Bibliografische Informationen der Deutschen Bibliothek

Bibliografische Informationen der Deutschen Bibliothek Die Deutsche Bibliothek verzeichnet diese Publikation in der Deutschen Nationalbibliografie; Detaillierte bibliografische Daten sind im Internet über http://dnb.ddb.de abrufbar. 1. Auflage 2011 2011 by Verlag: Deutsche Veterinarmedizinische Gesellschaft Service GmbH, Giessen Printed in Germany ISBN 978-3-86345-038-0 Verlag: DVG Service GmbH Friedrichstraße 17 35392 Gießen 0641/24466 geschaeftsstelle@dvg.net www.dvg.net

University of Veterinary Medicine Hannover In vitro and ex vivo studies of biocompatibility of magnesium-silver alloys and their antimicrobial effects on bovine bacterial species Thesis Submitted in partial fulfillment of the requirements for the degree Doctor of Veterinary Medicine Doctor Medicinae Veterinariae (Dr. med. vet.) by Yousra Ahmed Reyad Nomier Alexandria / Egypt Hannover, Germany 2011

Academic supervision: Univ.-Prof. Dr. Manfred Kietzmann Department of Pharmacology, Toxicology and Pharmacy University of Veterinary Medicine Hannover, Germany 1. Referee: Univ.-Prof. Dr. Manfred Kietzmann 2. Referee: Univ.-Prof. Dr. Martina Hoedemaker, PhD Clinic for cattle University of Veterinary Medicine Hannover, Germany Day of the oral examination: 17.08.2011

To my parents, my husband; and my sons I am grateful for all of you, all my love and care

In the name of Allah, the Most Beneficent, the Most Merciful Verily, my salat (prayer), my sacrifice, my living and my dying are for Allah, the lord of the Alameen Al-An3am 162, the Holly Quran

Contents Contents 1 Introduction... 1 2 Literature Overview.....4 2.1 Bovine udder.....4 2.1.1 Anatomy of bovine udder and natural defense systems... 4 2.1.2 The teat canal barrier.......4 2.1.3 The streak canal.....6 2.1.4 Teat cistern (Sinus papillaris)...6 2.1.5 Fürstenberg's rosette....6 2.1.6 Cricoid rings (Annular folds)...7 2.2 Drying off period...7 2.3 Mammary epithelial cells..9 2.4 Mastitis. 11 2.4.1 Causative factors of mastitis. 12 2.4.2 Pathogenic agents of mastitis. 13 2.4.2.1 Staphylococal mastitis 14 2.4.2.2 Streptococcal mastitis. 14 2.4.2.3 Arcanobacterium pyogenes...14 2.4.2.4 Coliform mastitis....15 2.4.2.5 Mycoplasmal mastitis..15 2.4.3 Mastitis and somatic cell counts...........15 2.4.4 Mastitis in organic dairy herds.........16 2.4.5 The main environmental pathogens of mastitis........17 2.4.6 Clinical and subclinical mastitis...18 2.4.7 Treatment of mastitis...... 19 2.4.7.1 Parenteral treatment...19 2.4.7.2 Udder infusions....19 2.4.7.3 Treatment of dry cows...20 2.4.7.3.1 The aim of dry cow therapy...21 2.4.7.3.2 Dry cow preparations. 21 2.4.7.3.3 Systemic dry cow therapy..22 I

Contents 2.4.7.4 Drying-off chronically affected quarters...23 2.4.7.5 Supportive therapy. 23 2.4.8 Control of mastitis..23 2.5 Magnesium......25 2.5.1 Biochemistry of magnesium..25 2.5.2 Physiology of magnesium..27 2.6 Silver..28 2.6.1 Mode of action...28 2.6.2 Cytotoxicity of silver..30 2.6.3 Antibacterial activity of silver 31 2.6.4 Uses of silver..34 2.6.4.1 In health and medicine...34 2.6.4.2 Non medical uses of silver.36 3 Materials and Methods. 37 3.1 Experimental setting. 37 3.2 Materials.38 3.2.1 Cell culture...38 3.2.1.1 Culture media...38 3.2.2 Cell cultures.41 3.2.3 Cell culture reagents.........41 3.2.4 Cell culture disposable materials.42 3.2.5 Cell culture counting and viability...42 3.2.6 Cell culture equipments.....43 3.2.7 Magnesium-silver 1 % (MgAg1%) sticks and silver nitrate 43 3.2.8 Materials and reagents.44 3.2.8.1 Materials and reagents for Bio-Rad assay...44 3.2.8.2 Materials and reagents for measuring cytokines (interlekin-1beta (IL-1beta), interleukin- 6 (IL- 6), tumour necrotic factor (TNF-alpha).....44 3.2.8.2.1 Equipments used for measuring cytokines... 44 3.2.9 Materials and reagents used for measuring silver concentrations....44 II

Contents 3.2.10 Materials and reagents used for measuring calcium and Magnesium concentrations......44 3.2.11 Materials and reagents used for the brilliant black reduction test (BRT MRL screening test)...45 3.2.11.1 Equipments used for BRT MRL screening test...45 3.2.12 Materials and reagents used for measuring Prostaglandin E 2......45 3.2.12.1 Equipments used for measuring PGE 2.45 3.2.13 Materials and reagents used for the bouillion dilution test..45 3.2.14 Materials and reagents used for histology and histochemistry...45 3.2.14.1 Equipments used for histology and histochemistry.46 3.2.15 Materials used for succinate dehydrogenase test (SDH).47 3.2.16 Materials used for pyruvate kinase test (PK)......47 3.2.17 Materials used for isolated perfused bovine udder......47 3.2.17.1 Equipments used for isolated perfused bovine udder.........48 3.2.18 Materials and reagents used for the lactate dehydrogenase assay 48 3.2.19 Materials and reagents used for the glucose assay...48 3.2.20 Materials and reagents used for the lactate assay....48 3.2.21 Analytical equipments......48 3.2.22 Solutions and buffers.......49 3.3 Methods......53 3.3.1 Bovine udders.....53 3.3.2 Cell culture experiments...53 3.3.2.1 Primary mammary epithelial cell cultures........53 3.3.3 Separation of primary mammary epithelial cells contaminated with mammary fibroblasts...55 3.3.4 Culturing primary mammary fibroblasts 55 3.3.5 Cell counting... 56 3.3.6 Immunocytochemistry.... 57 3.3.7 Treating of the cells..... 57 3.3.7.1 AgNO 3 treatments...... 57 3.3.7.2 Incubation with MgAg1% sticks..........58 III

Contents 3.3.8 Degradations process of MgAg1% sticks...59 3.3.8.1 The isolated perfused udder.......59 3.3.8.1.1 Preparation of the udder... 59 3.3.8.1.2 Measuring parameters of viability of bovine udder.....60 3.3.8.2 The degedradation experiments.. 61 3.3.8.3 Histological examination............62 3.3.8.4 Degradation in dry off period secretions.....63 3.3.8.5 Determination of the silver concentrations in the degradation medium.....63 3.3.8.6 Determination of the magnesium and calcium concentrations in the degradation medium....64 3.3.9 Biocompatibility tests...66 3.3.9.1 Measurements of cell viability and proliferation...66 3.3.9.1.1 MTS assay (3 - (4, 5-dimethylthiazol -2-yl) -5- (3-carboxymethoxyphenyl)- 2- (4-sulfophenyl) - 2H-tetrazolium]).....66 3.3.9.1.2 Neutral red assay.........66 3.3.9.2 Measurements of metabolic activity...68 3.3.9.2.1 Measurement of the succinate dehydrogenase (SDH) activity in the supernatant 68 3.3.9.2.2 Measurement of the pyruvate kinase (PK) activity in the supernatant...69 3.3.9.2.3 Determination of the protein content............70 3.3.9.3 Histochemistry analysis...............71 3.3.10 Measurements of biomarkers of inflammatory reactions.......72 3.3.10.1 Preparations of the udder tissue for PGE 2 measurements.......72 3.3.10.2 Measurements of PGE 2 in the udder tissue supernatants........72 3.3.10.3 Measurements of bovine and mouse TNF-alpha in the culture medium supernatant 73 3.3.10.4 Measurements of IL-6 in the culture medium supernatant........73 3.3.10.5 Measurements of IL-1 in the culture medium supernatant........73 3.3.11 Detection of antibacterial activity.....74 3.3.11.1 Bouillion dilution testand cultivation of bacteria in petri dishes........74 IV

Contents 3.3.11.2 Brilliant black reduction (BRT-MRL screening test).......74 3.3.12 Statistical analysis........76 4 Results...77 4.1 Establishment of mammary cell culture..77 4.1.1 Isolation of primary mammary cells 77 4.1.2 Culturing of primary mammary cells......77 4.1.3 Verification of primary mammary cells using immunocytochemistry 78 4.2 Degradation process of MgAg1 % sticks......79 4.2.1 Isolated perfused udder 79 4.2.1.1 Measuring lactate production in the perfused udder...79 4.2.1.2 Measuring glucose consumption in the perfused udder..79 4.2.1.3 Measuring lactate dehydrogenase enzyme (LDH) amounts in the perfused udder..80 4.2.2 Degradation of MgAg1% sticks in bovine udder 80 4.2.3 Degradation of MgAg1% sticks in secretion samples from cows at dry off Period..82 4.2.4 Histological parameters......84 4.2.4. Udder tissue incubated with MgAg1% sticks...84 4.2.5 Silver concentrations in the degradation medium......85 4.2.6 Magnesium and calcium concentrations in the degradation medium..85 4.3 Biocompatibility tests.87 4.3.1 Cell viability and proliferation......87 4.3.1.1 MTS assay 87 4.3.1.2 Neutral red assay.....93 4.3.1.3 Measuring the amount of protein in the supernatant....99 4.3.2 Measurement of metabolic activities......100 4.3.2.1 Measurement of SDH activity in the supernatant..100 4.3.2.2 Measurement of PK activity in the supernatant.....103 4.3.2.3 Succinate staining of tissue and cells.........106 4.3.2.3.1 Teat tissues.........106 4.3.2.3.2 Primmary mammary epithelial cells........107 V

Contents 4.3.3 Biomarkers of inflammatory reactions......108 4.3.3.1 Measurement of IL-1 beta in the culture medium supernatant..108 4.3.3.2 Measurement of IL- 6 in the culture medium supernatant...109 4.3.3.3 Measurement of bovine TNF-alpha in the culture medium supernatant...110 4.3.3.4 Measurement of mouse TNF-alpha in the culture medium supernatant 110 4.3.3.5 PGE 2 concentrations in udder tissue......112 4.4 Antibacterial activity......113 4.4.1 Bouillion dilution test and cultivation of bacteria in petri dishes....113 4.4.2 Brilliant black reduction (BRT- MRL screening test)...115 5 Discussion.....117 5.1 Biocompatibility of silver ions and silver containing alloy..117 5. 1.1 Effect of AgNO 3 and MgAg1% sticks on the viability of different cells..117 5.1.2 Effect of AgNO 3 and MgAg1% sticks on the metabolic activities...118 5.1.3 Effect of AgNO 3 and MgAg1% sticks on the biomarkers of inflammatory reactions.. 120 5.2 Detection of the degradation of MgAg1% sticks...121 5.3 Antibacterial activity of MgAg1% sticks....122 5.4 Outlook..125 6 Summary..127 7 Zusammenfassung...129 8 Appendix..131 9 References 141 10 Abbreviations...177 11 Acknowledgements..179 VI

Introduction 1 Introduction Bovine mastitis remains the largest hazard in the global dairy industry. It has been investigated for 100 years, but it has been very complicated to achieve progress in control, because mastitis is caused by several types of infection and each one is the result of different etiology. Occurrence of mastitis depends on the interaction of host, agent, and environmental factors. Mastitis is defined as an inflammation of the mammary gland, which occurs primarily in response to intramammary bacterial infection, in addition to intramammary fungal or algal infections. Intramammary infection may also occur due to mechanical, thermal and chemical trauma. Mammary tissue damage has been shown to be induced by either apoptosis or necrosis. Mastitis is the most costly disease in dairy cattle, and reduces the number and activity of epithelial cells and consequently contributes to decreased milk production, as well as an influx of somatic cells, primarily polymorphonuclear neutrophils, into the mammary gland. Understanding the immune defenses of the mammary gland is instrumental in devising and developing measurements to control mastitis and interactions of mastitis-causing bacteria such as Escherichia coli (E. coli) or Staphylococcus aureus (S. aureus). The mammary gland represents a suitable model for studies on innate immunity at an epithelium frontier. Powerful new research tools are radically modifying the prospects for the understanding of the interplay between the mammary gland innate defenses and mastitis-causing bacteria. Mastitis induces at least minor but at most fatal illness to the affected animal, causes major economic losses due to reduction in milk yield and waste of milk unfit for consumption, and entails massive antibiotic use. The prevention and treatment of mastitis represent a serious burden to producers and are primary concerns of the dairy industry. In spite of the efforts deployed to control it, the incidence of mastitis continues to be one of the highest of all cattle diseases, and as a result of the long-lasting feature of subclinical mastitis, the most common form of the disease. Its prevalence in dairy herds remains at the forefront on the international scale. 1

Introduction Although antibiotics are very useful to treat the infection, they do not directly protect the gland from being damaged. Antibiotics do not provide a control, but they enable losses to be contained and subsequently made the partial prevention of infection. As they interfere with manufacturing process due to the presence of antibiotic residues in milk, they affect the human consumption of dairy products, in addition to labor and veterinary costs. The aim of the study was to evaluate the ability of MgAg1% alloys as a treatment of mastitis in dry off period. Although current permanent implants have not already reached a very high medical standard, there still exist some difficulties which remain to be resolved. Degradable MgAg1% alloys have some unique properties which make them attractive for certain applications. Among metals with antimicrobial activity, Ag + has raised the interest of many investigators because of its good antimicrobial action and low toxicity (BRUTEL DE LA RIVIERA et al., 2000; OLSON et al., 2002; KLASEN, 2000; HOLLINGER, 1996). As proven for Ag + treatments in wide range of medicine. S. aureus-derived α-toxin induced bovine mammary gland epithelial cells (BMEC) damage through DNA fragmentation, reactive oxygen species (ROS) generation, and dissipation of mitochondrial transmembrane potential (MTP). Recent study showed that Ag + ion treatment doses below 2 µg/ml inhibited the effect of α-toxin on cell death by blocking DNA fragmentation and reducing ROS generation. In addition, Ag ion concentrations below 2 µg/ml had no effect on DNA fragmentation and ROS generation, and therefore did not induce cell death of BMEC. It was reported that Ag ion doses lower than 2 µg/ml inhibit the S. aureus-derived α-toxin effect on BMEC and thus be used as a potential therapy against bovine mastitis, particularly in cases induced by S. aureus (SOEL et al., 2009). Furthermore, Ag ions are used as health food additives and medicines without any toxic side effects on the human health. For magnesium compounds, their main advantage is the fact that they are supposed to be degraded over a certain period of time. They are not removed in a second surgery, so magnesium implants do not act as foreign bodies, in addition to the good 2

Introduction biocompatibility, good clinical tolerance in animals and its low toxicity, too (SCHUMACHER et al., 2011). In the present project, our target was to determine whether the degradation products of MgAg1% alloys have an impact on the biocompatibility of BMEC, metabolic activities of BMEC and the bacterial colonies from E. coli and S. aureus. MgAg1% alloys shall degrade over time during the dry off period to provide antibacterial effects without any further cytotoxic effects. If that could be accomplished, it would be a major advance in the treatment of mastitis during dry off period which is still up to now of unsatisfying results with current antibiotics. 3

Literature review 2 Literature review 2.1 Bovine udder 2.1.1 Anatomy of bovine udder and natural defense systems The cow s udder consists of two pairs of mammary glands that are attached to the body in the inguinal region and are called quarters. Histologically, the lactating udder is a lobulated and contains large exocrine gland with dilated alveoli that store milk (GRUET et al., 2001). Secretory cells are closely linked together at their apex by tight junctions, this structure is largely responsible for the selective diffusion of drugs between both compartments and also for the blood-milk barrier (GRUET et al., 2001). The alveoli drain into interlobular ducts which converge and unite into the major galactophorous ducts which open into the gland cistern. The gland cistern is connected to the teat cistern, which opens into the narrow teat canal. The synthesis and release of milk constituents is continuous until temporarily suspended by the distending pressure. (HIBBITT et al., 1992; TANHUANPÄÄ, 1995). Intraepithelial lymphocytes are profuse around the proximal opening of the teat canal (Fürstenberg's rosette) which is kept closed by a sphincter. Its lumen is composed of layers of keratinising epithelium, which plays a great role in control of mastitis as it acts as physical barrier for bacteria (Fig. 1). The udder is supplied with a huge amount of blood to enable milk production (KAARTINEN, 1995)]. 2.1.2 The teat canal barrier The functions of the teat is the only exit for the gland secretions and the only way for the calf to receive milk, it is the first line of defense for the bovine udder as it is the first way where the pathogens can enter through. This canal is sealed between milking and during the dry off period by a keratin plug derived from the stratified epithelial lining of the canal (RAINARD and RIOLLET, 2006). Teat size and shape are not related to the amount and shape of the milk production of the udder. Average size for the fore teats is about 6.6 cm long and 2.9 cm in diameter and for the rear teats is 5.2 cm long and 2.6 cm in diameter (HURLEY, 1989; HIBBITT et al., 1992) as shown in (Fig. 1). 4

Literature review SL= small lymphocytes; MØ= macrophages; PMN= Neutrophils; NCF= Neutrophil Chemotactic Factor Fig. 1: Teat canal barrier (from HIBBITT et al., 1992). A vertical section of a cow s teat, showing on the left, factors that protect against mechanical trauma during suckling or milking, and on the right the defenses against ascending infections by bacteria from the skin surface (curved arrow) entering the teat canal. 5

Literature review 2.1.3 The streak canal The streak canal is the main barrier against intramammary infection since it is lined with skin-like epidermis that forms the keratin material which has antibacterial properties. The streak canal is kept closed by sphincter muscles. Canal patency decreases and streak canal length increases with increasing lactation number (HURLEY, 1989). 2.1.4 Teat cistern (Sinus papillaris) The cavity within the teat continues into the gland cistern. It is lined with many circular and longitudinal folds in the mucosa which form pockets on the inner lining of the teat. During milk letdown, the teat cistern fills with milk (Fig. 2). Fig. 2: Structure of the mammary gland showing teat and gland cisterns, milk ducts, and glandular tissue. Glandular tissue is made up of many small microscopic sacs called alveoli that are lined by milk producing epithelial cells (http://www.ag.ndsu.edu/pubs/ansci/dairy/as1129w.htm). 2.1.5 Fürstenberg's rosette These are mucosal folds of the streak canal lining at the internal end of the canal. It may fold over the canal opening due to pressure when the udder is full. Leucocytes may leave the teat lining and enter the teat cistern through this entry (HARLEY, 1989). 6

Literature review 2.1.6 Cricoid rings (Annular folds) It is located at the proximal end of the teat cistern, it differentiates the boundary between the teat cistern and the gland cistern. These rings are not recognizable in the dissected gland. 2.2 Drying off period The dry off period can be divided into three distinct phases (active involution, steady involution and colostrogenesis). The risk of developing mastitis is greater during the periods of active involution and colostrogenesis. Active involution is characterized by regression of mammary tissue, changes in composition of mammary secretion and rapid decline in milk production. During the dry off period, susceptibility to intramammary infections is greatest the two weeks after drying off and the two weeks prior to calving (SMITH et al., 1985). Many infections acquired during the dry period persist to lactation and become clinical cases. The drying off period is the time of the greatest susceptibility to new environmental streptococci infections especially the first 1-2 weeks and the last 7-10 days before calving or early lactation.the rates of new intramammary infections caused by coliform are greater during the first and last quarter of the same period. In addition, the rates of coliform infections are greater during the dry off period than during lactation (EBERHART et al., 1979). Research has shown that 65% of coliform clinical cases that occur in the first two months of lactation are intramammary infections that originated during the dry period (SMITH et al., 1985). Coliforms are adept at infecting the mammary gland during the transitional phase from lactating to fully involuted mammary gland. Infections during the early drying off period are controllable by dry cow antibiotic therapy, but those in the late dry off period are not. The dry off period is an important focus for mastitis control strategies in dairy herds (NEAVE et al., 1950; SMITH et al., 1985a; OLIVER and SORDILLO, 1988; BURVENICH et al., 2003), because many intramammary infection that occur during the dry off period carry into the next lactation causing clinical mastitis (HOGAN and SMITH, 2003; SORDILLO, 2005). In 7

Literature review dairy cows the dry off period is important to replace senescent mammary epithelial cells (MEC) to maximize milk production in the ensuing lactation (HURLEY, 1989; CAPUCO et al., 1997). It is also important to facilitate cell turnover in the bovine mammary gland and to optimize milk production in the next lactation (PEZESCHKI et al., 2009), although shortening the dry off period has been reported to cause negligible milk production loss (GULAY et al., 2003; SCHAIRER, 2001; BACHMAN, 2002; ANNEN et al., 2004a). Recent studies demonstrated that milk yield is reduced in cows with short dry off periods (MADSEN et al., 2004; GULAY et al., 2005; KUHN et al., 2005 and 2007; RASTANI et al., 2005; KUHN et al., 2006; PEZESHKI et al., 2007, 2008; CHURCH et al., 2008; GALLO et al., 2008; WATTERS et al., 2008). The dry off period is also an important time to control intramammary infection for many reasons, because it is well known that many clinical coliform mastitis cases occuring during early lactation originate from new intramammary infection at the end of the dry off period (colostrogenesis), so it is an ideal period to treat intramammary infection (SMITH et al., 1985b). Mammary defense may be affected by modifying the dry off period length as there will be poorer quality of colostrum for calves of continuous milking cows. In addition, the colostrogenesis period is not sufficient for gamma globulin accumulation in these cows (REMOND et al., 1997a). Bovine mammary glands are protected from intramammary infection during mid dry off period, when fluid volume is reduced (NEAVE et al., 1950). The opportunity for new intramammary infection is increased, when milk accumulates in the glands. This is inversely related to mastitis resistance (BURVENICH et al., 2007). The mammary gland reduces its capacity to secrete milk in response to intramammary infection (HARMON, 1994). Increased yield at drying off period has been associated with high risk of new intramammary infection during calving and that refers to level of milk components, leaking milk and intramammary pressure (HUXLEY et al., 2002; BRADLEY and GREEN, 2004; RAJALA-SCHULTZ et al., 2005). Four hypotheses have been proposed to explain the need for a non-lactating period between successive lactations in dairy cows (SWANSON, 1965; SMITH et al., 1967; SWANSON et al., 1967; CAPUCO et al., 1997). The first hypothesis is nutritionally 8

Literature review based and suggests that a dry off period is required for cows to have sufficient body reserves before calving to support optimal milk production in the subsequent lactation. Determination of dry peroid lenght depends on productivity and body condition of the cow. Afterwards, it was reported that a pronounced reduction in milk yield was observed in undernourished cows with short dry period (DICKERSON and CHAPMAN, 1939). This hypothesis was subsequently disproven by results of other studies, these studies were applied on cows exhibited improved body weights and lower milk production (SWANSON, 1965; LOTAN and ALDER, 1976). Furthermore, in a half-udder study, reduced milk yield for continuous milking quarters was observed despite equal nutrient availability to all quarters (SMITH et al., 1967). The second hypothesis is hormonally based and proposes that reduced milk production in cows with short or no dry periods is resulting from continuous influence of lactopoietic hormones. This hypothesis was disproved by utilizing an udder design demonstrating reduced milk yield in continous milking quarters compared with control quarters, despite exposure of all quarters to the same endocrine milieu (SMITH et al., 1967). The third hypothesis was based on cell number, suggesting reduced mammary epithelial cell number as a cause for depressed milk yield in cows with modified dry peroid lenght (SWANSON et al., 1967; CAPUCO et al., 1997). This was invalidated, as no differences in dry fat-free tissue weight, DNA concentration, total DNA content, or the number of alveoli per tissue section was observed in quarters with 6-week differences in dry peroid lenght. These authors, therefore, suggested that reduced milk in continuous milking quarters can be attributed to decreased secretory activity per unit of mammary secretory tissue and physiological factors affecting the cells during lactogenesis, rather than systemic hormonal regulation or mammary epithelial cells numbers. Using 3H-thymidine incorporation to evaluate mammary cell proliferation, CAPUCO et al. (1997) demonstrated 80% greater incorporation in mammary tissue from control (60-day dry) cows compared with continuous milking cows. They also reported that total mammary DNA content increased two fold from 5 to 7 days prepartum, but was not affected by lactation status. Therefore, a fourth hypothesis was proposed, suggesting that a dry off period 9

Literature review of appropriate length was necessary for promoting cell turnover and replacement of senescent mammary epithelial cells during late gestation (CAPUCO et al., 1997). 2.3 Mammary epithelial cells Mammary tissue cells or explants have been widely used over the years as models to understand the physiological function of mammary glands (HU et al., 2009). The bovine mammary gland epithelial cell culture is an established in vitro model that is beneficial for studying the different functions of the mammary gland. These cell cultures and cell lines were developed to study hormonal influences on milk protein expression (BAUMRUCKER, 1980). In addition, mammary epithelial cells were infected with S. aureus to study events including the induction of apoptosis, cell tropism of bacteria and bacterial adherence and invasion (WESSON et al., 2000). To overcome most of the difficulties they faced, all efforts have been placed on cell culture methods for studying enzymatic activities, biochemical properties, hormonal responses and growth of mammary epithelial cells. Some of these previous works have led to the development of stable epithelial cell lines of bovine mammary gland (ZAVIZION et al., 1992). Mammary epithelial cell proliferation is higher in glands that are permitted to have typical dry period than in those continuously milked prepartum (CAPUCO et al., 1997). Mammary epithelial cell proliferation is reduced in continuous milking cows throughout the last 35 days of gestation, however, net mammary growth in these animals was not impaired (CAPUCO et al., 1997). In common, it has been stated in a number of studies that it is more difficult to establish primary cultures in vitro from tissue taken from a lactating mammary gland, but it is better to be taken from a developing mammary gland, to avoid loosing the ability of the cells to differentiate (ROSE et al., 2002). At birth, bovine mammary parenchyma consists of a rudimentary duct network connected to a small cisternal cavity which connects to the teat cistern (CAPUCO and ELLIS, 2005). At the beginning, bovine mammary terminal ductal unit consists of solid cords of epithelial cells that penetrate into the mammary stroma. As the primary cord of epithelial cells within the terminal ductal unit (and surrounding loose connective tissue) extends into the mammary fat pad, the terminal ductal unit contains 5-10 separate ductule outgrowths that are arranged around the 10

Literature review central epithelial cord, while each epithelial cord contains approximately 4-8 layers of epithelial cells (CAPUCO et al., 2002) as shown in (Fig. 3). Fig. 3: Structure of mammary alveolus regarding to mammary epithelial cells (HURLEY, 1989) 2.4 Mastitis Mastitis is defined as an inflammation of the mammary gland (ZHAO and LACASSE, 2008). It is clearly associated with causing apoptosis or necrosis of mammary epithelial cells. There is likely a substantial impact of this disease in preventing the realization of an animal s full genetic potential to produce milk (KERR and WELLNITZ 2003). Mammary tissue damage reduces the activity and the number of epithelial cells and normally leads to decreased milk production (ZHAO and LACASSE, 2008). It is also characterized by an influx of somatic cells, primarily polymorphonuclear neutrophils (PMN) into the mammary gland and an increase in milk protease content (KERR and WELLNITZ, 2003). Mastitis is recognized as the most costly disease in dairy cattle. About 70 % of total cost of mastitis is due to decreased milk production. The affected quarters suffers 30 % reduction in productivity while the cow looses about 15 % of its production. The infection in dry period causes about 35 % reduction in the production of the next lactation, while the infection in late lactation is about 48% reduction in the yield (RADOSTITS et al., 2000). Mastitis is the most dreadful disease confronting the dairy industry throughout the world (BILAL et al., 2004). The 11

