Longitudinal Evaluation of Bovine Mammary Gland Health Status by Somatic Cell Counting, Flow Cytometry, and Cytology

Abstract

Abstract. Bovine mastitis phases induced by Staphylococcus aureus were assessed in 6 lactating cows before challenge and at 1, 4-8, and 9-14 days postinoculation (dpi). Milk lymphocytes, macrophages, and polymorphonuclear cells (PMN) were counted by conventional (manual) cytology, identified by CD3ϩ and CD11bϩ immunofluorescence and counted by flow cytometry (based on leukocyte forward and side light scatter values). Somatic cell counts (SCC) and recovery of bacteria were recorded at the same times. Preinoculation samples showed a lymphocyte-dominated composition. At 1 dpi, the percentage of PMN increased and that of lymphocytes decreased. At 4-8 dpi, PMN were predominant, but the percentage of mononuclear cells increased above that at 1 dpi and further increased by 9-14 dpi (when lymphocytes approached prechallenge values). Based on leukocyte percentages, 3 indices were created from the data: 1) the PMN/lymphocyte percentage ratio (PMN/L), 2) the PMN/macrophage percentage ratio (PMN/M), and 3) the phagocyte (PMN and macrophage)/ lymphocyte percentage ratio (Phago/L). Significant correlations were found between cytologic and flow cytometric data in all of these indicators (all with P Յ 0.01). These indices identified nonmastitic, early inflammatory (1-8 dpi), and late inflammatory (9-14 dpi) animals. In contrast, SCC and bacteriology did not. Although sensitivity of the SCC was similar to that of Phago/L, the specificity of SCC was almost half that of the Phago/ L index. Based on flow cytometry indicators, an algorithm for presumptive diagnosis of bovine mastitis was developed. Flow cytometry provides results as valid as those obtained by conventional (manual) cytology, shows greater ability to identify mastitic cases than does SCC, and may identify 3 mammary gland healthrelated conditions.Infectious bovine mastitis is a major health problem of dairy cattle that results in decreased milk production and decreased milk quality. Staphylococcus aureus is a major bacterial pathogen associated with this disease. 18 Identification of bovine mastitis has historically been based on counting of all cells present in milk (leukocytes and epithelial cells), i.e., somatic cell counts (SCC). Counts Ͼ500,000 cells/ml are usually associated with bovine mastitis, which results in reduced milk production and reduced shelf life of dairy products. 8,32 The reduction of bovine mastitis prevalence is a major goal of the dairy industry throughout the world. To achieve this goal, most countries ban from the market milk with SCC Ͼ 500,000 cells/ml or charge fees for milk deliveries that approach that figure (Booth JM: 1996, Annu Meet Natl Mastitis Counc).In spite of these policies, it is questionable whether measures based on lower SCC will result in decreased prevalence of bovine mastitis. Although high SCC (Ͼ1 ϫ 10 6 cells/ml) is regarded as an accurate indicator of bovine mastitis, both mastitic and healthy cows can yield SCC below that figure. 25 Low SCC may also be associated with milk of poor industrial value. 8 The SCC is a quanti...

Aberrant promoter methylation of the CD4 gene in peripheral blood cells of mastitic dairy cows

“…Our results showed that the cows with CM had distinctly increased DNA methylation level in CD4 promoter but downregulated expression of the gene in the peripheral blood cells of Chinese Holstein cows, while healthy cows showed the opposite. The negative correlation of DNA methylation and CD4 expression was consistent with the significant decrease in CD4 + T cells observed in the cows with mastitis (Rivas et al, 2001;Rivas et al, 2007). Thus DNA methylation level of CD4 promoter could be a helpful molecular marker for selection against mastitis in dairy cattle.…”

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

“…A CCS diferencial permite a diferenciação das populações celulares presentes no leite e, desta forma, possibilita a detecção da mastite logo no início do processo infeccioso, quando a CCS ainda é baixa (Rivas et al, 2001;Dosogne et al, 2003;Koess e Hamann, 2008). Entre os métodos aplicados para a contagem diferencial de leucócitos, podem-se citar a contagem microscópica direta por esfregaços, a técnica de citocentrifugação e a citometria de fluxo.…”

“…A técnica de citocentrifugação apresenta as vantagens da rapidez de sua execução e da menor variabilidade em relação à contagem direta em esfregaços (Dulin et al, 1982). Do mesmo modo, a contagem diferencial pela citometria de fluxo tem sido associada à maior repetibilidade devido ao expressivo número de células avaliadas (Rivas et al, 2001;Dosogne et al, 2003;Koess e Hamann, 2008).…”

“…O presente estudo corrobora esses dados, em que os neutrófilos foram a população celular predominante apenas nas amostras com alta celularidade (<200.000 células/mL). Rivas et al (2001) também relataram correlação positiva entre a porcentagem de leucócitos polimorfonucleares (PMNL) (CD11b + ) pela citometria de fluxo e a porcentagem de PMNL pela citocentrifugação (r = 0,82). No entanto, o CD11b é uma das cadeias α da β 2 -integrina, que está envolvida no processo de migração celular (Paape et al, 2003), e pode ser expressa também por outras populações celulares, como exemplo a de linfócitos (Della Libera et al, 2012 Os resultados mostram que a citometria de fluxo pode ser uma ferramenta útil no diagnóstico da mastite bovina.…”

“…Embora a contagem microscópica de células somáticas seja um método comum para a determinação da contagem diferencial de células presentes no leite, ela consome bastante tempo e apresenta baixa repetibilidade, devido a sua subjetividade (Koess e Hamman, 2008). Em contraste, a citometria de fluxo é o método automático e rápido, que permite a contagem de expressivo número de células em curto espaço de tempo (Rivas et al, 2001;Dosogne et al, 2003;Koess e Hamman, 2008;Piepers et al, 2009). Isto acarreta em resultados mais precisos e menos trabalhosos do que a citologia convencional.…”