O presente estudo teve como objetivo relacionar os níveis de indicadores sanguíneos energéticos e de cálcio na ocorrência de afecções uterinas, mastite clínica e mastite subclínica em vacas de raça Holandesa. A presença de mastite foi monitorada através do California Mastitis Test (CMT) e da contagem de células somáticas, para a identificação de mastite subclínica, e do Teste da Caneca de Fundo Preta (TCFP), para identificação de mastite clínica. A presença das afecções uterinas foi identificada mediante exame clínico. Foram feitas coletas de sangue para a determinação dos níveis de cálcio (Ca), glicose e β-hidroxi-butirato (BHB). O CMT foi realizado nas duas primeiras semanas após o parto, o TCFP foi realizado conforme a rotina de ordenha, estabelecida pela propriedade, e as coletas de sangue foram realizadas na semana anterior ao parto e duas coletas até os 21 dias posteriores ao parto. A ocorrência de hipocalcemia subclínica foi determinada quando o Ca no soro atingiu valores ≤ 8,00 mg/dL, hipoglicemia com valores de glicose ≤ 50 mg/dL e hipercetonemia com valores de BHB ≥ 1,2 mmol/L. Nas vacas com mastite clínica foi realizado tratamento conforme estabelecido pelo protocolo de ordenha e tratamentos da fazenda. Os dados de mastite subclínica obtidos através do teste de CMT, por quarto mamário, foram validados através de cultura microbiológica realizada em laboratório de referência e em laboratório na fazenda para identificar crescimento bacteriano, bem como através da contagem de células somáticas (CCS) e da contagem bacteriana total (CBT). Não foi encontrada relação significativa entre a hipocalcemia subclínica, a hipoglicemia e a hipercetonemia com afecções uterinas e da glandula mamária nas vacas após o parto. / This study aimed to relate the levels of energy and calcium in the occurence of both uterine diseases, clinic and subclinic mastitis in Holstein cows. The presence of mastitis was monitored through California Mastitis Test (CMT) and the counting of somatic cells (CSC), for the identification of subclinic mastitis, and strip cup test, for the identification of clinic mastitis. The presence of uterine disorders was identified by clinical examination. Blood collections were made for determining calcium, glicose and β-hydroxy-butyrate (BHB). CMT was realized in the first two weeks after calving, BBMT was realized according to the routine of milking settled by the farm and the blood collections were made in the week before the calving and also two collections were made after it. The occurrence of subclinic hypocalcemia was determined when serum Ca reached ≤ 8,00 mg/dL, hypoglycemia with glucose values ≤ 50 mg/dL and hyperketonemia values with BHB BHB ≥ 1,2 mmol/L. Data of clinic mastitis were obtained through the identification of altered milk in BBMT and subsequently realized treatment as settled by the milking protocol and treatments of the farm. Data of subclinic mastitis were obtained through CMT test, for mammary quarter, and validated by microbiological culture in reference laboratory, culture in the farm for identifying the bacterial growth, counting of somatic cells (CSC) and total bacterial counting (TBC). It was not identified in the study any relationship between subclinical hypocalcemia and uterine and mammary gland diseases in cows after calving.

The objective was to evaluate the effect of the type of milking and the environment in the quality of raw milk by identification the Somatic Cell Count - CCS in samples obtained from farms in the mesoregion of south goiano and correlate it with the different types of milk, and the milk components: fat, protein, lactose and total solids (ST). Furthermore, we sought to diagnose the prevalence of subclinical mastitis by CCS, evaluate the correlation between CCS and milking type with antimicrobial residues, and check the influence of season, temperature and humidity index (THI) and Equivalent Temperature Index (ITE) on the CCS. The research developed in farms with different types of milking, being divided into manual and mechanical milking. The datas were collected from January 2011 to December 2013, and in each property was collected a monthly sample in the expansion tank, a total of 8242 samples, which were later sent for analysis. The diagnosis of the milk quality held by electronic count of somatic cells, for the FossomaticTM® and the levels of the components were analyzed using the MilkoscanTM FT®, the detection of antibiotic residues was performed using the Delvotest-P and meteorological data were obtained from the National Institute of Meteorology - INMET in Catalão-GO station. Among the variables type of it milks and subclinical mastitis, there was dependent relationship (p <0.05). The milking mechanics was the one with a higher prevalence of subclinical mastitis with 58% (910) of the samples presented CCS> 300.000 cells/mL. Among the variables type of milking and CCS advocated by Instruction n°62/2011 (BRASIL, 2011), there was a significant relationship (p <0.05). Only 19,53% (1610) of the samples had CCS > 500.000 cells/mL. According to the statistical analysis, there was a positive correlation between CCS and fat and ST, however, there was a negative correlation between the CCS and the protein and lactose contents. The type of milking and the somatic cell count did not affect the presence or absence of antimicrobial residues. During the rainy season has become a CCS elevation compared to the dry season, but there was no correlation between CCS and environmental indices (ITE and ITU). Regarding environmental indices can be seen that with the increase of the ITU occurred a decrease in fat and an increase in ST and lactose. It can be concluded that the type of manual milking showed the lowest prevalence of subclinical mastitis in relation to milking and with elevated CCS is a reduction in the concentrations of protein and lactose and an increase in fat and total solids in the raw milk. / Objetivou-se avaliar o efeito do tipo de ordenha e do ambiente na qualidade do leite cru por meio da identificação da Contagem de Células Somáticas - CCS nas amostras obtidas das propriedades rurais da mesorregião do sul goiano e correlacioná-las com os diferentes tipos de