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Mastitis is a highly prevalent disease in dairy cows and economically costly to the dairy industry worldwide (Bannerman, 2009). Clinical mastitis is characterized by visible changes in milk from the gland including presence of clots, heat, pain or swelling of a gland. Rate of intramammary infection (IMI) is higher around parturition (Smith et al. 1985; Bradley and Green 2004). Susceptibility of the mammary gland to new intramammary (IMM) infections is markedly increased during drying off and the periparturient period (Burvenich et al. 2006).
The periparturient or transition period for a dairy cow begins 2-3Â weeks before calving and lasts until 2-3Â weeks after calving. The bovine mammary gland has been reported to be more susceptible to new intramammary infections during the early stages of the dry period and in the late dry period (colostrogenesis) than during the remainder of the dry period (Sordillo and Streicher, 2002, Smith et al. 1985). These increased rates of IMI during transition periods may be attributable to changes in the anatomy and physiology of the mammary gland, teat end and extent of exposure to mastitis pathogens (Oliver and Sordillo, 1989). At the time of dry off milk is no longer removed from the udder and the increased intramammary pressure may cause leakage of milk from the udder (Pyorala, 2008), creating an easy entry for bacterial pathogens. During the early phase of dry period, the increased susceptibility to mastitis with increasing milk stasis in the mammary gland at dry off may also be related to incomplete closing of the teat end (Dingwell et al. 2004). Defense of the mammary gland against mastitis causing pathogens depends on the concentration and function of the resident and incoming leukocyte population and determines the outcome of the imm infections (Mehrzad et al. 2004, 2005). The leukocytes (phagocytes) migrating into the mammary gland in the first week after dry off are accompanied by decreased phagocytic activity. The leukocytes start ingesting milk fat, and cell debris, which decreases their function and induces apoptosis.
Active involution of the mammary gland is completed by 3 to 4 wk after drying off and is followed by a phase of steady-state involution. This mid dry period shows the lowest incidence of new infections. During this time period, most teats have become sealed, the fluid volume in the udder cisterns is low, and the composition of medium is unfavorable for bacterial growth (Burvenich et al. 2006).
At the time of colostrogenesis the mammary gland becomes more susceptible to infections as the teat canal starts to open and leaks mammary secretions (Oliver and Sordillo 1988). At the same time, during colostrogenesis, calving and early lactation, cows experience many endocrine, nutritional and metabolic changes that could influence the immune function and susceptibility of the cow to new IMI (Goff et al., 2002). As a consequence, during the periparturient period the innate immunity of the cow is compromised. During these later stages of dry period the citrate: lactoferrin ratio is very high, which makes any bacteriostatic action of lactoferrin ineffective. Milk citrate can effectively compete with lactoferrin for iron binding, and the resulting iron-citrate complex can be utilized by bacteria (Burvenich et al. 2006). Periparturient immunosuppression is most likely a result of several entities (physiological and immunologically related) acting in a concert with profound effects on the function of mammary gland of the periparturient cow.
Figure 1. Infection risk throughout four quartiles of Dry period (Smith et al., 1985).
The incidence of clinical mastitis peaks on most farms immediately following parturition (Barkema et al., 1998), however several field studies showed the presence of bacteria isolated from the dry gland before parturition without clear inflammatory indicators of mastitis (Bradley and Green 2004; Green et al., 2007). Intramammary infections that are present during the dry period can be partitioned into those that were carried into the dry period from the previous lactation (existing infections) and those that enter between the time of drying off and calving (Green et al, 2007). During the second half of dry period (colostrogenesis) the cows become more susceptible to new IMI. The presence of S. dysgalactiae, S. faecalis, E. coli, Enterobacter spp., and Corynebacterium spp. at drying off increased the probability of subsequent lactational clinical mastitis (Green et al, 2002). Coliform infections presented at parturition are characterized by severe inflammatory signs and sepsis during first 70 days of lactation (Hogan et al, 1989; Smith et al., 1985). Based on the previous studies, it can be concluded that IMI with major pathogens in the late, dry, and post-calving period increase the risk of clinical mastitis, and this mastitis occurs at a greater rate after calving than mastitis not associated with dry period infections (Green et al., 2002).
Figure 2. Bacteria isolated in cases of clinical mastitis from dry period sampled quarters (Green et al., 2002).