Literature review economic losses of mastitis in relation to mortality rate are negligible, but the production losses are great due to lowered milk quality or quantity, unsuitable for human consumption and interfering with manufacturing process. Some cases have a public health importance such as streptococcal sore throat, food poisoning due to s. aureus, tuberculosis and brucellosis (ANDREWS, 1992). Destruction of affected quarters and increased charges of laboratory and veterinary treatment and culling processes are tremendous (NICKERSON, 1990). Mastitis is the most common reason for the use of antimicrobials in dairy cows (MIECHELL et al., 1998; GRAVE et al., 1999). Antimicrobials have been used to treat mastitis for more than fifty years, but consensus about the most efficient, safe, and economical treatment is still lacking. The concept of evidence based medicine has been introduced to veterinary medicine (COCKCROFT and HOLMES, 2003) and should apply also to treatment of mastitis. The impact on public health should be taken into account as dairy cows produce milk for consumption (OIE, 2008). 2.4.1 Causative factors of mastitis Our understanding of mastitis has developed in several stages over the last 100 years. An association between mastitis and pathogenic microorganisms was established in 1887 (MUNCH-PETERSEN, 1938). Most major pathogens were identified by the 1940s, when antimicrobial therapy became available for production animals in 1945. It was effective in the control of some, but not all mastitis pathogens (EDWARDS et al, 1946; DOWNHAM and CHRISTIE, 1946). This prompted further research into potential husbandry related causes of mastitis. In the 1960s, the multifactorial etiology of bovine mastitis was commonly recognized (NEAVE, 1969; FELL, 1964). Today, mastitis is considered to be a multifactorial disease, closely related to the production system and environment that the cows are kept in (ANDREWS, 1992). Mastitis risk factors or disease determinants can be classified into three groups: host, pathogen and environmental determinants. Many infective agents have been implicated as causes of mastitis and these are dealt with separately as specific entities. The common causes in cattle are streptococcus agalactia (Str. agalactiae) and S. aureus with E. coli becoming a significant cause in 12

Literature review housed or confined cattle principally in the northern hemisphere (RADOSTITS et al., 2000). 2.4.2 Pathogenic agents of mastitis Over 200 different organisms have been recorded in scientific literature as being causes of bovine mastitis. They can be grouped as in Tab. 1. Tab. 1: The different pathogenic agents of mastitis (RADOSTITS et al., 2000; ANDREWS, 1992) Major pathogens Minor pathogens - S. aureus - Corynebacterium bovis - Str. agalactiae, Str. uberis, Str. dysagalactiae - Staphylococcus epidermis - Str. zooepidermicus, Str. pyogenes - Staphylococcus hyicus - E. coli - Arcanobacterium (Corynebacterium) Minor pathogens are constant - Campylobacter jejuni inhabitants of teats and - Mycobacterium bovis mammary glands. - Pasteurella multocida, Mannheimia hemolytica - Mycoplasma bovis - Listeria monocytogenes Leptospira include: - Leptospira interrogens Fungal infections include: - Aspergillus fumigatus - A. nidulans Yeast infections include : - Candida spp. - Cryptococcus neoformans - Saccharomyces spp. - Trichosporon spp. 13

Literature review 2.4.2.1 Staphylococal mastitis It is caused by S. aureus infection, and is characterized by its poor response to antibiotic therapy due to the ability of the organism to survive inside polymorphonucleocytes, macrophages and epithelial cells. This protection from antibiotic action may significantly contribute to therapy resistance (RADOSTITS et al., 2000). Additionally, the pathological changes, as granulomas and fibrosis, induced in chronic staphylococcal infections render chronically infected cows essentially incurable. Most commonly, staphylococcal udder infection is chronic, while acute mastitis is less common than with other bacteria. However, acute gangrenous staphylococcal infections can arise, in which uncontrolled growth of the organism occurs, elaborating large quantities of alpha toxin. Such infections are probably not due to strains of increased virulence, but rather to failures by the host to mount an effective defense (HUNGERFORD, 1990). 2.4.2.2 Streptococcal mastitis It is much more easily eliminated by intramammary antibiotic therapy, and may be eliminated from herds employing teat disinfection and dry cow therapy effectively and routinely. The disease may exist as an acute clinical mastitis or persist as a subclinical infection. The duration of infection is shorter due to its better response to therapy. Outbreaks indicate poor hygiene and therapy (BRAMLEY, 1984). In the absence of antibiotic dry cow therapy, the number of new Str. uberis infections increased markedly, especially during the early dry period and also near calving (OLIVER et al., 1996). 2.4.2.3 Arcanobacterium pyogenes It usually causes summer mastitis or heifer mastitis or dry cow mastitis, which is usually peracute or acute in nature. This syndrome occurs sporadically in dry cows or pregnant heifers and sometimes in lactating cows. It is always a serious disease with a high mortality rate if not treated; the disease is most common in the summer and early autumn, as well as calving season (HILLERTON, 1988). Flies have been incriminated in the transmission of summer mastitis. The disease is characterized by 14

Literature review the presence of severe systemic and local reactions and the presence of thick greenish yellow pus with a foul odour in the milk (RADOSTITS et al., 2000). 2.4.2.4 Coliform mastitis It is usually peracute and acute in nature and is caused by coliform species including E. coli, Enterobacter aerogenes, and K. pneumoniae. Infection is more common in housed cows and commonly occurs around the time of calving. The primary source of infection is bovine faeces (environmental mastitis). This can be treated by supportive therapy such as large volumes of isotonic fluids, while the use of antiinflammatory drugs may also be helpful (RADOSTITS et al., 2000). 2.4.2.5 Mycoplasmal mastitis The most common cause is Mycoplasma bovis and other species such as M. bovigentalium, M. canadense, and M. californicum. Infection with mycoplasma often involves multiple quarters and is refractory to antibiotic treatment. The secretion may remain normal at the onset, although a granular or flaky deposit is recognized (JASPER, 1981). Swelling and firmness are common, but after a few days the mammary gland may reduce in size. Milk secretion is severely reduced, swelling of the supramammary lymph nodes occurs and there may be pyrexia, transient malaise and arthritis (RADOSTITS et al., 2000). Secretion of mycoplasma in the milk frequently lasts for two months and often for longer. The diagnosis requires the application of specific microbiological and serological tests in specialized veterinary diagnostic laboratories. These involve culture from milk using selective media (JASPER, 1981). 2.4.3 Mastitis and somatic cell counts Somatic cell counts (SCC) have long been used as a way of measuring milk quality. Most dairy companies base their milk pricing policy, among other things, on SCC values of the milk. The somatic cells consist mainly of immune cells that enter the milk compartment of the udder. Only a minority of these cells are dead cells from the 15

Literature review udder tissue (DAY, 2004). There are always small quantities of immune cells in the cow s milk, and their function is to protect the udder against infection by bacteria. The older the animal gets, the more somatic cells it tends to have in its milk. Similarly, the SCC levels are higher immediately after calving and towards the end of each lactation. When bacteria enter the udder, the number of immune cells increases rapidly, as the immune system attempts to overcome the infection. Once the infection has been cleared, the SCC levels gradually drop to normal (HUNGERFORD, 1990). In cases of chronic infection, where the bacteria persist in the udder, the SCC levels can remain high throughout the lactation. High SCC levels in the milk cause deterioration of the milk quality. It has been shown that levels above 500 000 cells/ml decrease cheese yield and affect yoghurt making (RADOSTITS et al., 2000). The shelf life of milk is also affected, but at a higher level of SCC. Consistently high SCC levels in a herd are usually a sign of high levels of subclinical mastitis. Most cases of subclinical mastitis are caused by contagious mastitis bacteria (S. aureus or Str. agalactiae), even though Str. uberis is increasingly considered to cause chronic mastitis as well (DAY, 2004). 2.4.4 Mastitis in organic dairy herds Mastitis incidence and patterns were surveyed in 16 organic (O) and 7 conventionally (C) managed dairy herds in the south of England and Wales in 1997-1998 (HOVI and RODERICK, 1999). Clinical mastitis incidence in survey herds is presented in Tab.2 below. Overall mastitis incidence was significantly lower (P<0.001) in O herds than in C herds, the incidence rates during the dry period were significantly higher in O herds than in C herds (P<0.001). There was a wide variation in incidence rates amongst both O and C herds. The lower incidence in O herds was related to a very low incidence in one large herd as listed in Tab. 2 16

Literature review Tab. 2: Mastitis incidence (cow cases/100 cow years) O herds C herds Overall 36.4 48.9 Lactation 37.6 54.5 Dry period 28.9 9.2 O herds = Organic managed dairy herds; C herds = conventionally managed dairy herds Average individual cow SCC levels were significantly higher in O herds (135.000 cells/ml) than in C herds (84.000 cells/ml; P<0.001), resulting in high subclinical mastitis levels in O herds (individual cow SCC> 200.000 cells/ml in 34% of all measurements). Another UK survey of dairy farms converting to organic milk production (WELLER and COOPER, 1996) found average levels of 45.8 cases of clinical mastitis/100 cows on 11 farms at the end of the conversion period, with mean annual somatic cell counts of 299.000. A German study of 268 organic dairy herds identified mastitis as the most important health problem. While incidence rates for mastitis were similar to those on conventional farms, the culling rates for mastitis were higher than on conventional farms (KRUTZINNA et al., 1996). Studies of health and disease control on 14 organic dairy herds in Denmark found similar levels of mastitis incidence to comparable conventional herds (VAARST, 1995). A Dutch study has identified somatic cell count control as a critical area in mastitis control under organic production standards. In the study, S. aureus mastitis was seen as the main mastitis problem on organic dairy farms, and the difficulty in controlling was attributed to poor diagnosis and non-use of antibiotic in dry cow therapy (BAARS and BARKEMA, 1997). 2.4.5 The main environmental pathogens of mastitis There are many examples of the cow s surroundings (environmental pathogens) which can cause mastitis, such as bedding, manure, soil, etc. Contagious mastitis pathogens such as S. aureus, Str. agalactiae are spread from infected udders to clean udders during the milking process through contaminated teat cup liners, hand 17

Literature review milkers, paper or cloth towels used to wash or dry more than one cow, and possibly by flies. While E. coli and Klebsiella infection can take place during or between milking, they are found in sawdust that contains bark or soil. They are usually associated with an unsanitary environment (manure and/or dirty, wet conditions). Approximately 70-80% of coliform infections become clinical (JONES and WARD, 1989). 2.4.6 Clinical and subclinical mastitis Clinical mastitis: It depends on the resistance of the mammary tissue and the virulence of the invading bacteria. The clinical findings of mastitis include abnormalities of secretion, abnormalities of the size, consistency and temperature of the mammary glands and frequently a systemic reaction (DAY, 2004). The clinical forms of mastitis are usually classified according to their severity, severe inflammation of the quarter with a marked systemic reaction is classified as peracute, severe inflammation without a systemic reaction as acute, mild inflammation with persistent abnormality of the milk as subacute and recurrent attacks of inflammation with little change in the milk as chronic (RADOSTITS et al., 2000). Subclinical mastitis: It is the most common form of mastitis. For every clinical case of mastitis, there will be 15 to 40 subclinical cases. There are no visible signs of the disease. There is no gross inflammation of the udder, no gross changes in the milk. It is 15-40 fold more common than clinical mastitis (RADOSTITS et al., 2000). SCC of the milk elevates and decreases the quality and production of milk. In addition, causing the greatest financial losses to dairy farmers, staphylococci produce toxins that help in migration of PMN to chemo attractants which destroy the alveolar structure, then the alveolar structure is replaced by connective and scar tissue. In addition, clogging the milk ducts, secretary cells revert to non producing state and alveoli begin to shrink. Clots are formed by the aggregation of PMN and blood clotting factors block small ducts and prevent complete milk removal (JONES and BAILEY, 2009). 18

Literature review 2.4.7 Treatment of mastitis Special bacterial types of mastitis require specific treatments. The degree of response depends on the causative agent, and the speed with which treatment is commenced, but general guidelines for treatment are divided into parenteral treatment, udder infusions, treatment at dry off period, drying-off chronically affected quarters and supportive therapy. 2.4.7.1 Parenteral treatment Parenteral treatment is advisable in all cases of mastitis in which there is a marked systemic reaction, to control or prevent the development of a septicaemia or bacteraemia and treatment of the infection in the gland. Parenteral treatment is indicated when the gland is badly swollen and intramammary antibiotic are unlikely to diffuse to all part of the glandular tissue, although diffusion of antibiotic from the blood stream into the milk is relatively poor (RADOSTITS et al., 2000). So, to produce therapeutic levels of antibiotic in the mammary gland by parenteral injection and to control systemic reaction, higher dosages are given twice daily for 5 successive days (RADOSTITS et al., 2000). 2.4.7.2 Udder infusions Udder infusions are the preferable way of treatment because of convenience and efficiency by using disposable tubes containing suitable drugs. Complete emptying of the quarter before infusion by parenteral injection of oxytocin is advisable. In cases of acute mastitis, this can be further aided by hourly stripping of the quarter, leaving the intramammary infusions until immediately after the last stripping. After intramammary infusions, emptying the gland (thus losing the antibiotic or other drugs) should be avoided for as long as possible. The aminoglycosides neomycin and framycetin or cephalosporins are the drugs of choice for use in case in which the infection may be either Gram positive or negative. Penicillin G is the drug of choice for Gram positive bacteria especially S. aureus (RADOSTITS et al., 2000). Different antibiotics are widely used for intramammary treatment of mastitis with wide range of cure rate as shown in Tab. 3. 19

Literature review Tab. 3: Comparative efficiencies of intramammary treatment of mastitis in lactating Preparation quarters (LELOUDEC, 1978) Dose Cure rate % Staphylococcus Streptococcus Coliform Recommended uses Penicillin G 100000 40-70 100 Nil In slow release units base, 2 infusions (48 h) interval Cloxacillin 500 mg 30-60 Up to 100 - In long acting Cloxacillin + Ampicillin 200 mg 75 mg 64 94 97 base,1 infusion 3 infusions, once daily for 3 days (NEWBOULD, 1977) Spiramycin 500 mg 45-82 56-3 infusions (24 h) interval Rifamycin 250 mg 59-73 74-3 infusions (24 h) interval Streptomycin + Pencillin 1mg 100000 units 40-70 100 80 3 infusions (24 h) interval Tetracyclines 200 50 Up to 100 Poor Daily 2-3 days 400 mg Chloramphenicol 200 mg 28 24 50 Daily for 24 days Neomycin 500 mg 36 30-67 25 Daily (or 48 h intervals) for 2 infusions 2.4.7.3 Treatment of dry cows The dry cow therapy is an intramammary treatment of the udder with antibiotics administrated at the end of lactation (NEAVE et al., 1966). This is carried out at the end of the last milking before the cow is turned out. In seasonal areas, farmers would prefer to dry off their cows over 2-3 weeks. This method permits a large number of infections to develop in the period right after drying off. A teat dip should be used on cows after treatment, and animals should be observed daily for a week or until the mammary gland has begun to involute and is not secreting milk. Cows with udders or quarters that become hard and swollen during the dry period may need additional treatment (JANOSI and HUSZENICZA, 2001). The use of teat sealants is another effective strategy directed toward reducing exposure. There are two types of teat selants, external teat sealants which generate 20

Literature review a latex, acrylic, or other polymer-based film over the teats that prevents entry of pathogenic bacteria into the teat canal (TIMMS et al., 1997) and the other type is internal teat sealants, it is an inert viscous paste composed of bismuth subnitrate. It is administrated into the teat sinus after dry off with the objective of preventing the pathogens from entering. The teat sealants resides in the teat canal for the duration of the dry off period and is removed at calving by manual stripping (GODDEN et al., 2003) 2.4.7.3.1 The aim of dry cow therapy During the dry period, elimination of the infection with an antibiotic is more likely than during lactation as the drug is not milked out and higher and more uniform concentrations of antibiotics are maintained in the udder. In addition, there are no economic losses due to discarding of antibiotic containing milk (SANDHOLM and PYÖRÄLÄ, 1995). Experimental evidence suggests that dry cow therapy is effective in controlling intramammary infection dueare to Str. agalactiae and S. aureus (NATZKE, 1971, 1981; BRAMLEY and DODD, 1984; SCHUKKEN el al., 1990). In low SCC herds, the administration of antibiotics at drying off period resulted in lower clinical mastitis incidence in the dry period. WILLIAMSON et al. (1995) examined the prophylactic effect of a dry cow antibiotic against Str. Uberis, and the therapy reduced significantly the incidence of both dry period and post calving infections. Other studies suggest that dry cow therapy can play an important role in the prevention of new infections with theses environmental organisms during the dry period (JANOSI and HUSZENICZA, 2001). Dry cow therapy is very effective against the contagious organisms Str. agalactiae and S. aureus, while most dry cow therapy products are reasonably effective against environmental streptococci, they are not effective against coliform bacteria such as E. coli (WALDNER, 1990). 2.4.7.3.2 Dry cow preparations The udder is most susceptible to new infections during the first weeks mostly caused by environmental pathogens as Str. Uberis, and last weeks caused by coliform 21

Literature review bacteria. Therefore, the therapy should be extended over the whole dry period (SMITH et al., 1985; OLIVER and SORDILLO, 1988). Dry cow antibiotic preparations including ß-lactames require good activity against S. aureus including ß-lactames producing strains, Str. uberis, Str. dysagalctie, Str. agalactiae (JANOSI and HUSEZNICA, 2001). As udders are not milked during the dry period, pathogens are not flushed out of the lower portion of the teat cistern. This may lead to new intramammary infections especially by skin colonizing staphylococci. The number of new infections is related to the bacterial population on teat ends. Therefore, exercise lots, loafing areas, stalls and maternity pens should be clean and dry. Animals on pasture should not be allowed in ponds and muddy areas. Although chlortetracycline is widely used in the treatment of lactating cows, it should not be used in dry cows as it tends to cause chemical mastitis especially when the udder is completely dry. It is better to sample the cows before drying off and treat only the infected quarters by using high efficiency products such as benzathine cloxacillin, while the unaffected quarters should be treated with less effective and much cheaper product as procaine penicillin with streptomycin. Intramammary injectors which contain narrow spectrum penicillin are widely used such as penicillin, cloxacillin, oxacillin, nafcillin, cephalosporins and spiramycin (RADOSTITS et al., 2000). It is advantageous if antibiotic bound to the tissues for an extended period and did not immediately diffuse from the udder into blood. The antimicrobial effect must be long lived, as the purpose is to form a deposit in the milk ducts of the udder from which the antibiotic is slowly released (SANDHOLM and PYÖRÄLÄ, 1995). 2.4.7.3.3 Dry cow therapy BOLOURCHI et al. (1996) found that systemic enrofloxacin or tylosin at drying off approached but did not exceed the efficacy of the local treatment with nafcillin, penicillin and dihydrostreptomycin. Norfloxacin-nicotinate was reported as effective drug for systemic treatment of S. aureus intramammary infection. 22

Literature review In general, the systemic administrations of antibiotics at drying off (JOHANSSON et al., 1995) or some weeks before parturitions (ZECCONI et al., 1999) seem to be an effective supplementary of S. aureus intramammary infection. Treatments in chronic cases, especially those caused by S. aureus, are often best cleared up by treatment when the cow is not lactating. Treatment at this time is a good prophylaxis. Most dry cow preparations maintain an adequate minimum concentration in the quarter for about 4 weeks, and some persist for 6 weeks (FRANCIS, 1991). Cloxacillin and cephalosporin are popular for this purpose. Dry period treatment is a part of the control program for bovine mastitis. SANDHOLM and PYÖRÄLÄ (1995) stated that there are many adverse effect of dry cow therapy such as discarded meat and milk. A random antibiotic therapy kills the normal bacterial flora of the teat end and teat canal giving a chance for antibiotic resistant bacteria to colonize. Using antibiotics in large scale increases the spreading of antibiotic resistant bacterial strains and irritation of the teat ends. 2.4.7.4 Drying-off chronically affected quarters If a quarter does not respond to treatment and is classified as incurable, the affected animals should be isolated from the milking herd. The affected quarter may be permanently dried-off by producing a chemical mastitis via udder infusion of 30-60 ml of 3% silver nitrate solution, 20 ml of 5% copper sulphate solution, 100-300 ml of 1/500 or 300-500 ml of 1/2000 acriflavine solution (RADOSTITS et al., 2000). If severe local inflammation occurs, the quarter should be milked out and stripped frequently until the reaction subsides. If no reaction occurs, the quarter is stripped out 10-14 days later to infusion. 2.4.7.5 Supportive therapy Supportive therapy includes the parenteral injection of large quantities of isotonic fluids, especially those containing glucose. Antihistamine drugs are indicted in cases where extensive tissue damage and severe toxaemia are present. Crushed ice in a bag suspected around the udder may reduce absorption of toxins (RADOSTITS et al., 2000). 23

Literature review 2.4.8 Control of mastitis The recommended mastitis control programme is based on the following points: Udder and teat sanitation: Before each milking, the udder and the teat should be washed with running water and soap, then individual paper towels are used for drying, and each teat should be dipped or sprayed with suitable teat disinfectant to reduce rates of new infection, before and after each milking. ROGER and PETER (1995) recommended chlorhexidine 0.2 % or teat iodophores as teat disinfectants. Monitoring the infection rate: Monitoring the infection rate is carried out by detecting of either clinical or subclinical infected quarters, by the use of preliminary screening test such as California mastitis tests (CMT), individual cow milk cell count (ICCC) or N.acetyl B-D glucosaminidase (NAGase) test. RADOSTITS et al. (2000) stated that in normal healthy cows, the cell counts are less than 100.000 cells/ml, while counts of less than 250.000 cells/ml are considered to be below the limit indicative of inflammation, while counts of more than 250.000 cells/ml on an individual basis and 400.000 in bulk milk samples are considered a mastitis case. Mastitis has been also monitored in milk by measuring concentrations of the enzyme NAGase. The higher the NAGase concentration the more likely the presence of pathogens and clinical infections (RADOSTITS et al., 2000). Treat clinical case of mastitis: Treating clinical cases of mastitis is used to assist the elimination of infection and the resolution of clinical signs of the disease. Dry period treatment: Dry period treatment is applied by infusion of long acting antibiotics into all quarters at drying off, to help eliminate a high proportion of subclinical infections present at the end of lactation and to prevent many new dry off period infections (ROGER and PETER, 1995). An annual milking machine test and appropriate maintenance: The annual milking machine test and appropriate maintenance is intended to ensure efficient milking and prevent machine induced infections. 24

Literature review Culling chronic cases: Culling chronic cases is applied, when cows have more than three clinical cases per lactation, all cows that do not respond to dry cow therapy and a chronically affected cow. Treatment of mastitis at dry off period with antibiotics was widely used, as it is the most important period for treatment. Many infections acquired during the dry off period persist to lactation and become clinical cases. Treatment with antibiotics showed high laboratory and medical costs and some resistance problems, so an alternative way of treatment using magnesium and silver 1% alloys at dry off period was applied in this study. 2.5 Magnesium 2.5.1 Biochemistry of magnesium Magnesium (Mg ++ ) is an attractive material for biodegradable implants because of its low thrombogenicity and well-known biocompatibility (PEUSTER et al., 2006). Mg ++ was chosen in many engineering applications because of its low corrosion resistance and also for biomaterial applications, where the in vivo corrosion of Mg ++ based implants involves the formation of soluble non toxic oxide which is excreted in the urine (STAIGER et al., 2006). It stimulates the growth of new bone tissue (YAMASAKI et al., 2003; REVELL et al., 2004), and it is well known that Mg ++ and its alloys were applied for its lightweight, degradable function, and load bearing orthopaedic implants (WEN et al., 2001; WITTE et al., 2005). Mg ++ is the most abundant divalent cation within the cell. The majority of Mg ++ is bound to proteins and cellular metabolites while the rest, which is a small fraction, is free in the cytosol and within intracellular organelles (VELSO et al., 1973; CORKEY et al., 1986). Mg ++ is the main intracellular earth metal cation with a free concentration in the cytosol around 0.5 mmol/l (GRUBBS and MAGUIRE, 1987; WILLIAMS, 1970; FLATMAN, 1991; SHILS, 1994; QUANME, 1997). Mg ++ is a smaller ion that attracts water molecules more rapidly (WIILIAMS, 1970; JUNG and BRIERLEY, 1994). Mg ++ binds to neutral nitrogen groups such as amino-groups and imidazol in addition to oxygen especially in acidic groups (WILLIAMS, 1970). The normal range of plasma Mg concentration is 0.75-1 mmol/l (WEISINGER and BELLORIN-FONT, 1998). In animal 25

Literature review experiments, it has been shown that a reduction of total intracellular Mg ++ can only be achieved by feeding fast growing animals a severely magnesium deficient diet (VORMANN et al., 1998). Only if plasma Mg ++ concentrations were reduced below 0.2 mmol/l, a slight reduction of intracellular Mg ++ could be detected indicating that the intracellular Mg ++ concentration is not in equilibrium with the extracellular space and effects of magnesium deficiency are mainly effects restricted to the extracellular functions of Mg ++ (VORMANN, 2003). In growing animals, Mg ++ deficiency induced a loss of bone Mg ++ within a few days (VORMANN et al., 1997). Therefore, bone Mg ++ represents a Mg ++ reservoir that buffers extracellular magnesium concentration. Animal experiments (RUDE et al., 1998) and human studies (LINDBERG et al., 1990; DIMAI et al., 1998) showed a positive effect of supplementing magnesium on bone density and bone absorption parameters. Mg ++ is an exceptionally lightweight metal with a density of 1.74 g/cm 3 and Mg ++ is 1.6 and 4.5 times less dense than aluminium and steel, respectively (DEGARMAO, 1979). The fracture toughness of magnesium is greater than ceramic biomaterials such as hydroxyapatite. Moreover, Mg ++ is essential to human metabolism and is naturally found in bone tissue (HARTWIG, 2001). Mg ++ is a cofactor in hundreds of enzymatic reactions (GRUBBS and MAGUIRE, 1987; WACKER and PARISI, 1986; ROMANI and SCARPA, 1992), and is especially important for those enzymes that use nucleotides as cofactors or substrates.the form of Mg ++ complex is the actual cofactor or substrate for the phosphotransferases and hydrolases such as ATPases which are of a higher importance in the biochemistry of the cell. It is really important, especially in energy metabolism. In addition, Mg ++ regulates signal transduction and the cytosolic concentrations of Ca ++ and K and also ion transport by pumps, carriers and channels (AUGUS and MORAD 1991; FALTMAN 1991; ROMANI and SCARPA, 1992). Positively charged Mg ++ is able to bind electrostatically to the negatively charged groups in membranes, proteins and nucleic acids. Accordingly, Mg ++ and mitochondria which contain large amount of Mg ++ (BRIERLEY et al., 1987; ROMANI et al., 1991) may influence the binding of other cations like Ca ++ and polyamines depending on their concentrations (SARIS and KHAWAJA, 1996). However, it is not well known, if the change in Mg ++ within the mitochondrial matrix can regulate the 26

Literature review activities of dehydrogenase and the rate of respiration. Generally, due to the electrical effects, Mg ++ is considered to have a membrane-stabilizing and protecting effect (BARA et al., 1990) and in many biochemical reactions it has the ability to inhibit phospholipase (SARIS and KHAWAJA, 1996). Mg ++ and polyamines bind efficiently to the negatively charged groups in nucleic acids, ribosome and membranes (KHAWAJA, 1871; ROWAT and WILLIAMS, 1992). The two types of cations are involved in the synthesis of DNA, RNA and proteins. It stabilizes the structures of DNA and RNA (HARTWIG, 2001). When the level of Mg ++ in the extracellular fluid exceeds the normal level, homeostasis is maintained by the kidneys and intestine (HARTWIG, 2001). While when serum Mg ++ levels exceed the normal levels, lead to muscular paralysis, hypotension, respiratory distress and cardiac arrest (SARIS et al., 2000). 2.5.2 Physiology of magnesium Mg ++ is important for normal neurological and muscular function. Hypomagnesaemia leads to hyperexcitability due mainly to cellular Ca ++ transport and signalling (WACKER and PARISI, 1986; GRUBBS and MAGUIRE, 1987; SHILS, 1994), The development of Mg ++ deficiency is usually linked either to disturbances in the intestinal Mg ++ absorption and/or to an increased renal Mg ++ excretion (WOOD et al., 1992). Mg ++ deficiency induces severe vascular damage in the heart and kidney, accelerates the development of atherosclerosis, causes vasoconstriction of the coronary arteries, increases blood pressure and induces thrombocyte aggregations (GULLESTAD et al., 1994). Mg ++ is required for protein, nucleic acid synthesis, the cell cycle, cytoskeletal and mitochondrial integrity and for the binding of substances to the plasma membrane (BEYENBACH, 1990). Mg ++ has vasodilatatory effects and also acts as a cofactor of adenosintriphosphatase and as a physiologic antagonist of calcium. Its low thrombogenicity is due to its fibrinolytic and anticoagulative properties (PEUSTER et al., 2006). Mg ++ is absorbed mainly in the ileum and in the colon (BEYENBACH, 1990; SHILS, 1994; QUAMME, 1997; KAYNE and LEE, 1993) and this process is carried out by a passive paracellular mechanism (BEYENBACH, 1990; SHILS, 1994; QUANME, 1997). 27

