A.M.M.P. Della Libera

Function of milk polymorphonuclear neutrophil leukocytes in bovine mammary glands infected with Corynebacterium bovis

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.

Dinâmica da celularidade do colostro de vacas da raça Holandesa no pós-parto imediato

Avaliou-se a celularidade do colostro de 53 vacas da raca Holandesa, utilizando-se 171 amostras, colhidas apos a primeira e a segunda ordenha pos-parto. Para a analise citologica quantitativa e qualitativa, foram utilizadas as tecnicas de microscopia direta e citocentrifugacao. O total de leucocitos obtido por contagem microscopica direta foi de 0,878x106 e 1,260x106, antes da primeira e da segunda ordenha, respectivamente, e os valores da mediana dos fagocitos obtidos antes da primeira e da segunda ordenha foram de 0,657 e 0,828x106 leucocitos mononucleares, e 0,178 e 0,392x106 leucocitos polimorfonucleares (P<0,0001). Os valores da mediana dos tipos leucocitarios, antes da primeira e da segunda ordenha, foram: 0,640x106 e 0,772 monocitos/celulas epiteliais; 0,136x106 e 0,098 linfocitos; 0,045x106 e 0,203x106 neutrofilos, respectivamente. Concluiu-se que o colostro bovino da segunda ordenha apresentou aumento da quantidade de celulas somaticas e leucocitos polimorfonucleares/mL de colostro. Essas variacoes podem estar relacionadas a importância do colostro aos neonatos, ou a modificacoes fisiologicas e de defesa da glândula mamaria, durante o periodo de adaptacao dela.

Lactation stage and udder health status of Santa Ines ewes

Avaliou-se o risco de infeccao em diferentes fases da lactacao em 33 ovelhas da raca Santa Ines. Inicialmente a glândula foi submetida ao exame fisico e a prova de fundo escuro. Posteriormente, amostras de leite foram coletadas assepticamente para a realizacao de exame bacteriologico - California Mastitis teste -, e contagens microscopica e automatica de celulas somaticas. Nenhuma diferenca foi observada entre as distintas fases de lactacao. Observou-se alta persistencia de infeccoes intramamarias, e tendencia a maior contagem de celulas somaticas no ultimo periodo de lactacao, que pode ser oriunda da maior resistencia a infeccoes neste periodo.

Interaction between bovine-associated coagulase-negative staphylococci species and strains and bovine mammary epithelial cells reflects differences in ecology and epidemiological behavior

Bacteria adherence seems to be an essential first stage for the internalization of bacteria into the cytoplasm of the host cell, which is considered an important virulence strategy enabling bacteria to occupy a microenvironment separated from host defense mechanisms. Thus, this study aimed to explore the difference in the capacity of 4 bovine-associated staphylococci species or strains to adhere to and internalize into bovine mammary epithelial cells (MEC). Three different isolates of coagulase-negative staphylococci (CNS) were used: one strain of Staphylococcus fleurettii isolated from sawdust and considered an environmental opportunistic bacterium, and 2 dissimilar Staphylococcus chromogenes isolates, one cultured from a heifers teat apex (Staph. chromogenes TA) and the other originating from a chronic intramammary infection (Staph. chromogenes IM). Also, one well-characterized strain of Staphylococcus aureus (Newbould 305) was used for comparison with a major mastitis pathogen. The CNS species and strains adhered to and internalized into MEC slower than did Staph. aureus. Still, we observed high variation in adhesion and internalization capacity among the different CNS, with Staph. chromogenes IM showing a greater ability to adhere to and internalize into MEC than the 2 CNS strains isolated from extramammary habitats. In conclusion, the 3 well-characterized bovine-associated CNS species and strains originating from distinct habitats showed clear differences in their capacity to adhere to and internalize into MEC. The observed differences might be related to their diversity in ecology and epidemiological behavior.

Antimicrobial susceptibility of coagulase-negative staphylococci isolated from meat-producing ewes with mastitis

Avaliou-se a sensibilidade antimicrobiana in vitro de 121 cepas de estafilococos coagulase-negativa isolada de leite de ovelhas Santa Ines, aos farmacos: penicilina, amoxicilina, ampicilina, estreptomicina, oxaciclina, neomicina, cefalotina, gentamicina e sulfonamida. A resistencia a sulfonamida foi a mais frequente (27,3%), seguida pela estreptomicina (14,0%) e pela oxaciclina (14,0%), enquanto da gentamicina (1,6%) foi a menos frequente. Todas as cepas foram sensiveis a pelo menos um antimicrobiano, e 20,3% das cepas apresentaram resistencia multipla. Os resultados mostram a importância de Staphylococci coagulase-negativas como agentes causadores de mastite em ovinos, e o perfil de resistencia multipla indica a importância da determinacao da resistencia a oxaciclina como indicador da presenca de ilhas de patogenicidade que contem fatores de virulencia e resistencia a outros antimicrobianos que contribuem para a sobrevivencia da bacteria ao tratamento.

Flow cytometric analysis: Interdependence of healthy and infected udder quarters

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.

