days (15.4 for intramammary compounds, and 2.2 for systematically administered antimicrobials). Among intramammary drugs, aminoglycosides had the highest ATI (11.7 DDD per 1,000 lactating cow-days), while for systematically administrated antimicrobials, fluoroquinolones (4.2 DDD per 1,000 lactating cow-days) were the most frequently used antimicrobials. Herd size and BMSCC were positively associated with ATI. In addition, herd-level ATI was higher in freestall herds than in compost bedded-pack barns. In the Study 3, we determined the phylogeny of E. coli strains isolated from CM in dairy cows and the association of most frequent phylogroups with antimicrobial susceptibility. A total of 100 E. coli isolates recovered from CM cases described in the Study 1 were categorized according to their phylogenetic group using a quadruplex PCR method; antimicrobial susceptibility pattern was also evaluated. Most isolates were assigned to phylogenetic group A (52%), followed by B1 (38%), B2 (2%), C (4%), D (3%), and E (1%). Resistant isolates were observed for all evaluated antimicrobials. Overall, more than 96% of E. coli isolates were resistant to ampicillin, and more than 23% were resistant to cephalothin, sulphadimethoxine or tetracycline. High levels of resistance (&gt;70%) were also found to erythromycin, oxacillin, penicillin, penicillin associated with novobiocin, and pirlimycin. In contrary, high susceptibility was observed to ceftiofur (96.8%) among E. coli isolates. Difference in the antimicrobial susceptibility among phylogenetic groups was observed only for cephalothin, in which E. coli strains belonging to the phylogroup A were inhibited at lower antimicrobial concentrations than strains assigned to the phylogroup B1. In Study 4, we evaluated the genotypic diversity among Strep. agalactiae and Strep. uberis isolates recovered from CM in dairy cows; in addition, the study evaluated the association of genotypes clustered by genetic similarity with antimicrobial susceptibility pattern. Isolates were subtyped using randomly amplified polymorphic DNA (RAPD) analysis. A great genotypic diversity was found for both Strep. agalactiae (45 subtypes out of 89 isolates) and Strep. uberis (56 subtypes out of 88 isolates). For evaluation of antimicrobial susceptibility, subtypes of Strep. agalactiae were clustered into three groups (Ia, Ib and II), while Strep. uberis subtypes were clustered into two groups (I and II) according to their genetic similarity. Overall, Strep. agalactiae isolates showed high susceptibility to most antimicrobials, except to tetracycline and erythromycin. Differences in the antimicrobial susceptibility among clusters of Strep. agalactiae were observed for ampicillin, ceftiofur, erythromycin, pirlimycin, sulphadimethoxine and tetracycline. In contrary, Strep. uberis isolates were categorized as resistant to most antimicrobials, except to cephalothin and penicillin+novobiocin. No differences were observed among clusters for all antimicrobials in the analysis of Strep. uberis. In conclusion, the results of this thesis indicated a high IRCM in the evaluated herds, and although environmental pathogens were the most common cause of CM in these herds, contagious pathogens such as Strep. agalactiae and Staph. aureus, are still a concern in some dairy herds of Brazil. Furthermore, high frequencies of AMU and off-label protocols were observed among the evaluated herds. The non-judicious use of antimicrobials can become a risk factor for the development of antimicrobial resistance, which was even observed for isolates belonging to the three most prevalent bacterial species identified from CM cases in our study (E. coli, Strep. agalactiae and Strep. uberis). Finally, because there were some herd-level descriptors associated with the IRCM and AMU in our study, there may be opportunity for management strategies aiming to improve the control of CM in dairy herds of southeastern Brazil. / Os objetivos gerais desta tese foram: (i) determinar o perfil etiológico e molecular da mastite clínica (MC) em 20 rebanhos leiteiros do Sudeste do Brasil; e, (ii) quantificar os antimicrobianos usados para tratamento da MC na população estudada. Para alcançar esses objetivos, quatro estudos foram realizados. No Estudo 1, foi caracterizada a frequência de patógenos causadores de MC e a gravidade das infecções nos rebanhos leiteiros. Além disso, foi determinada a taxa de incidência de mastite clínica (TIMC) e sua associação com as seguintes variáveis em nível de rebanho: contagem de células somáticas em leite de tanque (CCSLT), contagem bacteriana total em leite de tanque (CBTLT), tamanho (número de vacas em lactação), produção de leite, sistema de alojamento e estação do ano. A associação entre as variáveis em nível de rebanho e a TIMC foi determinada por dois grupos de modelos de regressão logística multivariada: um baseado na TIMC geral, e cinco baseados nos seguintes grupos específicos de patógenos: contagiosos, outros Gram-positivos, Gram-negativos, outros patógenos (composto de leveduras e Prototheca spp.), e cultura negativa. Um total de 5.957 casos de MC em nível de quarto mamário foi registrado e os patógenos mais prevalentes foram Escherichia coli (6,6% de todas as culturas), Streptococcus uberis (6,1%), e Streptococcus agalactiae (5,9%). A maioria dos casos de MC foi de gravidade leve (60,3%), enquanto 34,1% dos casos foram moderados e 5,6% foram graves. A TIMC geral foi de 9,7 casos por 10.000 quartos-dia em risco (QDR), e o único parâmetro em nível de rebanho associado com a