Literature review The kidney occupies a central role in magnesium balance. In turn, magnesium balance affects numerous intracellular and systemic processes (ALLEN et al., 1997; NINOMI, 2002). About 75 % of the total plasma Mg ++ is filtered through the glomerular membrane and only 15 % of the filtered Mg ++ is reabsorbed in the proximal tubules, and 50-60% in the thick ascending loop of Henle (SHILS, 1994; QUAMME, 1997). Under normal conditions only 3-5 % of the filtered Mg ++ is excreted in the urine (QUAMME, 1997), while transcellular Mg ++ transport takes place mainly in intestinal absorption and renal excretion (BEYENBACH, 1990). Many reviews state that Mg ++ is an orphan ion due to the absence of specific endocrine control as for Ca ++, Na + and K + (KELEPOURIS and AGUS, 1998). The main organ responsible for the regulation of Mg ++ is the kidney in addition to several hormones which are related to the influence of Mg ++ balance like parathyroid hormone (PTH) and calcitonin, vitamin D, insulin, glucagon, antidiuretic hormone, aldosterone and steroids (DEROUFFIGNAC et al., 1993; KELEPOURIS and AGUS, 1998). Mg ++ is involved in nerve conduction, muscle contraction, potassium transport, and calcium channels. Because turnover of magnesium in bones is low, the short-term body requirements are met by a balance of gastrointestinal absorption and renal excretion (HARTWIG, 2001). 2.6 Silver 2.6.1 Mode of action Silver (Ag + ) is a broad spectrum antibiotic and also characterized by having antiseptic and anti-inflammatory properties (LANSDOWN, 2002; DUNN and EDWARDS- JONES, 2004; ORVINGTON, 2004; FONG, 2005). Ionic Ag + is considered to be effective against a broad range of microorganisms with low concentrations in addition to its therapeutic activity (RUSSELL and HUGO, 1994, LANSDOWN et al,. 2002). Ag + has the ability to destroy microorganisms immediately by disturbing the bacterial cell membrane function and stop its cellular respiration (TREDGET et al., 1998; WRIGHT et al., 1998; DEMLING and De SANTI, 2001; LANSDOWN, 2002; THOMAS, 2003 a, b; DUNN and EDWARDS-JONES, 2004). 28

Literature review Ag + cations have the ability to bind to tissue proteins causing change in the structure and can also bind and denature the bacterial DNA and RNA, thus inhibiting cell replication (TREDGET et al., 1998; WRIGHT et al, 1998; DEMLING and De SANTI 2001; LANSDOWN, 2002; THOMAS, 2003a, b; DUNN and EDWARDS-JONES, 2004). In bacteria, Ag + ions are known to react with nucleophilic amino acid residues in proteins and attach to sulfhydryl (SH) groups as shown in (Fig. 4), amino, imidazole, phosphate and carboxyl groups of membrane or enzymes leading to the inactivation and denaturation of cellular proteins and the inhibition of bacterial oxygen metabolism (RUSSELL and HUGO, 1994; KAUR and VADEHRA, 1986; LIAU et al. 1997; LANSDOWN, 2002; HOSTYNEK et al., 1993). Active enzyme Inactive enzyme Fig. 4: Replacement of silver to sulphydryl group of active enzyme (SEOL et al., 2009) Recent studies propose that Ag + ions react with the thiol group of vital enzymes in order to inactivate them (Russell and HUGO 1994; LIAU et al., 1997; LANSDOWN, 2002). Ag + is also known to inhibit a number of oxidative enzymes such as yeast alcohol dehydrogenase (SNODGRASS et al., 1972; YUDKIN 1937; SNODGRASS et al., 1960), the uptake of succinate by membrane vesicles (RAYMAN et al., 1972) and the respiratory chain of E. coli. It interferes with DNA replication (FENG et al. 2000; RUSSELL and HUGO, 1994; SCHREURS and ROSENBERG 1982; RAYMAN et al., 1972) resulting in the marked enhancement of pyrimidine dimerization through photodynamic reactions and the potential prevention of DNA replication (RUSSELL and HUGO, 1994; MODAC and FOX, 1973). Ag + cations have also been shown to be associated with the bacterial 29

Literature review cell wall, membrane (ROSENKRANZ and CARR, 1972), cytoplasm and the cell envelope (GODDARD and BULL 1989). CHAPPELL and GREVILLE (1954) acknowledged that low levels of Ag + collapsed the proton motive force on the membrane of bacteria, and later work by DIBROV et al. (2002) showed that low concentrations of Ag + induced a massive proton leakage through the bacterial membrane, resulting in complete deenergization, and ultimately, cell death. Overall, there is consensus that surface binding and damage to membrane function are the most important mechanisms for killing of bacteria by Ag +. As silver is strongly binds to the bacterial cell surface with toxic effects and inhibiting the bacterial respiratory transport (LANSDOWN et al., 1997). However, the mechanisms underlying the antimicrobial effect of Ag + are still not fully understood (SEOL et al., 2010). Ag + is found in most tissues, but has no known physiologic function (GABRIELE and ROSEMARIE, 1992). 2.6.2 Cytotoxicity of silver Ag + has been described as being oligodynamic because of its ability to exert a bacteriocidal effect at minute concentrations (CLEMENT and JARRETT, 1994). That is why many healthcare products now contain Ag +, mainly due to its antimicrobial activities and low toxicity to human cells (SAMPATH et al., 1995; DASGUPTA, 1994). The word oligodynamic describes any metal which exhibits bactericidal properties of metal ions on living cells, virus and bacteria even in relatively low concentrations (CLEMENT and JARRETT, 1994) oligos = small - dynamis = power (BERK 1947; VON NAGELI, 1893). Studies in humans and animals indicate that Ag + compounds are absorbed through the oral and inhalation routes of exposure with some absorption occurring through both intact and damaged skin (ATSDR, 1990). A result of this absorption, patients may suffer argyria which is defined as permanent gray or blue-gray discoloration of the skin due to deposits of Ag + granules in dermis especially in regions around hair follicles and sweat ducts (LEGAT et al., 1998; BOUTS, 1999; SILVER, 2003). Among metals with antimicrobial activity, Ag + has raised the interest of many investigators because of its good antimicrobial action with low toxicity (KLASEN, 2000; HOLLINGER, 1996). Cytotoxicity of Ag + is considered to 30

Literature review below, while bactericidal power is high (COOPER et al., 1991). Some other literature stated that there is no cytotoxic effect of Ag + in addition of having good biocompatibility (BOSWALD et al., 1999). A toxic effect of Ag ions may be due to blockage of the electron transport system, depolarization or alteration of permeability of the mitochondrial membrane, or to mitochondrial DNA damage (HIDALGO and DOMINGUEZ, 1998). It is of the upmost importance to consider their potential cytotoxic action on target cells (fibroblasts, endothelial cells and keratinocytes) which are basically responsible for tissue regeneration (HIDALGO and DOMINGUEZ, 1998). There is no evidence of Ag + toxicity reported to cultured fibroblasts until concentrations reached levels of 1200 µg/ml (TWEDEN et al., 1997). Since toxicity of Ag + ions is poorly characterized, different aspects of cellular toxicity mechanisms in cultured human fibroblasts were studied (HIDALGO and DOMINGUEZ, 1998). Toxicity of Ag + to human cells is considerably lower than to bacteria, as animals and human cells have thick cell membrane which can not be easily disturbed by bacteria (CLEMENT and JARRETT, 1994). In one of the studies, toxicological side effects were analyzed in rabbits with Ag + coated megaprosthesis. The results showed that the Ag + concentrations around the prosthesis and in the organs did not cause histological changes or functional deficits of the organs. Furthermore, concentrations in the blood were on average 1.88 µg/ml (GOSHEGER et al., 2003), knowing that the concentrations below 10 µg/ml were considered normal (BRUTEL DE LA RIVIERE et al., 2000). Toxic side effects were described for blood concentrations of 300 µg/ml in the form of argyrosis, leukopenia, liver and kidney damage (BRUTEL DE LA RIVIERE et al., 2000; TWEDEN et al., 1997; CHAMBRES et al., 1962). In other studies using Ag + -coated heart valves, the blood concentration did not exceed 22 µg/ml (BRUTEL DE LA RIVIERA et al., 2000) and results showed no toxicological side effects. In fact, the Ag + concentration was significantly increased in all analyzed organs, especially in the liver and the spleen with mean values of 90.4 and 27.9 µg/ml, respectively, although the functional parameters and histological examination of the organs revealed no pathologic findings. 31

Literature review It is well known that Ag + toxicity is a dose-dependent process. Elementary Ag + is an inert metal and is ionized slowly in the organism (PERRELLI and PIOLATTO, 1992; BRUTEL DE LA RIVIERA et al., 2000). 2.6.3 Antibacterial activity of silver Ag + has been used as an antimicrobial agent since the 1800s. But since the discovery of systemic antibiotics in the early 20 th century, the use of Ag + has declined. In the last two decades interest in Ag + or wound treatment resurged. Ag + was known to be of antimicrobial activity centuries ago where ancient people used to keep water, wine milk and vinegar in Ag + vessels to stay fresh during long sea voyage, while Roman and Greek dropt silver ions into water to serve as disinfectants (FONG, 2005). This practice by the Imperial Russian army was continued through World War I, and by some units in the Soviet Army in World War II in 1939-1945 (www.naturdc.com/library/medsmats/silver.). One recent in vivo study reported on a protocol involving puncture, aspiration, injection, and re-aspiration with AgNO3 directly into hepatic hydatid cysts with beneficial long term results (ODEV et al., 2000). Antimicrobial coatings for the inside and outside of medical catheters using Ag + have been developed for latex, polyurethane, and Teflon devices. These Ag + coatings are very effective at blocking bacteria such as E. coli and S. aureus from entering the body along a catheter pathway (ZHAO and STEVENS, 1998). Bacteriocidal activity of Ag + has been reported at concentrations below 35 µg/ml (CHAMBRES et al., 1962). Ag + has an important antimicrobial effect (BRETT, 2006). This effect is dependent on superficial contact, in that Ag + can inhibit enzymatic systems of the respiratory chain and can alter DNA synthesis (HIDALGO et al., 2006). The antibacterial activity of Ag + has long been known. However, the mechanisms by which Ag + kills the cells are not known to date (CLEMENT and PENELOPE, 1994). Some studies showed that Ag + ions released from the surface coating and reach the antimicrobial effect in the surrounding tissue (ILLINGWORTH et al., 2000) and the antimicrobial effect of Ag + is caused by the Ag + ions which bind to membranes, enzymes and to nucleic acid (HOLLINGER, 1996; BRUTEL DE LA RIVIERE, 2000). 32

Literature review Antiseptics (such as Ag + ) and antibiotics have differing modes of action. Antiseptics tend to target multiple sites on or within bacterial cells in addition to having broad spectrum activity. Antibiotics tend to target specific sites on or within a bacterial cell and have a narrower spectrum of activity (PERCIVAL et al., 2007). In general, antiseptics or biocides resistance can be acquired through mutations in normal cellular genes, plasmids or transposons (DAVIES and ORIGINS, 1997). Plasmid-mediated biocide resistance has been documented (SONDOSSI et al., 1985; RUSELL, 1997) as occurring in S. aureus, coagulase-negative staphylococci, and members of the Enterobacteriaceae and Pseudomonas spp. (TENNENT et al., 1985, KUCKEN et al., 2000). The majority of biocides act on cell surface components of the bacteria and/or the cytoplasmic membrane. Therefore, intrinsic resistance would involve natural resistance through the structure of the cell surface and its chemical composition (TATTAWASART et al., 2000). But its not the same for Ag + as SILVER (2003) suggested that the probability of transfer of Ag + resistance genes is considered to be very low (HUGH et al., 1975; ANNEAR et al., 1976), unstable and difficult to maintain and transfer (HENDRY and STEWART 1979; DESHPARD and CHOPADE 1994). Recently, the importance of evaluating the combination of S. aureus virulence factors has been emphasized both in human and veterinary medicine (PEACOCK et al., 2002; BECKER et al., 2003; VON EIFF et al., 2004; HASLINGER-LOFFLER et al., 2005). Ag + exhibits potent antibacterial activity against a variety of bacterial species with maintaining low toxicity for mammalian cells (BERGER et al., 1976). S. aureus is a common cause of bacterial infection in animal mastitis (BRAMLEY et al., 1989). The production of alpha- and beta-toxins is very common among isolates from bovine mastitis (BRAMLEY et al., 1989). The majority of bovine S. aureus isolates carry the gene for alpha- and beta-hemolysin (AARESTRUP et al., 1995). In most of cases, α- toxin is used, because it is produced by most strains of S. aureus isolated from peracute, acute, and chronic cases of bovine mastitis (MATSUNGA et al., 1993), and because it produces extensive tissue damage (BHAKDI and TRANUM-JENSEN, 1991). S. aureus may damage the skin through production of reactive oxygen species (ROS) (OKAYAMA, 2005). Staphylococcal α-toxin is a powerful lytic toxin 33

Literature review with the ability to lyse a variety of cell types (ANDERSON and MASON, 1974). Knowledge regarding the effect of Ag ions on bovine mastitis caused by S. aureus is still lacking (SEOL et al., 2009). Recent study was able to clarify the effect of Ag + ions on α-toxin-induced cell death in bovine mammary epithelium cells (BMEC), its results stated that α-toxin (1 μg/ml) and Ag + ion treatment at 2 µg/ml did not induce cell death. The cell morphology also clearly distinguished cell death resulting from different Ag + ion doses. In addition, the ability of Ag + ions to block DNA fragmentation induced by α-toxins. That proved that Ag + ions could inhibit α-toxin-induced apoptotic cell death in BMEC. Also of interest is that ROS generation, dissipation of MTP, which facilates the release of cytochrome-c into cytosol resulting from α-toxin treatment, was reduced by the Ag + ion treatment. Results stated that Ag + ion doses lower than 2 µg/ml can inhibit the S. aureusderived α-toxin effect and thus be used as a potential therapy against bovine mastitis, particularly in cases induced by S. aureus (SEOL et al., 2009). In another study, a comparison between antibiotic surface coating and Ag + coated megaprostheses, Ag + coated might have major advantages, as there was no resistance had been described before. The effect of Ag + ions is continues and long lasting due to the oligodynamic effect of elementary Ag + (JANSEN et al., 1994, SCHIERHOLZ et al., 1998), and finally the ability of any bacteria to produce a biofilm is reduced and the likelihood of bacterial colonization is decreased (BOWSWALD et al., 1999, OLSON et al., 2002). In another study, Ag + coated external fixation pins and conventional stainless steel pins were placed in the iliac crest of sheep and inoculated with S. aureus. The pin sites were examined for inflammation with scanning electron microscopy (SEM). 84 % of the uncoated pins were infected in comparison to 62 % of the silver-coated pins (COLLINGE et al., 1994). In another study, for endoprosthetic replacement of the diaphyseal femur in rabbits. The infection rate was established in 15 rabbits with a titanium-vanadium prosthesis without Ag + coating and in 15 rabbits with a Ag + -coated endoprosthesis, after injecting a bacterial suspension S. aureus, the superficial Ag + coated endoprosthetic resulted in a significantly reduced infection rate (7% versus 47% in comparison with titanium- 34

Literature review vanadium without Ag + coating ). S. aureus was not able to cause infection with systemic illness (GOSHEGER et al., 2004). One of the researches stated that the use of Ag + coated endotracheal tubes in dogs significantly lowered colonization rates and reduced lung inflammation after bacterial challenge with Pseudomonas aeruginosa (OSLON et al., 2002). 2.6.4 Uses of silver 2.6.4.1 In health and medicine Dental amalgams, so-called Ag + fillings, contain about 35% Ag + and 50% Hg, but there is no evidence that sufficient Ag + is released and oxidized to Ag + which has an antimicrobial effect. The amounts of mercury released are sufficient to select for mercury-resistant bacteria in gut flora of animals with silver/mercury amalgams (LORSCHEIDER et al., 1995). Ag + containing consumer products include Ag + coated mints called Jintan in Japan. They are used for heartburn, nausea and vomiting, motion sickness, hangover, dizziness, bad breadth, choking, indisposition, and sunstroke and are also used as health food additives in Florida. Ag + is familiar in laboratory use as a stain for proteins in polyacrylamide gels, but the silver in stained gels is reduced polymeric Ag + (HEUKESHOREN and DEMICK, 1985). Ag + coated bandages are widely used in prophylactic treatment of burns (LO et al., 2002) wounds, diabetic wounds and traumatic injuries of humans (BECKER, 1999) and large animals (ADAMS et al., 1999; SWAIM and LEE, 1987). It was also used as dressing foams, gauze and antiseptic agents. Ag + salts have traditionally been administered to the eyes of newborn infants to prevent neonatal eye infections, with concentraton of 1.5 % Ag + nitrate as required by law in some countries (SILVER, 2003; CREDE, 1901), and it is also widely used in America for treatment of blindness up to date. Topical cream of 1% Ag + sulfadiazine plus 0.2 % chlorhexidine digluconate in a water immiscible cream base is the most widely used product for human use and veterinary medicine in the USA and the UK. In addition, Ag + was used in bandages for burned skin surfaces and large open wounds (WRIGHT et al., 1998; KLAUS et al., 1999). Clinical uses of Ag + nitrate include aseptic coverings for plastic surgery, traumatic wounds, leg ulcers, skin 35

Literature review grafts, incisions, abrasions, and minor cuts. Another new product of considerable interest is silverzeolite which is a hydrated aluminosilicate powder which can bind up to 40 % of its weight as Ag +. It can be incorporated into medical and dental objects. Ag + is subsequently released slowly to result in antibacterial activity (INOUE et al., 2002; KAWAHARA et al., 2000). Also, Ag + proteinate preparations were used to cure up to 680 various diseases, including lupus and AIDS (METCALF, 2001). Ag + - impregnated polymers of medical devices such as catheters, heart valves, orthopaedic devices, endotracheal tubes and cardiac pacemakers have widely been used to prevent the growth of bacterial biofilms of E. coli and S. aureus (SAMPATH et al,. 1995; DASGUPTA 1997; GATTER et al., 1998; GREENFELD et al., 1995). Ag + -zeolite is also elective against anaerobic oral bacteria (KAWAHARA et al., 2000). In North America, a preparation called Argyrol defined as a trademark for a mild Ag + protein compound which used as a local antiseptic, and it was marketed from 1902 to 1996 by a Philadelphia based company (FARBER, 1997). Ag + is used as health additive in traditional Chinese and Indian ayurvedic medicine. 2.6.4.2 Non medical uses of silver Ag + was used for water purification cartridges in the USA called Brita and supermarket-available colloidal silver-gelatine for washing salad vegetables and drinking water. In Mexico, a product called Microdyn is used for water disinfection, swimming pools and drinking water. Supermarket home-water purification units in the USA contain silverized activated carbon filters and ion-exchange resins (CHAMBRES et al., 1962, BELL, 1991). Also in Japan, a Ag + compound Amenitop silica gel microspheres containing a Ag + -thiosulfate complex is mixed into plastics for lasting antimicrobial protection of telephone, receivers, calculators, toilet seats, and children s plastic (http://www.yourlifewell.com/index4.shtml). Supermarket surfaces used Ag + for meat storage as a possibly useful biocide. Metallic Ag + coppercontaining ceramic disks are marketed as an alternative for users who might be allergic to laundry detergents. Ag + combined with copper is used as disinfectant for hospitals and hotels to control infectious agent as legionella (GUPTA and SILVER, 36

Literature review 1998). In addition, the ability of Ag + to inhibit bacterial and fungal growth in chicken farms, that is why it can be used in post harvest cleaning of oysters. 37

Materials and methods 3 Materials and methods 3.1 Experimental setting An overview of the experimental setting for this study is given in Tab. 4. Tab. 4: Experimental design 1. Degradation process of MgAg 1 % alloy Degradation of MgAg1% sticks Isolated perfused Histology Degradation in Silver 3 test Magnesium bovine udder dry off secretion Nanocolor and calcium Nanocolor 2. Biocompatibility experiments Cell lines - L929 - Murine macrophages - raw macrophages - Mammary epithelial cells - Mammary fibroblasts Isolation and cultivation of mammary cells Characterization of primary mammary cells Detection of biomarkers of inflammatory reactions Cell culture Immunocytochemistry ELISA Detection of the viability of murine fibroblasts, raw macrophages, mammary epithelial cells, and mammary fibroblasts Measuring metabolic activities MTS assay, neutral red assay, Bio-Rad test Pyruvate kinase test, histochemistry, succinate dehydrogenase 3. Antibacterial effects Detection of antibacterial activity Bouillon dilution test and cultivation of bacteria in petri dishes Brilliant black reduction test 38

Materials and methods 3.2 Materials All purchased materials and equipments are listed in the following chapters. 3.2.1 Cell culture 3.2.1.1 Culture media The basic culture medium used for culturing murine fibroblasts (L929 cells) was RPMI 1640 (PAA, Pasching, Germany). All ingredients of this basic culture medium are listed in Tab. 5. Tab. 5: Ingredients of the RPMI 1640 [mg/ml] Inorganic Salts Calcuim Nitrate 4 H 2 O 100.00 Potassuim Chloride 400.00 Magnesuim Sulphate 48.80 Sodium Chloride 6000.00 Di-Sodium Hydrogen 800.00 Phosphate anhydrous Sodium Hydrogen Carbonate 2000.00 Amino Acids L- Alanyl-L-Glutamine 445.90 L- Arginine HCl 241.86 L- Asparagine H 2 O 50.00 L-Aspartic Acid 20.00 L-Cystine 50.00 L-Glutamine Acid 20.00 L-Glutamine 00.00 Glycine 10.00 L-Histidine 15.00 L-Hydroxyproline 20.00 L-Isoleucine 50.00 L-Leucine 50.00 Vitamins P-Aminobenzoic Acid 1.00 D(+)-Biotin 0.20 Cholin Chloride 3.00 Folic Acid 1.00 Myo-Inositol 35.00 Nicotinamide 1.00 D-Pantothenic Acid 0.25 Pyridoxine HCl 1.00 Riboflavin 0.20 Thiamine HCl 1.00 Vitamin B12 0.005 L-Serine 30.00 L-Threonine 20.00 L-Tryptophan 5.00 L-Tyrosine 20.00 L-Valine 20.00 39

Materials and methods Tab. 5: Continued L-Lysine.HCl 40.00 L-Methionine 15.00 L-Phenylalanine 15.00 L-Proline 20.00 Other Components D- Glucose anhydrous 2000.00 Glutathione (red) 1.00 HEPES, Phenol Red 57.50 The basic culture medium used for culturing the raw murine macrophages was Dulbecco s MEM (DMEM), low Glucose (1g/l) (PAA, Pasching, Germany). All ingredients of this basic culture medium are listed in Tab. 6. Tab. 6: Ingredients of the DMEM, low Glucose (1g/ l). Inorganic Salts Calcium Chloride anhydrous 200.00 Ferric(III)-Nitrate 9 H 2 O 0.10 Potassium Chloride 400.00 Magnesium Sulphate anhydrous 97.70 Sodium Chloride 6400.00 Sodium Dihydrogen Phosphate H 2 O 125.00 Sodium Hydrogen Carbonate 3700.00 Amino Acids L-Arginine HCl 84.00 L-Cystine 48.00 Glycine 30.00 L-Histidine HCl H 2 O 42.00 L-Isoleucine 105.00 L-Leucine 105.00 L-Lysine HCl 146.00 L-Methionine 30.00 L-Phenylalanine 66.00 L-Serine 42.00 L-Serine 42.00 L-Threonine 95.00 L-Tryptophan 16.00 L-Threonine 95.00 L-Tryptophan 16.00 L-Tyrosine 72.00 L-Valine 94.00 Vitamins D-Calcium-Pantothenate 4.00 Choline Chloride 4.00 Folic Acid 4.00 Myo-Inositol 7.20 Nicotinamide 4.00 Pyridoxal HCl 4.00 Riboflavin 0.40 Thiamine HCl 4.00 Other Components: D-Glucose anhydrous 1000.00 Phenol Red 15.00 40

Materials and methods The basic culture medium used for culturing the mammary epithelium and fibroblasts was DMEM / Ham s F-12 Medium with L-glutamine (PAA, Pasching, Germany). All ingredients of this basic culture medium are listed in Table 7. Tab. 7: Ingredients of the DMEM / Ham s F-12 Medium. Inorganic Salts Calcium Chloride anhydrous 116.60 Ferric(III)-Nitrate 9 H 2 O 0.05 Ferric(III)-Sulphate 7 H 2 O 0.417 Potassium Chloride 311.80 Cupric(II)-Sulphate 5 H 2 O 0.0013 Magnesium Chloride 6 H 2 O 61.20 Magnesium Sulphate anhydrous 48.84 Sodium Chloride 6996.00 Sodium Dihydrogen Phosphate H2O 62.50 Di-Sodium Dihydrogen Phosphate 71.02 Anhydrous Zinc Sulphate 7H 2 O 0.432 Sodium Hydrogen Carbonate 1200.00 Amino Acids L-Alanine 4.45 L-Arginine HCl 147.50 L-Asparagine H 2 O 7.50 L-Aspartic Acid 6.65 L-Cystine HCl H 2 O 31.29 L-Cysteine 2 HCl 7.56 L-Glutamic Acid 7.35 L-Glutamine 365.00 Glycine 18.75 L-Histidine HCl H 2 O 31.48 L-Phenylalanine 35.48 L-Proline 17.25 L-Serine 26.25 L-Threonine 53.45 L-Tryptophan 9.02 L-Tyrosine 38.70 L-Valine 52.85 Vitamins D(+)-Biotin 0.0035 D- Calcium Pantothenate 2.24 Choline Chloride 8.98 Folic Acid 2.65 Myo-Inositol 12.60 Nicotinamide 2.02 Pyridoxal HCl 2.00 Pyridoxine HCl 0.031 Riboflavin 0.219 Thiamine HCl 2.17 Thymidine 0.365 Vitamin B12 0.68 Other components D-Glucose anhydrous 3151.00 Hypoxanthine 2.10 41

Materials and methods Tab. 7: Continued L-Isoleucine 54.47 L-Leucine 59.05 L-Lysine HCl 91.25 L-Methionine 17.24 DL-68-Lipoic Acid 0.105 Linoleic Acid 0.042 Phenol Red 8.10 Putrescine 2 HCl 0.081 Sodium Pyruvate 55.00 3.2.2 Cell cultures Murine fibroblast (L929) cell line Raw murine macrophages Cell line service, Eppelheim, Germany Gift from Microbiology Institute, Hannover, Germany 3.2.3 Cell culture reagents - Fetal calf serum - EDTA (Versen) 1 % - Trysin/EDTA (0.05 %/ 0.02 %) - L-Glutamine - EGF - Penicillin - Streptomycin - Amphotericin B - Insulin Transferrin-Selenium A for adherent cultures - LPS (E.coli, 0111:B4) - Collagen (type I, rat tail) - Gentamicin (10mg\ml) - Protease enzyme - Braunol - ß- Mercaptoethanol Biochrom AG, Berlin, Germany Biochrom AG, Berlin, Germany Biochrom AG, Berlin, Germany Sigma-Aldrich, Steinheim, Germany PAA Laboratories GmbH, Pasching, Germany Biochrom AG, Berlin, Germany PAA Laboratories GmbH, Pasching, Germany PAA Laboratories GmbH, Pasching, Germany Invitrogen7GIBCO,Darmstadt, Germany Sigma-Aldrich, Steinheim, Germany Roche Diagnostics GmbH, Mannheim, Germany PAA Laboratories GmbH, Pasching, Germany Sigma-Aldrich, Steinheim, Germany B. Braun Melisungen, AG Sigma-Aldrich, Steinheim, Germany 42

