Lactoferrin gene expression in dairy buffaloes affected by mastitis

Economic losses due changes in the milk production and composition, making it less suitable for consumption and processing, are caused mainly by mastitis, an infection of the mammary gland related to metabolic and physiological changes, trauma, or microorganisms' invasion. In the early stages of infection, in which there is a predominance of innate immunity, lactoferrin is an important protein with antimicrobial property that can act by two mechanisms: direct interaction with the infectious microorganism or sequestration of free iron ions preventing the development of bacteria that use iron as a metabolic cofactor. Thus, the aim of this study was to evaluate the expression level of lactoferrin gene (LTF) in buffaloes with mastitis, from the RNA extraction of somatic cells in the milk. Milk samples of 24 animals, 12 with mastitis and 12 without mastitis, were collected in tubes free of RNases and immediately conditioned in ice. The verification of mastitis was performed using the California Mastitis Test and somatic cell count (SCC). The total RNA was extracted using the Trizol(R) reagent (Invitrogen, USA). The quality and RNA integrity were measured by spectrophotometer at a wavelength of 260 nm, 280 nm and 230 nm. The absence of contamination of the samples by genomic DNA was confirmed in a Qubit(R) 2.0 Fluorometer (Invitrogen, USA). The cDNA was amplified by quantitative real-time PCR using specific primers described on the literature to bovine studies and redesigned from the homologous mRNA sequence of buffalo, obtained in the GenBank database (www.ncbi.nlm.nih.gov). The GAPDH, EEF1A1, RPLP0 and HPRT1 genes were used as endogenous controls for normalization of LTF gene expression. Data were analyzed using the SAS statistical software (SAS Inst. Inc., Cary, NC). The 2^-?Ct method was used for gene expression analysis and the means were compared by the Student's t-test. The mRNA expression level of LTF gene was 9.6 times higher in buffalos with mastitis when compared to animals without mastitis (p-value = 0.004). This result demonstrates the importance of this gene for the study of mastitis in dairy buffaloes, where LTF gene can be considered a candidate gene associated with resistance to this disease.