ordenha, bem como os componentes do leite: gordura, proteína, lactose e sólidos totais (ST). Ademais, buscou-se também diagnosticar a prevalência da mastite subclínica por meio da CCS, avaliar a correlação entre CCS e tipo de ordenha com os resíduos de antimicrobianos, verificar a influência do período do ano, Índice de Temperatura e Umidade (ITU) e Índice de Temperatura Equivalente (ITE) sobre a CCS. A pesquisa desenvolveu-se em fazendas com diferentes tipos de ordenha, sendo essas divididas em ordenha manual e mecânica. O trabalho de campo foi realizado de janeiro de 2011 a dezembro de 2013, sendo que em cada propriedade foi coletada uma amostra mensal no tanque de expansão, perfazendo um total de 8242 amostras, que posteriormente, foram enviadas para análise. O diagnóstico da qualidade do leite realizou-se por meio da CCS, pelo aparelho FossomaticTM®, os teores dos componentes foram analisados utilizando-se o MilkoscanTM FT®, a detecção de resíduos de antimicrobianos foi realizada através do Delvotest-P e os dados meteorológicos foram obtidos do Instituto Nacional de Meteorologia- INMET na estação de Catalão-GO. Entre as variáveis tipo de ordenha e mastite subclínica, houve relação de dependência (p<0,05). A ordenha mecânica foi a que obteve maior prevalência de mastite subclínica, 58% (910) com CCS > 300.000 células/mL. Entre as variáveis tipo de ordenha e a CCS preconizada pela Instrução Normativa n°62/2011 (BRASIL, 2011), houve relação significativa (p<0,05). Apenas 19,53% (1610) apresentaram CCS > 500.000 células/mL. De acordo com a análise estatística, existiu correlação positiva entre a CCS e o teor de gordura e ST, porém houve uma correlação negativa entre a CCS e os teores de proteína e lactose. O tipo de ordenha e a contagem de células somáticas não interferiram na presença ou ausência de resíduos de antimicrobianos. No período da chuva tem-se uma elevação de CCS se comparado ao período da seca, porém não houve correlação entre CCS e os índices ambientais (ITE e ITU). Em relação aos índices ambientais pode-se observar que com a elevação do ITU ocorreu uma redução nos teores de gordura e ST e um aumento no teor de lactose. Pode-se concluir que o tipo de ordenha manual obteve a menor prevalência de mastite subclínica em relação à ordenha mecânica e com a elevação da CCS tem-se uma redução nas concentrações dos teores de proteína e lactose e um aumento nos teores de gordura e sólidos totais do leite cru. / Mestre em Ciências Veterinárias

Os objetivos do presente estudo foram os de verificar a validade do método de reação em cadeia da polimerase em tempo real (qPCR) para detectar e quantificar o Staphylococcus aureus em amostras de leite conservadas com bronopol oriundas de quartos mamários bovinos subclinicamente infectados, e de avaliar os efeitos da presença e da quantidade de células da bactéria sobre a contagem de células somáticas (CCS), a composição do leite (lactose, gordura, proteína bruta, proteína verdadeira e caseína), e a produção de leite de quartos mamários bovinos subclinicamente infectados pelo patógeno. Para a quantificação do S. aureus e das células somáticas bovinas por meio do qPCR, foi utilizado leite cru bovino para o preparo dos padrões como meio de diluição da inoculação seriada de células somáticas e do S. aureus ATCC 29213, e construídas as equações log10UFC = 37,86 23,54 log10CtSAU e log10CCS = 49,3 - 34,0 log10CtBMCB, com base nos resultados obtidos pelas metodologias de referência para cada procedimento. Para testar a equivalência dessas equações aos respectivos métodos de referência, determinar a sensibilidade e especificidade analíticas e a repetibilidade do método proposto, foram coletadas amostras de leite dos quartos mamários de 60 animais de 2 rebanhos leiteiros da região de Pirassununga dos quais se determinou previamente a ocorrência de casos subclínicos de mastite por S. aureus. Dos quartos mamários também foram mensuradas as produções e coletadas amostras de leite para análise de composição, diagnóstico da mastite, e determinação da sensibilidade e especificidade diagnósticas do procedimento de qPCR estabelecido no estudo. Cada amostra foi submetida à análise de composição, CCS, cultura microbiológica, contagem em placas do S. aureus, processadas para a extração do DNA genômico bovino e do S. aureus, e submetida à reação de qPCR. Para análise da concordância entre os resultados obtidos pelos métodos de referência para o diagnóstico da mastite por S. aureus e o de qPCR foi utilizado o teste Kappa. Para avaliação da equivalência das contagens obtidas pelos métodos de referência do S. aureus e de células somáticas bovinas, foi utilizado o teste das diferenças de Bland-Altman. Para a identificação do efeito da infecção subclínica pelo S. aureus sobre a composição e produção de leite do quarto mamário afetado foi utilizada a análise da variância num delineamento em parcelas subdivididas em faixas. Para estimar o grau de relação entre as contagens de S. aureus, a CCS, produção e composição do leite produzido pelo quarto mamário afetado foi utilizado o coeficiente de correlação de Pearson. A correlação entre os resultados de contagem de células somáticas bovinas determinados pelos métodos de rotina e de qPCR para a quantificação de células somáticas apresentou coeficiente r = - 0,978 (P < 0,001). A correlação entre os resultados da contagem do S. aureus ATCC 29213 determinados pelos métodos de rotina e de qPCR para a quantificação do patógeno apresentou coeficiente r = - 0,989 (P < 0,001). A especificidade analítica do qPCR para a detecção do S. aureus em amostras de leite frente a Escherichia coli, Enterococcus sp., Pseudomonas aeruginosa, Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus uberis, os estafilococos coagulase-negativa, e as espécies coagulase-positiva Staphylococcus hyicus e Staphylococcus intermedius foi de 100%. O método de qPCR aplicado à detecção de Staphylococcus aureus ATCC 29213 em amostras de leite é replicável e apresentou sensibilidade analítica com limite de detecção para a faixa de 10 UFC/mL à 4,2 x 106 UFC/mL. Em amostras de leite conservadas com bronopol provenientes de quartos mamários subclinicamente infectados, o S. aureus pôde ser detectado, mas