Materials and methods 3.2.4 Cell culture disposable materials - 6-well flat bottom tissue culture plate - 96-well flat bottom tissue culture plate - 25 cm² tissue culture flask, 50 ml - Sterile cell scraper - Scalpel blade - Syringes (2, 5, 10, 20 ml; Omnifix ) - Minisart filter unit - Terumo needle (0.9 x 40 mm/ 0.6 x 25 mm) - Cryovials (1 ml, Cryo.s) - Cell culture tubes 50 ml - Cell culture tubes 15 ml - Biopsy bunch 6 mm - Petri dish - Cover glasses 12 mm Greiner BIO-ONE GmbH, Frickenhausen, Germany Greiner BIO-ONE GmbH, Frickenhausen, Germany Greiner BIO-ONE GmbH, Frickenhausen, Germany TPP,Omnilab, Mettmenstetten, Germany Bayha, Tuttlingen, Germany B. Braun, Melsungen, Germany Millipore, Carrigtwohill, Ireland Terumo Europe, Leuven, Belgium Greiner BIO-ONE GmbH, Frickenhausen, Germany Greiner BIO-ONE GmbH, Frickenhausen, Germany Corning Incorporated, USA pfm, Köln, Germany Sarsdet AG & Co.Nümbrecht, Germany VWR International GmbH, Hannover 3.2.5 Cell culture counting and viability - Trypan Blue - Celltiter 96 Aqueous One Solution - Cell Proliferation Heamocytometer 0.0025 m 2 - Cover glasses for haemocytometer 24 x 24 mm - Neutral red Sigma, St. Louis, USA Promega, Mannheim, Germany LO-Labor Optik GmbH,Bad Homburg, Germany Menzel GmbH &CO,Braunschweig, Germany Sigma-Aldrich, Steinheim, Germany 43

Materials and methods 3.2.6 Cell culture equipments Incubator - Hera cell 150 CO 2 incubator - VaroLab VL 150 DES CO 2 incubator Thermo Electron LED GmbH, Langenselbold, Germany VaroLab GmbH, Gieβen, Germany Sterile work bench - Heraeus LaminAir 2448 - Thermoscientific MSC-Advantages - Phase contrast microscope (Axiovert 25) - Sterilizer AESCULAP Iso 100 - Canon Power Shot A70 - Water bath CH-9230FLAWIL\SG - Centrifuge 5804R - magnetic stirrer Mini MR 0.1500 1/min Heraeus-Kulzer, Hanau, Germany Thermo Electron LED GmbH, Langenselbold-Germany Zeiss, Oberkochen, Germany AESCULAP-Werke TUTTLINGEN, Germany Canon Deutschland GmbH, Krefeld, Germany BÜCHI Laboratoriums-Technik AG, Essen, Germany Eppendorf, Hamburg, Germany IKA WERKE, Breisgau, Germany 3.2.7 Magnesium-silver1% (MgAg1%) stick and silver nitrate The MgAg1% alloy as shown in (Fig. 5) is made from 99% magnesium and 1% silver and has a weigh of 10-70 mg. It was produced with a length of 1 cm and a diameter of 0.2 cm (Institute of Materials Science, Leibniz University, Hannover, Germany). Silver nitrate was purchased from Merck, Darmstadt, Germany. Fig. 5: MgAg1 % stick 1cm 44

Materials and methods 3.2.8 Materials and reagents 3.2.8.1 Materials and reagents for Bio-Rad assay - BSA SIGMA-ALDRICH Chemie GmbH, Steinheim, Germany - Bio-Rad Reagent BioRad Laboratories GmbH, Munich, Germany 3.2.8.2 Materials and reagents for measuring cytokines (Interlekin-1beta (IL- 1beta), interleukin- 6 (IL- 6), tumour necrotic factor (TNF-alpha) - Mouse TNF-alpha/TNFSF1A DuoSet R&D Systems, Inc., Minneapolis, USA ELISA Development System - Mouse IL-1beta/IL-1F2 DuoSet R&D Systems, Inc., Minneapolis, USA ELISA Development System - Mouse IL-6 DuoSet R&D Systems, Inc., Minneapolis, USA ELISA Development System 3.2.8.2.1 Equipments used for measuring cytokines - 96- well micro plate R&D Systems, Inc., Minneapolis, USA - ELISA plate sealers R&D Systems, Inc., Minneapolis, USA 3.2.9 Materials and reagents used for measuring silver concentrations NANOCOLOR MACHEREY-NAGEL GmbH & Co. KG, Düren, Germany Silver 3 test kits 3.2.10 Materials and reagents used for measuring calcium and magnesium Concentrations - NANOCOLOR MACHEREY-NAGEL GmbH & Co. KG, Düren, Hardness 20 test kits Germany - Magnesium chloride Merck, Darmstadt, Germany 45

Materials and methods 3.2.11 Materials and reagents used for the brilliant black reduction test (BRT- MRL screening test) - BRT MRL Screening test kits AM GMBH, Munich, Germany 3.2.11.1 Equipments used for BRT MRL screening test - Microtiter plate with coated wells AM GMBH, Munich, Germany - Thermo block AM GMBH, Munich, Germany 3.2.12 Materials and reagents used for measuring PGE 2 - PGE 2 assay Kits - Indometacin - Dimethylsulphoxide R&D Systems, Inc., Minneapolis, USA Sigma-Aldrich, Steinheim, Germany Merck, Darmstadt, Germany 3.2.12.1 Equipments used for measuring PGE 2 - Goat anti-mouse microplate - Plate covers-adhesive strips - Biopsy punch - ULTRA-TURRAX 20000 UpM, Tp 18-10 - Polypropylene tubes R&D Systems, Inc., Minneapolis, USA R&D Systems, Inc., Minneapolis, USA pfm, Köln, Germany IKA WERKE, Breisgau, Germany Sarstedt AG & Co. Nümbrecht, Germany 3.2.13 Materials and reagents used for the bouillion dilution test - NaCl - Buffered peptone water The test was made by the Milchtierherden Betreuungs- und Forschungsgesellschaft mbh (MBFG), Wunsdorf, Germany 3.2.14 Materials and reagents used for histology and histochemistry - Methanol AppliChem GmbH, Darmstadt, Germany - Acetone Sigma-Aldrich, Seelze, Germany - Ethanol Riedel-de Häen, Seelze, Germany 46

Materials and methods - Eosin Y - 100 % acetic acid - Hydrochloric acid Primary antibodies - Anti-cytokeratin (pan-antibody clone PCK-26) - Anti-vimentin (clone V-9) Secondary antibodies - F(ab)2 Goat anti-mouse IgG: FITC - Mayer s hemalum solution - Fluromount - Tissue freezing medium - Disodium succinate - Nitrotertazolium blue - Bisbenzimid Merck, Darmstadt, Germany AppliChem GmbH, Darmstadt, Germany Merck, Darmstadt, Germany Sigma-Aldrich, Steinheim, Germany Sigma-Aldrich, Steinheim, Germany Serotec GmbH, Düsseldorf, Germany Merck, Darmstadt, Germany Sigma-Aldrich, Steinheim, Germany Leica Microsystems GmbH, Nussloch, Germany SAFC Supply solutions,st.louis, USA Sigma-Aldrich, Steinheim, Germany Sigma-Aldrich, Steinheim, Germany 3.2.14.1 Equipments used for histology and histochemistry - Microme HM 560 M - Leica microscope 020-519.511DMLB 100T - Axiocam - Microscope glass cover slips - Microscope slides - Hybridization oven - Surgical blade Microm GmbH, Walldorf, Germany Leica Mikroskopie & Systems GmbH, Wetzlar, Germany Zeiss, Oberkochen, Germany Chance propper LTD, Warley, England Gerhard Menzel GmbH, Braunchweig, Germany Gesellschaft für Labortechnik mbh, Burgwedel, Germany Bayha, Tuttlingen, Germany 47

Materials and methods 3.2.15 Materials used for succinate dehydrogenase test (SDH) - KCl Merck, Darmstadt, Germany - Tris-HCl Merck, Darmstadt, Germany - EDTA Merck, Darmstadt, Germany - BSA Sigma-Aldrich, Steinheim, Germany - KCN Merck, Darmstadt, Germany - Disodium succinate SAFC Supply solutions, St.Louis, USA - Cytochrome C Sigma-Aldrich, Steinheim, Germany 3.2.16 Materials used for pyruvate kinase test (PK) - Triethanolamine - MgSO 4 - NADH - KCl - PEP - LDH - ADP Merck, Darmstadt, Germany Merck, Darmstadt, Germany Sigma-Aldrich, Steinheim, Germany Merck, Darmstadt, Germany Sigma-Aldrich, Steinheim, Germany Sigma-Aldrich, Steinheim, Germany Sigma-Aldrich, Steinheim, Germany 3.2.17 Materials used for isolated perfused bovine udder - NaCl Merck, Darmstadt, Germany - Glucose H 2 O Merck, Darmstadt, Germany - NaHCO 3 Merck, Darmstadt, Germany - KCl Merck, Darmstadt, Germany - NaH2PO 4 2H 2 O Merck, Darmstadt, Germany - MgCl 2 6H 2 O Merck, Darmstadt, Germany - CaCl 2 2H 2 O Merck, Darmstadt, Germany - Oxygenated carbon (95 % O 2, 5 % CO 2 ) Westfalen AG, Göttingen, Germany 48

Materials and methods 3.2.17.1 Equipments used for isolated perfused udder - Water bath Mod. SWBD 5030 - Flexible pump tube 9553-75 - Surface thermometer (HI 93530) with surface sensor (HI 766B2) - Adhesive tapes - Surgical blade - Perfusat reservoir Stuart Scientific Co. Ltd., Exeter, Great Britain Cole-Parmer, Illinois, Vernon Hills, USA Hanna Instruments, Milano, Italy Leukotape, Beiersdorf, Heidenheim Bayha, Tuttlingen, Germany Schott, Jena, Germany 3.2.18 Materials and reagents used for the lactate dehydrogenase assay (LDH) - LDH cytotoxicity assay kit Cayman Chemical Company, Ann Arbor, USA 3.2.19 Materials and reagents used for the glucose assay - Glucose assay kit Cayman Chemical Company, Ann Arbor, USA 3.2.20 Materials and reagents used for the lactate test - Lactate test Roche GmbH, Mannheim, Germany 3.2.21 Analytical equipments - MRX Microplate-Reader Germany - Centrifuges 5403 - Reax top 2000 - Orbital shaker SO3 - ph-meter (ph 320) - Scale SBA 53 Scaltec Dynatech Laboratories, Denkendorf, Germany Eppendorf, Hamburg, Germany Heidolph, Kehlheim, Germany Stuart scientific Co, Ltd, Staffordshire, Great Britian WTW, Weilheim, Germany 49

Materials and methods - Precision Scale ALS 120-4 - Water bath 1083 - Sonication Sonorex.RK52 - Magnetomix M1 stirrer Labor-u.Analysen-Technik, GmbH, Garbsen, Germany Kern & Sohn GmbH, Balingen, Germany Gesellschaft für Labortechnik, m.b.h & CO, Hannover, Germany Bandelin electronic, Berlin, Germany Colora Messtechnik GmbH, Lorch, Württ, Germany 3.2.22 Solutions and buffers PBS (0.01 mol/l; ph 7.4) - 137 mmol/l NaCl Merck, Darmstadt, Germany - 2.7 mmol/l KCl Merck, Darmstadt, Germany - 6.5 mmol/l Na 2 HPO 4 2H 2 O Merck, Darmstadt, Germany - 1.5 mol/l KH 2 PO 4 Merck, Darmstadt, Germany Block-Ag-Retrievel - 100 ml PBS - 0.25 % BSA Sigma-Aldrich Chemie GmBH, Steinheim, Germany - 1 % Triton X 100 Sigma-Aldrich, Steinheim, Germany Histochemistry buffer 0.2 M phosphate buffer (PH 7.4) contains - 0.2 M Na 2 HPO 4-0.2 M KH 2 PO 4-200 mm Succinic acid - 1.34 mm Nitrozolium blue Tyrode solution - 136 mmol/l NaCl - 11.9 mmol/l NaHCO 3 50

Materials and methods - 5.5 mmol/l Glucose H 2 O - 2.7 mmol/l KCl - 1.8 mmol/l CaCl 2 2H 2 O - 1.05 mmol/l MgCl 2 6H 2 O - 0.461 mmol/l NaH 2 PO 4 SDH reaction solution - 50 mm KCl - 10 mm Tris HCl - 1 mm EDTA - 1 mg/ml BSA - 1 mm KCN ; ph 7.4-10 mm Succinate - 6.16 mm Cytochrome C PK reaction solution - 97.5 mm triethanolamine - 13 mm MgSO 4-74 mm KCl - 185 µm NADH - 1 mm PEP - 2.5 U/ml LDH - 3 mm ADP Neutral red solution 1 (1 % CaCl 2, 0.5 % formaldehyde) - 6.5 ml Formaldehyde - 50 ml CaCl 2-445 ml Aqua bidest. Neutral red solution 2 (1 % acetic acid, 50 % ethanol) - 4.75 ml acetic acid - 250 ml ethanol (95 %) - 245 ml Aqua bidest. 51

Materials and methods Wash Buffer (IL-1beta, TNF-alpha, IL- 6) IL-1beta TNF-alpha IL-6 Tween 20 0.250 ml 0.250 ml 0.250 ml PBS 500 ml 500 ml 500 ml Reagent Diluent (IL- 1beta, IL- 6) TNF-alpha IL-6 BSA 0.8 g 0.8 g PBS 80 ml 80 ml Reagent Diluent (IL-1beta) - 0.05 g BSA - 0.438 g NaCl - 0.121 g Tris HCl - 25 µl Tween 20 Added in 50 ml Aqua bidest. Blocking Buffer (IL-1beta) - 0.3 g BSA - 0,015 g NaN 3 Added in 30 ml PBS Eosin solution 2.5 g eosin in 250 ml Aqua bidest. + 3 drops of acetic acid Dendritic cell medium (DC-medium) - ß- Mercaptoethanol - RPMI supplemented with FCS + penicillin/streptomycin 52

Materials and methods 3.3 Methods 3.3.1 Bovine udders All bovine udders (used for mammary epithelium and mammary fibroblast cell cultures) were obtained from recently slaughtered cows. The first study was performed to establish a protocol to culture mammary epithelium cells. Later, when contamination of fibroblasts appeared, the second protocol was established to separate mammary epithelium cells from mammary fibroblasts. The bovine udders were obtained immediately after slaughtering, at different ages from the slaughterhouse in Minden-Lübbecke, Germany. The udders were transported to the laboratory within 75 minutes. All the experiments of the udder nearly started within half an hour after receiving the udder. 3.3.2 Cell culture experiments Murine fibroblast (L929) cells: They are monolayer fibroblast cells obtained from male mice at the age of 100 days and were obtained from connective tissue, subcutaneous, areolar and adipose tissue. Raw murine macrophages: They are monocytes/macrophages from adult male mice with ascites. During passaging, no trypsin or EDTA was added to detach the cells. They were detached using cell scrapers. Primary murine macrophages: Primary murine macrophages originated from bone marrow of the mice femur; therefore, the bone marrow was collected from the mice femur containing dendritic cells and primary macrophages. After incubation in DC medium, primary macrophages were attached to the bottom of the petri dish and were thereby separated from the dendritic cells. 3.3.2.1 Primary mammary epithelium cell cultures Mammary epithelium cells were cultured by leaving the cells to grow out of the explants. Bacterial and fungal contamination initially appears after culturing the explants. For this reason, a new protocol was established using collagen as a coating for 6-well plates in order to have best results in terms of differentiation when using collagen gels which allow the cells to be confluent. When the collagen released from 53

Materials and methods the bottom of the well, cells shrink which in other terms stimulate the cells to differentiate (EMERMAN and PITELKA, 1977). Washing process was done as follow: Tissues taken from teats and mammary glands were cut in small pieces (1 cm 3 ); the tissues were incubated in Braunol (1:10 dilution in PBS) for 15 minutes, they were washed in sterile PBS up to 5 times, then they were washed in DMEM-F12 supplemented with 20 % FCS, 10 % amphotericin B, 10 % gentamicin and 10 % penicillin/streptomycin. Afterwards, they were placed on an orbital shaker for 10 min, washed again in sterile PBS up to 5 times, and were washed with DMEM-F12 supplemented with 10 % FCS, 1 %, amphotericin B, 1 % gentamicin and 1 % penicillin/streptomycin. According to HU et al. (2009) culturing was done with some modifications: The tissues were incubated in DMEM-F12 supplemented with 10 % FCS, 1 %, amphotericin B, 1 % gentamicin and 1 % penicillin/streptomycin and protease 0.6 mg/ml in a petri dish over night in a fridge; the tissues were taken out to a new petri dish containing DMEM-F12 with 10 % FCS, 1 %, amphotericin B, 1 % gentamicin and 1 % penicillin/streptomycin. The tissues were agitated gently and were incubated at 37 C, 5 % CO 2 for one hour to attach fibroblasts and detach epithelium cells in the supernatants.the supernatants contained the detached epithelial cells were collected, and then the collected supernatants were centrifuged at 600 x g for 5 min. The medium was removed, fresh medium was added, and they were centrifuged again. Afterwards, they were washed 3 times, and then 5 ml of DMEM-F12 supplemented with 10 % FCS, 10 % penicillin/streptomycin, 10 % gentamicin, 10 % amphotericin B, 25 µg/ml EGF, 10 % L-Glutamine and 5 ml Insulin-transferrin- Selenium A were added. Afterwards, 6-well plates were coated with rat tail collagen (2 mg/ml) by spreading 33 µl per well using a sterile cell scraper. They were left to dry under the sterile bench for one hour. Afterwards, the plates were flushed with sterile PBS. Afterwards, the cells were counted and seeded in 6-well plates at a density of about 500.000 to 1000.000 cells per well. Later, the medium was changed after 48 hours, then every 24 hours after washing with sterile PBS. Cells were confluent after 8-10 days and were passaged into 25 cm² culture flasks at a density 54

Materials and methods of 1000.000-2000.000 cells/well. Cells were detached by adding 1 % EDTA solution for 5 min at 37 C and 5 % CO 2, then 0.05 % trypsin/0.02 % EDTA solution for 10 min at 37 C and 5 % CO 2 until cells were detached and separated followed by adding DMEM-F12 with 10 % FCS to inactivate the trypsin. The cells were centrifuged for 10 min at 600 x g at 4 C, and seeded at a density of 10.000-20.000 cells/well using a 96-well plate. For the experiments, primary mammary epithelium cells were used at passages 4-7. 3.3.3 Separation of primary mammary epithelium cells contaminated with mammary fibroblasts In the case of using trypsin digestion, epithelial cells were isolated after 3 passages. The degree of digestion and culture conditions seemed to be important for cell development. For detaching epithelial cells from culture dishes or culture flasks, it took 10 to 15 minutes, while the fibroblasts needed less than 1 to 2 minutes, but this method only succeeded for one or two passages (HU et al., 2009). According to FRESHNEY and FRESHENEY (2002), primary mammary epithelium cells were separated from primary mammary fibroblasts as follow: The cell mixture was seeded into a flask for 30 min to 1 hour. Then the mixture was transferred into another new flask which was done for intervals of 1-3 hours up to 24-48 hours. Commonly the fibroblasts tend to attach first at the bottom of the flask, while the epithelial cells remain in the suspension and attach in the later seeded flask. 3.3.4 Culturing primary mammary fibroblasts Tissues taken from the teats and mammary glands were sterilized and washed as mentioned in 3.3.2.2, then culturing followed according to HU et al. (2009): the tissue explants were cut into small pieces about 1 cm 3 in 6-well plates, and 1-1.5 ml DMEM-F12 supplemented with 10 % FCS, 10 % penicillin/streptomycin, 10% gentamicin, 10 % amphotericin B, 25 µg/ml EGF, 10 % L-Glutamine, 5 ml Insulintransferrin-Selenium A was added. The explants were incubated at 37 C and 5 % CO 2 for 45-60 min, monitored every 30 min. If the adjacent area surrounding the tissue was dry, several drops of medium were added ensuring the tissue would not 55

Materials and methods float and separate from the bottom of the culture dish. After 4 hours, 0.5 ml medium was added to every culture dish and 1 ml medium was added every 48 hours until cells were visibly spread across the bottom of the culture dish. When the cells became confluent after 10-15 days, they were passaged into 25 cm² culture flasks at a mean density of 1000.000 to 2000.000 cells/well, using 1 % EDTA for 1 min, and 0.25 % trypsin, 0.02 % EDTA for 3 min, 37 C and 5 % CO 2. DMEM-F12 containing 10 % FCS was added to inactivate the trypsin, then centrifugation took place for 10 min at 2357 x g at 4 C. Finally, the cells were seeded in 96-well plates at a mean density of 10.000-20.000 cells/well in fresh whole medium. For the experiments, primary mammary fibroblast cells were used at passages 5 to 8. 3.3.5 Cell counting To determine the cell count, cells were stained with trypan blue and counted using a Neubauer chamber: 50 μl of a cell suspension were mixed with 100 μl trypan blue suspensions (40 mg trypan blue in 10 ml Aqua bidest.) and 10 μl of this suspension was placed in the counting chamber. The cell count was determined in four quadrants with a 100-fold magnification under the light microscope. Trypan blue enables a differentiation of viable and dead cells. All cells incorporate the stain, but only viable cells are able to eliminate the blue stain. When viewed under the microscope, dead cells have a blue cytoplasm and nucleus, whereas viable cells are not stained. The total cell count can be calculated with the following formula (1): Formula (1): calculation of total cell count. Number of counted cells x dilution x 5 x 10 4 Number of counted squares = cell absolute Cells absolute = absolute cell count in 1 ml of culture medium Number of counted cells = number of counted cells in 4 quadrants 5 = the cell were suspended in 5 ml medium 10 4 = cells under the glass slide 56

Materials and methods 3.3.6 Immunocytochemistry Primary mammary epithelium and primary mammary fibroblasts were verified by indirect immunofluorescence staining. The monoclonal mouse anti-vimentin antibody was used in a 1:100 dilution, the monoclonal mouse anti-cytokeratin antibody was used in a 1:300 dilution, and both were diluted in blocking-ag-retrieval solution. The secondary antibody (FITC) was used in 1:200 dilution and bisbenzimid was used in a 1:100 dilution in Aqua bidest. The staining protocol was done as follow: Cells were grown in 6-well plates on sterile cover slips up to 70 % confluence; they were washed twice with PBS and fixed with cold methanol/acetone (50/50; v/v) for 5 min.. Afterwards, the slides were either stored in sterile PBS at 4 C until further processing or stained immediately. The slides were washed with PBS (5 min, max. 2 times), and were incubate with blocking-ag-retrieval solution for 30 minutes at room temperature. Afterwards, they were incubated with the primary antibody (antivimentin & anti-cytokeratin) for 1 h at 37 C, then they were washed with PBS (5 min, max. 2 times), and were incubated with the fluorescence-labelled secondary antibody (FITC) for 30 min at 37 C in the dark. After washing with PBS (5 min, max. 2 times), the slides were stained with bisbenzimid at room temperature for 15 min in the dark, and were washed again with PBS for 5 min. 100 µl of the antibody and bisbenzimid solutions were added per slide and 1 ml of blocking-ag-retrieval solution were added per slide for the washing process. During the entire process of the staining, drying of the wells and slides was strictly avoided. 3.3.7 Treating of the cells 3.3.7.1 AgNO 3 treatment A stock solution of 1 mmol/l AgNO 3 was prepared and different concentrations of AgNO 3 (control (medium), 0.0001, 0.0003, 0.001, 0.003, 0.01, 0.03, 0.1, 0.3, 1 mmol/l) in different media or NaCl solution according to the cell type were prepared; the cells were treated and were incubated at 37 C and 5 % CO 2 for 24 h. Afterwards the supernatants of the cell incubation experiments were collected and frozen -20 C for measurement of biomarkers of inflammatory reactions by ELISA. The samples were in 7 duplicate numbers per dilution. 57

Materials and methods 3.3.7.2 Incubation with MgAg1% sticks MgAg1% sticks were sterilized and incubated in different types of media according to the cell type or NaCl with different volumes (1, 3 and 10 ml), in addition to one tube of medium as negative control; they were incubated in a water bath at 37 C and shaken for 5 days. Afterwards, the supernatants were taken to treat the different cell types, which were incubated at 37 C and 5 % CO 2 for 24 h. The supernatants of the cell incubation experiments were collected and frozen at -20 C for biomarkers of inflammatory reactions by ELISA. The samples were in 7 duplicate numbers per dilution. 58

Materials and methods 3.3.8 Degradation process of MgAg1% sticks 3.3.8.1 The isolated perfused udder 3.3.8.1.1 Preparation of the udder Within 10-15 min post-slaughter, the udders were transported over 45 min in a plastic sacks to the laboratory. Blood clots in the vessels of the glands were cleared using 1 l heparinised (1 %) tyrode solution for each half udder. The udder was fixed in a natural position to a metal frame. Within a few minutes, the large arteries of the udder (the right and left A. pud. ext) were supplied with the perfusion fluid delivered via silicone tubes with suitable diameter using a peristaltic pump, while the larger veins (cranial and caudal superficial epigastric vein) were cannulated to allow sampling and removal of the perfusate (Fig. 6). Smaller veins were closed using artery forceps (KIETZMANN et al., 1993; EHINGER and KIETZMANN, 2000 a, 2000 b). A B 3.3.11.1.2 Fig. 6: The Starting mammary the vein perfusation and mammary and artery controlling are shown the here viability (A) on the carcass of a cow after the udder has been cut away (B). The isolated perfused udder after preparation. To inhibit the development of tissue odema, each udder half was perfused with tyrode solution (Fig. 7). To ensure a perfusate flux of 100-120 ml/ min, the pressure was 50 mm Hg. The mammary glands were milked during 30 min equilibration phase. The viability of the perfused udder was controlled using biochemical parameters such as lactate dehydrogenase activity (LDH), glucose consumption and lactate production in the perfusate (KIETZMANN et al., 1993; EHINGER and KIETZMANN, 59

Materials and methods 2000 a, 2000 b). The udder skin temperature was measured every 60 minutes. Udders which exceeded the limits were not included in the test evaluation. Fig. 7: Schematic design for isolated perfused udder (EHINGER, 1998) 3.3.8.1.2 Measuring parameters of viability of bovine udder Lactate dehydrogenase enzyme (LDH) is a soluble enzyme located in the cytosol. LDH can be used as an indicator of cell membrane integrity and is also a measurement of cytotoxicity. The assay was performed according to the protocol of the manufacturer as follows: 100 µl of the perfusate from each udder were pipetted in 96-well plate. They were centrifuged at 400 x g for 5 min. 100 µl of the standards were prepared as mentioned in the manufactures protocol, then 100 µl of each supernatant from each well was transferred to the new plate, 100 µl of reaction solution was added to each well. Afterwards, the plate was incubated on an orbital shaker for 30 min. at room temperature. The measurements were performed using photometer with a wavelength of 490 nm. Glucose concentration: It is the most important carbohydrate in biology transported through blood stream. It is the primary source of energy for the body cells. The assay was applied according to the protocol of the manufacturer as follows: 60