Invasion of a host by pathogenic infectious agents triggers a battery of immune responses through interactions between a diverse array of pathogen-borne virulence factors and the immune surveillance mechanisms of the host. The innate immune system is a universal and ancient form of host defense against infection (Janeway and Medzhitov 2002). The innate immune system acts as first line of defense and functions by recognizing highly conserved sets of molecular structures specific to the microbes (pathogen-associated molecular patterns, or PAMPs) through a limited set of germ line encoded receptors called pattern-recognition receptors. However, vertebrates have evolved a second line of defense called the adaptive immune system. This system uses a diverse set of somatically rearranged receptors (T -cell receptors (TCRs) and B -cell receptors (BCRs)) with the ability to recognize a large spectrum of antigens (Pasare and Medzhitov 2004).
The innate immune response is triggered at early stages of infections in order to recognize the invading pathogens. Among the more important cells that bear innate immune recognition receptors are macrophages, dendritic cells (DCs), mast cells, neutrophils, eosinophils, and the so-called NK cells. These cells can become activated during an inflammatory response. In the mammary gland, this inflammatory response is virtually always a sign of infection with a pathogenic micro organism. During bacterial infection of the bovine mammary gland, large numbers of leukocytes migrate into the udder, resulting in the establishment of a host response against the pathogen. Thymocytes are divided into 2 main categories, the cytotoxic T cells and the helper T cells (TH). The function of activated cytotoxic T cells (CD8+) is to kill host cells infected with a pathogen, as detected by antigens expressed on the surface of infected cells. Helper T cells (CD4+) have a more indirect but equally important effect on the infection. When a TH cell matures, it develops into 1 of 4 types of TH cells. Stimulation of these mature TH cells can cause the expression a large variety of cytokines that can direct the immune response toward a pro-inflammatory cytotoxic T cell-mediated (TH1), B-cell-mediated (TH2), neutrophil-mediated response (TH17), or to counter-regulate the response (Treg). Classification is based on the repertoire of cytokines that are produced by these subpopulations of T-cells (Shafer-Weaver et al, 1999). The TH-1 lymphocytes mainly secrete interleukin-2 (IL-2) and interferon gamma (IFN-Î³) and promote cellular response. The TH-2 lymphocytes mainly secrete interleukin-4 (IL-4) and promote humoral response by stimulating IgG1 and IgE production. TH17 produce immunosuppressive cytokine transforming growth factor (TGF)-Î², and Treg cells also produce immunosuppressive cytokine interleukin-10 (IL-10). Thus a balance between immunostimulation and immunosuppression is essential to maintain homeostasis. Günther et al. (2009) reported that upon recognizing the pathogen, bovine Mammary Epithelial cells are able to send out a strong signal of chemokines into the organism to recruit cellular factors of immune defense (e.g. monocytes, granulocytes, macrophages) from the blood stream into the infected mammary gland.
Pregnancy presents a major challenge to maternal immune system, both in normal and pathologic state (Denney et al., 2011). The immune response is modulated to allow establishment and maintenance of a viable pregnancy without rejection. Pregnancy constitutes a time of distinctive challenges for the human immune system. The growth and development of a semi-allogeneic fetus must be tolerated by the mother, whilst both maternal and fetal protection against infection, as well as immunological processes crucial for tissue growth, remodelling and differentiation, must be maintained. Based on human and mice studies, various paradigms have been proposed for the modulation of the immune system that maintains viable pregnancy (Raghupathy, 2001). Maternal T lymphocytes at fetomaternal interface play an important role in the fetal survival and development (Piccinni, 2002). TH-2 cytokines (IL-4) are detectable at fetomaternal interface during all period of gestation, whereas TH-1 cytokine IFN-Î³ is transient, detected only in first period (Chaouat, 2007). A TH-2 shift in pregnancy is also characterized by reduction in IFN-Î³ and IL-2 producing CD4+ and CD8+ T cells (Raghupathy, 2001). The predominance of a particular cytokine in the given microenvironment at the time of antigen presentation is an important factor in driving the naÃ¯ve T cells towards TH-1 and TH-2 dominated populations. The differentiation of TH cells into polarized TH-1 and TH-2 cells can be influenced by hormones (Piccinni, 2002). Progesterone present at a concentration comparable to those during pregnancy is a potent inducer of TH-2 cytokines (IL-4).
