Corynebacterium bovis is one of the most commonly isolated bacteria from aseptically collected bovine milk samples. The objective of the current study was to characterize the bovine innate immune response by evaluating milk polymorphonuclear neutrophilic leukocytes (PMNL) in mammary glands infected with C. bovis. Twenty quarters infected with C. bovis and 28 culture-negative quarters (with milk somatic cell count <1×10(5) cells/mL) were used. The percentages of milk PMNL and the PMNL expression of L-selectin (CD62L), β2-integrin (CD11b), and one of the endothelial-selectin ligands (CD44), as well as the levels of intracellular reactive oxygen species (ROS) and the phagocytosis of Staphylococcus aureus, were evaluated by flow cytometry. The apoptosis and necrosis rates of the PMNL were quantified using dual-color flow cytometry with fluorescein-labeled annexin and propidium iodide. The present study revealed a higher percentage of PMNL in the milk from C. bovis-infected quarters, although no significant differences were found in levels of CD44, CD62L, or CD11b expression among the PMNL. A lower percentage of apoptotic PMNL was observed in C. bovis-infected quarters, as well as higher percentages of viable PMNL and of PMNL that produced intracellular ROS. However, no alterations were observed in phagocytosis of Staph. aureus by the PMNL or in intensity of intracellular ROS production by PMNL. Thus, results from this investigation of the PMNL function support, at least in part, the fact that intramammary infections by C. bovis may offer protection against intramammary infections by other bacteria.

The effects of bovine leukemia virus (BLV) on the immune response have been extensively investigated; however, its effects on mammary gland immunity are only speculative. Although BLV has a tropism for B cells, it can affect both adaptive and innate immunities because these systems share many effector mechanisms. This scenario is the basis of this investigation of the effects of BLV on mammary gland immunity, which is largely dependent upon neutrophilic functions. Thus, the present study sought to examine neutrophilic functions and the lymphocyte profile in the milk of naturally BLV-infected cows. The viability of the milk neutrophils and the percentage of milk neutrophils that produced reactive oxygen species (ROS) or phagocytosed Staphylococcus aureus were similar between BLV-infected and BLV-uninfected dairy cows. Furthermore, the expression of CD62L and CD11b by the milk neutrophils and the percentage of milk neutrophils (CH138+ cells) that were obtained from the udder quarters of the BLV-infected cows were not altered. Conversely, the median fluorescence intensity (MFI) representing intracellular ROS production and the phagocytosis of S. aureus, the expression of CD44 by the milk neutrophils and the percentage of apoptotic B cells were lower in the milk cells from BLV-infected dairy cows, particularly those from animals with persistent lymphocytosis (PL). The lymphocyte subsets were not different among the groups, with the exception of the percentage of CD5−/CD11b− B cells, which was higher in the milk cells from BLV-infected cows, particularly those with PL. Thus, the present study provides novel insight into the implications of BLV infection for mammary gland immunity.

Streptococcus dysgalactiae is a bacterium that accounts for a notable proportion of both clinical and subclinical intramammary infections (IMIs). Thus, the present study explores the function of milk neutrophils and the lymphocyte profile in mammary glands naturally infected with Streptococcus dysgalactiae. Here, we used 32 culture-negative control quarters from eight clinically healthy dairy cows with low somatic cell counts and 13 S. dysgalactiae-infected quarters from six dairy cows. Using flow cytometry, we evaluated the percentage of milk monocytes/macrophages and neutrophils, expression of CD62L, CD11b and CD44 by milk neutrophils, the levels of intracellular reactive oxygen species (ROS) production and phagocytosis of Staphylococcus aureus by milk neutrophils, and neutrophil viability. Furthermore, the percentages of B cell (CD21(+)) and T lymphocyte subsets (CD3(+)/CD4(+)/CD8(-); CD3(+)/CD8(+)/CD4(-); and CD3(+)/CD8(-)/CD4(-)), and the expression of CD25 by T milk lymphocytes (CD3(+)) and T CD4(+) milk cells were also assessed by flow cytometry using monoclonal antibodies. The present study showed a higher SCC and percentage of milk neutrophils, and a decrease in the percentage of milk monocytes/macrophages from S. dysgalactiae-infected quarters when compared to uninfected ones. We also observed a higher expression of CD11b by milk neutrophils and a tendency toward a decrease in neutrophil apoptosis rate in S. dysgalactiae-infected quarters. In addition, the S. dysgalactiae-infected quarters had higher percentages of milk T cells (CD3(+)) and their subset CD3(+)CD8(+)CD4(-) cells. Overall, the present study provided new insights into S. dysgalactiae IMIs, including distinct lymphocyte profiles, and a tendency toward an inhibition of apoptosis in milk neutrophils.

