Apoptosis quantification in uninfected and Staphylococcus aureus infected mammary gland of cows treated with a biological response modifier at drying off.

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

San Antonio, Texas, USA.

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

National Mastitis Council

Introduction The bovine mammary gland is highly susceptible to new intramammary infections (IMI) during physiological transition from lactation to involution. Milk yield in infected quarters can decrease 30% compared with uninfected quarters (1). Increased susceptibility to IMI has been related to changes during the involution process that may facilitate bacterial penetration of the streak canal, interfere with natural defense mechanisms and enhance bacterial growth. Intramammary antibiotic therapy administered at drying off is an integral part of mastitis control; however, limitations of this practice determined the need to explore new control measures, including manipulation of the mammary gland immune mechanisms, to enhance bacterial clearance during involution. Biological response modifiers (BRM) are compounds capable of interacting with the immune system to regulate specific aspects of host response, and have been used as an adjunct 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. Mammary involution is promoted by milking interruption in dairy cows or weaning in other species. Programmed cell death or apoptosis occurs during mammary involution. Apoptosis is an ATP-dependent cell death pathway, characterized morphologically by chromatin condensation, nuclear fragmentation and cell size decrease (3). Apoptosis results in cell fragmentation in membrane-limited apoptotic bodies that are removed by phagocytic cells (4). Although the use of mouse models has increased the understanding of apoptosis during mammary involution, studies performed in other animals indicated the existence of marked differences between species (2). The objective of this study was to quantify apoptosis in mammary tissue 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. Detection of apoptotic cells in situ was performed utilizing terminal deoxynucleotidyl transferase dUTP nick en labeling (TUNEL). All cells in each field were conunted (at least 1500 cells) using Image Pro-Plus 3.0.1® (Media Cybernetics, Silver Spring, MA, USA). Cells were classified as epithelial or labeled epithelial cells. Cells counted as epithelial were part of the secretory tissue, including epithelial and myoepithelial cells, and possibly leukocytes if present in the alveolar lumen. 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 Results of BRM treatment effect on percent apoptotic cells in mammary tissue of uninfected and S. aureus-infected quarters during involution is shown in table 1. Table 1: Effect of treatment with a biological response modifier on percent of apoptotic cells in uninfected and S. aureus-infected mammar quarters during involution.                                                                Quarters infected with S. aureus                           Uninfected quarters Involution BRM PLACEBO BRM PLACEBO 7 d 2.443 (0.204)a 1.597 (0.033)c,d 1.171 (0.010)e,f 1.466 (0.084)c,d,e 14 d 1.823 (0.091)b,c 1.236 (0.108)d,e,f 1.076 (0.032)f 1.604 (0.257)c,d 21 d 2.063 (0.144)b 2.019 (0.020)b 1.041 (0.048)f 1.593 (0.046)c,d Values represent means of percent apoptotic cells ± SEM; a,b,c,d,e,f  Means for each treatment and days of involtion without a commos superscript differ (p<0.05). BRM treatment in S. aureus-infected mammary quarters affected percent apoptosis in mammary gland parenchyma components. A significant increase of apoptotic cells was observed at 7 and 14d of involution compared with uninfected control quarters (P<0.05). In addition, placebo-treated infected quarters showed increased percent of apoptotic cells at 21d of involution compared with placebo-treated uninfected quarters (P<0.05). BRM-treated uninfected mammary quarters showed a significant decrease in percent apoptotic cells at 14 and 21d of involution compared with placebo-treated quarters  (P<0.05). References 1. Sordillo, L., Nickerson, S., Akers. (1989) Pathology of S. aureous mastitis during lactogenesis: relationship with bovine mammary structure and function. J. Dairy Sci. 77:228-236. 2. Bramley, A. and M. Dodd. (1984) Reviews of the progress of dairy science: mastitis control-progress and prospects. J. Dairy Res. 51:481-492. 3. Campos, M.; Godson, D.; Hughes, H.; Babiuk, L.; Sordillo, L. (1993) The role of biological response modifiers in disease control. J. Dairy Sci. 76:2407-2417. 4. Monks, J.; Geske, F.; Lehman, L.; Fadok, V.A. (2002) Do Inflammatory Cells Participate in Mammary Gland Involution?  J. Mammary Gland Biol. 7:163-176. 5. Reed, J.C. (2000) Mechanisms of apoptosis. Am. J. Pathol. 157:1415-1430. 6. Sladek, Z.; Rysanek, D. (2000) Apoptosis of polymorphonuclear leukocytes of the juvenile bovine mammary gland during induced influx. Vet. Res. 31:553-563.