A third subset of T-lymphocyte population TReg cells are reported to be capable of inhibiting allogeneic reactions in human peripheral blood, thereby inducing a long term antigen specific anergic state (Bach, 2001).These TReg cells depend on IL-10 cytokine for their growth and function. TReg cells play an important role in the induction of tolerance to paternal antigens during pregnancy (Saito et al., 2007). Regulatory natural T cells (TReg) are defined as a subpopulation of thymus-derived T lymphocytes representing about 1-3% of T CD4+ cells. Treg cells also play an important role for regulating fetal rejection by maternal immune cells in human pregnancy by actively suppressing self reactive lymphocytes (Leber et al., 2010). Expansion of TReg cells during pregnancy probably depends on hormonal changes, and sex steroid hormone estradiol has been reported to up regulate TReg cells function (Prieto and Rosenstein, 2006). The forkhead box transcription factor (Foxp3) acts as a master switch in the regulation of their development and function (Leber et al., 2011).
There is very little information about changes in the systemic and local immune function in the peripartum period in cows. During the transition period, the dairy cow experiences many endocrine and sudden metabolic changes, in addition acute deficiencies of nutritional factors required for maintenance of healthy immune function occurs when parturition approaches. Lymphocyte function and numbers is reduced during pregnancy due to negative energy balance (Pezeshki et al., 2010). The phagocytic activity of the macrophages is also compromised during parturition due to negative energy balance (Kehrli et al., 1989). A hormone-cytokine-T cell network at fetomaternal interface could possibly interact with other immune cells and alter the immune function in the transition period in dairy cows. Progesterone, present at a high level at the fetomaternal interface may be in part responsible for a TH-2 switch during late gestation period (Piccinni, 2002). There is also systemic decrease in blood T-cell population followed by impaired function and response to antigens (Kehrli et al., 1989; Kimura et al., 1999). Defective leukocyte functions also have been correlated to shifts in leukocyte trafficking patterns during the transition period (Shafer-Weaver et al, 1999). The percentage of T-lymphocytes decrease from 45% of circulating lymphocyte population to 20% in transition period (Shafer-Weaver et al., 1996).
Data obtained from previous studies (Shafer-Weaver et al, 1999), indicated a predominance of TH-2 lymphocyte population in the dry off period as compared to TH-1 lymphocyte population in the postpartum period. The CD4+:CD8+ cells ratio in the blood varies during different stages of lactation, and this ratio is 3:1 around calving (Shafer-Weaver et al., 1996; VanKampen and Mallard, 1998). It can be concluded that the bovine immune system is less capable of battling pathogens in transition period than in the mid lactation period (Burvenich et al., 2003). Limited inflammatory response during the dry period could reflect an adaptation in maternal immune signaling during late gestation to protect the fetus from severe pro-inflammatory responses, and improve fetal survival chances in a genetically dissimilar maternal environment (Dietert and Piepenbrink 2008).
The early postpartum period is a period of high risk for severe clinical mastitis because of extreme sensitivity for a number of animals for IMI during transition period (Green et al., 2002). The PMN population represents a major line of defense against infection in the bovine mammary gland. PMN in blood and milk are important components of the mammary gland defense against coliforms (Burvenich et al., 1994). At parturition and onset of lactation there is an increase in the PMN population in the mammary gland due to high levels of cortisol at parturition. The PMN dysfunction influences the balance between the inflammatory reactions, bactericidal capacity and tissue damage, boosting the severity of E. coli mastitis.
Figure 3. Viability of PMN during different stages of Lactation (Burvenich et al., 2003).
The lymphocyte pattern in both blood and milk also changes around parturition. The proportion of T-cells and the number of T-cell subsets (CD4, CD8) vary significantly during this period (VanKampen and Mallard, 1997). The proportion of CD4+ cells decrease postpartum, both in blood and mammary tissue due to decrease in IFN-Î³ concentrations (Shafer-Weaver et al., 1996). Moreover, the authors observed preferential trafficking of CD8+ cells of the suppressor type rather than cytotoxic type into the mammary gland around calving. This observation was supported by increase concentration of IL-4 during late dry period. The decreased levels of pro inflammatory cytokines (IFN- Î³) around calving might contribute to the increased susceptibility of mammary glands to new IMI. Other factors such as hormonal changes alter the functional capacity of leukocytes during parturition. Increased levels of beta-hydroxybutyrate during early lactation, reduces the respiratory burst activity of leukocytes by impairing the hydrogen peroxide production and thereby inhibit lymphocyte function (Pezeshki et al., 2010). Based on these studies, it could be concluded that severity of the E. coli mastitis depends on the speed at which PMN are recruited from the peripheral blood, as well as its opsonic activity in the mammary gland.