Materials and methods 5 µl of each perfusate sample was added in tubes, 500 µl of the enzyme mixture was added and then mixed well. The tubes were incubated at 37 C for 10 min., and finally 150 µl was taken away from each tube to a 96-well plate. The measurements were performed using photometer with a wavelength of 520 nm. Lactate concentrations: Anaerobic glycolysis increases blood lactate and pyruvate levels. The test was done as follows: A standard calibration curve was prepared from 5 mg/ml tyrode with concentrations of zero, 0.1, 0.3, 0.6, 0.9, 2 and 1 mg/ml lactate, 833 µl of reagent 1 was mixed with 167 µl of reagent 2, then 2 µl of each standard was added per well. 2 µl of the perfusate was added per well. 100 µl of the reaction medium was added per well, the plate was incubated for 10 min in room temperature. The measurements were performed using photometer with a wavelength of 520 nm. 3.3.8.2 The degradation experiments The bovine udder was perfused with tyrode solution, sterile MgAg1% sticks (52-56 mg) were administrated intracisternally and left in the teat cistern for 5-6 hours, while another teat was used as a control. The teats were cut and frozen at -20 C for histological experiments. The sticks were weighed to calculate the degradation percentage. 61

Materials and methods 3.3.8.3 Histological examination The frozen teats were thawed, cut into small pieces using a scalpel blade, then liquid freezing medium was added and a freezing microtome was used to cut sections of 8 um thickness at -20 C. They were dried and stained as follow in (Fig. 8). 30 seconds in alcohol 100 % 1 30 seconds in alcohol 96 % 2 30 seconds in alcohol 90 % 3 30 seconds in alcohol 80 % 4 30 seconds in alcohol 70 % 5 30 seconds in Eosin 15 min in tape water 20 seconds HCl-alcohol 12 min in fresh hemalum 3 min Aqua bidest. Repeat the steps from 5 to 1 Leave them to dry, cover with cover slide after adding tylon, then examine under microscope Fig. 8: Scheme for haematoxylin eosin staining protocol 62

Materials and methods 3.3.8.4 Degradation in dry off period secretion Milk samples were received from the Clinic for Cattle, University of Veterinary Medicine, Hannover, Germany from different cows at dry off period over different intervals of time. Sterile MgAg1% sticks were incubated in 5 ml milk samples in a water bath at 37 C for 21 days. The sticks were weighed at day of 5, 10, 15 and 21 to measure the loss percentage of the sticks. 3.3.8.5 Determination of the silver concentrations in the degradation medium The amount of Ag + in the degradation medium of MgAg1% was measured using the kit NANOCOLOR Silver 3 test using a photometric procedure determination. Ag + could be measured between 0.20-3.00 mg/l Ag +, as Ag ions react with an indicator to form a blue dye. Briefly, for the determination of Ag + concentrations in degradation medium the following was done: A stock solution of 1 mmol/l AgNO 3 was prepared and a calibration standard curve of 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6 and 0.8 mmol/l was prepared (Fig. 9). Sterile MgAg 1% sticks were incubated in different volumes of medium (1, 3 and 10 ml) or NaCl without adding any other supplements, in addition to one tube of medium as negative control. They were incubated in a water bath at 37 C with shaking process for 25 days. The test was applied according to the manual manuscript with slight modifications. 28 µl NANOCOLOR Silver 3 were added per well of a 96-well plate. 10 µl of the NANOCOLOR Silver 3 R2 was added. Then 80 µl of tested sample was added and all the reagents were shaken. Afterwards, 10 µl NANOCOLOR Silver 3 R3 was added per well, then it was mixed again and incubated for 10 min. The samples were in duplicate number. The measurements were performed on day 5, 10, 15, and 25 using a photometer with a wavelength of 620 nm. 63

Materials and methods Optical density 0.14 0.12 0.1 0.08 0.06 0.04 0.02 y = 0.0704x + 0.0512 R 2 = 0.9956 0 0 0.2 0.4 0.6 0.8 1 1.2 Concentration of Silver nitrate solution (mmol/l) Fig. 9: Calibration standard curve for the determination of Ag + concentrations, n=6. - Calculated from the measured calibration function of the optical standards. - Densities of total hardness measurement and the measurement of silver. - Y = optical density, X = salt concentration, R2 = coefficient of determination (> 0.95) 3.3.8.6 Determination of the magnesium and calcium concentrations in the degradation media The Mg ++ and Ca ++ concentration in the degradation media were measured by the test kit NANOCOLOR hardness 20 using a photometric procedure. The test is based on measuring the total hardness of the medium, which is primarily maintained by Mg ++ and Ca ++, but is also determined by barium and strontium ions. The color intensity depends on the total hardness of the surrounding medium. The calcium content can be obtained from the measured values of total hardness and the Mg ++ content can be calculated. A stock solution of MgCl 2 was prepared (10 mmol/l) and a calibrations standard curve of 0.1, 0.25, 0.5, 0.75, 1, 2, 3, 4 and 5 mmol/l was prepared (Fig. 10). The samples were prepared from the degradation media by incubating MgAg1% sticks in different volumes of 10, 3 and 1 ml of medium or NaCl. One tube of pure medium or pure NaCl served as negative control. Subsequently, 6 µl of tested samples were added in each well of a 96-well plate, and then 180 µl of NANOFIX hardness 20 R2 was added per sample and 6 µl of hardness 20 R3 solution was added per sample. It was mixed 64

Materials and methods by pipetting up and down. The samples were in duplicate number. The samples were measured at 570 nm. Samples of higher concentrations above the calibration curve were diluted. The measurements were performed at day 5, 10, 15 and 25. 0.45 0.4 Optical density 0.35 0.3 0.25 0.2 0.15 0.1 0.05 y = 0.0542x + 0.1464 R 2 = 0.991 0 0 1 2 3 4 5 6 Concentration of magnesium chloride (mmol/l) Fig.10: Calibration standard curve for the determination of Mg ++ concentrations, n=6. - Calculated from the measured calibration function of the optical standards - Densities of total hardness measurement and the measurement of magnesium. -Y = optical density, X = salt concentration, R2 = coefficient of determination (> 0.95). 65

Materials and methods 3.3.9 Biocompatibility tests 3.3.9.1 Measurement of cell viability and proliferation 3.3.9.1.1 MTS assay Cell viability and proliferation for each cell type was evaluated using a modified MTS test (3 - (4, 5-dimethylthiazol -2-yl) -5- (3-carboxymethoxyphenyl) -2- (4-sulfophenyl) - 2H-tetrazolium]) according to the manufacturer s protocol. It is a colorimetric test for measuring the enzyme activity depending on the reduction equivalent of the tetrazolium salt. By using enzymatic activities of viable cells, viable cells reduce the yellow water soluble inner salt MTS to the brown water soluble at 490 nm absorbing formazan compound. Phenazine ethosulfate (PES) serves as an electron coupling reagent. To obtain a growth curve, 10.000 cells/well were plated in a 96-well plate up to confluence (5 days), during incubation with different concentration, of AgNO 3 and degradation media of Mg/Ag1% sticks for 24 hours as mentioned before in 3.3.7. The test was applied according to the manufacturer s protocol, the reagent solution was diluted in 1:4 in culture medium and 100 µl of this solution was added to each well. The measurement of the extinction at a wavelength of 490 nm was performed after one hour of incubation at 37 C and 5 % CO 2. Optical density of formazan was measured in this assay. 3.3.9.1.2 Neutral red assay Cell viability and proliferation for each cell type were evaluated using the neutral red assay according to the manufacturer s protocol. It is a colorimetric test for detecting cytotoxicity by inclusion of neutral red dye in lysosomes of vital cells. Living cells are coloured red, while dead cells are not stained as they do not have intact lysosomes any more. To obtain a growth curve, cells were plated and treated the same way as mentioned in 3.3.7; then the test was applied according to the manufacturer s protocol: The reagent solution was diluted 1:10 with the culture medium. 200 µl of the neutral red solution was added per well and the cells were incubated for 3 hours at 37 C and 5 % CO 2. The cells were washed, then 100 µl neutral red solution 1 (1 % CaCl 2, 0.5 % formaldehyde) were added for fixation for few minutes and were discarded. 66

Materials and methods Afterwards, 100 µl neutral red solution 2 (1 % acetic acid, 50 % ethanol) were added to extract the incorporated neutral red dye. The plates were placed on an orbital shaker for 10 min. The measurement of the extinction with a wavelength of 570 nm was performed after one hour of incubation at 37 C and 5 % CO 2. Neutral red optical density was measured in this assay. 67

Materials and methods 3.3.9.2 Measurement of metabolic activities 3.3.9.2.1 Measurement of the succinate dehydrogenase (SDH) activity in the supernatant SDH binds to the inner mitochondria membrane of mammalian and many bacterial cells. It takes an active part in the citric acid cycle and electron transport chain. Succinate is the most efficient energy source, so the SDH activity assay is an important method for measuring of metabolic activities (SAMOKHVALOV et al., 2004). In many cell types, mitochondria are the primary source of reactive oxygen species (ROS) (CADENS and DAVIES, 2000). The two major superoxide producing enzymes are considered to be the respiratory chain complexes nicotinamide adenine dinucleotide (NADH) and cytochrome C oxidoreductase (SUN and TRUMPOWER, 2003). Succinate oxidoreductase was discovered in 1909 in aerobic cells (THUNBERG, 1909). The enzyme has several interesting properties, for example that SDH is a membrane bound dehydrogenase linked to the respiratory chain and a member of the Krebs cycle (Fig. 11), beside its activity which is modulated by several activators and inhibitors. SDH is a complex enzyme containing nonheme iron, acidlabile sulfur, and covalently bound flavin adenine dinucleotide (FAD) (HEDERSTEDT and RUTBERG, 1981). SDH Fig. 11: Citric acid cycle (HEDERSTEDT and RUTBERG, 1981). 68

Materials and methods According to MRACEK et al. (2009), SDH was determined with some modifications spectrophotometrically in isolated mitochondria. The cells were plated and treated in the same way as mentioned in 3.3.7, and then the protocol of measuring SDH was done as follow: Cells were washed twice with PBS, 100 µl of Aqua bidest. was added in each well. The cells were lysed for 10 min by heat and cold, then were sonicated for 15 seconds and were centrifuged at 2000 x g at 4 C for 15 min. Afterwards, the supernatants were used for protein determination and 50 µl SDH reaction solution was added per well. The measurements of the extinction were performed at a wavelength of 340 nm at different time points (zero time, 10 min, 20 min and 30 min). Enzyme activities were expressed as nmol X min -1 /mg protein -1, using the molar absorption coefficient (e550 = 19.6 mm -1 cm). 3.3.9.2 Measurement of the pyruvate kinase (PK) activity in the supernatant Pyruvate is the end product of the glycolysis. Under aerobic conditions pyruvate enters the mitochondria and catalyzes the conversion of pyruvate to acetyl coenzyme A (acetyl-coa) producing NADH and CO 2. Acetyl-CoA subsequently enters into the Krebs (citric acid) cycle, providing energy adenosine triphosphate (ATP) to the cell (Fig. 12). This reaction is catalyzed by the enzyme-coenzyme complex pyruvate dehydrogenase (PDH) (HARRIS et al., 2002). PDH activity is under the control of pyruvate dehydrogenase kinases (PDKs). Under hypoxic conditions, conversion of pyruvate to lactate occurs. Lack of PK leads to a decrease in the glycolysis process. According to IORI et al. (2008), cells were plated and treated in the same way as described in 3.3.7, and then the protocol of measuring pyruvate was done as follow: The medium was removed from the cells, they were washed with PBS. 100 µl PBS were added and cells were lysed for 10 min by heat and cold, and then sonicated for 2 min and cells were centrifuged at 2000 g for 15 min at 4 C. The supernatants were used for protein determination (BRADFORD, 1976), The cells were collected using 250 μl of 100 mm triethanolamine, 0.5 mm EDTA/Na+ buffer (ph 7.6), supplemented with protease inhibitors. The enzyme assay starts by adding 100 μl of PK reaction solution. PK activity was estimated by a modification of the spectrometric method (GUTMANN and BERNT, 1974). The PK activity was measured as the 69

Materials and methods change in absorption of NADH at 340 nm (25 C) due to the coupled conversion of pyruvate to lactate catalyzed by lactate dehydrogenase (LDH). The measurements were performed on different time points (zero time, 10 min, 20 min and 30 min). Fig. 12: Pyruvate kinase pathway (YAISH, 2009) 3.3.9.2.3 Determination of the protein content The protein content of the cells was measured using Bio-Rad assay based on the method of Bradford. Bio-Rad is a dye binding assay, in which a differential colour change of a dye occurs in response to various concentrations of protein. The maximum absorbance of an acidic solution of Coomassive Brilliant Blue G-250 dye shifts when binding to protein. Coomassive blue dye binds to primary basic acids and aromatic amino acids residues especially arginine. Subsequent measurements with a spectrophotometer and comparing the results to a standard curve provide a relative measurement of protein concentration. The test was done as follow: Frozen samples were thawed and 160 µl of standard calibrations were added in each well of a 96-well plate, then 159 µl PBS was added to each sample (1 µl). Afterwards, 40 µl Bio-Rad reagents were added for the whole plate and all wells were mixed well 10 times. The plate was left at room temperature for 5-60 min. The measurements of the extinction took place at a wavelength of 570 nm. 70

Materials and methods 3.3.9.3 Histochemistry analysis To evaluate SDH activity by histochemistry (SELIGMAN and RUTENBURG, 1951; BROUILLET et al., 1998) frozen teat sections were cryosectioned at 8 µm at a temperature of -20 C. The udder tissue sections were transferred to glass slides and air-dried for 60 min before histochemical staining. Each tissue section was then incubated for 1 h at 37 C in a dark and humidified chamber in SDH phosphate buffer containing succinic acid (as a substrate) and nitroblue tetrazolium (SELIGMAN and RUTENBURG, 1951; TANJI and BONILLA, 2001; KIYOMOTO et al., 2008). After this procedure, the slices were rinsed with Aqua bidest. to stop the chemical reaction. The slides were then cover slipped. Endogenous SDH activity resulted in dark blue diformazan deposits from the NBT reduction through succinate oxidation. No blue deposits are formed in the absence of succinate substrate or in the presence of 3NP in the incubation. To quantify SDH, an image of each section was captured using camera connected to an optical microscope. The protocol was applied on cell culture samples by culturing and treating in the same way as mentioned before in 3.3.7. The medium was removed and cells were washed twice with PBS. 100 µl of histochemistry buffer was added and incubated for 4 h at 37 C and 5 % CO 2. The measurements were performed using a spectrophotometer with a wavelength of 570 nm. 71

Materials and methods 3.3.10 Measurement of biomarkers of inflammatory reactions 3.3.10.1 Preparation of the udder tissue for PGE 2 measurements The teats were cut in small pieces of approximately 1 cm 3 (25-30 mg) using a biopsy punch and were transferred in polypropylene tubes over crushed ice. A mixture of 5 mg indometacin + 5 ml DMSO (dimethylsufoxide) + 1 ml PBS was added and the samples were mixed by an ULTRA TURRAX for 30 min, followed by centrifugation at 3000 x g for 10 min. The supernatants were collected for the PGE 2 measurement. 3.3.10.2 Measurement of PGE 2 in the udder tissue supernatant The PGE 2 concentration in the culture medium supernatant was measured by a competitive enzyme immunoassay. This assay is based on the forward sequential competitive binding technique, in which PGE 2 competes with horseradish peroxidase (HRP) - labelled PGE 2 for a limited number of binding sites on a mouse monoclonal antibody. PGE 2 in the sample is allowed to bind to the antibody in the first incubation. During the second incubation, HRP- labelled PGE 2 binds to the remaining antibody sites. The amount of PGE 2 tracer that is able to bind to the monoclonal antibody is inversely proportional to the concentration of PGE 2 in the sample. This antibody- PGE 2 complex binds to goat polyclonal anti-mouse IgG that has been previously attached to the well of the assay kit. The plate is washed to remove any unbound material, and a substrate solution is added to the wells to determine the bound enzyme activity. The product of this enzymatic reaction has a distinct yellow colour and absorbs strongly at 412 nm, when the colour development has been stopped. The intensity of this colour is determined spectrophotometrically and is proportional to the amount of PGE 2 tracer bound to the well, which is inversely proportional to the amount of free PGE 2 present in the sample during the incubation. A standard curve was performed in each ELISA assay. The percentage of binding was used to establish a calibration curve. The measurements for the extinction of each sample were put in relation to these curves and the PGE 2 concentration was calculated. The samples were usually measured in duplicates and the following two controls were included in each. One of the samples was stimulated by the addition of 5 ml lipopolysaccharide (LPS) working solution (500 ng/ml LPS, final concentration) while 72

Materials and methods the other control was a pure medium. The supernatant was collected 24 h later and stored at -20 C until determination. 3.3.10.3 Measurement of bovine and mouse TNF-alpha in the culture medium supernatant The TNF-alpha concentration in the culture medium supernatant was measured. Briefly, the wells of the assay plates were preincubated with a monoclonal goat antimouse or bovine TNF-alpha antibody which binds any TNF-alpha of the sample (bovine or mouse). The bound TNF-alpha is detected using a biotinylated goat antimouse or bovine TNF-alpha antibody, and addition of TMB-substrate (tetramethylbenzidine) leads to a color reaction which stopped by using stop solution H 2 SO 4 (2N). TNF-alpha was measured spectrophotometrically at 450 nm. The intensity of the colour is proportional to the concentration of TNF-alpha in the sample. 3.3.10.4 Measurement of IL- 6 in the culture medium supernatant The IL-6 concentration in the culture medium supernatant was measured similarly to the TNF-alpha concentration with an enzyme immunoassay. Briefly, the wells of the assay plates were preincubated with rat anti-mouse IL-6 antibody, which binds any IL-6 of the sample. The bound IL-6 is detected utilizing a biotinylated goat anti-mouse IL-6 (detection antibody) and TMB-substrate. The colour reaction was stopped using stop solution H 2 SO 4 (2N) and was measured spectrophotometrically at 450 nm. The intensity of the colour is proportional to the concentration of IL- 6 in the sample. 3.3.10.5 Measurement of IL-1 beta in the culture medium The IL-1 beta concentration in the culture medium supernatant was measured similarly to the IL-6 concentration with an enzyme immunoassay using the same capture and detection antibody. All those assays are established methods at the Institute of Pharmacology, Toxicology and Pharmacy, Hannover, Germany. 73

Materials and methods 3.3.11 Detection of antibacterial activity 3.3.11.1 Bouillion dilution test and cultivation of bacteria in petri dishes Bouillion dilution test and cultivation of bacteria in petri dishes were done by the Milchtierherden-Betreuungs- und Forschungsgesellschaft mbh (MBFG). The aim of the tests is to determine the effect of Mg-Ag-NaCl solutions on the growth of bacteria (E. coli and S. aureus). Briefly, MgAg1% sticks were incubated in 5 ml NaCl solution in a water bath for 15 days and the degrading supernatants (MgAg1% sticks incubated in NaCl) were used for the bouillion dilution test and the cultivation of bacteria (E. coli and S. aureus). Firstly, bouillion dilution test was done by preparing a bacterial suspension by incubating about 2 E. coli and S. aureus colonies (GK) in 100 ml buffered peptone water at 37 C for 24 hours on the shaker. The bacterial dilution series was770 X 10 5, 910 X 10 5, 100 X10 6 and 210 X10 6 KbE/ml. Then 2 test tubes were filled by 5 ml of bacterial suspension, then degrading supernatants (MgAg1% sticks incubated in NaCl) were added to one of the tubes, and the other one serves as a negative control. Later, the number of bacterial colonies was determined. Secondly, E. coli and S. aureus colonies were cultivated in petri dishes. Afterwards, the bacterial colonies were incubated with the degrading supernatants (MgAg1% sticks incubated in NaCl), and the number of bacterial colonies was determined. 3.3.11.2 Brilliant black reduction (BRT- MRL Screening test) The BRT was first described by KRAACK and TOLLE (1967). The test medium is a mixture of nutrients, test bacteria Geobac. Stearothermophilus var. calidolactis C953 (B. stearothermophilus), brilliant black and other supplements which help to improve detection sensitivity towards chosen inhibitors (Fig. 13). Penicillin G serves as a positive control and milk as a negative control. 74

Materials and methods Cavities containing test medium + other supplements Fig.13: The BRT- MRL screening test cavities MgAg1% sticks were incubated with different volumes of DMEM medium (10, 3 and 1 ml). One tube of medium serves as a negative control, they were incubated in a water bath for 5 days. In addition, different concentrations of AgNO 3 solution (control, 0.0001, 0.0003, 0.001, 0.003, 0.01, 0.03, 0.1, 0.3, 1 mmol/l) were incubated with DMEM medium in test tubes without adding any supplements in a water bath for 5 days. 100 µl of each sample were added per well. Adhesive tapes were chosen to cover the wells during incubation for 5-6 hours at 65 C in a special incubator (Fig. 14). Fig.14: Incubator of BRT- MRL Screening test During the incubation time, the growing test bacteria shift the redox indicator (brilliant black) to its yellow or colourless reduction stage through the division of double azocompounds. Thus test medium changes from blue to yellow or colourless, if inhibitors are not present in the sample, therefore, the sample has no antibacterial effect. While the growth of bacteria will be minimal or non-existent, when there will be no reduction of the colouring agent or to a very small degree and the test medium will 75

Materials and methods remain blue, if the inhibitors are present in the medium, therefore, the sample has antibacterial effect. 3.3.12 Statistical analysis Values are means ± SD. A randomized block design was used in all of the experiments. Samples were usually measured at least in duplicates. The experiments were performed at least 4-6 times.to enable better comparison of the experiments, results are expressed in optical density and percent in comparison to the negative control. For statistical calculation of differences in the viability of isolated perfused udder (glucose consumption, lactate production and LDH activity), Two-way ANOVA test was performed. For the biocompatibility tests (MTS, neutral red, SDH, PK, IL-1 beta, IL-6 and TNF-alpha), One-way ANOVA test was performed followed by a Dunn s Multiple comparison test. For the degradation process of MgAg1% sticks in dry off period secretion and in the teats of isolated bovine udder, t-test was performed. For silver and magnesium concentrations in degradation medium, Two-way ANOVA test was performed. Statistical calculations were performed with GraphPad Prism 5.03 (GraphPad Software Inc., La Jolla). P values < 0.05 were considered statistically significant. 76

Results 4. Results 4.1 Establishment of mammary cell culture 4.1.1 Isolation of primary mammary cells Mammary cells were enzymatically and mechanically isolated and taken into culture. Both primary mammary fibroblasts and primary mammary epithelial cells were morphologically distinct (Fig. 15 A, B). Each cell type exhibited the typical morphological characters. The fibroblasts showed a spindle shaped morphology and the epithelial cells grew in a cobblestone-like pattern. The purity of each cell population was ensured by the specific isolation method and the typical morphological appearance of the cells, respectively. A B Fig. 15: Primary mammary fibroblasts (A) and primary mammary epithelial cells (B) under light microscope showed a spindle shaped morphology and cobblestone-like pattern, respectively cells; magnification = 20x; bar = 100 μm. 4.1.2 Culturing of primary mammary cells Epithelial cells were first passaged every 7 to 10 days, thereafter cells could be passaged every 3 to 5 days, while fibroblasts were passaged every 12 to 15 days. Then cells could be passaged every 8 to 10 days with an average number of 3-8 passages without changes in cell morphology. 77

Results 4.1. 3 Verification of primary mammary cells using immunocytochemistry Immunofluresence was performed to verify the morphological distinction of the two cell types. Fibroblasts were detected using a monoclonal anti-vimentin antibody (Fig. 16, B), while adding anti-cytokeratin to these cells did not result in staining of them. On the other hand, epithelium cells were detected using a monoclonal anti-pan cytokeratin which resulted in staining (Fig. 16, D), whereas the isotype control with the anti-vimentin antibody was negative. A B C D Fig. 16: Verification of the primary mammary cells: phase contrast microscopy (A, C) and fluorescence microscopy of immunocytological stains (B, D). Mammary epithelial cells (C, D) have a cobblestone-like growth and stain for cytokeratin (D), whereas the fibroblasts (A, B) show a typical spindle-like morphology and are vimentin-positive (B) ; magnification = 40x; bar = 50 μm. 78

Results 4.2 Degradation process of MgAg1% sticks 4.2.1 Isolated perfused udder 4.2.1.1 Measuring lactate production in the perfused udder The udder viability was measured by calculating the amount of lactate production after the perfusation process; the amount ranged from 0.1-0.5 mg/ml which indicates that the udder was viable during the experiment. Lactate production did not show a significant difference on both sides over time (Fig. 17). mg/ml 0.6 0.4 Left side Right side 0.2 0.0 0 2 4 6 Time (h) Fig. 17: Mean lactate concentration (mg/ml) in the perfusate of perfused udder. There is no significant difference of lactate production of left and right sides of perfused udder, n=6. 4.2.1.2 Measuring glucose consumption in the perfused udder The glucose consumption during the perfusation process ranged from 45-77 mg/dl on both sides of the udder. Glucose concentration did not show a significant difference on both sides over time (Fig. 18). 150 Left side Right side mg/dl 100 50 0 0 2 4 6 Time (h) Fig. 18: Mean glucose concentration (mg/dl) in the perfusate of perfused udder. There is no significant difference of glucose consumptions lactate production of left and right sides of perfused udder, n=6. 79

Results 4.2.1.3 Measuring lactate dehydrogenase enzyme (LDH) activity in the perfused udder The lactate dehydrogenase production during the perfusation process ranged from 1000-1750 mu/l on both sides of the udder. LDH concentration did not show a significant difference on both sides over time (Fig. 19). 3000 2000 Left side Right side mu/l 1000 0 0 2 4 6 8 Time (h) Fig. 19: Mean LDH concentration in the perfusate of perfused udder (mu/l). There is no significant difference of (LDH) production of left and right sides of perfused udder, n=6. 4.2.2 Degradation of MgAg1% sticks in bovine udder The MgAg1% sticks degraded in bovine udder after being incubated for 5-6 hours in the teat of isolated bovine udder (Fig. 20). A B Fig. 20: MgAg1% sticks. The stick before incubation in the teat of bovine udder (A), while (B) showed the stick after the incubation. 80

Results The initial and the final weight of MgAg1% sticks incubated in the teat of isolated bovine udder for 5-6 hours for 7 separated experiments was listed in (Tab. 8). The mean percentage of weigh loss during incubation of MgAg1% sticks was about 3 % of the initial weight (Fig. 21). Tab. 8: The initial and final weight of MgAg 1% sticks incubated in the teat of bovine udder for 5-6 hours (n=7) Trials 1 2 3 4 5 6 7 Initial weight/mg 56.06 53.27 53.97 54.43 54.63 53.3 52.6 Final weight/mg after 6 h 51.43 52.06 53.56 51.33 53.87 53.23 51.93 Weight in mg 60 55 50 * 45 Initial weight Weight after loss Fig. 21: Mean weight of MgAg1% sticks before and after incubation in the teat of bovine udder. There is a significant decrease of weight of MgAg1% sticks incubated 6 h in the teat of bovine udder (* P< 0.05, n= 7). 81

Results 4.2.3 Degradation of MgAg1% sticks in secretion samples from cows at dry off Period MgAg 1% stick was cutted into three parts nearly of the same weight.the initial and final weight of MgAg1% sticks after incubation in secretion from dry cows for 21 days was listed in (Tab. 9). The mean loss percentage for 6 separated experiments after incubation was 60 % of the initial weight, regarding that the stick of the highest weight was completely degradable after 21 days. Tab. 9: The initial and final weight of MgAg1% sticks incubated in dry secretion for 21 days (n=6) Trials 1 2 3 4 5 6 Initial weight 15.7 mg 14.0 mg 10.3 mg 9.5 mg 10.1 mg 8.6 mg Final weight Completely degraded 3.8 mg 6.6 mg 4.0 mg 4.7 mg 5.3 mg The mean final weigh of 6 separated experiments of MgAg1% sticks, after incubation in dry secretion for 21 days, revealed a significant difference compared to the initial weight (Fig. 22). 20 weight in mg 15 10 5 * 0 initial weight Final weight Fig. 22: Mean weight of MgAg1% sticks before and after incubation in dry off period secretion. There is a significant decrease of weight of MgAg1% sticks incubated 21 days in dry secretion (* P< 0.05, n=6). 82