não pôde ser quantificado pelo método de qPCR. Nessas amostras, a CCS pôde ser determinada de forma equivalente ao método de rotina. A CCS independe da contagem de S. aureus viáveis, mas foi observada correlação linear e negativa entre o número total de células do patógeno e a CCS. A mastite subclínica pelo S. aureus aumentou a CCS nos quartos mamários, mas não alterou a composição do leite. A doença diminuiu a produção de leite e de gordura dos quartos mamários anteriores acometidos pela infeção, mas não se observou efeito da interação entre o posicionamento da glândula e a infecção sobre a produção de leite. Houve correlação entre as concentrações de lactose (r = 0,42; P = 0,0051), de gordura (r = 0,46; P = 0,0016), de produção de gordura (r = 0,49; P = 0,001), e de leite com produção ajustada para o teor de 3,5% de gordura (r = 0,41; P = 0,006), e o número de S. aureus presentes na amostra de leite. / The objectives of this study were to verify the validity of the real-time polymerase chain reaction (qPCR) to detect and quantify Staphylococcus aureus in bronopol-preserved milk samples from subclinically infected mammary quarters, and to assess the effects of the presence and amount of the pathogen on the somatic cell count (SCC), the composition of milk and milk yield of bovine mammary quarters subclinically infected by the pathogen. In order to quantify S. aureus and bovine somatic cells through qPCR, raw bovine milk was used as a means of serial inoculation media of somatic cells and S. aureus ATCC 29213. From that, equations based on the reference methods for each procedure were built, log10UFC = 37,86 23,54 log10CtSAU and log10CCS = 49,3 - 34,0 log10CtBMCB, respectively. To test their equivalence with the reference methods, determine the analytical sensitivity and specificity, and repeatability of the proposed method, milk was sampled from quarters of 60 animals from two dairy herds in Pirassununga, where subclinical S. aureus mastitis cases had been previously diagnosed. Also, quarter milk yield had been measured and samples collected for milk composition analysis, diagnosis of mastitis, sensitivity and specificity of the procedure established in the study had been determined. Each sample was subjected to composition analysis, SCC, microbiological culture, plate counting of S. aureus, DNA extraction, and subjected to qPCR reaction. Agreement between results from reference methods and qPCR for the diagnosis of mastitis by S. aureus was assessed by Kappa test. Equivalence between S. aureus, SCC scores obtained by reference and qPCR was assessed with Bland-Altman procedures. The effect of S. aureus subclinical infection on milk composition and milk yield of affected quarters was measured using a strip plot design. To estimate the degree of relationship between the counts of S. aureus, SCC, yield and composition of the milk from affected quarters was assessed by the Pearson Correlation. Correlation between SCC determined by routine methods and qPCR was r = - 0.978 (P <0.001). Correlation between S. aureus ATCC 29213 determined by routine methods and qPCR was r = - 0.989 (P <0.001). Analytical specificity of qPCR to detect S. aureus in milk samples against Escherichia coli, Enterococcus sp., Pseudomonas aeruginosa, Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus uberis, coagulase-negative staphylococci and coagulase-positive species, Staphylococcus hyicus and Staphylococcus intermedius was 100%. The use of the qPCR to detect S. aureus ATCC 29213 in milk samples is replicable. Analytical sensitivity detection limit of the method ranged from 10 CFU/mL to 4.2 x 106 CFU/mL. S. aureus could be detected, but not quantified by qPCR in bronopol-conserved milk samples from subclinically infected quarters. In these samples, SCC could be determined by qPCR as it had been done by routine method. SCC was not dependent on S. aureus viable cells, but a negative linear correlation between the total number of cells of the pathogen and SCC was observed. S. aureus subclinical mastitis increased quarters SCC, but did not change milk composition. The disease decreased quarter milk and fat yield, but no interaction effect was observed between the gland positioning and S. aureus subclinical infection on milk production. Correlations between lactose (r = 0.42, P = 0.0051), fat (r = 0.46, P = 0.0016), fat yield (r = 0.49, P = 0.001), and 3.5% fat adjusted milk yield (r = 0.41, P = 0.006), and the number of S. aureus present in the milk sample were observed.

A mastite subclínica (MS) é a doença mais prevalente em rebanhos leiteiros e pode ser identificada pela contagem de células somáticas (CCS) individual das vacas. A partir disso, indicadores podem ser calculados como, por exemplo, prevalência e incidência. A prevalência diz respeito à proporção do rebanho infectada em determinado momento, enquanto que a incidência estima a taxa de novas infecções intramamárias ao longo do tempo. Altos valores de prevalência e incidência acabam resultando em elevada CCS do tanque de resfriamento (CCST), que por consequência leva a penalizações oriundas dos programas de pagamento por qualidade do leite (PPQL). Desta forma, estimar os valores atuais de prevalência e incidência de MS relacionados com valores futuros de CCST e as perdas financeiras oriundas dos PPQLs se torna importante para estimular a melhoria da qualidade do leite produzido no Brasil. Portando, o objetivo deste trabalho é estimar os indicadores epidemiológicos de MS em rebanhos leiteiros brasileiros, além prever a CCST relacionando-a as perdas financeiras devidas ao PPQL. Para isso, foram utilizados dois bancos de dados, sendo o primeiro com dados de CCS individual de vacas para estimação da prevalência e incidência de MS, e o segundo com dados de CCST para a previsão e cálculo de perdas financeiras devido ao PPQL. Como análise estatística, foi utilizado o modelo linear generalizado misto para comparar a prevalência e incidência de MS entre anos, regiões, tamanho de rebanhos e categorias de número de testes enviados durante o período de estudo, além de modelos de séries temporais para previsão da CCST e regressão linear múltipla para prever a probabilidade de mudança de classe de pagamento