Figure 4. Variations in CD4+/CD8+ T lymphocyte ratios in the mammary gland secretions during the drying off and parturition periods.
Parturition is also associated with low levels of serum IgG concentrations (Kehrli et al., 1991) and previous investigations suggested low levels of IgG2 was associated with an increased incidence of pyogenic mastitis (Solbu, 1984). Although the concentrations of Ig bearing plasma cells increase during IMI, its ability to protect against mastitis has been vastly debated (Mallard et al., 1998). While, number of integrative factors mediate host defense, leukocytes or their secreted products (cytokines, chemokines) derived from blood or locally contribute to protection of the mammary tissue against infectious agents. Based on the previous reports, altered lymphocyte responsiveness around parturition coupled with peripartum hormone changes is linked to increased mastitis susceptibility but those studies do not necessarily confirm a cause and effect relationship. More definitive studies need to be designed to determine the precise effects of various immune factors, hormones on the immune status during the peripartum period.
Genome wide profiling techniques are now available to monitor all infection related changes in the transcriptome and proteome, promising deep insight into the molecular mechanisms of host-pathogen interactions. The development and use of microarray and quantitative reverse transcription pcr to identify molecular pathways and gene networks affected by IMI would yield mechanistic information of the underlying tissue adaptations to infection. Moyes et al., (2009) reported wide transcriptional response of the mammary tissue 20 h after S. uberis challenge. Overall, the functional analysis uncovered up regulation of immune response followed by induction of cell proliferation/cycle/death and transport of protein and ions pathways. The use of well-established pathways (i.e., canonical pathways) together with information about single genes could provide additional means to unravel the mechanisms controlling the mammary response to IMI before peak clinical signs. In another study, Lutzow et al., (2008) employed transcriptional profiling to measure changes in gene expression occurring in bovine mammary tissues sampled from three dairy cows after brief and graded intramammary challenges with S. aureus. The authors observed secretion of a number of proinflammatory cytokines and chemokines from mammary epithelial cells stimulated by the bacteria that triggered the recruitment and activation of neutrophils in mammary tissue. Future research should be focused on developing and implementing strategies to enhance periparturient defense mechanisms of the dairy cow as a means of disease prevention. Identifying the dominant inflammatory mediators invoked in the mammary gland upon bacterial infection during late gestation will provide a better understanding of the potential gaps in the immune response necessary for defense of the gland against bacterial invasion.
In another study Günther et al. (2011), identified genes that were strongly and specifically activated by the E. coli challenge were part of a regulatory network controlled by pro-inflammatory cytokines IL-1 and TNF-Î±. The regulated genes are encoding for antimicrobial and chemotactic proteins S100A8 and S100A9; the Î²-defensin LAP; the anti-apoptotic factors BIRC2, BIRC3, and BCL2A1; the complement factors C2 and CFB; as well as enzymes degrading the extracellular matrix (MMP9) or the key regulator of prostaglandin synthesis (PTGS2). Based on these reports it could be concluded that a characteristic early expression of pro-inflammatory cytokines IL-1 and TNF-Î± in E. coli-challenged cells, could be attributed to the likely causes of the severe inflammatory symptoms often observed for udder infections with E. coli. One aim of the study is to identify the network of genes that becomes activated in mammary tissue in response to a brief intramammary challenge with E. coli. The identification of these poorly characterized genes and in-depth characterization of their corresponding proteins may be important for the development of novel strategies to enhance resistance of cows to mastitis and the development of novel therapeutic agents.
Research directed at understanding the status of innate and adaptive immune function of a dairy cow in the transition period may provide important data concerning interaction of dry period and immune function.