Results The MgAg1% sticks degraded after incubation in dry off period secretion for 21 days compared to the initial stick (Fig. 23). A B Fig. 23: Cutted MgAg1% sticks. The stick before incubation (A). The stick after the 21 days of incubation in dry off period secretion (B). 83

Results 4.2.4 Histological parameters 4.2.4.1 Udder tissue incubated with MgAg1% sticks Both unstained teat tissue (Fig. 24, B) and teat tissues stained with hematoxylin and eosin (H&E) treated with MgAg1% sticks (Fig. 24, D) showed black deposits precipitated on the upper layer, which is more prominent compared to the unstained and stained tissues without treatment (Fig. 24, A & C). This indicates the degradation of MgAg1% sticks during incubation A B C D Fig. 24: The unstained teat tissue treated with MgAg1% sticks (B) and stained teat tissue with hematoxylin and eosin (H&E) treated with MgAg1% sticks (D) showed black deposits precipitated on the upper layer compared to control teat (A, C); magnification = 40x; bar = 50 μm. 84

Results 4.2.5 Silver concentrations in the degradation medium The amount of silver ions increased after 25 days of incubation in the degradation medium. The 1 ml volume of degradation medium contains higher concentration of silver than the volume of 3 and 10 ml. The mean concentration of Ag + ranged from 0.02-1 mmol/l (Fig. 25) Amount of silver (mmol\l) 1.4 1.2 1 0.8 0.6 0.4 0.2 0 5 10 15 20 25 Days of incubation Degradation medium Fig. 25: Mean silver concentration (mmol/l) in degradatiom medium after incubation of MgAg1% sticks in 1, 3 or 10 ml medium. There is a significant increase of silver concentration in degradation medium after 25 days of incubation (* p< 0.05, n=6). * * * 1ml 3ml 10ml 4.2.6 Magnesium concentrations in the degradation medium The amount of Mg ++ in the degradation medium increased after 10 days up to 25 days of incubation without showing any significant differences between 1, 3 and 10 ml volume of degradation medium. The mean concentration of magnesium ranged from 0.2-9 mmol/l (Fig. 26). 85

Results Amount of Mg (mmol/l) 14 12 10 8 6 4 2 0 * * * * * * * * * * * * 5 10 15 20 25 Degradation medium 1 ml 3 ml 10 ml Days of incubation Fig. 26: Mean Mg ++ concentration (mmol/l) in degradation medium after incubation of MgAg 1% sticks in 1, 3 or 10 ml medium. There is an enhanced concentration of magnesium in degradation medium on day 10 up to 25 (* P< 0.05, n=6). 86

Results 4.3 Biocompatibility tests 4.3.1 Cell viability and proliferation 4.3.1.1 MTS assay 1. Murine cells a. Incubated with silver ions Murine fibroblasts (L929 cells) were treated with different concentrations of AgNO 3 solution. A significant reduction in the viability of murine fibroblasts of 50 % was observed, when incubated with concentrations equal or above 0.1 mmol/l and more than 75 %, when incubated with concentrations above or equal to 0.3 mmol/l compared to the negative control cells (Fig. 27). Optical density 2.5 2.0 1.5 1.0 0.5 0.0 control 0.0001 0.0003 0.001 0.003 0.01 0.03 Fig. 27: Cell viability (MTS) of murine fibroblasts as indicated by the mean optical density of formazan after incubation in various concentration of AgNo 3. A significant reduction in the viability of murine fibroblasts at 0.1 mmol/l of AgNO 3 solution compared to control was observed (* p< 0.05, n= 6). 0.1 0.3 Concentration of AgNO 3 (mmol/l) * * * 1 L929 cells Raw murine macrophages and primary murine macrophages were treated with different concentration of AgNO 3 solution. A significant reduction in the viability of raw macrophages was observed by 30 %, when incubated with concentrations equal or higher than 0.3 mmol/l, while primary macrophages showed a significant reduction in viability by 35 % when incubated with concentrations equal or below 1 mmol/l (Fig. 28). 87

Results 1.5 Optical density 1.0 0.5 0.0 0.8 control 0.0001 0.0003 0.001 0.003 0.01 0.03 0.1 0.3 1mmol Concentration of AgNO 3 (mmol/l) * * Raw macrophages Optical density 0.6 0.4 0.2 0.0 control 0.0001 0.0003 0.001 0.003 0.01 0.03 Concentration of AgNO 3 (mmol/l) 0.1 0.3 1mmol Fig. 28: Cell viability (MTS) of raw macrophages and primary macrophages as indicated by the mean optical density of formazan after incubation in various concentration of AgNo 3. A significant reduction in the viability in raw macrophages at 0.3 mmol/l AgNo 3 solution compared to control, while primary macrophages showed a significant reduction in the viability at 1 mmol/l AgNo 3 solution compared to control (* p< 0.05, n= 5). * Primary macrophages 88

Results b. Incubated with MgAg1% sticks Murine fibroblasts were treated with degradation medium (MgAg1% sticks incubated in 1, 3, 10 ml medium). A significant reduction in viability of about 75 % was observed, when murine fibroblasts were incubated with volumes equal or below 1 ml of degradation medium (1 ml = 0.5 mmol/l Mg ++, 0.2 mmol/l Ag + ) (Fig. 29). 3 Optical density 2 1 * L929 0 control 10 ml 3 ml 1 ml Different volumes of degradation medium (ml) Fig. 29: Viability test (MTS assay) of murine fibroblasts as indicated by the mean optical density of formazan after treatment with degradation medium (MgAg1% sticks incubated in 1, 3, 10 ml medium). A significant reduction in the viability of murine fibroblasts at 1ml compared to control regarding that, 10 ml 0.2 mmol/l Mg ++, 0.02 mmol/l Ag +, 3 ml 0.3 mmol/l Mg ++, 0.04 mmol/l Ag + and 1 ml 0.5 mmol/l Mg ++, 0.2 mmol/l Ag + (* p< 0.05, n= 5). 89

Results Both raw murine macrophages and primary murine macrophages did not show a significant reduction in the viability, when treated with degradation medium (MgAg1% sticks incubated in 1, 3, 10 ml medium) (Fig. 30). 2.5 2.0 Raw macrophages Optical density 1.5 1.0 0.5 0.0 Control 10 ml 3 ml 1 ml Different volumes of degradation medium (ml) 2.5 Optical density 2.0 1.5 1.0 0.5 Primary macrophages 0.0 Control 10 ml 3 ml 1 ml Different volumes of degradation medium (ml) Fig. 30: Cell viability (MTS asaay) of raw macrophages and primary macrophages as indicated by the mean optical density of formazan after treatment with degradation medium (MgAg1% sticks incubated in 1, 3, 10 ml medium). No significant reduction in the viability of raw macrophages and primary macrophages compared to control regarding that, 10 ml 0.2 mmol/l Mg ++, 0.02 mmol/l Ag +, 3 ml 0.3 mmol/l Mg ++, 0.04 mmol/l Ag + and 1 ml 0.5 mmol/l Mg ++, 0.2 mmol/l Ag + (n= 5). 90

Results 2. Bovine cells a. Incubated with silver ions Primary mammary epithelial cells were treated with different concentrations of AgNO 3 solution and showed a slight tendency toward reduction, when incubated with concentrations equal or below 1 mmol/l (Fig. 31). 1.4 Optical density 1.2 1.0 0.8 Primary epithelial cells 0.6 control 0.001 0.003 0.01 0.03 0.1 0.3 Concentration of AgNO 3 (mmol/l) 1 Fig. 31: Cell viability (MTS assay) of primary mammary epithelial cells as indicated by the mean optical density of formazan after incubation in various concentration of AgNo 3. A tendency towards reduction in the viability was observed at 1 mmol/l AgNO 3 solution compared to control (* p< 0.05, n= 6). Primary mammary fibroblasts were treated with different concentrations of AgNO 3 solution. A significant reduction in the viability of about 75 % was observed, when incubated with concentrations equal or below 1 mmol/l (Fig. 32) 0.8 Optical density 0.6 0.4 0.2 * Primary mammary fibroblasts 0.0 control 0.001 0.003 0.01 0.03 0.1 0.3 Concentration of AgNO 3 (mmol/l) 1 Fig. 32: Cell viability (MTS) of primary mammary fibroblasts as indicated by the mean optical density of formazan after incubation in various concentration of AgNo 3. A significant reduction in the viability at 1 mmol/l of AgNO 3 solution compared to control (* p< 0.05, n= 6). 91

Results b. Incubated with MgAg1% sticks Primary mammary epithelial cells were treated with degradation medium (MgAg1% sticks incubated in 1, 3, 10 ml medium) did not show a reduction in the viability (Fig. 33). 1.4 optical density 1.2 1.0 0.8 Primary mammary epithelial cells 0.6 control 10 ml 3 ml 1 ml Different volumes of degradation medium (ml) Fig. 33: Cell viability (MTS assay) of primary mammary epithelial cells as indicated by the mean optical density formazan after treatment with degradation medium (MgAg1% sticks incubated in 1, 3, 10 ml medium). No significant reduction in the viability was observed compared with control regarding that, 10 ml 0.2 mmol/l Mg ++, 0.02 mmol/l Ag +, 3 ml 0.3 mmol/l Mg ++, 0.04 mmol/l Ag + and 1 ml 0.5 mmol/l Mg ++, 0.2 mmol/l Ag + l (n= 5). Primary mammary fibroblasts were treated with degradation medium (MgAg1% sticks incubated in 1, 3, 10 ml medium), while there was no significant reduction in the viability of primary mammary fibroblasts (Fig. 34). 0.5 Optical density 0.4 0.3 0.2 0.1 Primary mammary fibroblasts 0.0 control 10 ml 3 ml 1 ml Different volumes of degradation medium (ml) Fig. 34: Cell viability (MTS assay) primary mammary fibroblasts as indicated by the mean optical density of formazan after treatment with degradation medium (MgAg1% sticks incubated in 1, 3, 10 ml medium). They showed no significant reduction in the viability compared with control regarding that, 10 ml 0.2 mmol/l Mg ++, 0.02 mmol/l Ag +, 3 ml 0.3 mmol/l Mg ++, 0.04 mmol/l Ag + and 1 ml 0.5 mmol/l Mg ++, 0.2 mmol/l Ag + (n= 5). 92

Results 4.3.1.2 Neutral red assay 1. Murine cells a. Incubated with silver ions Murine fibroblasts were treated with different concentrations of AgNO 3 solution. A significant reduction in the viability of murine fibroblasts of 50 %, 70 % and 85 % was observed, when incubated with concentrations equal or higher than 0.1, 0.3 and 1 mmol/l, respectively (Fig. 35). Optical density 1.5 1.0 0.5 0.0 control 0.0001 0.0003 0.001 0.003 0.01 0.03 * 0.1 0.3 1mmol Concentration of AgNO 3 (mmol/l) L929 cells Fig. 35: Cell viability (Neutral red assay) of murine fibroblasts as indicated by the mean optical density of neutral red after incubation in various concentrations of AgNo 3. A significant reduction in the viability of murine fibroblasts at 0.1 mmol/l compared with control was observed, (* p< 0.05, n= 6). * * Both raw murine macrophages and primary murine macrophages were treated with different concentrations of AgNO 3. A significant reduction in the viability of 60 % and 85 % was observed, when raw macrophages were treated with concentrations above 0.03 and 0.3 mmol/l, respectively (Fig. 36). 93

Results Optical density Optical density 1.5 1.0 0.5 0.0 2.5 2.0 1.5 1.0 0.5 0.0 * control 0.0001 0.0003 0.001 0.003 0.01 0.03 0.1 0.3 Concentration of AgNO 3 (mmol/l) control 0.0001 0.0003 0.001 0.003 0.01 0.03 0.1 0.3 Concentration of AgNO 3 (mmol/l) Fig. 36: Cell viability (Neutral red assay) of raw macrophages and primary macrophages fibroblasts as indicated by the mean optical density of neutral red after incubation in various concentrations of AgNo 3. A significant reduction in the viability of raw macrophages at 0.1 mmol/l of AgNO 3 solution was observed compared with control, while primary macrophages showed significant reduction in the viability at 0.3 mmol/l compared with control (* p< 0.05, n= 6). * * 1 * 1 * Raw macrophages Primary macrophages 94

Results a. Incubated with MgAg1% sticks Murine fibroblasts did not show a reduction in viability, when treated with degradation medium (MgAg1% sticks incubated in 1, 3, 10 ml medium) (Fig. 37). 0.55 Optical density 0.50 0.45 0.40 0.35 L929 cells 0.30 Control 10 ml 3 ml 1 ml Different volumes of degradation medium (ml) Fig. 37: Cell viability (Neutral red assay) of murine fibroblasts as indicated by the mean optical density of neutral red after treatment with degradation medium (MgAg1% sticks incubated in 1, 3, 10 ml medium). No significant reduction in the viability was observed, when compared with control regarding that, 10 ml 0.2 mmol/l Mg ++, 0.02 mmol/l Ag +, 3 ml 0.3 mmol/l Mg ++, 0.04 mmol/l Ag + and 1 ml 0.5 mmol/l Mg ++, 0.2 mmol/l Ag + (n= 4). 95

Results Raw macrophages and primary macrophages did not show a reduction in the viability, when treated with degradation medium (MgAg1% sticks incubated in 1, 3, 10 ml medium/) (Fig. 38). 0.4 Optical density 0.3 0.2 0.1 Raw macrophages 0.0 control 10 ml 3 ml 1 ml Different volumes of degradation medium (ml) 0.6 Optical denisty 0.4 0.2 Primary macrophages 0.0 Control 10 ml 3 ml 1 ml Different volumes of degradation medium (ml) Fig. 38: Cell viability (Neutral red assay) of raw macrophages and primary macrophages fibroblasts as indicated by the mean optical density of neutral red after treatment with degradation medium (MgAg1% sticks incubated in 1, 3, 10 ml medium). No significant reduction in the viability of raw macrophages and primary macrophages were observed compared to control regarding that, 10 ml 0.2 mmol/l Mg ++, 0.02 mmol/l Ag +, 3 ml 0.3 mmol/l Mg ++, 0.04 mmol/l Ag + and 1 ml 0.5 mmol/l Mg ++, 0.2 mmol/l Ag + (n= 4). 96

Results 2. Bovine cells a. Incubated with silver ions Primary mammary epithelial cells were treated with different concentrations of AgNO 3 solution. A significant reduction in the viability of primary mammary epithelium cells of 20 % was observed, when incubated with concentrations equal or below 1 mmol/l (Fig. 39). Optical density 0.50 0.45 0.40 0.35 0.30 0.25 control 0.001 0.003 0.01 0.03 0.1 0.3 Concentration of AgNO 3 (mmol/l) Fig. 39: Cell viability (Neutral red assay) of primary mammary epithelial cells fibroblasts as indicated by the mean optical density of neutral red after incubation in various concentrations of AgNo 3. A significant reduction in the viability was observed at 1 mmol/l of AgNo 3 solution compared to control (* p< 0.05, n= 4). 1 * Primary mammary epithelial cells Primary mammary fibroblasts were treated with different concentrations of AgNO 3 solution. A significant reduction in viability of 80 % was observed, when incubated with concentrations equal or below 1 mmol/l (Fig. 40). 0.4 Optical density 0.3 0.2 0.1 * Primary mammary fibroblasts 0.0 control 0.001 0.003 0.01 0.03 0.1 0.3 1 Concentration of AgNO 3 (mmol/l) Fig. 40: Cell viability (Neutral red assay) of primary mammary fibroblasts as indicated by the mean optical density of neutral red after incubation in various concentrations of AgNo 3. A significant reduction in the viability of primary mammary fibroblasts at 1 mmol/l was observed compared to control (* p< 0.05, n= 6). 97

Results b. Incubated with MgAg1% sticks Primary mammary epithelium cells did not show a reduction in the viability, when treated with degradation medium (MgAg1% sticks incubated in 1, 3, 10 ml medium) (Fig. 41). 0.6 Optical density 0.5 0.4 0.3 0.2 0.1 0.0 control 10 ml 3 ml 1 ml Different volumes of degradation medium (ml) Primary mammary epithelial cells Fig. 41: Cell viability (Neutral red assay) of primary mammary epithelium cells as indicated by the mean optical density of neutral red after treatment with degradation medium (MgAg1% sticks incubated in 1, 3, 10 ml medium). No significant reduction in the viability was observed compared to control regarding that, 10 ml 0.2 mmol/l Mg++, 0.02 mmol/l Ag+, 3 ml 0.3 mmol/l Mg++, 0.04 mmol/l Ag+ and 1 ml 0.5 mmol/l Mg++, 0.2 mmol/l Ag+ (n= 4). Primary mammary fibroblasts did not show a reduction in viability, when treated with degradation medium (MgAg1% sticks incubated in 1, 3, 10 ml medium) (Fig. 42). 0.20 Optical density 0.15 0.10 0.05 Primary mammary fibroblasts 0.00 control 10 ml 3 ml 1 ml Different volumes of degradation medium (ml) Fig. 42: Cell viability (Neutral red assay) of primary mammary fibroblasts as indicated by the mean optical density of neutral red after treatment with degradation medium (MgAg1% sticks incubated in 1, 3, 10 ml medium). No significant reduction in the viability was observed compared with control regarding that, 10 ml 0.2 mmol/l Mg++, 0.02 mmol/l Ag+, 3 ml 0.3 mmol/l Mg++, 0.04 mmol/l Ag+ and 1 ml 0.5 mmol/l Mg++, 0.2 mmol/l Ag+ (n= 4). 98

Results 4.3.1.3 Measuring the amount of protein in the supernatants Primary mammary epithelium cells showed a reduction in the protein amounts when treated with different concentrations of AgNO 3 solution. A significant reduction was observed at concentrations equal or above 0.3 mmol/l. When they were treated with degradation medium (MgAg1% sticks incubated in 1, 3, 10 ml medium), they did not show a significant decrease in the protein amount (Fig. 43) 25 Optical density Optical density 20 15 10 5 0 10 9 8 7 6 * control 0.0001 0.0003 0.001 0.003 0.01 0.03 0.1 0.3 Concentrations of AgNO 3 (mmol/l) 1 * Primary mammary epithelial cells 5 control 10 ml 3 ml 1 ml Different volumes of degradation medium (ml) Fig. 43: Bio-Rad assay of primary mammary epithelial cells. Mean optical density for Coomassive Brilliant Blue G-250 dye as an indicator of protein concentration. A significant decrease in the protein amount of primary mammary epithelial cells at concentrations equal or above 0.3 mmol/l was observed. While no significant difference was observed when treated with degradation medium (MgAg1% sticks incubated in 1, 3, 10 ml medium) compared to control (* p< 0.05, n= 4). 99

Results 4.3.2 Measurement of metabolic activities 4.3.2.1 Measurement of SDH activity in the supernatant 1. Murine cells Murine fibroblasts showed an increase in SDH activity, when incubated with different concentrations of AgNO 3 solution equal or higher than 0.3 mmol/l, while they showed no significant differences when treated with degradation medium (MgAg1% sticks incubated in 1, 3, 10 ml medium) (Fig. 44). SDH OD/ g protein 0.5 0.4 0.3 0.2 0.1 0.0 control 0.0001 0.0003 0.001 0.003 0.01 0.03 0.1 0.3 Concentration of AgNO 3 (mmol/l) * 1 * L929 cells 0.5 SDH OD/ g protein 0.4 0.3 0.2 0.1 L929 cells 0.0 control 10 ml 3 ml 1 ml Different volumes of degradation medium (ml) Fig. 44: Mean SDH activity of murine fibroblasts. A significant increase in the SDH activity of L929 at 0.3 mmol/l was observed compared to control, while no significant difference were observed with different volumes of degradation medium of MgAg1% sticks compared to control regarding that, 10 ml 0.2 mmol/l Mg ++, 0.02 mmol/l Ag +, 3 ml 0.3 mmol/l Mg ++, 0.04 mmol/l Ag + and 1 ml 0.5 mmol/l Mg ++, 0.2 mmol/l Ag + (* p< 0.05, n= 6). 100

Results 2. Bovine cells Primary mammary epithelium cells showed an increase in the SDH activity by 65%, when incubated with concentrations of AgNO 3 solution equal or below 1 mmol/l. Furthermore, they showed the highest SDH activity, when treated with volume equal or below 1 ml degradation medium of MgAg1% sticks (Fig. 45). SDH OD/ g protein 40 30 20 10 * Primary mammary epithelial cells 0 control 0.001 0.003 0.01 0.03 0.1 0.3 Concentration of AgNO 3 (mmol/l) 1 SDH OD/ g protein 8 6 4 2 * Primary mammary epithelial cells 0 control 10 ml 3 ml 1 ml Different volumes of degradation medium (ml) Fig. 45: Mean SDH activity of primary mammary epithelial cells. A significant increase in the SDH activity of primary mammary epithelial cells at 1 mmol/l compared to control was observed, in addition, a significant increase was observed at 1 ml of the degradation medium of MgAg1% compared to control regarding that, 10 ml 0.2 mmol/l Mg ++, 0.02 mmol/l Ag +, 3 ml 0.3 mmol/l Mg ++, 0.04 mmol/l Ag + and 1 ml 0.5 mmol/l Mg ++, 0.2 mmol/l Ag + (* p< 0.05, n= 6). 101

Results Primary mammary fibroblasts did not show differences in SDH activity, when incubated with different concentrations of AgNO 3 solution, while they showed the highest SDH activity, when treated with volume equal or below 1 ml degradation medium of MgAg1% sticks (Fig. 46). 0.35 SDH OD/ g protein 0.30 0.25 0.20 0.15 control 0.001 0.003 0.01 0.03 0.1 0.3 Concentration of AgNO 3 (mmol/l) 1 SDH OD/ g protein 5 4 3 2 1 * Primary mammary fibroblasts 0 control 10 ml 3 ml 1 ml Different volumes of degradation medium (ml) Fig. 46: Mean SDH activity of primary mammary fibroblasts. There is no significant difference of SDH activity of primary mammary fibroblasts compared to control when treated with concentrations of AgNO 3 solution, while a significant increase was observed at 1 ml volume of the degradation medium of MgAg1% sticks compared to control regarding that, 10 ml 0.2 mmol/l Mg ++, 0.02 mmol/l Ag +, 3 ml 0.3 mmol/l Mg ++, 0.04 mmol/l Ag + and 1 ml 0.5 mmol/l Mg ++, 0.2 mmol/l Ag + (* p< 0.05, n= 6). 102

Results 4.3.2.2 Measurement of PK activity in supernatant 1. Murine cells Murine fibroblasts cells showed an increase in the PK activity by about 60 %, when incubated with concentrations equal or higher than 0.3 mmol/l, while they showed no significant difference, when incubated with degradation medium (MgAg1% sticks incubated in 1, 3, 10 ml medium) (Fig. 47). PK OD/ g protein 0.5 0.4 0.3 0.2 0.1 0.0 control 0.0001 0.0003 0.001 0.003 0.01 0.03 0.1 0.3 Concentration of AgNO 3 (mmol/l) * 1 * L929 cells 0.6 PK OD/ g protein 0.4 0.2 0.0 control 10 ml 3 ml 1 ml Different volumes of degradation medium (ml) Fig. 47: Mean PK activity of murine fibroblasts. A significant increase in the PK activity of L929 at 0.3 mmol/l compared to control was observed, while no significant difference was observed with different volumes of degradation medium of MgAg1% compared to control regarding that, 10 ml 0.2 mmol/l Mg ++, 0.02 mmol/l Ag +, 3 ml 0.3 mmol/l Mg ++, 0.04 mmol/l Ag + and 1 ml 0.5 mmol/l Mg ++, 0.2 mmol/l Ag + (* p< 0.05, n= 6). 103

Results 2. Bovine cells Primary mammary epithelium cells showed an increase in the PK activity by about 60 %, when incubated with concentrations equal or below 1 mmol/l. Furthermore, they showed the highest PK activity when incubated with 1 ml volume of the degradation medium of MgAg1% (Fig. 48). 30 * PK OD/ g protein 20 10 0 control 0.001 0.003 0.01 0.03 0.1 0.3 Concentration of AgNO 3 (mmo/l) 1 PK OD/ g protein 8 6 4 2 * Primary mammary epithelial cells 0 control 10 ml 3 ml 1 ml Different volumes of degradation medium (ml) Fig. 48: Mean PK activity of primary mammary epithelial cells. A significant increase in the PK activity of primary mammary epithelium at 1 mmol/l compared to control, in addition to a significant increase was observed at 1 ml of the degradation medium of MgAg1% sticks compared to control regarding that, 10 ml 0.2 mmol/l Mg ++, 0.02 mmol/l Ag +, 3 ml 0.3 mmol/l Mg ++, 0.04 mmol/l Ag + and 1 ml 0.5 mmol/l Mg ++, 0.2 mmol/l Ag + (* p< 0.05, n= 6). 104

Results Primary mammary fibroblasts did not show significant difference of PK activity, when incubated with different concentrations of AgNO 3 solution, while they showed highest PK activity, when incubated with 1 ml of degradation medium of MgAg1% sticks (Fig. 49). 0.35 PK OD/ g protein 0.30 0.25 0.20 0.15 control 0.001 0.003 0.01 0.03 0.1 0.3 Different concentration of AgNO 3 (mmol/l) 1 Primary mammary fibroblasts PK OD/ g protein 4 3 2 1 * 0 control 10 ml 3 ml 1 ml Different volumes of MgAg1% (ml) Fig. 49: Mean PK activity of primary mammary fibroblasts. No significant increase in the PK activity of primary mammary fibroblasts was observed, when treated with different concentrations of AgNO 3 solution compared to control. In addition, a significant increase was observed at 1 ml of the degradation medium of MgAg1% sticks compared to control regarding that, 10 ml 0.2 mmol/l Mg ++, 0.02 mmol/l Ag +, 3 ml 0.3 mmol/l Mg ++, 0.04 mmol/l Ag + and 1 ml 0.5 mmol/l Mg ++, 0.2 mmol/l Ag + (* p< 0.05, n= 6). 105

Results 4.3.2.3 Succinate staining of tissue and cells 4.3.2.3.1 Teat tissues After succinate staining, the teat tissues, which were incubated with MgAg1% sticks, showed a higher SDH activity presented by a violet or dark blue diformazan from nitrozolium blue reduction through succinate oxidation (Fig. 50 B) compared to the untreated teat (Fig. 50 A). Furthermore, after staining with succinate and H&E, higher SDH activity was also observed in the teat tissues which were incubated with MgAg 1% sticks (Fig. 50 D) compared to the untreated teat (Fig. 50 C). A B C D Fig. 50: Tissue sections of untreated teat (A, C) and the treated teat with MgAg1% sticks for 5-6 hours (B, D). The treated teat stained by succinate stain (B) and succinate stain / H&E (D) showed a dense dark blue colour compared to untreated teat; magnification = 40x; bar = 50 μm. 106

Results 4.3.2.3.2 Primary mammary epithelial cells Primary mammary epithelial cells showed a higher SDH activity compared to control, when incubated with higher concentrations of AgNO 3 solution at 1 mmol/l (Fig. 51). A B Fig. 51: Light microscopic pictures of primary mammary epithelium cells stained with succinate and nitrozolium blue. Primary mammary epithelium cells treated with 1 mmol/l of AgNO 3 showed darker blue colour (B) compared to untreated control cells (A) which showed less colour; magnification = 20x; bar = 100 μm. Primary mammary epithelial cells stained with succinate and nitrozolium blue did not show significant differences, when incubated with either different concentrations of AgNO 3 or different volumes of degradation medium of MgAg1% (Fig. 52). Primary mammary epithelial cells 0.4 0.30 Optical denisty 0.3 0.2 0.1 Optical denisty 0.25 0.20 0.0 control 0.001 0.003 0.01 0.03 0.1 0.3 Concentration of AgNo 3 (mmol\l) 1 0.15 Control 10 ml 3 ml 1 ml Different volumes of degradation medium (ml) Fig. 52: Mean SDH activity of primary mammary epithelial cells treated with succinate and nitrozolium blue. There is no significant difference in incubated primary epithelium cells with different concentration of AgNO 3 and degradation medium of MgAg1% sticks (n= 4). 107