por qualidade do leite do PPQL. Os resultados encontrados indicam que, ambos, prevalência e incidência de MS permaneceram elevados e com leve tendência de aumento ao longo dos anos estudados, sendo que não houve diferenças entre regiões e tamanho de rebanhos. Por outro lado, rebanhos que realizaram mais testes ao longo do período estudado apresentaram menor prevalência, mostrando a importância da análise rotineira de CCS individual. Para CCST, os valores tem se mantido altos nos recentes anos e sem tendência de melhora. A probabilidade de mudança de classe foi fortemente afetada pela média e desvio padrão de CCST para as classes 1 e 2 (1.000 a 200.000 e 201.000 a 400.000 céls/mL, respectivamente). Os modelos de séries temporais indicaram que em algum ponto do ano, as fazendas não podiam permanecer na sua classe atual do PPQL e acumulavam perdas financeiras. Por fim, este estudo permite concluir que prevalência e incidência de MS estão elevadas e não melhoram ao longo dos anos, sendo que isto acaba elevando os valores de CCST. Além disso, existem perdas financeiras em todas as classes do PPQL, mostrando que os produtores não tem sido eficientes em captar a bonificação máxima dos PPQLs. Por isso, o desenvolvimento de programas de qualidade do leite deve ser uma prioridade em busca da melhoria da qualidade do leite de rebanhos brasileiros. / Subclinical mastitis (SM) is the most prevalent disease in dairy herds and can be identified by individual somatic cell counts (SCC) of cows. From this, indicators can be calculated as, for example, prevalence and incidence. Prevalence refers to the number of infected cows in a herd at a given time, while the incidence estimates the rate of new intramammary infections over time. High prevalence and incidence values result in high bulk tank SCC (BTSCC), which consequently leads to penalties from payment programs based on milk quality (PPBMQ). In this way, estimating current values of prevalence and incidence of SM related to future values of BTSCC and financial losses from PPBMQs becomes important to stimulate the improvement of the quality of milk produced in Brazil. Therefore, the objective of this study is to estimate the epidemiological indicators of SM in Brazilian dairy herds, besides to predicting the BTSCC and relating it to the financial losses due to the PPBMQ. For this, two databases were used, the first one with individual SCC data from cows to estimate the prevalence and incidence of SM, and the second with BTSCC data for prediction and calculation of financial losses due to PPBMQ. For statistical analysis, the mixed generalized linear model was used to compare the prevalence and incidence of SM between years, regions, herd size and number of test-day categories sent during the studied period, as well as time series models for prediction of BTSCC and multiple linear regressions to predict the probability of changing the current payment class for milk quality. The results indicate that both prevalence and incidence of SM remained high and with a slight tendency to increase over the years studied, and there were no differences between regions and herd sizes. On the other hand, herds that performed more test-day during the studied period presented a lower prevalence, showing the importance of the routine analysis of individual SCC. For BTSCC, values have remained high in recent years and no trend for improvement. The probability of class change was strongly affected by the mean and standard deviation of BTSCC for classes 1 and 2 (1,000 to 200,000 and 201,000 to 400,000 cells/mL, respectively). Time series models indicated that at some point of the year, farms could not remain in their current class and accumulated financial losses due to PPBMQ. Finally, this study allows to conclude that the prevalence and incidence of SM are high and do not improve over the years, and this ends up increasing the BTSCC values. In addition, there are financial losses in all classes of PPBMQ, showing that producers have not been efficient in capturing the maximum PPBMQs bonus. Therefore, the development of milk quality programs should be a priority in order to improve the quality of milk from Brazilian herds.

Le virus d'Epstein-Barr (EBV), est un gamma herpes virus exclusivement humain qui infecte près de 95% de la population adulte. EBV établit un cycle de latence dans les cellules B mémoires et les cellules épithéliales. EBV entre périodiquement dans une réplication lytique avec sécrétion des virions dans la salive. L’infection EBV est associée à l’apparition de lymphomes, de cancers et de maladies auto-immunes. Les conditions favorisant le développement de ces pathologies restent mal connues mais l’immunodépression et potentiellement l’activation des lymphocytes B nécessaire à la réplication d’EBV jouent un rôle clé.Nous avons examiné l’association entre activation des cellules B dans le compartiment systémique et le contrôle sur le réservoir de l’EBV. Deux situations cliniques d’activation chronique des cellules B ont été explorées ; le syndrome de Gougerot-Sjögren et la mastite subclinique dans le contexte de l’infection par le VIH.Dans ce travail de thèse, nous avons tout d’abord développé une PCR en temps réel pour la quantification de l'ADN de EBV ciblant la région répétée BamHI-W du génome. Le travail de validation de la technique a permis d’évaluer précisément le gain de sensibilité comparativement à une qPCR LMP2 (cible unique). L’impact des variations de répétition de la séquence BamHI-W suivant les souches et isolats EBV a également été analyse.Dans un deuxième travail, nous avons montré que, la mastite subclinique était fréquente au cours de l’allaitement et qu’elle était un facteur indépendant associé à une augmentation de l'excrétion EBV par le lait maternel. Cette sécrétion est associée localement à l’inflammation et à l’excrétion du VIH ce qui témoigne de phénomène de synergie entre les deux virus. Nous avons également démontré que l'ADN EBV dans le lait maternel peut être résistant à la DNase et que le virus était est probablement encapsidé dans le lait, donc potentiellement infectieux.Enfin, bénéficiant d’un accès aux prélèvements de la cohorte ASSESS nous avons recherché de possible