The long term goal of our research is to understand the immune response to invading bacterial pathogens in the mammary gland and to optimize the immune response to these invading pathogens. Understanding the role of specific T-cell subset in mastitis in different stages of gestation will have a significant impact on the development of effective preventative programs, treatments or vaccination schemes. Our research will also contribute to our understanding of the periparturient immunosuppression. This improved understanding of the dynamics of periparturient immune response could lead to the development of novel interventions that modulate the inflammatory response in periparturient cows. Ultimately leading to better vaccine efficacy and less clinical and subclinical disease in early lactation.
CURRENT WORK PERTINENT TO THE PROPOSED RESEARCH
To provide a proof of concepts, we have performed some initial intramammary experimental challenges with live Escherichia coli in late gestation dairy cows. The cows became infected after challenge and remained infected after calving and into lactation. After challenge, the cows responded with an anti-inflammatory cytokine profile, reflecting a potentially Th2 / Th3 biased response to intramammary challenge in late gestation. Cows showed signs of clinical mastitis after calving associated with presence of the challenge organism (Figure 6. RAPD). Clinical mastitis symptoms in early lactation were accompanied by a conventional pro-inflammatory cytokine profile.
Figure 4. Cytokine response in relation to an intrammamary challenge with E. coli in late gestation.
Flow cytometry assay for phenotypic analysis of immune cells (CD4, CD8) and presence of intracellular cytokines in dry cow secretion or milk cells (IFN-Î³, IL-10) was developed. A RT-PCR assay for RNA quantification of cytokine genes (IFN-Î³, IL-10, IL-4) in dry cow secretions and milk cells was also developed. With the RAPD analysis of our challenge strain and the recovered strains from different time points, the RAPD profile before and after challenge matched in our cows. We observed that 80% of challenged quarters became infected as judged by the presence of the challenge organism at 24 hours after the challenge. The levels of anti-inflammatory cytokine IL-10 and IL-4 were elevated after challenge in late gestation. Ratios of CD4+ and CD8+ lymphocytes were high in dry period, indicating a clear CD4 predominance. The CD4+ to CD8+ ratio dropped quickly post freshening.
Figure 5. T-cell subpopulations in mammary secretum during late gestation and early lactation.
Figure 6. RAPD patterns of E. coli isolates obtained before and after imm challenge
Figure 7. Expression of MHC-II in low and high CD14+ Monocytes in cows during late gestation and in non pregnant cows.
The innate immune response as evaluated by the upregulation of the immune response genes in MEC to an in vitro and an in vivo E. coli challenge is different between late gestation and non-pregnant early to mid lactation cows. Specifically:
We hypothesize that peripheral blood monocyte derived dendritic cells have lower MHC expression in late gestation cows as compared to mid to early lactation cows.
The in-vitro cytokine profiles in response to a standardized challenge of monocytes and monocyte derived dendritic cells from cows in late gestation versus non pregnant mid to late lactation is significantly different.
Differentiation of CD4+ T-cells is different in naÃ¯ve CD4+ cells co-cultured with blood monocyte derived dendritic cells from late gestation cows versus non-pregnant early to mid lactation cows.
The innate immune response to an in-vivo imm challenge in late gestation cows is biased towards a down regulated Th2/Th3/TReg response whereas non pregnant early to mid lactation cows show a biased innate immune response towards a pro-inflammatory Th1 response.
Animal Selection and Housing:
Twelve cows (six late gestation and six non-pregnant early to mid lactation) will be selected from the Cornell University Veterinary College Dairy herd based upon examination of mastitis history and milk somatic cell count collected monthly.
Hundred colony forming units of a persistent strain of E. coli (Dogan et al. 2006) suspended in phosphate buffered saline will be introduced into the teat canal, below Furstenburg's Rosette, of 2 contra lateral quarters of each animal. A third quarter will receive a similar infusion of vehicle only, and the fourth quarter will receive no infusion.
Samples will be collected aseptically for evaluation of bacterial presence within the glandular secretions and 100Î¼l samples were plated immediately on MacConkey's Agar plates and incubated overnight at 37Â°C. Colonies will be counted and individual colonies will be isolated and stored at -70Â°C for molecular strain typing via random amplification of polymorphic DNA (RAPD) analysis.
Dry cow secretions, colostrum, and milk samples will be centrifuged at 40,000g for 30 minutes at 4Â°C. Fat layer will be removed with a sterile wooden stick, and translucent whey moved to another tube and maintained at -70Â°C until cytokine analysis.
