ABSTRACT The present study explored the predictive values of milk leukocyte differentials (MLD) as a basis for improving the diagnosis of intramammary infections (IMIs) and subclinical mastitis. Quarter milk samples were collected for bacteriological analysis, quarter somatic cell count (qSCC), and MLD. The MLD were assessed using the cytospin technique, direct microscopic smears, and flow cytometry. The predictive values of each single leukocyte population and useful potential indices that could better reflect immune complexity were also calculated. Changes in the percentage of any leukocyte alone failed to substantially improve the predictive value of qSCC in diagnosing IMIs. Although certain parameters increased the area under the receiver operating characteristic curve (ROC curve) as a result of increased specificity values, a slight reduction in sensitivity was observed. The so-called CD8 complex was a unique parameter which improved both the sensitivity (78.79 %) and the specificity (80.77 %) in IMI diagnosis, resulting in the highest area under the ROC curve (0.87). To diagnose subclinical mastitis, the percentage of macrophages and the sum of the percentage PMNLs and T CD8+ cells divided by the percentage of macrophages showed the highest predictive values (sensitivity = 79.63, specificity = 73.68, and area under the ROC curve = 0.83) in the differentiation of the inflammatory condition status of cows. In conclusion, this study provides further insights into using T CD8+ lymphocytes in diagnosing bovine IMIs, combined with PMNLs and macrophages. The antidromic trend of macrophages vs. PMNLs and T CD8+ lymphocytes due to the increasing qSCCs was crucial to differentiating quarters under both inflammatory and non-inflammatory conditions.

Abstract Bovine infectious mastitis is largely resistant to antibacterial treatment, mainly due to mechanisms of bacterial resistance in the biofilms formed by Staphylococcus aureus. Melaleuca (MEO) and citronella essential oils (CEO) are promising agents for reducing or eliminating biofilms. Free melaleuca oil presented a medium Minimum Inhibitory Concentration (MIC) of 0.625% and a Minimum Bactericidal Concentration (MBC) of 1.250%, while free citronella oil showed medium MIC and MBC of 0.313%. Thus, free CEO and MEO demonstrate bacteriostatic and bactericidal potential. We generated polymeric nanocapsules containing MEO or CEO and evaluated their efficacy at reducing biofilms formed by S. aureus. Glass and polypropylene spheres were used as test surfaces. To compare the responses of free and encapsulated oils, strains were submitted to 10 different procedures, using free and nanoencapsulated essential oils (EOs) in vitro. We observed no biofilm reduction by MEO, free or nanoencapsulated. However, CEO nanocapsules reduced biofilm formation on glass (p=0.03) and showed a tendency to diminish biofilms on polypropylene (p=0.051). Despite nanoencapsulated CEO reducing biofilms in vitro, the formulation could be improved to modify the CEO component polarity and, including MEO, to obtain more interactions with surfaces and the biofilm matrix.

Resumo A ocorrência de células viáveis em secreções das glândulas mamárias atestam a funcionalidade do sistema de defesa. Este fato, juntamente com o medicamento aplicado, é fundamental para a cura do úbere. O objetivo deste trabalho foi avaliar a viabilidade in vitro de leucócitos do leite desafiados com quatro drogas anti-mastite intramamárias. Foram coletadas oito amostras de leite de vaca negativas no exame bacteriológico. As suspensões de células foram submetidas à avaliação da viabilidade pelo método de exclusão do Azul de Trypan pré-incubação e pós-incubação com os medicamentos. A taxa média de viabilidade pré-incubação (± desvio-padrão) das células do leite foi 50,17% (± 6,50). As taxas médias de viabilidade pós-incubação (± desvio-padrão) de grupo controle (desprovido de qualquer droga), Drogas 1 (cefalônio anidro), 2 (gentamicina), 3 (benzilpenicilina procaína associada a diidroestreptomicina naficilina) e 4 (cloxacilina benzatina) foram, respectivamente, 31,36% (± 3,61); 26.23% (± 4,49); 7,82% (± 2,33); 3,10% (± 0,94); e 30,33% (± 4,14). Os maiores prejuízos foram encontrados quando as células do leite foram desafiadas com as drogas 2 e 3. Embora a atividade antimicrobiana das drogas testadas não tenha sido avaliada, e um único aspecto da função das células tenha sido testado, concluiu-se que os tratamentos 2 e 3 poderiam prejudicar a funcionalidade de leucócitos do leite e fornecer uma resposta imunológica ineficaz, e uma resposta eficaz é indispensável para controlar as infecções mamárias.