No abstract

An important question about intramammary infections that is still debated in the literature is the independence or interdependence of the quarters of dairy cows. The present study sought to explore milk neutrophil function and the milk lymphocyte profile of uninfected quarters from uninfected and infected (one infected quarter per cow) udders to evaluate interdependence of the quarters. Thus, 32 (8 cows) and 18 (6 cows) uninfected quarters from uninfected and infected udders were used, respectively. Using flow cytometry, we evaluated the percentage of milk neutrophils and their expression of adhesion molecules L-selectin (CD62L), β2-integrin (CD11b), and an endothelial-selectin ligand (CD44); levels of intracellular reactive oxygen species (ROS); phagocytosis of Staphylococcus aureus by milk neutrophils; and neutrophil viability. Furthermore, we assessed the percentage of B-cell (CD21(+)) and T-lymphocyte subsets (CD3(+)/CD4(+)/CD8(-), CD3(+)/CD8(+)/CD4(-), CD3(+)/CD4(+)/CD25(-), CD3(+)/CD4(+)/CD25(+), and CD3(+)/CD4(-)/CD25(-)) using flow cytometry with monoclonal antibodies. The infected quarter did not affect somatic cell count or the percentage of neutrophils in the neighboring uninfected quarters. Furthermore, the infected quarter did not influence neutrophil viability, intracellular reactive oxygen species production, or phagocytosis of S. aureus by milk neutrophils. Conversely, the expression of adhesion molecules CD11b, CD62L, and CD44 by milk neutrophils differed between uninfected quarters from infected versus uninfected udders. The lymphocyte subsets did not differ between groups, except for a higher percentage of B cells in uninfected quarters from infected udders than in those from uninfected udders. Thus, our study strongly supports the hypothesis of interdependence of quarters based on the influence of infection on both the percentage of B cells and the expression of adhesion molecules by milk neutrophils in the neighboring uninfected quarters.

RESUMO.-[Expressão de CD14 e dos receptores do tipo toll 2 e 4 por neutrófilos lácteos provenientes de glândulas infectadas por Corynebacterium bovis.] O presente estudo objetivou avaliar alterações na expressão de CD14, e dos receptores do tipo toll (TLR) 2 e 4 na superfície de neutrófilos lácteos provenientes de glândulas mamárias infectadas por Corynebacterium bovis. O presente estudo utilizou 23 quartos negativos no exame bacteriológico, sem alterações na prova de fundo escuro e com contagem automática de células somáticas menor que 1 x10 5 células/mL, e 14 quartos mamários infectados por C. bovis A identificação de neutrófilos, assim como a porcentagem de neutrófilos lácteos que expressaram CD14, TLR2 e 4 foram avaliadas por citometria de fluxo utilizando anticorpos monoclonais. A porcentagem de neutrófilos que expressaram TLR2 e TLR4 nos quartos mamários infectados por C. bovis não diferiu dos quartos mamários sadios, assim como na expressão de TLR4. No entanto, a intensidade de fluorescência do TLR2 na superfície dos neutrófilos foi menor nos quartos mamários infectados por C. bovis. A porcentagem de neutrófilos que expressaram CD14 e a intensidade de fluorescência da molécula de CD14 foi menor na superfície dos neutrófilos lácteos dos quartos infectados por C. bovis. This study evaluated the expression of CD14, toll-like receptor (TLR) 2 and TLR4 on the surface of milk neutrophils in bovine mammary glands infected with Corynebacterium bovis. Here, we used 23 culture-negative control quarters with no abnormal secretion on the strip cup test and milk somatic cell count lower than 1x10 5 cells/mL, and 14 C. bovis infected quarters. The identification of neutrophils, as well as, the percentage of neutrophils that expressed CD14, TLR2 and TLR4 were analyzed by flow cytometry using monoclonal antibodies. The present study encountered no significant difference in the percentages of milk neutrophils that expressed TLR2 and TLR4 or in the expression of TLR4 by milk neutrophils. Conversely, a lower median fluorescence intensity of TLR2 in milk neutrophils was observed in C. bovis-infected quarters. The percentage of neutrophils that expressed CD14 and the median fluorescence intensity of CD14 in milk neutrophils was also lower in C. bovis-infected quarters.INDEX TERMS: Immune response, pattern recognition receptor, mastitis, milk, polymorphonuclear neutrophil leukocyte.

Introdução: A Síndrome Pré-menstrual (SPM), com início de uma a duas semanas antes da menstruação, engloba um padrão recorrente de sintomas físicos, afetivos e comportamentais, podendo ser agravados por fatores externos, como hábitos de vida e condição socioeconômica, afetando negativamente as atividades diárias. Objetivo: Analisar a relação entre a síndrome pré-menstrual e a possibilidade de reabilitação. Método: Revisão integrativa da literatura realizada através da leitura de portais oficiais de órgãos públicos e da busca eletrônica na Biblioteca Virtual em Saúde (BVS), no PubMed e na Scientific Electronic Library Online (SciELO). Resultados: Nessa revisão integrativa foram selecionados 41 estudos primários publicados nos últimos 5 anos, disponíveis gratuitamente e nos idiomas inglês e português. Discussão: Com relação às técnicas não farmacológicas, observou-se que os métodos de exercício físico, dieta, medicina tradicional iraniana, suplementação de vitamina D e terapia cognitivo-comportamental obtiveram boa eficácia na atenuação dos sintomas da SPM, como dismenorreia e alterações de humor; o tratamento com auriculoterapia mostrou resultados semelhantes ao uso de placebo; outros como acupuntura e acupressão não trouxeram resultados conclusivos.  Entre os métodos farmacológicos, o tratamento padrão preconizado é por meio de anticoncepcionais orais combinados. Além disso, o uso de inibidores seletivos da recaptação da serotonina (ISRSs) pode aumentar a quantidade de serotonina para neurotransmissão e consequentemente melhorar os sintomas da SPM. Os moduladores seletivos do receptor de estrogênio oferecem uma estratégia segura para manejo de sintomas, inclusive os de menopausa. O uso de moduladores do receptor de progesterona, por outro lado, mostrou maior controle de comportamentos agressivos. Por fim, um estudo multicêntrico realizado na Suécia mostrou resultado promissor do neuroesteroide endógeno em comparação ao uso de anticoncepcionais orais combinados. Conclusão: Há fortes indícios de melhora dos sintomas da SPM diante das diversas possibilidades para sua reabilitação, sejam elas farmacológicas ou não-farmacológicas.