For the preparation of plasma, tail vein blood samples will be collected in Vacutainer glass tubes (Becton Dickinson Corp., Franklin Lakes, N.J.) and centrifuged at 1,500 x g for 15 min, and the clear plasma supernatant collected, aliquoted, and stored at -70Â°C.
A sandwich ELISA will be used to quantify pro inflammatory cytokine IFN-Î³ and anti inflammatory cytokines IL-4 and IL-10 levels in milk and serum. Recombinant bovine IFN- and IL-4, IL-10 (Serotec, Inc., Raleigh, N.C.) will be used to generate a standard curve for the ELISA. The concentration of IFN- and IL-4 in milk calculated by extrapolating from the respective standard curves, and the values will be expressed as biological units of activity per milliliter. A background correction reading at 565Â nm will be subtracted from the 450-nm absorbance readings.
RAPD strain typing:
Individual isolates from bacterial culture plates will be grown in Luria-Burtani (LB) broth at 37Â°C for 12 hours. DNA will be isolated from samples using QiaQuick DNAeasy isolation kit (Qiagen, Valencia, CA). RAPD primers designed specifically for Random amplified polymorphic DNA typing of Gram negatives, resulting in the following primers: forward 5'-AGTAAGTGACTGGGGTGAGCG-3' and reverse 5'TACATTCGAGGACCCCTAAGTG-3' will be used to run the RAPD PCR. These primers have shown to provide discernment between mastitis E. coli bacterial strains (Dogan et al. 2006). PCR products will be evaluated using gel electrophoresis in a 1Â½% agarose gel at 60V for 1Â½hours.
(a) CD4/CD8 cell surface staining:
Milk (or dry cow secretion) samples will be centrifuged at 550Â Ã-Â g for 15Â min, and the top layer aspirated down to remove as much of the cream fat layer as possible (if milk spin ~100cc, if dry cow secretion dilute ~5ml with equal volume of PBS). The pellet re-suspended in 2ml PBS and transferred to FACS tube (5 ml round-bottom falcon tube #352054) and spun and wash again twice for 5 minutes. The pellet re-suspended in 2ml PBS, passed through BD falcon 40Âµm cell strainer (# 352340) into new FACS tubes. 1 ml FACS buffer (0.5%BSA, 0.05%Na-azide in PBS) will be added and the tube spun for 5 minutes at 550Â Ã-Â g. Following a second centrifugation at 500Â Ã-Â g for 5Â min, the supernatant removed by aspiration. The cells will be fixed by exposure to 1ml/tube of fixation/permeabilization medium BD Cytofix/CytoPerm Plusâ„¢ (BD, Franklin Lakes, N.J) for 20Â min at 4Â°C in the dark. Following fixation, ~106 cells in 50 Î¼l of FACS buffer will be stained with the appropriate amount of a fluorochrome-conjugated monoclonal antibody specific for a cell surface antigen such as CD4 (#IL-A11, VMRD, Pullman, WA) CD8 (# BAQ111A, VMRD, Pullman, WA) (30 min, 4Â°C). After staining, the cells will be washed twice with FACS buffer (1 ml/wash) and pellet by centrifugation (250 Ã- g). For intracellular cytokine staining of IFN-Î³ and IL-10 thoroughly resuspend fixed/permeabilized cells in 50 Î¼l of 1Ã- BD Perm/Washâ„¢ buffer containing a pre-determined optimal concentration of a fluorochrome-conjugated anti-cytokine antibody IFN-Î³ (MCA1783) and IL-10 (MCA2111EL), (Serotec, Inc., Raleigh, N.C.). The cells will be incubated at 4Â°C for 30 minutes in the dark. After incubation the cells will be washed twice with 1Ã- BD Perm/Washâ„¢ buffer (1 ml/wash) and resuspended in 1 ml FACS buffer prior to flow cytometric analysis on BD FACS Canto IIâ„¢ flow cytometer. The flow cytometric data will be analyzed by FACSDiva softwareâ„¢.