anomalie du contrôle de l’infection EBV dans le compartiment sanguin au cours du syndrome de Gougerot-Sjögren primaire dans lequel les lésions glandulaires salivaires et lacrymales sont associées à la présence d’EBV. Nous avons démontré que le réservoir EBV et la réplication EBV dans le compartiment systémique sont bien contrôlées au cours du syndrome de Gougerot-Sjögren primaire. La réponse anticorps contre l’antigène précoce d’EBV (EA) n'était pas associée à une augmentation de l'ADN de EBV.Ce travail de thèse, souligne le lien entre l'activation des lymphocytes B, l'inflammation chronique et le contrôle sur le réservoir d’EBV. Dans la glande mammaire le contrôle de l’infection EBV est perturbé en cas de mastite subclinique. Au contraire dans le syndrome de Gougerot-Sjögren l’infection reste bien contrôlée dans le compartiment sanguin malgré l’activation des lymphocytes B et la présence renforcée du virus dans les lésions glandulaires. / TEpstein-Barr virus (EBV), an ubiquitous human gammaherpesvirus affects 95% of adult human population and establishes a lifelong latency in memory B cells periodically entering into lytic replication with further propagation in oropharyngeal epithelia and shedding through saliva. Latent EBV infection is associated with lymphomas, carcinomas and autoimmune diseases. Although the conditions favoring the development of these pathologies are not completely understood, the immunosuppression and B cell activations play an important role in EBV-associated diseases.We examined the control over the EBV reservoir in a diseases associated with B cell activation. Two clinical situations associated with altered B cell activation were explored: primary Sjogren’s syndrome and subclinical mastitis in HIV-infected mothers.Primarily, we developed an in-house real-time PCR for EBV DNA quantification targeting the repetitive BamHI-W region of EBV DNA. The validation analyses enabled to evaluate the gain in sensitivity of the BamHI-W test relative to single repeat LMP2 qPCR. The impact of the variations in BamHI-W reiteration on EBV DNA quantification was further assessed on different EBV strains and clinical samples.In a second study, we showed that subclinical mastitis was common during breastfeeding, and it was an independent factor associated with increased EBV breast milk shedding. This secretion was associated with local inflammation and HIV shedding, reflecting synergy between the two viruses. We have also demonstrated that breast milk EBV DNA may be resistant to DNase, and the virus was probably encapsidated in the breast milk and thus potentially infectious.Finally, with an access to ASSESS cohort we looked for possible abnormal control over EBV infection in the blood compartment in primary Sjögren's syndrome, where salivary and lacrimal gland lesions were shown to be associated with the local activation of EBV. We have demonstrated that in primary Sjogren's syndrome EBV reservoir and replication in the systemic compartment are well controlled. The antibody response against EBV early antigen (EA) was not associated with increased DNA EBV.This thesis points out the link between the activation of B cells, chronic inflammation and control over the reservoir of EBV. In the mammary gland disturbed control of EBV infection is linked with subclinical mastitis. In contrast, in primary Sjogren’s syndrome EBV infection remains well controlled in the blood compartment despite the activation of B cells and the increased presence of the virus in glandular lesions.

In der vorliegenden Studie wurde die Sanierung einer durch S. aureus verursachten Eutergesundheitsstörung in einem hessischen Milchviehbestand mittels klassischer Sanierungsmaßnahmen über einen Zeitraum von 18 Monaten begleitet. Durch konsequente Einhaltung der Sanierungsmaßnahmen nach dem Fünf-Punkte-Plan und die räumliche Trennung der Herde in eine S. aureus-positive und -negative Gruppe sank die S. aureus-Prävalenz im Betrieb innerhalb von 15 Monaten von 30% auf <1%. Nach 18 Monaten waren erstmalig keine Neuinfektionen mehr mit S. aureus zu verzeichnen. Zusätzlich zu den im Abstand von drei Monaten durchgeführten Viertelanfangsgemelk (VAG)- Gesamtbestandsuntersuchungen wurden Umweltproben bakteriologisch auf das Vorhandensein von S. aureus untersucht. Diese Untersuchungen verliefen ausschließlich mit negativem Ergebnis. Die im Untersuchungszeitraum asservierten 218 S. aureus-Isolate aus diesem Betrieb wurden genotypisch mittels PCR untersucht. Thermonuklease-Gen, Protein A-Gen (IgG-bindende Region) und Polymorphismen bei Protein A-Gen (Xr-Region) sowie Koagulase-Gen ermöglichten die Unterscheidung in sechs Typen. Zusätzlich wurden 80 dieser Isolate mittels Pulsfeldgelelektrophorese (Pfge, Gold Standard) typisiert. Es konnten zwei Pfge-Typen gefunden werden: Pfge-Typ I mit insgesamt 10 Subtypen (bei 78 Isolaten) und Pfge-Typ II (bei zwei Isolaten). Der Pfge-Typ II wurde ausschließlich bei einem Zukaufstier nachgewiesen, das vor der Einstallung in diesen Betrieb nicht zytobakteriologisch untersucht worden war. Die 12 unterschiedlichen Pfge-Typen bzw. -Subtypen wurden zusätzlich mittels Staphylokokken-Protein A- (spa)-Typisierung untersucht. Dabei zeigte sich, dass alle Subtypen des Pfge-Typs I dem spa-Typ t2067 zugeordnet werden konnten und der Pfge-Typ II dem spa-Typ t2112 entsprach. 92 weitere bovine S. aureus-Mastitisisolate wurden möglichst randomisiert über das Landesgebiet Hessens entnommen und mittels dieser Typisierungsmethode untersucht. Die Isolate stammten ebenfalls aus S. aureus-Problembetrieben und wurden aus VAG von (sub-) klinischen Mastitiden in Reinkultur isoliert. Insgesamt konnten 28 spa-Typen unterschieden werden. Durch den Algorithmus BURP wurden 57 dieser Isolate dem Clonal Complex CC543 zugeordnet und waren demnach genetisch eng miteinander verwandt. Der in dem Sanierungsbetrieb vorherrschende spa-Typ t2067 war dem CC543 nicht zuzuordnen. Die Anwendung der spa-Typisierung in der Routinediagnostik und damit die Möglichkeit der laborübergreifenden, möglichst zentralisierten Datendokumentation der Ergebnisse könnten zukünftig die Identifizierung besonders häufig vorkommender und damit hochkontagiöser S. aureus-Stämme ermöglichen.