Abstract The occurrence of viable cells in mammary gland secretions attests the functionality of the defense system. This fact, along with applied medicine, is fundamental for the healing udder. The objective of this study was to evaluate the in vitro viability of milk leukocytes challenged with four intramammary anti-mastitis drugs. We collected eight cow milk samples that were negative after a bacteriological examination. Cell suspensions were submitted to an evaluation of viability through Trypan Blue exclusion before and after incubation with the drugs. Mean pre-incubation viability rate (± standard deviation) of milk cells was 50.17% (± 6.50). Mean post-incubation viability rates (± standard deviation) of control group (devoid of any drug), Drug 1 (anhydrous cephalonium), Drug 2 (gentamicin), Drug 3 (procaine benzylpenicillin associated with dihydrostreptomycin nafcillin), and Drug 4 (benzathine cloxacillin) were, respectively, 31.36% (± 3.61); 26.23% (± 4.49); 7.82% (± 2.33); 3.10% (± 0.94); and 30.33% (± 4.14). The uppermost impairments were found when milk cells were challenged with Drug 2 and Drug 3. Although antimicrobial activity of tested drugs was not appraised, and a single aspect of cell function was tested, we concluded that treatments 2 and 3 could impair the functionality of milk leukocytes and could provide an ineffective immunological response, and an effective response is essential to control mammary infections.

O presente estudo objetivou avaliar a correlação entre a contagem de células somáticas (CCS) automática e a CCS microscópica direta por diferentes colorações no leite de ovelhas da raça Santa Inês. Avaliou-se a CCS automática por citometria de fluxo, e a CCS microscópica foi realizada por esfregaços lácteos corados com as colorações de Broadhurst-Paley (BP), Hematoxilina-eosina (HE) e Rosenfeld. Os valores logarítmicos da CCS microscópica pela coloração de BP e Rosenfeld foram superiores aos valores logarítmicos da CCS automática e a CCS microscópica pela coloração de HE (P<0,0001), e os valores logarítmicos da CCS pela coloração de HE foram inferiores aos valores logarítmicos da CCS automática (P<0,0001). As correlações entre os valores logarítmicos da CCS automática e os valores logarítmicos das CCS microscópica pelas colorações de HE, BP e Rosenfeld foram de 0,774, 0,803 e 0,859 (P<0,0001), respectivamente. Os valores logarítmicos reais da CCS microscópica e aqueles estimados pela CCS automática, considerando as equações das curvas por regressão quadrática, não foram estatisticamente diferentes pelas colorações de HE (P=0,90), BP (P=0,09) e Rosenfeld (P=0,23). Conclui-se que a metodologia influencia a CCS no leite de ovinos e propõe-se a utilização da equação da curva para estimação da CCS automática a partir dos valores da CCS microscópica, quando colorações não específicas para o ácido desoxirribonucleico são aplicadas para avaliação da celularidade do leite no diagnóstico da mastite em ovinos.

The present study was designed to assess the correlation among the automatic somatic cell count and the microscopic somatic cell count using the Broadhurst-Paley (BP), Hematoxilin-eosin (HE) and Rosenfeld dyes. The milk smears were stained with BP, HE and Rosenfeld and the automatic cell count was performed by flow cytometry. The mean logarithmic microscopic SCC by BP and Rosenfeld was higher than the values from automatic SCC and microscopic SCC by HE (P<0.0001). Indeed, the mean values from microscopic SCC using the HE was lower than the automatic cell count (P<0.0001). The correlation among the automatic cell count and the microscopic SCC using the HE, BP and Rosenfeld dyes were 0.774, 0.803 e 0.859 (P<0.0001), respectively. The automatic SCC values and the estimated automatic SCC applying the quadratic equation using the results of the microscopic SCC using the HE (P=0.90), BP (P=0.09) and Rosenfeld (P=0.23) dyes were not different. Thus, it can be concluded that the SCC was influenced by the methodology applied, and nonspecific stains used for microscopic SCC can be used to assess udder health in ewes if an equation is applied to estimate automatic SCC.

O objetivo do presente estudo foi avaliar a capacidade de liberação de peróxido de hidrogênio (H2O2) por fagócitos oriundos de glândulas mamárias bovinas sadias e infectadas. Desse modo, 73 amostras de leite provenientes das glândulas mamárias foram classificadas em sadias e infectadas de acordo com a cultura bacteriológica e a contagem de células somáticas (CCS). Após o isolamento das células do leite, procedeu-se à contagem diferencial de leucócitos e determinação da liberação de H2O2 pela oxidação da solução de vermelho fenol. Foi observada menor liberação de H2O2 pelos fagócitos oriundos dos quartos mamários infectados, assim como houve correlação negativa entre a liberação de H2O2 por fagócitos e a CCS (r=-0,34; P=0,0025), e a porcentagem de neutrófilos (r=-0,24; P=0,04). Além disso, houve tendência de menor liberação de H2O2 pelos fagócitos estimulados por forbol 12-miristato 13-acetato nas glândulas mamárias infectadas. Entretanto, observou-se maior liberação de H2O2 pelos fagócitos em 1mL de leite nos quartos mamários infectados, ao considerar a CCS mL-1. Pode-se concluir que fagócitos de quartos mamários infectados apresentaram menor liberação de H2O2, o que indica menor capacidade microbicida. Por outro lado, observou-se maior liberação de H2O2 pelos fagócitos em 1mL de leite nos quartos infectados, fato que pode contribuir com o maior recrutamento de leucócitos para a glândula mamária e/ou a persistência do processo inflamatório.