Results pg/ml pg/ml 4.3.3 Biomarkers of inflammatory reactions 4.3.3.1 Measurement of IL-1 beta in the culture medium supernatant Primary macrophages, raw macrophages and L929 did not show indications of an enhance of IL-1 beta release after incubation with different concentrations of AgNO 3 solution or different volumes of degradation medium MgAg1%, although there was a significant response for stimulation with LPS (Fig. 53). pg/ml 1500 1000 500 0 1500 1000 500 0 50 40 30 20 10 0 control primary macrophage control 0.001 0.003 0.01 0.03 0.1 0.3 L929 cells 1 LPS Concentration of AgNO 3 (mmol/l) raw macrophage control 0.001 0.003 0.01 0.03 0.1 0.3 1 LPS Concentration of AgNO 3 (mmol/l) 0.0001 0.0003 0.001 0.003 0.01 0.03 0.1 0.3 1 LPS Concentration of AgNO 3 (mmol/l) * Pg/ ml Pg/ ml Pg/ ml L929 cells Fig. 53. Detection of IL-1 beta of primary macrophage, raw macrophage and L929 cells. primary macrophage, raw macrophage and L929 cells treated with AgNO 3 and degradation medium of MgAg1% showed no significant difference of IL-1 beta release compared to control (* p< 0.05, n= 4). 800 600 400 200 0 control 10 ml 3 ml 1 ml LPS Different volumes of degradation medium (ml) 1500 1000 500 0 1500 1000 500 0 control control primary macrophage raw macrophage * * * 10 ml 10 ml 3 ml 3 ml 1 ml 1 ml LPS Different volumes of degradation medium (ml) LPS Different volumes of degradation medium (ml) * * 108

Results pg/ml 4.3.3.2 Measurement of IL- 6 in the culture medium supernatant Primary macrophages and raw macrophages did not show indication of an enhance of IL-6 release after incubation with different concentrations of silver nitrate or different volumes of degradation medium, although there was a significant response for stimulation with LPS (Fig. 54). pg/ml pg/ml 1500 1000 500 0 100 80 60 40 20 0 2000 1500 1000 500 0 control primary macrophages control 0.001 0.003 0.01 0.03 0.1 0.3 1 LPS Concentration of AgNO 3 (mmol/l) raw macrophage below detection control 0.001 0.003 0.01 0.03 0.1 0.3 1 LPS Concentration of AgNO 3 (mmol/l) L929 cells * 0.001 0.003 0.01 0.03 0.1 0.3 1 LPS Concentration of AgNO 3 (mmol/l) * * pg/ml raw macrophages below detection Fig. 54: Detection of IL-6 of primary macrophage, raw macrophage and L929 cells. Primary macrophage, raw macrophage and L929 cells treated with AgNO 3 and MgAg1% showed no significant difference of IL-6 release compared to control (* p< 0.05, n= 4). pg/ ml pg/ml 1500 1000 150 100 50 0 control below detection 10ml 3ml 1ml LPS Different volumes of degradatiom medium (ml) 500 0 150 100 50 0 control control primary macrophages 10ml L929 cells 10ml 3ml 3ml 1ml 1ml LPS Different volumes of degradatiom medium (ml) * LPS Different volumes of degradation medium (ml) * * 109

Results 4.3.3.3 Measurment of bovine TNF-alpha in the culture medium supernatant Primary mammary epithelium cells did not show an enhance of TNF-alpha production after incubation with different concentrations of AgNO 3 solution, while there was a significant stimulation with LPS (Fig. 55). Primary mammary epithelial cells 30 * 20 * pg/ml 20 10 0 control below detection 0.001 0.003 0.01 0.03 0.1 0.3 1 LPS Concentration of AgNO 3 (mmol/l) pg/ml 15 10 5 0 control below detection 10 ml 3 ml 1 ml LPS Different volumes of degradation medium (ml) Fig. 55: Detection of TNF-alpha of primary mammary epithelium cells. Primary mammary epithelium cells treated with different concentrations of AgNO 3 solution and degradation medium (MgAg1% sticks incubated in 1, 3, 10 ml medium) showed no significant difference of TNF-alpha release compared to control, regarding that, 10 ml 0.2 mmol/l Mg ++, 0.02 mmol/l Ag +, 3 ml 0.3 mmol/l Mg ++, 0.04 mmol/l Ag + and 1 ml 0.5 mmol/l Mg ++, 0.2 mmol/l Ag + (* P< 0.05, n= 4). 4.3.3.4 Measurement of mouse TNF-alpha in the culture medium supernatant Primary macrophages, raw macrophage and L929 cells did not show an indication of an enhance of TNF-alpha release after incubation with different concentrations of silver nitrate or different volumes of degradation medium, although there was a response for stimulation with LPS (Fig. 56). 110

Results pg/ml pg/ml 2000 1500 1000 500 0 2000 1500 1000 500 0 primary macrophages control 0.001 0.003 0.01 0.03 0.1 0.3 1 LPS Concentration of AgNO 3 (mmol/l) raw macrophages control 0.001 0.003 0.01 0.03 0.1 0.3 1 LPS Concentration of AgNO 3 (mmol/l) 5000 4000 * * L929 cells pg/ml pg/ml 600 400 200 0 250 200 150 100 50 0 control control primary macrophages * 10 ml 10 ml 3 ml 3 ml 1 ml 1 ml LPS Different volumes of degradation medium (ml) LPS Different volumes of degradation medium (ml) * * raw macrophages pg/ml 3000 2000 1000 0 control 0.001 0.003 0.01 0.03 0.1 0.3 Fig. 56: Detection of TNF-alpha of primary macrophage, raw macrophage and L929 cells. Primary macrophage, raw macrophage and L929 treated with AgNO 3 and different volumes of the degradation medium of MgAg1% showed no significant difference of TNF-alpha release compared to control (* P< 0.05, n= 4). 1 LPS Concentration of AgNO 3 (mmol/l) 111

Results 4.3.3.5 PGE 2 concentration in teat tissue The teat tissue did not show a release of PGE 2 after incubation with MgAg1% sticks for 5-6 hours (Fig. 57). 2000 1500 pg/ml 1000 500 0 untreated teats MgAg1% treated teats Fig. 57: Mean of PGE2 concentration of bovine teat tissues. There is no significant difference in PGE2 release, when teats treated with MgAg1% sticks compared to the untreated teats. 112

Results 4.4 Antibacterial activity 4.4.1 Bouillion dilution test and cultivation of bacteria in petri dishes The mean concentration of Mg ++ in degrading supernatants (MgAg1% sticks incubated in NaCl) which was used in the bouillion dilution test was about 0.8 mmol/l after 15 days of incubation (Fig. 58). Amount of Mg (mmol/l) 1 0.8 0.6 0.4 0.2 0 5 10 15 Days of incubation Fig. 58: Mean magnesium concentration (mmol/l) in the degrading supernatants (MgAg1% sticks incubated in NaCl). The amount of Mg ++ showed no significant differences at different time interval when incubated in MgAg1% degradation-nacl (n=6). The mean concentration of Ag + in degrading supernatants (MgAg1% sticks incubated in NaCl) which was used in bouillion dilution test was about 0.3 mmol/l after 15 days of incubation (Fig. 59). * * Fig. 59: Mean silver concentration (mmol/l) in degrading supernatants (MgAg1% sticks incubated in NaCl). The concentration of silver showed a significant increase after 10 days up to 15 days of incubation n MgAg1% degradation NaCl (* p< 0.05, n=6). 113

Results MgAg1% sticks were incubated in NaCl for 15 days, then the degrading supernatants (MgAg1% sticks incubated in NaCl) were incubated with E. coli and S. aureus for bouillion dilution test with series of bacterial dilutions. The mean number of the bacterial colonies without incubation with the degrading supernatants (MgAg1% sticks incubated in NaCl) was 1195 X 10 5 KbE/ml (Fig. 60 B), while after incubation with the degrading supernatants (MgAg1% sticks incubated in NaCl), the mean number of the bacterial colonies decreased to 0 X 10 5 KbE/ml (Fig. 60 A). Preliminary studies of bouillion dilution test showed antibacterial effect of the degrading supernatants of MgAg1%-NaCl on the growth of E. coli and S. aureus. A B Fig. 60: Bouillion dilution test of the degrading supernatants (MgAg1% incubated in NaCl). The degrading supernatant of MgAg1%-NaCl has antibacterial effect on the growth of E. coli and S. aureus (A) compared to the untreated bacteria (B). Therefore, the growth of E. coli and S. aureus was studied on petri dishes.the number of the bacterial colonies of E. coli without incubation with the degrading supernatants (MgAg1% stick incubated in NaCl) was 1299 colonies (Fig. 61 A), while after incubation (15 days of incubation) with the degrading supernatants (MgAg1% incubated in NaCl), bacterial colonies decreased to 269 colonies (Fig. 61 B). In 114

Results addition, the number of the bacterial colonies of S. aureus was 1194 colonies (Fig. 61 C), while after incubation with degrading supernatants (MgAg1% sticks incubated in NaCl), bacterial colonies decreased to 177(Fig. 61 D). The MgAg1% sticks have antibacterial activity with 5 times less colonies. E. coli A B S. aureus C D Fig. 61: Growth of E. coli and S. aureus cultivated on petri dishes. The number of the bacterial colonies of E. coli and S. aureus incubated with degrading supernatants (MgAg1% sticks incubated in NaCl) was decreased 5 times(b, D) compared to non incubated ones(a,c). 4.4.2 Brilliant black reduction (BRT- MRL screening test) Geobacillus stearothermophilic bacteria and other supplements were incubated with different concentrations of AgNO 3 solution or degradation medium (MgAg1% sticks incubated in 1, 3, 10 ml medium). Geobacillus stearothermophilic bacteria shifted from brilliant black colour to yellow or colourless, when treated with concentrations equal or below 0.003 mmol/l of AgNO 3 solution (Fig. 62). While, the colour of the bacteria shifted to blue colour at volume 115

Results higher than 3 ml of degradation medium (MgAg1% sticks incubated in 1, 3, 10 ml medium) (Fig. 63). Conc. of AgNo 3 Antibacterial activity solution (mmol/l) 1 +++ 0.3 +++ 0.1 +++ 0.03 +++ 0.01 ++ 0.003 + 0.001 + 0.0003-0.0001 - +ve +ve 0.1 0.1 0.001 0.001 -ve -ve 0.03 0.03 0.0003 0.0003 1mmol 0.01 0.0001 0.0001 1mmol 0.01 0.0001 0.3 0.003 medium 0.3 0.003 medium Fig. 62: BRT-MRL screening test of different concentrations of AgNO 3 + ve wells indicate positive control - ve wells indicate negative control 0.01-1 mmol/l AgNO 3 solution showed antibacterial activity 0.001-0.003 mmol/l AgNO 3 solution showed a suspicious result 0001-0.0003 mmol/l AgNO 3 solution showed no antibacterial activity Conc. of MgAg1% Antibacterial activity degradation medium (ml) 1 +++ 3 ++ 10 - Fig. 63: BRT- MRL screening test of degradation medium of MgAg 1% + ve wells indicate positive control - ve wells indicate negative control 3-1 ml of MgAg 1% degradation medium showed antibacterial activity 10 ml of MgAg 1% degradation medium showed no antibacterial activity 116

Discussion 5 Discussion 5.1 Biocompatibility of silver ions and silver containing alloy 5.1.1 Effect of AgNO 3 and MgAg1% sticks on the viability of different cells Ag + salts have been shown to exert an inhibitory effect on proliferation and differentiation of several cell lines: bone marrow cells and keratinocytes (HOLLINGER, 1996), hepatocytes (LIU et al., 1991), lymphocytes (HUSSAIN et al., 1992) or leukocytes (JANSSON and HARMS-RINGDAHL, 1993). Our experiments provided detailed information about the biocompatibility of AgNO 3 and degradation medium of MgAg1% sticks on primary cell cultures (fibroblasts and epithelial cells), murine fibroblasts and macrophages. AgNO 3 is known to have a good antimicrobial action with low toxicity (BRUTEL DE LA RIVIERA et al., 2000; OLSON et al., 2002; KLASEN, 2000; HOLLINGER, 1996). Antiseptics such as AgNO 3, which are potent broad-spectrum antimicrobial agents, are administered until there is closure of the epithelium to minimize the risk of infection. COOPER et al. (1991) stated that cytotoxicity of Ag + is considered to be low while the bactericidal power is high and that is one of the reasons why we choose Ag + for our alloy, in addition to BOSWALD et al. (1999), GREIT et al. (1999), OLOFFS (1994) who stated that there is no cytotoxic effect of Ag in addition to its good biocompatibility. In the present study, several end-point parameters (MTS, Neutral red) were used to indicate the cytotoxic action of AgNO 3 and degradation medium of MgAg1% sticks. Protein content was used as an indirect method for assessing cell number revealed that AgNO 3 produces an evident loss of cell protein content depending on the concentration of AgNO 3. Our cell viability results classify AgNO 3 as low cytotoxic, since there was an affect on the viability of different cells either primary or cell lines, but only at high concentrations. When MTS and neutral red tests were conducted, AgNO 3 solution had a slight influence on the viability of primary mammary epithelium cells at high concentrations, while MgAg1% degradation medium did not show an influence on on the viability of primary mammary epithelium. AgNO 3 showed a weak influence on the viability of murine fibroblasts at 0.3 mmol/l, but primary mammary fibroblasts were more resistant as AgNO 3 affected its viability at 1 mmol/l. MgAg1% degradation medium did not show an influence on the viability of both murine and 116

Discussion mammary fibroblasts cell. Murine and raw macrophages viability was not affected by MgAg1% degradation medium, while they showed a decrease in the viability at 0.3 mmol/l of AgNO 3. If the results obtained in our study are compared with those presented by TEEPE et al. (1993) in similar conditions (24-hour incubation with AgNO 3 in 10 % FCS), but performed in human keratinocytes, they found that AgNO 3 concentrations which are totally toxic for fibroblasts are approximately 40-50 times lower than those found by those authors for keratinocytes. In addition, when FCS 10 % is added to the fibroblast culture, AgNO 3 toxicity decreased markedly and only began to appear at 6.5 mmol/l. However, this AgNO 3 concentration with FCS 5 % or 2 % produced a total loss of protein content (TEEPE et al., 1993) This would indicate that fibroblasts are far more susceptible to AgNO 3 than keratinocytes. We can conclude that degradation medium of MgAg1% did not have an influence on the viability of our cell cultures (mammary epithelium and fibroblasts, murine fibroblasts and murine and raw macrophages), while AgNO 3 solution had a slight affect on our cell cultures at high concentrations, as silver is well known to be dose dependent (PERRELLI and PIOLATTO 1992; BRUTEL DE LA RIVIERA et al., 2000). 5.1.2 Effect of AgNO 3 and MgAg1% sticks on the metabolic activities Ag ions are also actively involved in the cellular energy metabolism for the most part by influencing many enzymes participating in glycolysis, the citric acid cycle and the respiratory chain (WOLF and TRAPANI, 2008; COWAN, 2002). Pyruvate kinase and succinate dehydrogenase were chosen as marker enzymes for mitochondria and cytosol. Inhibition by Ag ions of succinate uptake into membrane vesicles of E. coli has been noted (RAYMAN et al., 1972). Quite surprisingly, our results indicate an enhanced activity of SDH and PK activity in a tissue in contact with MgAg1% sticks implant as well as the different cell cultures treated with AgNO 3 solution and degradation medium of MgAg1% sticks. SDH and PK activity was found to be significantly increased in case of higher concentrations of AgNO 3 solution and degradation medium of MgAg1% sticks in the culture medium. The reason for this outcome is yet unknown. 117

Discussion Our results indicate a higher activity by mitochondrial dehydrogenase. To our best knowledge, these findings are described here for the first time and refer to a basic aspect of cell physiology. SCHREURS and ROSENBERG (1981) stated that if Ag + damaged the cell membrane, making it freely permeable esterified phosphate should appear outside the cell after Ag + treatment and that probability could be applied on succinate and pyruvate enzymes, but we found that there was no decrease in SDH and PK activity after treatment with either AgNO 3 solution or degradation medium of MgAg1%. In contrast to this, increased amounts of AgNO 3 concentrations showed a stimulation of SDH and PK activity, but unfortunately the reason is still unknown. The results showed that SDH and PK activity of primary mammary epithelium and fibroblasts was high after treatment with either AgNO 3 solution at 1 mmol/l or degradation medium of MgAg1% at 1 ml. similary, primary mammary epithelium cells showed a high SDH activity under light microscope too, when treated with AgNO 3 solution at 1 mmol/l, furthermore, histologically, bovine teats treated with MgAg1% sticks showed a high SDH activity in comparison to the untreated bovine teats, while murine fibroblasts showed a higher SDH and PK activity than primary mammary cell cultures at 0.3 mmol/l, when treated with AgNO 3 solution. Interestingly, the histochemical SDH activity showed the same results for the bovine teats tissues after treatment with MgAg1% sticks in comparison to the untreated teats. It showed a higher SDH activity represented in a dark blue colour in comparison to the control untreated teat. The definite explanation for this point still can not be stated, but whatever the explanation for this increase is, we did not have any decrease in the metabolic activity which means that the mitochondrial functions and lysosomes were not affected by our MgAg1% alloys or with different concentration of AgNO 3. HIDALGO and DOMINGUEZ (1998) demonstrated that Ag ions impaired mitochondrial activity in human fibroblasts assessed by succinate dehydrogenase activity. 118

Discussion 5.1.3 Effect of AgNO 3 and MgAg1% sticks on the biomarkers of inflammatory reactions The aim was to measure the inflammatory reaction in ex vivo and in vitro experiments, as potential inflammation is a major concern in the context of biomaterials and its constitutes are an important element of biocompatibility (WITTE et al., 2008; PURNAMA et al., 2010). PGE 2 is an important indicator for measuring the inflammatory reactions in vitro as well as ex vivo; BÄUMER and KIETZMANN (2000) showed that isolated udder is a suitable model for inflammatory skin reactions. In our experiments, teat tissues from isolated udder showed no enhanced production of PGE 2 in reaction to MgAg1% sticks regarding that PGE 2 in the treated teat was nearly of the amount in comparison to the untreated teat for 6 experiments. On the other hand, it was determined in one of the isolated udder experiments an enhanced production of PGE 2, but it might be related to an already persisting infection in the udder, while there was no enhanced production of PGE 2 in other isolated udder experiments, that might be referred to short duration of the experiments which trigger PGE 2 production, but the absence of other increased inflammatory markers released from the other isolated udders would increase the probability towards the first reason. We found that primary mammary epithelium cells and primary mammary fibroblasts also did not show an enhanced production of TNF-alpha in comparison to untreated control, while they were stimulated with 100 μg/ml LPS as a positive control. LPS is a part of the bacterial membrane of gram-negative bacteria and reacts as a bacterial endotoxin. This endotoxin, like other microbial components mediates its proinflammatory effects through binding to toll-like receptors (TLRs). LPS is recognized by the TLR4 (SONG et al. 2001), which has been detected in epithelial cells and keratocytes, but not in endothelial cells (SONG et al., 2001; JOHNSON et al. 2005; KUMAGAI et al. 2005). We did not measure the other cytokines compared to the other cell lines, as TNF-alpha was the only cytokine available for measurement for bovine cell line. In addition, neither murine fibroblasts nor murine macrophages showed an enhanced production of IL-1beta, IL-6 and TNF-alpha, although these cells were stimulated with 100 μg/ml LPS as a positive control. LPS treated cells 119

Discussion showed a significant increase of TNF-alpha, IL-1beta and IL-6 compared to the treated control. The fact that none of these markers were found after the incubation with degradation medium of MgAg1% or different concentrations of AgNO 3 confirms our last step towards the biocompatibility experiments. 5.2 Detection of the degradation of MgAg1% sticks Another aim of our study was to investigate the degradation behavior of MgAg1% stick in an ex vivo isolated perfused bovine udder model and during the dry off period. Our results showed a statistically significant release of MgAg ions after 5-6 hours of incubation which started early and continued to raise over the incubation period either in vitro or ex vivo. These results were confirmed by histological experiments, which showed black precipitated particles of MgAg1% sticks. The degradation kinetics using the ex vivo model which included histological experiments and weight loss methods were quite similar from those we obtained in vitro using dry off period secretion. We showed that in the udder teat treated for 5-6 hours, the percentage of weigh loss was about 3 % of the initial weight. Interestingly, these results were confirmed by the histological experiments. In addition, the loss percentage of the sticks in dry off period secretion of about 60 % after 21 days of incubation. Moreover, one stick was completely degraded with visible particles still present in the medium after 21 days, and the reason was still unknown. To our best knowledge, this matter of degradation was not discussed before. That is why we were not able to obtain information about the end point for the degradation behavior of this alloy ex vivo or the longer term effects and reactions. Additionally, there is the yet unanswered question whether the degradation process of MgAg1% sticks is referred to the presence of Mg ++ as mentioned in many reports before about the use of degradable orthopaedic implants (STAIGER et al., 2006; XU et al., 2007), absorbable cardiovascular stents (PEUSTER et al., 2006), the ability of pure magnesium to corrode rapidly in body fluids (SONG 2007), or whether the degradation process referred to Ag ions or the combination of the alloy. However, we can conclude that the degradation duration depends on the size of the implant, so it is possible to determine the degradation period. 120

Discussion For estimating the amount of Ag + during incubation process either in degradation medium or in NaCl using silver 3 test Nanocolor, the amount of Ag + in degradation medium range from 0.02 up to 1 mmol/l after 25 days of incubation without showing any significant differences between 1, 3 and 10 ml volume of degradation medium, while in NaCl the Ag + amount ranged from 0.2 up to 0.3 mmol/l with a significant increase after 10 days of incubation. In addition to the estimation of the Mg ++ amount in the degradation medium which ranged from 0.8 up to 1 mmol/l after 25 days without showing significant differences between 1, 3 and 10 ml volume of degradation medium, the amount of Mg ++ ranged from 0.2 up to 9 mmol/l in NaCl without showing any significant differences during different intervals of time. On the other hand, we observed a significant increase in both Ag + and Mg ++ amount at 1 ml volume of degradation medium and that may be referred to the fact that the stick is in a small amount of medium (1 ml). Our experiments showed the ability of MgAg1% sticks or alloys to degrade in bovine udder showing a degradable behavior for the first 6 hours. In addition, the ability of MgAg 1% sticks to degrade in the dry off period secretion during 21 days. 5.3 Antibacterial activity of MgAg1% sticks Ag + has an important antimicrobial effect (BRETT, 2006). This effect depends on the superficial contact between Ag + and the enzymatic systems of the respiratory chain with altered DNA synthesis (BRETT, 2006; HIDALGO and DOMINGUEZ 1998). Nevertheless, Ag ions continue to be used as antibacterial agents. They constitute excellent bacteriostatic or bactericidal agents, the main advantage of which is their activity against Gram-negative bacilli (particularly Pseudomonas spp. and Proteus spp.). Their antimicrobial power and their non-accidental exposure to Ag + is increasing their use in drugs, dental amalgams, and covered catheters. Since toxicity of Ag + ions is poorly characterized, different aspects of cellular toxicity mechanisms produced by this antiseptic in cultured human fibroblasts were studied further (LIEDBERG and LUNDEBERG, 1898; HOLLINGER, 1996). AgNO 3 greatly inhibits the growth of bacteria. Ag + can be effective against a wide range of microorganisms, including aerobic, anaerobic, Gram-negative and Gram- 121

Discussion positive bacteria, yeast, fungi, and viruses. The antimicrobial effect of Ag + can be explained by various mechanisms. Ag + interferes with the respiratory chain in the cytochromes of microbacteria; additionally, Ag ions also interfere with components of the microbial electron transport system, bind DNA, and inhibit DNA replication (LOK et al., 2006; LANSDOWN, 2002) and that was discussed before in literature review. The soluble form of Ag + is the most toxic to bacteria (TILTON and ROSENBERG, 1978; TREVORS, 1987) and Ag + toxicity can be reduced by precipitation of the metal by phosphates, sulphides and chloride ions (TREVORS, 1987). Our results showed that the number of the bacterial colonies (E. coli and S. aureus) incubated with the supernatants of degradation of MgAg1% in NaCl, decreased 5 times compared to the control, when cultivated on petri dishes. The measured amount of silver which induced the antibacterial effect was 0.2-0.3 mmol/l as measured before after 15 days of incubation. Furthermore, the preliminary results of bouillion dilution test showed antibacterial effect of the degrading supernatants (MgAg1% sticks incubated in NaCl) the on the growth of bacterial colonies (E. coli and S. aureus). Interestingly, another study by STARODUB and TREVORS (1989) stated that E. coli strain R1 cultures treated with AgNO 3 concentrations of 1, 0.5 and 0.3 mm, did not have an extended lag phase, but the final cell biomass was slightly reduced in the presence of any of AgNO 3 concentrations. Strain Rl was capable of growth in the presence of 0.1 mm AgNO 3, but not at 0.5 or 1.0 mm, even when the incubation period was extended to 48 h. In addition, we could observe that cells grown in the presence of AgNO 3 were capable of binding more Ag +. Ag + is not an essential metal and it is unlikely that there is a specific energy dependent transport system for it, but Ag + could enter cells via a transport system for an essential metal. Moreover, the results we obtained from BRT-MRL screening test was clear that Geobacillus stearothermophilus shift brilliant black colour to yellow or colourless at concentrations equal or below 0.003 mmol/l of AgNO 3 solution, which indicates that concentrations of AgNO 3 solution starting from 1 up to 0.01 mmol/l has antibacterial effect. Moreover, different volume of degradation medium of MgAg1% shifted the 122

Discussion blue colour to yellow or colourless at volume equal or above 3 ml, which indicates the presence of antibacterial effect at 1 and 3 ml volume of degradation medium of MgAg1%. Quite interestingly, the measured Ag + amounts at 1 and 3 ml volume were about 0.02-1 mmol/l. Surprisingly, these amounts of Ag + are the same amounts which had antibacterial effect, when the bacteria were treated with different concentrations of AgNO 3 solution. JANSEN et al. (1994) and SCHIERHOLZ et al. (1998) stated that the effect of Ag ions is continuous and long lasting due to the oligodynamic effect of elementary Ag +. Moreover, BOWSWALD et al. (1999) and OLSON et al. (2002) stated that the ability of many bacteria to produce a biofilm is reduced and the likelihood of bacterial colonization is decreased when Ag + is introduced. This result supports our idea that MgAg 1% sticks may have antibacterial effects. That is why our idea could be attractive for further applications and interesting for other investigation. Hopfully, our study could be a useful aid for the treatment of mastitis. MgAg1% sticks would share in the treatment of mastitis at dry off period alone or combined with some antibiotics depending on the concentration of Ag +. As a result, we can help in saving some of the economical losses (vet. costs, labour costs, antibiotics costs and intreferring with the manufacturing of dairy products) and culling rates, when mastitis appeared. In addition, protecting the animal wealh and providing a safer human consumption Finally, our hypothesis was confirmed, namely that MgAg1% alloys are of a good biocompatibility action, did not have any cytotoxic effect on primary mammry cell cultures, while MgAg1% alloys had a slight cytotoxic effect on murine cell lines at high concentrations, or affect the metabolic activities and did not enhance the production of some inflammatory mediators. In addition to its ability to be degraded, however, the solubility of Ag + was known to be poor and at the end the most valuable knowledge is about the antibacterial effect of the alloy which makes it of interest to use it in wider applications for treating mastitis in dry off period, whether to be used in some drugs or as ancillary treatment. 123