Einleitung: Euterinfektionen sowohl mit kuh-, vor allem mit umweltassoziierten Streptokokken nehmen bei Milchkuhherden eine dominierende Rolle ein. Ziel der Untersuchungen: Die Beziehungen zwischen der Innerherdenprävalenz (IHP) von subklinischen Mastitiden mit Streptokokken zur Haltungshygiene und zur Melkhygiene wurden erfasst. Dabei standen laktierende Kühe mit Nachweis von äskulinpositiven Streptokokken und Streptococcus agalactiae in Viertelanfangs-gemelksproben im Mittelpunkt. Tiere, Material und Methoden: Von September 2009 bis Dezember 2010 fanden in 34 Thüringer Milchviehherden zweimalig im Abstand von etwa 100 Tagen bakteriologische Untersuchungen der Viertelanfangsgemelke von allen zu diesem Zeitpunkt laktierenden und klinisch eutergesund erscheinenden Kühen statt. Der Medianwert der Herdengröße der laktierenden Kühe betrug 246. Die bakteriologischen Untersuchungen von 81.567 Viertelanfangsgemelken erfolgten entsprechend den Leitlinien der Deutschen Veterinärmedizinischen Gesellschaft durch das Labor des Tiergesundheitsdienstes der Thüringer Tierseuchenkasse. Während des ersten Bestandsbesuchs wurden die Haltungsbedingungen sowie die Melktechnik und die Melkhygiene im Betrieb mittels eines dreiteiligen, strukturierten Fragebogens erfasst. Die Bewertung der Merkmale erfolgte durch die Einteilung in zwei oder drei vorgegebene Kategorien. Mittels multivariabler Varianzanalyse wurde anschließend geprüft, ob eine Beziehung zwischen der IHP intramammärer Infektionen mit Streptococcus agalactiae bzw. äskulinpositiven Streptokokken und dem jeweiligen Risikofaktor besteht. Ergebnisse: Infektionen mit äskulinpositiven Streptokokken traten in allen Herden auf, während Galtstreptokokken in nur sieben der 34 Herden nachgewiesen werden konnten. Der Median der IHP der Infektionen mit äskulinpositiven Streptokokken lag zur Untersuchung 1 bei 8,5 %, zur Untersuchung 2 bei 4,6 %, für Infektionen mit Streptococcus agalactiae bei 4,9 % bzw. 3,1 %. Signifikante Beziehungen zur IHP von subklinischen Mastitiden mit äskulinpositiven Streptokokken oder Streptococcus agalactiae finden sich für die Sauberkeit der Liegeflächen im Bereich der Trockensteher und der kalbenden Kühe, für die Art der Laufflächen im Bereich der Trockensteher und der milchliefernden Kühe und für die regelmäßige Nutzung der Handwaschgelegenheiten durch das Melkpersonal. Für Galt-Nachweise zeigten sich signifikante Beziehungen und niedrige IHP im Zusammenhang mit der Verwendung einer Rohrmelkanlage für die Frischmelker, guter Sauberkeit der Euter der zum Melken kommenden Kühe und der Verwendung einer Tauchdesinfektion als Melkzeugzwischendesinfektion. Für subklinische Mastitiden mit äskulinpositiven Streptokokken gilt dies für Herden, in denen die Kühe der Leistungsherde mit kaum verschmutzten Eutern zum Melken kommen und stark verschmutzte Euter in der Leistungsherde vor dem Melken intensiv gereinigt werden. Schlussfolgerungen: Die Häufigkeit der Infektionen mit äskulinpositiven Streptokokken steht mit der Sauberkeit der Euter in Beziehung. Das Bereitstellen von sauberen und trockenen Liegeflächen und Laufgängen für alle Kühe in der Herde, auch für die trockenstehenden Tiere, trägt zu einer Senkung der IHP intramammärer Infektionen mit umweltassoziierten Streptokokken bei. Dabei geht es um die Vermeidung von Verschmutzungen der Euter. Weiterhin stellen das Melken der frischmelkenden Kühe in einem separaten Melkstand und die Melkzeugzwischendesinfektion durch Tauchen geeignete Maßnahmen zur Unterbindung der Erregerverbreitung von Euterinfektionen mit Streptococcus agalactiae dar.

Einleitung: Euterinfektionen sowohl mit kuh-, vor allem mit umweltassoziierten Streptokokken nehmen bei Milchkuhherden eine dominierende Rolle ein. Ziel der Untersuchungen: Die Beziehungen zwischen der Innerherdenprävalenz (IHP) von subklinischen Mastitiden mit Streptokokken zur Haltungshygiene und zur Melkhygiene wurden erfasst. Dabei standen laktierende Kühe mit Nachweis von äskulinpositiven Streptokokken und Streptococcus agalactiae in Viertelanfangs-gemelksproben im Mittelpunkt. Tiere, Material und Methoden: Von September 2009 bis Dezember 2010 fanden in 34 Thüringer Milchviehherden zweimalig im Abstand von etwa 100 Tagen bakteriologische Untersuchungen der Viertelanfangsgemelke von allen zu diesem Zeitpunkt laktierenden und klinisch eutergesund erscheinenden Kühen statt. Der Medianwert der Herdengröße der laktierenden Kühe betrug 246. Die bakteriologischen Untersuchungen von 81.567 Viertelanfangsgemelken erfolgten entsprechend den Leitlinien der Deutschen Veterinärmedizinischen Gesellschaft durch das Labor des Tiergesundheitsdienstes der Thüringer Tierseuchenkasse. Während des ersten Bestandsbesuchs wurden die