The purpose of the present study was to evaluate the hydrogen peroxide (H2O2) release by phagocytes from infected and uninfected mammary glands in dairy cows. Thus, milk samples from 73 quarters were divided in healthy and infected samples according to bacteriological culture and somatic cell count (SCC). After separation of milk cells, the samples were submitted to differential SCC and hydrogen peroxide release by oxidation of phenol red. There was a lower H2O2 release by phagocytes from infected quarters, as well as, a negative correlation between the H2O2 release by phagocytes and SCC (r=-0.34; P=0.0025) and percentage of neutrophils (r=-0.24; P=0.04). Furthermore, it was observed a tendency toward a lower H2O2 release by phagocytes stimulated by phorbol 12-myristate 13-acetate in the infected quarters. However, a higher H2O2 release by phagocytes was observed in milk samples from infected quarters by the estimation of the H2O2 release by phagocytes in 1mL of milk according to SCC mL-1 of each sample. The present study showed a decrease in H2O2 release by the infected quarters which indicate lower microbicidal activity. However, the higher H2O2 release by phagocytes in 1mL of milk in the infected quarters may contribute to recruitment of leukocytes to the site of infection and the persistence of infection.

A pesquisa avaliou a influência do número de ordenhas, número de parições e quarto mamário na concentração de imunoglobulinas (Ig) do colostro de vacas hígidas da raça Holandesa. Foram colhidas duas amostras de colostro por ordenha manual, obtendo-se os primeiros jatos para a realização do exame bacteriológico e determinação dos níveis de imunoglobulinas pelo teste de imunodifusão em gel de ágar. As amostras positivas ao exame bacteriológico foram eliminadas desta investigação. Os valores medianos obtidos para a concentração de imunoglobulinas das classes G, A e M do colostro colhido antes da primeira e segunda ordenha foram, respectivamente de 9.200 e 6.400mg/dL (p=0,0029); 400 e 200mg/ dL (p=0,0018); 800 e 400mg/dL (p=0,0001), respectivamente. Os valores medianos da concentração de imunglobulinas, nos animais com apenas 1 parto, 2, 3 ou nas vacas com 4 a 6 partos foram de 6.400, 6.400, 3.200 e 11.200mg/ dL para a IgG; 100, 200, 100 e 800mg/dL para a IgA; e 400, 400, 100 e 800mg/dL para a IgM, respectivamente. As concentrações de IgG, IgA e IgM foram superiores nos animais com mais de 4 partos (p<0,05). Os valores medianos de IgG, IgA e IgM obtidos nos quartos mamários anterior direito (AD), posterior direito (PD), anterior esquerdo (AE) e posterior esquerdo (PE) foram respectivamente 7.800, 6.400, 7.800, 6.400mg/dL; 200, 200, 200, 200mg/dL; e 400, 400, 400 e 400mg/dL, não observando-se diferenças estatísticas (p>0,05) entre os quartos mamários. Os teores de Igs do colostro de vacas Holandesas sofrem influência do número de ordenhas pós-parto e número de lactações. Estas variações podem ser consideradas fatores de risco associados à falha na transferência de imunidade passiva em bezerros neonatos, predispondo-os às doenças e ocasionando perdas produtivas á pecuária.

This study analyzed the influence of the number of milkings, number of births, and udder quarter in immunoglobulin (Ig) concentration in the colostrum of healthy Holstein cows. It was collected two samples of colostrum by manual milking, getting the first jets to completion of bacteriological examination and immunoglobulin levels by radial immunodiffusion test in agar gel. Positive samples for bacteriological examination were excluded from this investigation. Medians of immunoglobulin's G, A and M in the colostrum collected before the first and second milking were respectively 9,200 and 6,400mg/dL (p=0.0029); 400 and 200mg/dL (p=0.0018); 800 and 400mg/dL (p=0.0001). Median immunoglobulin concentration in animals that calved once, twice or three times or in cows that calved 4 to 6 times were 6,400; 6,400; 3,200 and 11,200mg/dL IgG; 100, 200, 100 and 800mg/dL IgA ; and 400, 400, 100 and 800mg/dL IgM, respectively. Concentrations of IgG, IgA and IgM were greater in animals that calved more than 4 times (p<0.05). Medians of IgG, IgA and IgM in the right fore quarter (RF), right hind quarter (RH), left fore quarter (LF) and left hind quarter (LH) were, respectively, 7,800; 6,400; 7,800 and 6,400mg/dL; 200, 200, 200 and 200mg/dL; and 400, 400, 400 and 400mg/dL. Ig concentrations in the colostrum of Holstein cows were influenced by the number of milkings after delivery and number of lactations. These variations may be considered risk factors to passive immunity transfer to newborn calves, predisposing them to diseases and causing economic losses to dairy production.