(b) Expression of CD14 and MHC class II on blood monocytes:
PBMC (3Â·6 Ã- 106 mlâˆ’1) from late pregnant and mid lactation cows will be resuspended in complete RPMI medium 1640 (Invitrogen, Carlsbad, CA). Briefly, monocytes will be allowed to adhere for 2 h; supernatants and non-adherent cells removed to eliminate lymphocytes. The monocytes will be harvested and processed further for flow cytometric analysis for identification of expression of bovine leukocyte antigen D-related (BoLA-DR) on the surface of monocytes using monoclonal antibody TH14B (VMRD, Pullman, WA) and also for the expression of monocyte marker CD14 (VMRD: MM61A).
(c) Monocyte differentiation into DC:
PBMC (3Â·6 Ã- 106 mlâˆ’1) from late pregnant and mid lactation cows will be resuspended in complete RPMI medium 1640 (Invitrogen, Carlsbad, CA). Briefly, monocytes will be allowed to adhere for 2 h; supernatants and non-adherent cells removed to eliminate lymphocytes, and fresh complete RPMI medium supplemented with rBo IL-4 and rBo GM-CSF, preheated at 37Â°C, added. At days 3 and 6, half the culture medium replaced by fresh preheated complete RPMI medium with rBo IL-4 and rBo GM-CSF. Cells harvested at D7 will be defined as immature DC (iDC). Mature DC (mDC) will be obtained after additional 48 h stimulation with 10 ng/ml sonicated lipopolysaccharide (LPS). To monitor the differentiation into DC of monocytes isolated from cows and cultured in the presence of IL-4 and GM-CSF, we will analyze the expression of CD14 (VMRD: MM61A) and CD11c (VMRD: BAQ153A), which are monocyte and DC markers, respectively. Expression will be analyzed by flow cytometry at days 1, 2, 3 and 7 of the culture and the percentage of cells within the cell population that was positive for CD14, CD11c or both markers will be determined.
RNA from milk (or dry cow secretion) cells will be extracted using RNeasy Mini Kit (Qiagen) according to the manufacturer's protocol. RNA will be further treated with DNase to eliminate contaminating genomic DNA. The purified RNA will be quantified by spectrophotometric measurements and its purity and integrity verified by the OD260/OD280 ratio (> 1.8). Controls without reverse transcriptase will also be included for qRT-PCR analyses to validate the absence of genomic DNA. Quantitative Real-Time Reverse Transcription PCR (qRT-PCR) using the QuantiTect SYBR Green RT-PCR Kit (Qiagen) and the ABI Prism 7900 Sequence Detection System (PE Applied Biosystems, Forster City, CA), will be used to measure mRNA abundance. Primers will be designed with the aid of publicly available bovine sequences for cytokine IL-4, IL-10 and IFN-Î³. Glyceraldehyde 3-Phosphate Dehydrogenase will be used as the reference gene. A constant amount of RNA (~10 ng) will be used for qRT-PCR measurements.
Affymetrix gene chip assay:
A 10-20 g sample of mammary tissue will be surgically removed from each udder quarter in less than 10 min after euthanasia of the cows, snap frozen in three segments in liquid nitrogen and stored at -80Â°C. The secretory tissue samples will be taken from carefully selected regions of each udder quarter avoiding large blood vessels and the cisternal region. Frozen mammary tissue (~3 g) will be placed in liquid nitrogen, pulverized with a hammer, followed by addition of 25 ml of Trizol reagent (Invitrogen, Carlsbad, CA). The tissue will be homogenized and RNA extracted according to the manufacturer's protocol. The purified RNA quantified by spectrophotometric measurements and its purity and integrity verified by the OD260/OD280 ratio (> 1.8). Hybridization and gene array analyses will be performed using a previously described array (Tao et al., 2004). Briefly, the array will contain 167 immune and endocrine genes grouped into the following categories: cytokine genes, chemokines and their respective receptor genes, immune regulatory genes, T-cell associated genes, pattern recognition or innate host defense genes, immunglobin (Ig) or Ig-related genes, complement genes, antigen processing or presenting genes, and genes encoding endocrine hormones or their receptors. As positive controls, the array will include five "housekeeping" genes (GAPDH, HPRT, PRL19, Î²-actin and Î²2-microglobulin).
Results will be analyzed using a SAS statistical analysis program (SAS Institute Inc., Cary, NC). The difference between control and challenged quarters analyzed using ANOVA and Student's t-test. Differences are considered statistically significant when the probability of a type I error is < 0.05.