TNF-alpha expression in uninfected and Staphylococcus aureus-infected mammary glands of cows treated with a biological response modifier at drying off.

DALLARD, B.; HEFFEL, S.; RUFFINO, V.; CANAVESIO, V.; NEDER, V.; CALVINHO, L.

San Antonio, Texas, USA.

Congreso; NMC (National Mastitis Council) 46th Annual Meeting, Proceedings; 2007

National Mastitis Council

Introduction Innate immunity represents the basic resistance to infection, which includes anatomic, physiologic and phagocytic barriers, as well as inflammation. Changes in expression of IL-1α, IL-8 and TNF-α during intramammary infections (IMI) have been described (1). Resident mammary cells, including mammary epithelial cells and macrophages, have been identified as the primary source of cytokines (2). Sthapylococcus aureus is the most frequently isolated contagious pathogen and the origin of significant economic losses to dairy industry. This organism causes IMI characterized by extensive tissue damage, its chronic nature and poor response to antibiotic therapy. Persistent S. aureus IMI has been associated with immune response impairment, mediated both by bacterial and host factors (3). Intramammary antibiotic therapy administered at drying off is an integral part of mastitis control programs (4); however, therapeutic success against S. aureus may vary due to factors associated with the cow, the immunologic response of the mammary gland has led researchers to address alternative approaches to classic mastitis control measures, such as manipulation of local immune responses to mastitis pathogens. Biological response modifiers (BRM) or immunomodulators are compounds capable of interacting with the immune system to regulate specific aspects of host response. BRM have been used as an adjuvant to antibiotic therapy or to enhance specific immune responses; however, the exact mechanism of action and effect of compounds employed on the bovine mammary tissue is not fully understood. The objective of this study was to evaluate expression of TNF-α in uninfected and S. aureus-infected bovine mammary glands following a single intramammary infusion of a lipopolysaccharide (LPS)-based BRM at drying off. Material and Methods Holstein nonpregnant cows in late lactation were used. Infection of mammary quarters with S. aureus was considered as co-variable. Infectious status of mammary quarters was determined within six months previous to initiation of the experiment and confirmed 20 and 3 days prior to inoculation. Infections were naturally acquired either in the dry period of first two months of lactation previous to initiation of the study. Only two mammary quarters from either infected or uninfected animals were included in the experiment. BRM and placebo, consisting in vehicle alone, were each infused in mammary quarters of three groups of 12 cows that were sacrificed at 7, 13 and 21d post infusion. Uninfected (n=6) an S. aureus-infected (n=6) mammary quarters were included in each group (7, 14 and 21d). In all cases milking was interrupted after intramammary infusion. BRM contained LPS of an Escherichia coli strain (LN02) at 0.35 µmoles concentration and 4.5 mg of membranous and ribosomal fractions of the same strain incorporated into liposomes contained in a vehicle of 10 ml. TNF-α expression was determined by a streptavidin-biotin immunoperoxidase method and immunohistochemical stained area (IHCSA) for antibody reaction was calculated as a percentage of total area evaluated through color segmentation analysis using program Image Pro-Plus 3.0.1®. Data were analyzed by one-way ANOVA using Duncan´s multiple range test. All statistics were calculated using the SPSS software (version 11.0 for Windows, SPSS Inc. ©). Results and Discussion TNF-α expression was associated with mammary parenchyma and stroma structures both in infected an uninfected quarters. Intense immunostaining of alveolar epithelial cells cytoplasm and ducts was observed. Intraepithelial macrophages, neutrophils and lymphocytes, as well as endothelium and vascular smooth muscle cells, showed intense immunostaining. Effects of BRM treatment on TNF-α expression in S. aureus infected and uninfected quarters during involution is shown in table 1. In chronically infected quarters TNF-α expression increased significantly throughout involution compared to uninfected quarters. TNF-α expression in BRM-treated infected quarters was higher only at 21 d of involution. BRM treatment of uninfected quarters did not alter TNF-α expression during involution. Staphylococcus aureus infection rather than BRM infusion appeared to be the main factor affecting TNF-α expression in mammary tissue during early involution. Table 1: Effect of treatment with a biological response modifier on percent of immunohistochemical stained area (IHCSA) for TNF-α in uninfected and  S. aureus-infected mammary quarters during involution.                                                                Quarters infected with S. aureus                           Uninfected quarters Involution BRM PLACEBO BRM PLACEBO 7 d 11.88 (0.31)b 11.11 (0.36)b,c 7.22 (0.73)e,f 8.55 (0.45)d,e 14 d 9.75 (0.74)c,d 8.71 (0.57)d,e 4.90 (0.79)g 5.84 (0.46)f,g 21 d 17.20 (0.50)a 12.28 (0.61)b 8.20 (0.42)d,e 8.81 (0.44)d,e Values represent means of percent IHCSA ± SEM; a,b,c,d,e,f  Means for each treatment and days of involtion without a commos superscript differ (p<0.05). References (1) Riollet, C., P. Rainard, and B. Poutrel. 2000. Differential induction of complement fragment C5a and inflammatory cytokines during intramammary infections with Escherichia coli and Staphylococcus aureus. Clin. Diag. Lab. Immunol. 7:161–167. (2) Riollet, C., P. Rainard, and B. Poutrel. 2001. Cell subpopulations and cytokine expression in cow milk in response to chronic Staphylococcus aureus infection. J. Dairy Sci. 84:1077–1084. (3) Jones, G.M. (1998) Mastitis cost? Dairy Pipeline: December of 1998, Virginia Cooperative Extension. Virginia Polytechnic Institute and State University. Actualization 7 of November 2002. http://www.dasc.vt.edu/jones/mastcost.htm. (4) Sordillo, L.M. and K.L. Streicher. 2002. Mammary gland immunity and mastitis susceptibility. J. Mammary Gland. Biol. 7:135-146. (5) Bramley, A. and M. Dodd. 1984. Reviews of the progress of dairy science: mastitis control-progress and prospects. J. Dairy Res. 51:481-492.