Haltungsbedingungen sowie die Melktechnik und die Melkhygiene im Betrieb mittels eines dreiteiligen, strukturierten Fragebogens erfasst. Die Bewertung der Merkmale erfolgte durch die Einteilung in zwei oder drei vorgegebene Kategorien. Mittels multivariabler Varianzanalyse wurde anschließend geprüft, ob eine Beziehung zwischen der IHP intramammärer Infektionen mit Streptococcus agalactiae bzw. äskulinpositiven Streptokokken und dem jeweiligen Risikofaktor besteht. Ergebnisse: Infektionen mit äskulinpositiven Streptokokken traten in allen Herden auf, während Galtstreptokokken in nur sieben der 34 Herden nachgewiesen werden konnten. Der Median der IHP der Infektionen mit äskulinpositiven Streptokokken lag zur Untersuchung 1 bei 8,5 %, zur Untersuchung 2 bei 4,6 %, für Infektionen mit Streptococcus agalactiae bei 4,9 % bzw. 3,1 %. Signifikante Beziehungen zur IHP von subklinischen Mastitiden mit äskulinpositiven Streptokokken oder Streptococcus agalactiae finden sich für die Sauberkeit der Liegeflächen im Bereich der Trockensteher und der kalbenden Kühe, für die Art der Laufflächen im Bereich der Trockensteher und der milchliefernden Kühe und für die regelmäßige Nutzung der Handwaschgelegenheiten durch das Melkpersonal. Für Galt-Nachweise zeigten sich signifikante Beziehungen und niedrige IHP im Zusammenhang mit der Verwendung einer Rohrmelkanlage für die Frischmelker, guter Sauberkeit der Euter der zum Melken kommenden Kühe und der Verwendung einer Tauchdesinfektion als Melkzeugzwischendesinfektion. Für subklinische Mastitiden mit äskulinpositiven Streptokokken gilt dies für Herden, in denen die Kühe der Leistungsherde mit kaum verschmutzten Eutern zum Melken kommen und stark verschmutzte Euter in der Leistungsherde vor dem Melken intensiv gereinigt werden. Schlussfolgerungen: Die Häufigkeit der Infektionen mit äskulinpositiven Streptokokken steht mit der Sauberkeit der Euter in Beziehung. Das Bereitstellen von sauberen und trockenen Liegeflächen und Laufgängen für alle Kühe in der Herde, auch für die trockenstehenden Tiere, trägt zu einer Senkung der IHP intramammärer Infektionen mit umweltassoziierten Streptokokken bei. Dabei geht es um die Vermeidung von Verschmutzungen der Euter. Weiterhin stellen das Melken der frischmelkenden Kühe in einem separaten Melkstand und die Melkzeugzwischendesinfektion durch Tauchen geeignete Maßnahmen zur Unterbindung der Erregerverbreitung von Euterinfektionen mit Streptococcus agalactiae dar.

Alle laktierenden Ziegen aus 12 hessischen Milchziegen-Betriebe wurden über einen Zeitraum von 2 Jahren beprobt. 83,6 % der 2038 Hälftegemelksproben waren kulturell negativ. 10,7 % der bakteriologisch positiven Proben waren Koagulase-negative Staphylokokken. Zur KNS-Differenzierung wurden Kulturmorphologie, ID32 Staph-Test, in-vitro-Sensitivität gegenüber Antibiotika und die t-DNA-PCR angewandt. Keines dieser Verfahren konnte alleinig zur Identifizierung der KNS-Isolate erfolgreich angewandt werden, nur die Kombination der Verfahren war zielführend. Nachgewiesen wurden die Spezies S. caprae, S. epidermidis, S. simulans, S. hyicus, S. saprophyticus, S. chromogenes, S. lentus, S. xylosus und S. hominis. Weiterhin wurde nach Methoden gesucht, um die subklinische Mastitis der Ziege diagnostizieren zu können. Hier erscheint der Vergleich der Zellzahl beider Euterhälften einer Ziege geeignet, um eine subklinische Mastitis abgrenzen zu können. Aufgrund der erhöhten Zellzahl, der Erregernachweis in Reinkultur sowie des Vergleichs mit der kultur-bakteriologisch negativen Euterhälfte erscheint die ätiologische Bedeutung der nachgewiesenen KNS-Isolate als Mastitiserreger der Ziege wahrscheinlich. / All lactating goats out of 12 hessian Dairy-goat-farms were being tested over a period of 2 years. 83,6 % of 2038 half-milk-samples were bacteriological negative. 10,7 % of the bacteriological positive samples were Coagulase-negative staphylococci. For KNS-differentiation morphology of culture, ID 32 Staph Test, in-vitro-sensitivity against antibiotics and t-DNA-PCR were evaluated. None of these methods could be used alone to identify the CNS isolates, the combination of the methods led to results. The species S. caprae, S. epidermidis, S. simulans, S. hyicus, S. saprophyticus, S. chromogenes, S. lentus, S. xylosus and S. hominis were found. Further it was searched for methods to diagnose subclinical mastitis of goats. The comparison of cell amount of both udder halfs of a goat seems to be adequate to diagnose subclinical mastitis. Because of increased cell amount, proof of agent in pure culture and comparison with the bacteriological negative udder half the etiological impact of the detected CNS-Isolates as causative agents of goat-mastitis is likely.