Proliferação de linfócitos e apoptose de células CD5 + de bovinos infectados pelo vírus da leucose enzoótica bovina

The purpose of the present trail was to evaluate the lymphocyte proliferation and the apoptosis rates of CD5+ cells in dairy cows infected with bovine leukemia virus (BLV) with distinct lymphocyte profiles in infected animals known as alymphocytotic (AL) and persistent lymphocytosis (PL). A total of 100 Holstein cows were sera tested for bovine leukemia virus through agar gel immunodiffusion (AGID) and enzyme-linked immunosorbent-assay (ELISA). From these animals, 15 cows were selected and divided uniformly in 3 groups (negative, AL, LP). The lymphocyte proliferation was performed using flow cytometric measurement of CFSE-DA dye, where 2x106/mL lymphocytes were plated per well. The apoptosis of CD5+ cells from peripheral blood was performed using the annexin V-FITC to measure the apoptosis rates and the identification of CD5+ was accessed using monoclonal antibodies. Animals from the LP group showed lower lymphocyte proliferation and also lower apoptosis rates of CD5+ cells compared with negative and AL animals. The development of PL which resulted from an increase in B cell count, is due to the decrease in the apoptosis rates of CD5+ cells, and the higher lymphocyte proliferation appears to be limited only in the initial stages of development of LP.

Avaliação funcional de monócitos de bovinos naturalmente infectados pelo vírus da leucose bovina

Assuming that the bovine leukosis virus (BLV) alters quantitatively and qualitatively bovine circulating leukocyte subpopulations, thus influencing the innate immune response, monocytes function in BLV-infected cattle was assessed. Peripheral blood was obtained from 10 BLV-negative cows (SN), 10 naturally BLV-infected, non-lymphocytotic cows (AL), and 10 BLV-infected cows with persistent lymphocytosis (PL). Monocytes were isolated by density gradient and adherence to plates. Cells were submitted to Trypan Blue dye exclusion viability assay, phagocytosis of Zymosan and cell-spreading assays, and quantification of hydrogen peroxide (H2O2) and nitric oxide (NO) production. Monocytes from cattle with PL had the lowest viability (P<0.001), phagocytosis of Zymosan particles (P<0.001), and spreading (P=0.006) rates. Additionally, monocytes from cows with PL had the highest production of H2O2 , with no prior stimulus (P=0.001), and after in vitro stimulus with phorbol 12-myristate 13-acetate (P=0.006). Nonetheless, the boost in H 2 O 2 production, provided by in vitro stimulus, observed in monocytes from cows with PL was lower (P=0.015) than that observed in monocytes from SN and AL cattle. There was no difference in NO production among groups. Results show that BLV, despite infecting B lymphocytes, alters innate immune functions of monocytes isolated from BLV-infected cows expressing PL.

Evaluation of alveolar macrophage function after experimental infection with equine herpesvirus-1 in horses

O papel dos macrofagos alveolares (MA) nos mecanismos de defesa pulmonar foi estudado em cavalos infectados pelo herpesvirus equino tipo 1 (EHV-1). Cinco cavalos adultos foram inoculados com 106,6 TCID50 do EHV-1, por instilacao intranasal. A citologia do lavado broncoalveolar (LBA) foi feita usando-se citocentrifugacao das amostras e confeccao de lâminas coradas por Rosenfeld. A concentracao celular foi ajustada para 2´106 celulas/ml, para mensuracao da atividade macrofagica - espraiamento, fagocitose de particulas de zymosan e liberacao de peroxido de hidrogenio (H2O2). Observou-se soroconversao no 14o dia pos-inoculacao (DPI) e isolamento viral positivo no segundo, terceiro e quinto DPI. Na citologia do LBA observou-se linfocitose no 16o DPI. Os testes de mensuracao da atividade macrofagica demonstraram aumento significativo nos indices de espraiamento no 23o e 30o DPI e diminuicao no indice de fagocitose no segundo DPI, retornando a valores semelhantes aos observados antes da inoculacao a partir do 23o DPI. Houve diminuicao na liberacao de H2O2 no segundo DPI, e no 16o DPI esses valores retornaram aos observados antes da inoculacao. O declinio da atividade fagocitica dos MA, na fase aguda da infeccao, indicou que essas celulas atuam de forma importante nos mecanismos de defesa pulmonar contra esse agente.

Correlação entre a contagem automática de células somáticas e a porcentagem de neutrófilos pela citometria de fluxo e pela técnica de citocentrifugação

The purpose of the present study was to assess the correlation between the automatic somatic cell count (SCC) and the percentage of neutrophils through cytocentrifugation technique and flow cytometry. Thus, 102 milk samples from 28 Holstein dairy cows were collected and submitted to milk cell isolation procedures, and afterwards, the neutrophils were identified. After cytocentrigugation, the neutrophils were microscopically identified using the rosenfeld dye. The milk neutrophils were recognized by flow cytometry using primary mouse IgM monoclonal antibody (CH138A) and phycoerytrin (PE) goat anti-mouse IgM antibody. This study found a positive correlation between SCC and the percentage of neutrophils through cytocentrifugation (r= 0.267) and flow cytometry (r= 0.625). A positive correlation was also encountered between the percentage of neutrophils through cytocentrifugation and flow cytometry (r= 0.496), although the percentage of neutrophils was higher in samples submitted to cytocentrifugation. In conclusion, flow cytometry can be a useful tool in the diagnosis and control of mastitis.