TIMC geral foi a CCSLT, em que a TIMC mais alta foi observada em rebanhos com CCSLT &gt;600.000 × 103 células/mL. Nos modelos que avaliaram os grupos específicos de patógenos, a TIMC de patógenos contagiosos foi associada com a CCSLT, produção de leite e sistema de alojamento. Na avaliação de outros patógenos Gram-positivos, a TIMC foi maior na estação chuvosa de 2015 em comparação com as outras categorias referentes à estação do ano. Adicionalmente, para o modelo avaliando o grupo de patógenos Gram-negativos, a TIMC foi mais alta em rebanhos com CBTLT &gt;30.000 × 103 ufc/mL. O Estudo 2 teve como objetivo caracterizar o perfil de tratamento e o consumo de antimicrobianos em rebanhos leiteiros; e determinar a associação de uso de antimicrobianos (UAM) e as mesmas variáveis em nível de rebanho descritas no Estudo 1. Dados sobre as práticas terapêuticas e UAM foram obtidos de 19 rebanhos leiteiros durante um período de 12 meses por rebanho. A frequência de UAM para tratamento da MC foi quantificada mensalmente em unidades de doses definidas diárias (DDD) e expressa como incidência de tratamento antimicrobiano (ITA: número de DDD por 1.000 vacas em lactação-dia). A média de ITA mensal foi de 17,7 DDD por 1.000 vacas em lactação-dia (15,4 para compostos intramamários, e 2,2 para compostos sistêmicos). Entre os produtos intramamários, os aminoglicosídeos tiveram a ITA mais alta (11,7 DDD por 1.000 vacas em lactação-dia), enquanto que para os compostos administrados pela via sistêmica, as fluoroquinolonas (4,2 DDD por 1.000 vacas em lactação-dia) foram os antimicrobianos mais frequentemente usados. O tamanho do rebanho e CCSLT foram positivamente associados com a ITA. Além disso, a ITA foi mais alta em rebanhos com freestall do que em rebanhos com sistema tipo compost barn. No Estudo 3, determinou-se a filogenia de cepas de E. coli isoladas de casos de MC em vacas leiteiras, e a associação dos filogrupos mais frequentes com a susceptibilidade aos antimicrobianos. Um total de 100 isolados de E. coli identificados nos casos de MC descritos no Estudo 1 foram categorizados de acordo com os grupos filogenéticos por meio de um método de PCR quadruplex; o perfil de susceptibilidade aos antimicrobianos também foi avaliado. A maioria dos isolados pertenceram ao grupo A (52%), seguido dos grupos B1 (38%), B2 (2%), C (4%), D (3%), e E (1%). Foram encontrados isolados resistentes para todos os antimicrobianos avaliados. De forma geral, mais de 96% dos isolados de E. coli foram resistentes a ampicilina, e mais de 23% foram resistentes a cefalotina, sulfadimetoxina ou tetraciclina. Altos níveis de resistência (&gt;70%) foram encontrados também para eritromicina, oxacilina, penicilina e penicilina associada a novobiocina. Ao contrário, foi observado alta susceptibilidade ao ceftiofur (96.8%) entre os isolados de E. coli. Diferenças na susceptibilidade entre os grupos filogenéticos foi observada apenas para a cefalotina, em que os isolados de E. coli pertencentes ao filogrupo A foram inibidos em concentrações de antimicrobianas mais baixas que isolados pertencentes ao filogrupo B1. No Estudo 4, avaliou-se a diversidade genotípica entre isolados de Strep. agalactiae e Strep. uberis identificados em casos de MC em vacas leiteiras; adicionalmente, o estudo avaliou a associação dos genótipos agrupados de acordo com a similaridade genética com o perfil de susceptibilidade aos antimicrobianos. Os isolados foram genotipados por meio do método de amplificação randômica de DNA polimórfico (RAPD). Grande diversidade genotípica foi observada tanto para o Strep. agalactiae (45 subtipos de 89 isolados) quanto para Strep. uberis (56 subtipos de 89 isolados). Para a avaliação de susceptibilidade aos antimicrobianos, os subtipos de Strep. agalactiae foram agrupados em três clusters (Ia, Ib e II), enquanto que os subtipos de Strep. uberis foram agrupados em dois clusters (I e II) de acordo com a similaridade genética. De forma geral, os isolados de Strep. agalactiae apresentaram alta susceptibilidade à maioria dos antimicrobianos, exceto para tetraciclina e eritromicina. Diferenças na susceptibilidade aos antimicrobianos entre os clusters de Strep. agalactiae foram observadas para ampicilina, ceftiofur, eritromicina, pirlimicina, sulfadimetoxina e tetraciclina. Por outro lado, os isolados de Strep. uberis foram resistentes à maioria dos antimicrobianos, exceto para cefalotina e penicilina + novobiocina. Não foram encontradas diferenças entre os clusters para todos os antimicrobianos na análise de Strep. uberis. Em conclusão, os resultados desta tese indicaram alta TIMC nos rebanhos avaliados, e apesar de os patógenos ambientais serem a causa mais comum de MC nestes rebanhos, patógenos contagiosos como Strep. agalactiae e Staph. aureus, ainda são uma preocupação em alguns rebanhos do Brasil. Além disso, observaram-se altas frequências de UAM e de terapias não recomendadas em bula entre os rebanhos avaliados. O uso não judicioso de antimicrobianos pode se tornar um fator de risco para o desenvolvimento da resistência bacteriana aos antimicrobianos, o que foi inclusive observado para isolados pertencentes as três espécies bacterianas mais prevalentes nos casos de MC no nosso estudo (E. coli, Strep. agalactiae e Strep. uberis). Finalmente, pelo fato de algumas variáveis em nível de rebanho terem sido associadas com a TIMC e com o UAM em nosso estudo, é possível que hajam oportunidades para implementação de estratégias de manejo com o objetivo de melhorar o controle da MC em rebanhos leiteiros do sudeste do Brasil.