La litière de fumier recyclé (LFR) est utilisée dans les fermes laitières québécoises depuis quelques années, et ce, malgré le manque de connaissances scientifiques quant aux risques reliés à l’utilisation de ce produit pour la santé des animaux. Le premier objectif de cette thèse était de décrire les méthodes de production de LFR dans un contexte québécois et les pratiques de régie associées. Le deuxième objectif du projet consistait à déterminer le potentiel de recroissance de certaines espèces bactériennes dans la LFR lorsqu’elle est contaminée. Par la suite, le niveau de propreté et la prévalence de lésions aux jarrets chez les vaches laitières exposées ou non à ce produit ont été estimés. Finalement, l’association entre l’utilisation de LFR et l’incidence de mammite sous-clinique et clinique a été évaluée. Des études observationnelles cohorte ou transversale (selon l’objectif de recherche) ont été réalisées en 2018-2019 sur 27 fermes utilisant de la LFR et 61 fermes utilisant de la litière de paille, à titre comparatif. Les visites de fermes ont permis de constater que les méthodes utilisées pour produire de la LFR n’étaient pas standardisées et qu’elles ne permettaient pas un réel compostage de la fraction solide du fumier. Les essais de recroissance ont permis de démontrer que les différentes LFR (traitée en tas, contenant fermé ou cuve rotative) ne réagissaient pas de la même façon à une inoculation par des coliformes. La LFR de cuve rotative contenait une plus faible concentration initiale de Klebsiella spp. et a montré une croissance bactérienne significative dans les premières 24 h post-inoculation. Les LFR conditionnées dans des tas ou contenants avaient quant à elles une concentration initiale importante de Klebsiella spp. et n’ont pas démontré de croissance bactérienne significative suivant leur inoculation, ce qui suggère un effet de saturation de croissance bactérienne. Au cours de l’étude transversale, 30 vaches par troupeau, en moyenne, ont été notées à l’aide d’un score de propreté sur trois zones du corps ainsi que d’un score sur l’état des jarrets, afin d’estimer leur propreté et la présence de lésions aux jarrets. Le score de propreté attribué à chaque zone du corps des vaches allait de 1 à 4, 1 étant très propre et 4 très sale. Nous avons observé que les vaches logées sur LFR avaient, en général, une meilleure propreté du pis et du bas des pattes que celles logées sur paille. La LFR avait un effet protecteur pour le risque d’avoir un score de propreté du pis ≥ 3 (rapport de cotes (RC) : 0,43) ou d’avoir un score de 4 (RC : 0,29). Les vaches logées sur LFR avaient aussi le bas des membres plus propres que celles logées sur paille avec de plus faibles cotes d’avoir un score ≥ 2 (RC : 0,45), un score ≥ 3 (RC : 0,16) ou un score de 4 (RC : 0,07). Cependant, nous n’avons pas trouvé de différence entre les deux groupes d’animaux au niveau de la propreté du flanc et du haut des pattes. Lors de leur évaluation pour la propreté, les vaches ont aussi été notées à l’aide d’un score de lésions aux jarrets allant de 0 à 3. Un score de 0 représentait un jarret parfaitement sain alors qu’un score de 3 était attribué à un jarret présentant une enflure de plus de 2,5 cm. Les deux jarrets étaient évalués et le jarret ayant reçu le score le plus élevé était inclus dans les analyses. Nous n’avons pas identifié de différence quant aux scores de jarret entre les deux groupes d’animaux. Afin d’évaluer la santé de la glande mammaire, une étude cohorte d’une durée d’un an a été mise en place à partir de la visite de la ferme. Les dynamiques de comptages de cellules somatiques ont été suivies sur 11 031 vaches durant cette période afin d’analyser l’incidence de mammite sous-clinique. Nous n’avons pas été en mesure de détecter une différence d’incidence entre les deux groupes d’animaux. Au cours de l’étude cohorte, les producteurs laitiers ont identifié et fait parvenir au laboratoire 1 144 échantillons de lait provenant de vaches atteintes de mammites cliniques. L’incidence totale de mammite clinique n’était pas plus élevée dans les fermes LFR qu’au sein des fermes paille. Cependant, lorsque nous avons analysé l’incidence de mammite clinique par agent pathogène spécifique, nous avons pu constater que les vaches logées sur LFR étaient 7,0 fois plus à risque d’expérimenter une mammite clinique causée par Klebsiella pneumoniae que celles du groupe comparatif. / Recycled manure solids (RMS) bedding has been used on Quebec dairy farms for a number of years, despite the lack of scientific knowledge about the health risks associated with the use of this product for animals. The first objective of this thesis was to describe the RMS production methods in a Quebec context and the associated management practices. The second objective of the project was to determine the potential for regrowth of certain bacterial species in the RMS when it is contaminated. Subsequently, the level of cleanliness and prevalence of hock lesions in dairy cows exposed or not to this product were estimated. Finally, the association between RMS use and the incidence of subclinical and clinical mastitis was evaluated. Observational cohort or cross-sectional studies (depending on the research objective) were conducted in 2018-2019 on 27 farms using RMS and 61 farms using straw bedding for comparison. The farm visits highlighted that the methods used to produce RMS were not standardized and did not allow for true composting of the manure solid fraction. The regrowth trials showed that different RMS (treated in heap, closed container or rotating drum) did not react in the same way to coliform inoculation. The rotating drum RMS contained a lower initial concentration of Klebsiella spp. and experienced significant bacterial growth in the first 24 hours post-inoculation. The heap or closed container RMS had a high initial concentration of Klebsiella spp. and did not show significant bacterial growth following inoculation, suggesting a saturation effect on bacterial growth. In the cross-sectional study, an average of 30 cows were measured using a cleanliness score on three body areas as well as a hock lesion score to estimate their cleanliness and the presence of hock lesions. The cleanliness score assigned to each area of the cows' body ranged from 1 to 4, with 1 being very clean and 4 being very dirty. We found that cows housed on RMS generally had better udder and lower leg cleanliness than those housed on straw. Recycled manure solids bedding had a protective effect for the risk of having an udder cleanliness score ≥ 3 (odds ratio (OR): 0,43) or having a score of 4 (OR: 0,29). Cows housed on RMS also had cleaner lower legs than those housed on straw with lower odds of having a score ≥ 2 (OR: 0,45), a score ≥ 3 (OR: 0,16) or a score of 4 (OR: 0,07). However, we found no difference between the two groups of animals in flank and upper leg cleanliness. When they were evaluated for cleanliness, cows were also measured using a hock lesion score ranging from 0 to 3. A score of 0 represented a perfectly healthy hock, while a score of 3 was assigned to a hock with swelling greater than 2.5 cm. Both hocks were scored and the hock with the higher score was included in the analyses. We did not identify any difference regarding hock lesions between the two groups of animals. To assess mammary gland health, a one-year cohort study was set up from the farm visit. Somatic cell count dynamics were followed on 11 031 cows during this period to analyze the incidence of subclinical mastitis. We were not able to detect a difference in incidence between the two groups of animals. During the cohort study, dairy farmers identified and sent to the laboratory 1 144 milk samples from cows with clinical mastitis. The total incidence of clinical mastitis was not higher on RMS farms than on straw farms. However, when we analyzed the incidence of clinical mastitis by specific pathogen, we found that cows housed on RMS were 7.0 times more likely to experience clinical mastitis caused by Klebsiella pneumoniae than those in the comparison group.