Discussion 5.4 Outlook The primary mammary cells presented in this study are a useful model to examine the degradation behavior of MgAg1% stick intended for treating bovine mastitis. The use of murine fibroblasts and macrophages for the construction of the equivalent is advantageous for practical reasons, since the cells are commercially available, highly proliferative and unproblematic to cryoconserve compared to the slowly proliferating and difficult to passage primary mammary epithelial cells. Nevertheless, we succeeded to cultivate and isolate primary mammary epithelium and fibroblasts cells with adequate proliferation. For the inflammatory reactions, it is also of interest to measure more than one endpoint. When studying the degradation effect, it would be beneficial by giving a broader picture and thus a closer resemblance to the actual situation in an inflamed tissue or around an inflamed cell. Generally, the use of MgAg1% sticks as an initial point for treating mastitis for the cows will aid the approval and licence of many other Ag + alloys or drugs containing Ag + for veterinary use. Regarding toxicological studies, our results were really optimistic for the future of using Ag + in treating mastitis at dry off period, and it would be interesting to investigate the possible effects of degrading MgAg1% sticks on cell cascades and signalling. Moreover, the stick may not be able to save the udder from peracute and acute mastitis, because of the slow degradation of MgAg1% sticks or somehow the poor solubility of Ag + particles, so it would be of great interest to try higher concentrations of this alloy, reaching higher antibacterial effects in the udder, hopefully with promising results of low inflammatory responses and higher biocompatibility. Our results could be helpful to establish a screening tool and thus aid in the reduction of the economic losses which are caused by mastitis. Furthermore, another possible future application for this model includes basic research on cellular interactions with the advantage of including primary epithelium cells and fibroblasts. Such studies could provide in-depth knowledge of physiological as well as pathological mechanisms of the udder, and thus help to understand of mastitis. 124

Discussion Finally, ex vivo and in vitro experiments reflect some promising results for the in vivo experiments in bovine udder. 125

Summary 6 Summary In vitro and ex vivo study of the biocompatibility of magnesium-silver alloys and their antimicrobial effects on bovine bacterial species Yousra A. R. Nomier 2011 The aim of this study is to investigate the ability of MgAg1% alloys to inhibit bacterial growth during the dry off period. For this purpose, primary mammary epithelial cells and primary mammary fibroblasts were successfully isolated, cultured and verified by phase contrast microscopy and immunofluorescence. In addition, three main points (biocompatibility, degradation properties and antibacterial effects) were addressed in order to achieve the objective. In doing so, not only the the antibacterial effects of MgAg1% alloys could be evaluated but some light was shed on the impact of the alloys on the tissue response, too. In the in vitro models of differentiated primary mammary cells (epithelium and fibroblasts), murine fibroblasts and murine macrophages (primary as well as raw cell line) acceptable biocompatibility without a marked increase in mediators of inflammation was shown. However, a slight influence on the cell viability of the different concentrations of AgNO 3 solution was observed at high concentrations. While different volumes of the degradation medium of MgAg1% sticks did not show an influence on cell viability, using different assays such as (MTS assay, neutral red assay and Bradford assay). On the other hand, there was no effect on the activity of cytosolic and mitochondrial marker enzymes (PK and SDH, respectively). In the ex vivo study on isolated perfused udders MgAg1% sticks showed the ability to degrade within 5-6 hours of incubation in mammary teats, which was confirmed with the weight loss and histological examinations. Furthermore, MgAg1% sticks degraded during 21 days of incubation in dry off period secretion (60% of the initial weight) which represents a favourable base in pursuit of this approach. But the 127

Summary question remains whether this degradation kinetic will suffice for the whole dry off period and whether it is suitable for the treatment of acute and peracute mastitis. The isolated perfused udder goes beyond the in vitro models regarding the in vivo situation. Its use enabled the tissue reaction to be tested in a dynamic environment, which together with the in vitro models gives a more complete overview of the impact of MgAg1% sticks in an udder without the utilization of animal experiments. The antibacterial effect of different concentrations of AgNO 3 solution and different volumes of degradation medium of MgAg1% sticks was successfully evaluated. The bouillion dilution test showed antibacterial activity of the degrading supernatants (MgAg1% sticks incubated in NaCl) on the growth of E. coli and S. aureus. Furthermore, the cultivation of the bacterial colonies (E. coli and S. aureus) showed 5-fold reduction in the number of bacterial colonies, when incubated with the degrading supernatants (MgAg1% sticks incubated in NaCl) compared to the untreated control bacteria. The BRT-MRL screening test showed an inhibition of Geobacillus stearothermophilus bacteria at AgNO 3 concentraions equal to 0.01 mmol/l or above, and at concentration equal to 3 ml up to 1 ml of degradation medium of MgAg1% sticks. Regarding that the amount of silver ions in 3 ml volume up to 1 ml of volume of degradation medium was equal to 0.02-1 mmol/l which already showed antibacterial effect. In conclusion an ex vivo and in vitro system for the testing of MgAg1% alloys was successfully established. The results were satisfying concerning biocompatibility, degradation process and antibacterial effects. However, other questions are not answered yet, for instance, if the sticks should be used in parallel with or instead of antibiotics and for which types of mastitis they should be used. Although our present study showed a good biocompatibility, a good degradation process and high antibacterial effects, it may be of interest trying to achieve higher concentrations in further investigations. 128

Zusammenfassung 7 Zusammenfassung In vitro- und ex vivo-studie der Biokompatibilität von Magnesium-Silber- Legierungen und deren antimikrobielle Wirkung auf bovine Bakterienspezies Yousra A. R. Nomier 2011 Das Ziel dieser Studie ist es, die Eignung von MgAg1%-Legierungen zu untersuchen, bakterielles Wachstum während der Trockenstehperiode zu hemmen. Zu diesem Zweck ist es gelungen, primäre Euterepithelzellen und primäre Euterfibroblasten zu isolieren, in Kultur zu nehmen und mittels Phasenkontrast-Mikroskopie und Immunfluoreszenz zu verifizieren. Darüber hinaus wurden drei Hauptpunkte (Biokompatibilität, Degradationseigenschaften und antibakterielle Effekte) angegangen, um das gesetzte Ziel zu erreichen. Auf diesem Wege konnte nicht nur die antibakterielle Wirkung von MgAg1% Legierungen bewertet werden, sondern es wurde auch etwas Aufschluss gegeben über den Einfluss der Legierungen auf die Gewebereaktion. In den In-vitro-Modellen der differenzierten primären Euterzellen (Epithel und Fibroblasten), der murinen Fibroblasten und murinen Makrophagen (primäre sowie raw-zelllinie) zeigte sich eine annehmbare Biokompatibilität ohne deutlichen Anstieg der Entzündungsmediatoren. Dennoch wurde ein leichter Einfluss der unterschiedlichen Konzentrationen von AgNO 3 und der unterschiedlichen Volumina an Degradationsmedium der MgAg1%-Stäbchen auf die Zellvitalität (MTS-Assay, Neutralrot-Assay und Bradford-Assay) gesehen. Andererseits gab es keinen Effekt auf die Aktivität der cytosolischen und mitochondrialen Markerenzyme (PK bzw. SDH). In der Ex-vivo-Studie an isoliert perfundierten Eutern, zeigte sich die Degradationsfähigkeit der MgAg1%-Stäbchen während der 5-6 Stunden dauernden Inkubation in den Zitzen, welche anhand der Gewichtsabnahme und histologischen Untersuchungen bestätigt wurde. Darüber hinaus degradierten MgAg1%-Stäbchen 129

Zusammenfassung während einer 21-tägigen Inkubation in Trockenstehsekret (60% des ursprünglichen Gewichts) was eine erfreuliche Basis für die Weiterverfolgung dieses Ansatzes ist. Es bleibt jedoch die Frage, ob dieser Degradationsverlauf für die gesamte Trockenstehzeit ausreichend ist und ob er sich zur Behandlung von akuten und perakuten Mastitiden eignet. Das isoliert perfundierte Eutermodell geht im Hinblick auf die In-vivo-Verhältnisse über die In-vitro-Modelle hinaus. Sein Einsatz ermöglichte die Testung der Gewebereaktion auf die MgAg1%-Stäbchen in einem dynamischen Milieu, was zusammen mit den In-vitro-Modellen einen vollständigeren Überblick über Auswirkungen der MgAg1%-Stäbchen im Euter verschafft, ohne Tierversuche einzusetzen. Die antibakterielle Wirkung von unterschiedlichen Konzentrationen an AgNO 3 und verschiedener Volumina an Degradationsmedium der MgAg1%-Stäbchen wurde erfolgreich bewertet. Der zeigte eine 5-fach niedrigere Zahl an Bakterienkolonien (E. coli und S. aureus) im Vergleich zu nicht behandelten Kontrollbakterien. Der BRT- MRL-Screening-Test zeigte eine Hemmung von Geobacillus stearothermophilus ab AgNO 3 -Konzentrationen von 0,01 mmol / l. Die eine Degradationsmedium (3 ml) gemessene Silber Konzentrationen erreicht somit eine antibakterielle wirksame Konzentrationen. In Bezug auf die 3 ml Volumen bis zu 1 ml Volumen des Abbaus Medium gleich 0,02 war - 1 mmol / l, die bereits gezeigt, antibakterielle Wirkung. Zusammenfassend wurde ein Ex-vivo- und In-vitro-System zur Testung von MgAg1%- Legierungen erfolgreich etabliert. Die Ergebnisse hinsichtlich Biokompatibilität, und antibakterieller Wirkung waren zufriedenstellend. Weitergehende Fragen sind jedoch noch nicht beantwortet, beispielsweise ob die Stäbchen gleichzeitig mit oder anstelle von Antibiotika verwendet werden sollten und für welche Arten der Mastitis sie sich eignen. Obwohl unsere Studie eine gute Biokompatibilität, einen guten Degradationsverlauf und eine hohe antibakterielle Wirkung zeigt, könnte versucht werden, in weiteren Untersuchungen noch höhere Konzentrationen zu erzielen. 130

Appendix 8 Appendix Tab. 10: Data of neutral red assay for different cells treated with different volumes of degradation medium of MgAg1% sticks (ml). Results were measured using spectrophotometrical extinction; each value is a mean of 4-6 measurements. Cell types Control 10 ml 3 ml 1 ml Primary mammary epithelium 0,457± 0,11 0,447± 0,07 0,433± 0,05 0,343± 0,12 L929 cells 0,443± 0,06 0,463± 0,07 0,451± 0,07 0,422± 0,07 Primary mammary fibroblasts 0,139± 0,02 0,141± 0,02 0,154± 0,02 0,122± 0,02 Raw macrophages 0,274± 0,05 0,246± 0,09 0,245± 0,02 0,188± 0,12 Primary macrophages 0,341± 0,11 0,398± 0,13 0,379± 0,06 0,357± 0,03 Tab. 11: Data of SDH activity for different cells treated with different volumes of degradation medium of MgAg1% sticks (ml). Results were measured using spectrophotometrical extinction; each value is a mean of 4-6 measurements. Cell types Control 10 ml 3 ml 1 ml Primary mammary epithelium 2,617± 0,67 2,925± 0,27 3,727± 0,84 5,475± 1,03 L929 cells 0,208± 0,04 0,248± 0,15 0,246± 0,11 0,282± 0,09 Primary mammary fibroblasts 1,266± 0,22 1,465± 0,31 1,220± 0,16 3,886± 0,30 Tab. 12: Data of PK activity treated with different volumes of degradation medium of MgAg1% sticks (ml). Results were measured using spectrophotometrical extinction; each value is a mean of 4-6 measurements. Cell types Control 10 ml 3 ml 1 ml Primary mammary epithelium 2,676± 0,90 2,622± 0,54 3,313± 0,69 4,829± 0,93 L929 cells 0,295± 0,09 0,301± 0,17 0,308± 0,18 0,259± 0,13 Primary mammary fibroblasts 1,356± 0,22 1,401± 0,18 1,510± 0,39 3,069± 0,26 Tab. 13: Data of PGE 2 for isolated perfused bovine udder teats incubated with MgAg1% sticks for 4-6 hours. Trials Control teats Silver treated teats 1 1076,771 268,872 2 173,080 691,936 3 301,906 739,847 4 364,413 773,462 5 1546,981 1470,207 6 1397,667 735,742 MW± SD 810,136± 603,66 780,011± 387,04 131

Appendix Tab. 14: Amount of silver detected by the Silver 3 Nanocolor test in different volumes of degradation medium of MgAg1% sticks after different days of incubation; each value is a mean of 6 measurements. Days of incubation 1 ml 3 ml 10 ml 5 0,205 0,049 0,027 10 0,244 0,168 0,049 15 0,174 0,084 0,060 20 0,226 0,202 0,037 25 0,971 0,312 0,332 Tab. 15: Amount of silver detected by the Silver 3 Nanocolor test of different volumes of degraded MgAg1% sticks in NaCl after different days of incubation; each value is a mean of 6 measurements. Trials 5 days 10 days 15days 1 st 0,023 0,234 0,356 2 nd 0,019 0,219 0,298 3 rd 0,026 0,209 0,279 MW± SD 0,023± 0,004 0,221± 0,012 0,311± 0,040 Tab. 16: Amount of magnesium detected by the magnesium and calcium test of degraded MgAg1% sticks in NaCl after different days of incubation; each value is a mean of 6 measurements. Trials 5 days 10 days 15 days 1 st 0,922 0,774 0,798 2 nd 1,045 0,801 0,873 3 rd 0,898 0,801 0,699 MW± SD 0,955± 0,079 0,792± 0,016 0,790± 0,087 Tab.17: Amount of magnesium detected by the magnesium and calcium test in different volumes degradation medium of MgAg1% sticks after different days of incubation; each value is a mean of 6 measurements. Different volumes of degradation medium 5 days 10 days 15 days 20 days 1ml 0,450 8,300 5,000 8,100 3ml 0,300 4,600 5,600 7,500 10ml 0,200 3,300 3,900 3,900 132

Appendix Tab. 18: Data of IL-1 beta for different cells treated with different volumes of degradation medium of MgAg1% sticks; each value is a mean of 4-6 measurements. LPS is a positive control. Cell types Control 10 ml 3 ml 1 ml LPS Primary macrophage 6,333± 3,5 8,0± 9,5 13,8± 15,8 10,7± 6,50 489,5± 140,9 Raw macrophage 7,250± 3,1 5,0± 3,8 7,5± 7,7 10,5± 5,7 316,5± 479,6 L929 183± 352 25,17± 4 7,17± 7,8 20,5± 28,5 327,2± 471,9 Tab. 19: Data of IL-6 of different cells treated with different volumes of degradation medium of MgAg1% sticks; each value is a mean of 4-6 measurements. LPS is a positive control. Cell types Control 10 ml 3 ml 1 ml LPS Primary macrophage 0,0± 0,0 0,0± 0,0 0,0± 0,0 0,0± 0,0 77,3± 19,7 Raw macrophage 0,0± 0,0 0,0± 0,0 0,0± 0,0 0,0± 0,0 828,5± 487 L929 27,5± 41,6 32,3± 38,1 31,7± 24,5 17,3± 19,4 95,5± 15,4 Tab. 20: Data of bovine TNF-alpha for primary mammary epithelium cells treated with different volumes of degradation medium of MgAg1% sticks. Results were measured using spectrophotometrical extinction; each value is a mean of 4-6 measures. LPS is a positive control. Cell type Control 10 ml 3 ml 1 ml LPS Primary mammary epithelium 0,0± 0,0 0,0± 0,0 0,0± 0,0 0,0± 0,0 13,3± 4,5 Tab. 21: Data of IL-6 of different cells treated with different volumes of degradation medium of MgAg 1% sticks. Results were measured using spectrophotometrical extinction; each value is a mean of 4-6 measures. LPS is a positive control. Cell type Control 10 ml 3 ml 1 ml LPS Primary macrophage 33,0± 12,5 ± 15,9 59,2± 30,9 37,6± 19 109,4± 58,9 Raw macrophage 75,8± 30,4 99± 63,7 84,2± 72,2 71,2± 30,9 260,8± 239,3 Tab. 22: Data of Lactate production in the isolated perfused bovine udder over 6 hours. Results were measured using spectrophotometrical extinction; each value is a mean of 4-6 measurements. Left side Right side Hours Mean SD Mean SD 1 0,164 0,050 0,223 0,071 2 0,211 0,071 0,246 0,060 3 0,202 0,068 0,217 0,149 4 0,206 0,059 0,292 0,160 5 0,155 0,027 0,293 0,204 6 0,235 0,041 0,260 0,177 133

Appendix Tab. 23: Data of Glucose consumption of the isolated perfused bovine udder over 6 hours. Results were measured using spectrophotometrical extinction; each value is a mean of 4-6 measurements. Left side Right side Hours Mean SD Mean SD 1 77,250 18,000 45,950 12,000 2 67,033 17,000 56,992 8,000 3 55,908 16,000 63,617 11,000 4 66,533 16,000 56,200 7,000 5 73,850 5,000 63,117 7,000 6 60,617 7,000 67,158 9,000 Tab. 24: Data of LDH production of the isolated perfused bovine udder over 6 hours. Results were measured using spectrophotometrical extinction; each value is a mean of 4-6 measurements. Left side Right side Hours Mean SD Mean SD 1 1135,83 123,510 1090,000 11,785 2 948,333 23,570 1731,667 1084,23 3 990,000 82,496 1715,000 441,903 4 0,000 0,000 1190,000 200,347 5 965,000 0,000 1515,000 0,000 6 1148,33 0,000 973,333 11,785 Tab. 25: Data of MgAg1% sticks after incubation in the teat of the isolated perfused bovine udder within 4-6 hours, following by a data for MgAg1% sticks after incubation in dry off secretion for 21days. Sticks after incubation in udder Sticks after incubation in dry off secretion Trials Initial Weight Weight after loss Initial weight Weight after loss 1 56,066 51,433 15,733 0,000 2 53,266 52,060 13,967 3,833 3 53,960 53,566 6,300 4,600 4 52,600 51,933 9,467 3,967 5 54,430 51,333 10,033 4,733 6 54,630 53,866 8,633 5,333 7 53,300 53,233 15,733 0,000 MW± SD 54,036±1,142 52,489±1,045 10,689±3,510 3,744±1,914 134

Appendix Tab. 26: Data of MTS assay for different cells treated with different volumes of degradation medium of MgAg1% sticks (ml). Results were measured using spectrophotometrical extinction; each value is a mean of 4-6 measurements. Cell types Control 10 ml 3 ml 1 ml Primary mammary epithelium 0,921± 0,46 0,854± 0,44 0,822± 0,42 0,849± 0,45 L929 cells 1,538± 0,36 1,611± 0,72 1,301± 0,49 0,485± 0,24 Primary mammary fibroblasts 0,339± 0,04 0,345± 0,03 0,350± 0,03 0,201± 0,03 Raw macrophages 0,957± 0,67 1,017± 0,69 0,965± 0,74 0,775± 0,70 Primary macrophages 1,117± 0,39 1,617± 0,27 1,365± 0,324 0,975± 0,65 135

Appendix Tab. 27: Data of MTS assay for different cells treated with different concentrations of AgNO3 (mmol/l). Production of formazan measured as spectrophotometrical extinction; each value is a mean of 4-6 measurements. mmol/l Cell types Control 0.003 0.001 0.01 0.03 0.1 0.3 1 Primary mammary epithelium 0.98 ± 0.49 0.82 ± 0.42 0.84 ± 0.41 0.82 ± 0.41 0.84 ± 0.42 0.82 ± 0.40 0.83 ± 0.41 0.41 ± 0.47 L929 cells 1.85 ± 0.22 1.60 ± 0.58 1.80 ± 0.08 1.78 ± 0.13 1.78 ± 0.16 0.77 ± 0.53 0.35 ± 0.07 0.34 ± 0.03 Primary mammary fibroblasts 0.45 ± 0.03 0.42 ± 0.16 0.40 ± 0.14 0.37± 0.09 0,38 ± 0.14 0.36 ± 0.15 0.42 ± 0.17 0.16 ± 0.01 Raw macrophages 0.35 ± 0.09 0.29 ± 0.07 0.33 ± 0.17 0.41 ± 0.31 0.36 ± 0.08 0.43 ± 0,23 0.26 ± 0.08 0,22 ± 0,06 Primary macrophages 0.25 ± 0.09 0.19 ± 0.05 0.24 ± 0.15 0.23 ± 0.14 0.26 ± 0.08 0.33 ± 0.23 0.19 ± 0.06 0,14 ± 0,05 136

Appendix Tab. 28: Data of Neutral red assay for different cells treated with different concentrations of AgNO3 (mmol/l). Results were measured using spectrophotometrical extinction; each value is a mean of 4-6 measurements. mmol/l Cell types Control 0.003 0.001 0.01 0.03 0.1 0.3 1 Primary mammary epithelium 0.45 ± 0.02 0.43 ± 0.04 0.42 ± 0.04 0.42 ± 0.04 0.44 ± 0.03 0.44 ± 0.02 0.42 ± 0.02 0.34 ± 0.04 L929 cells 1.18 ± 0.06 1.12 ± 0.13 1.15 ± 0.07 1.18 ± 0.06 1.14 ± 0.08 0.56 ± 0.13 0.38 ± 0.40 0.18 ± 0.03 Primary mammary fibroblasts 0.28 ± 0.05 0.17 ± 0.04 0.16 ± 0.04 0.16 ± 0.05 0.15 ± 0.05 0.09 ± 0.09 0.09 ± 0.09 0.06 ± 0.02 Raw macrophages 1.18 ± 0.06 1.1 ± 0.13 1.15 ± 0.07 1.18 ± 0.06 1.14 ± 0.08 0.48 ± 0.44 0.56 ± 0.13 0.18 ± 0.03 Primary macrophages 1.69 ± 0.19 1.17 ± 0.50 1.16 ± 0.42 1.11 ± 0.54 0.94 ± 0.15 0.99 ± 0.59 0.74 ± 0.24 0.66 ± 0.32 137

Appendix Tab. 29: Data of SDH activity for different cell cultures treated with different concentrations of AgNO3 (mmol/l). Results were measured using spectrophotometrical extinction; each value is a mean of 4-6 measurements. mmol/l Cell types Control 0.003 0.001 0.01 0.03 0.1 0.3 1 Primary mammary epithelium 12,41± 1,66 7,99 ± 0,89 8,09 ± 0,72 14,77 ±1,80 13,20 ±1,57 18,67 ±2,17 17,72 ±1,17 27,35 ±0,98 L929 cells 0,26 ± 0,03 0,22 ± 0,02 0,21 ± 0,03 0,23 ± 0,04 0,24 ± 0,02 0,25 ± 0,06 0,23 ± 0,03 0,2 4± 0,04 Primary mammary fibroblasts 0,26 ± 0,03 0,22 ± 0,02 0,21 ± 0,03 0,23 ± 0,04 0,24 ± 0,02 0,25 ± 0,06 0,23 ± 0,03 0,24 ± 0,04 Tab. 30: Data of PK activity for different cells treated with different concentrations of AgNO3 (mmol/l). Results were measured using spectrophotometrical extinction; each value is a mean of 4-6 measurements. mmol/l Cell types Control 0.003 0.001 0.01 0.03 0.1 0.3 1 Primary mammary epithelium 8,80 ± 1,79 5,89 ± 1,93 6,56 ± 2,23 11,52 ± 2,63 10,64 ± 3,2 12,93 ±3,5 13,66 ±3,3 25,87±2,05 L929 cells 0,21 ± 0,04 0,23 ± 0,04 0,23 ± 0,04 0,23 ± 0,04 0,22 ± 0,04 0,19 ± 0,04 0,34 ± 0,02 0,34 ± 0,04 Primary mammary fibroblasts 0,27 ± 0,04 0,24 ± 0,02 0,23 ± 0,03 0,26 ± 0,03 0,27 ± 0,02 0,28 ± 0,01 0,27 ± 0,02 0,27 ± 0,02 138

Appendix Tab. 31: Data of IL-1 beta for different cells treated with different concentrations of AgNO3 (mmol/l). Results were measured using spectrophotometrical extinction; each value is a mean of 4-6 measures. LPS is a positive control. mmol/l Cell types Control 0.003 0.001 0.01 0.03 0.1 0.3 1 LPS Primary macrophages 9,67± 19,34 0,0 ± 0,00 0,0 ± 0,0 0,0 ± 0,00 8,05 ± 16,12 9,13 ± 18,27 8,05 ± 16,1 8,05 ±16,1 579± 537 Raw macrophages 42,59± 21,1 35,8 ± 14,7 35,9 ± 16 36,26 ± 14,6 29,84 ± 13,9 24,91± 7,47 24,87± 2,0 27,31± 7,8 1045± 17 L929 cells 0,0 ± 0,0 1,75 ± 3,50 2,0 ± 1,83 3,0 ± 3,83 1,50 ± 1,92 1,0 ± 2,0 3,50 ± 4,36 1,75± 3,50 10 ±20.5 Tab. 32: Data of IL-6 of different cells treated with different concentrations of AgNO3 (mmol/l). Results were measured using spectrophotometrical extinction; each value is a mean of 4-6 measures. LPS is a positive control. mmol/l Cell types Control 0.003 0.001 0.01 0.03 0.1 0.3 1 LPS Primary macrophages 50 ± 22,90 42,99 ± 12 42,99 ±15 43,5 ± 11,8 34,91± 14,7 28,99 ± 5,1 21,47± 14,6 32,22± 2,4 555± 565 Raw macrophages 0,00 ± 0,00 0,00 ± 0,00 0,00 ± 0,0 0,00 ± 0,00 0,00 ± 0,00 0,00 ± 0,00 0,00 ± 0,00 0,00± 0,0 65,7 ± 21 L929 cells 41,1± 67,29 24,2 ± 31,8 20,28 ± 24 33,99± 25,2 37,46 ± 40,81 26,13± 32,9 27,44± 40,2 26,12 ± 39 757± 604 139

Appendix Tab. 33: Data for the mouse TNF-alpha for different cells treated with different concentrations of AgNO3 (mmol/l). Results were measured using spectrophotometrical extinction; each value is a mean of 4-6 measurements. LPS is a positive control. mmol/l Cell types Control 0.003 0.001 0.01 0.03 0.1 0.3 1 LPS Primary macrophages 15,73± 37,2 23,18± 36,3 28,64 ± 31 34,46± 39,4 17,36 ± 16,8 20,46 ± 14 33,55± 16,6 34,3± 22,4 757± 534 Raw macrophages 28,46± 23,1 28,66± 24,4 26,82± 23 41,36± 46,6 23,00± 13,74 28,8± 28,79 36,82±10,1 45,2± 17,6 757± 534 L929 cells 2694 ± 290 2186 ± 362 2235± 266 2242 ± 224 2286 ± 378 2418 ± 289 2088 ± 396 2151± 464 2322± 1273 Tab. 34: Data for bovine TNF-alpha of primary mammary epithelium cells treated with different concentrations of AgNO3 (mmol/l). Results were measured using spectrophotometrical extinction; each value is a mean of 4-6 measures. LPS is a positive control. mmol/l Cell type Control 0.003 0.001 0.01 0.03 0.1 0.3 1 LPS Primary mammary epithelium 0,00 ± 0,00 0,00 ± 0,00 0,00 ± 0,00 0,00± 0,00 0,00 ± 0,00 0,00 ± 0,00 0,0 ± 0,00 0,0 ± 0,0 17,27± 8,28 140

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Abbreviations 9.1 Abbreviations Ag + Silver ANOVA Analysis of variance ATP Adenosine triphosphate BMEC Primary mammary epithelial cells BRT Brilliant Black Reduction Test Ca ++ Cl CO 2 CMT DC DMEM DMSO DNA Calcium Chloride Carbon dioxide California mastitis tests Dendritic cell medium Dulbecco s modified Eagle s medium Dimethylsufoxide Deoxyribonucleic acid E. coli Escherichia coli EDTA Ethylenediaminetetraacetic acid ELISA Enzyme-linked immunosorbent FAD Flavin adenine dinucleotide FCS Fetal calf serum Fig Figure FITC Fluorescence-labelled secondary antibody fluorescein isothocyanate isomer g Gram h Hour HRP Horseradish peroxidase ICCC Individual cow milk cell count IL Interleukin L Liter L929 Murine fibroblast LDH Lactate dehydrogenase enzyme LPS Lipopolysaccharide Mmol millimole µ micro 177

Abbreviations Mg ++ Min MTP MTS MØ N NADH NBT NCF NAGase PES PDKs Magnesiumm minute Mitochondrial transmembrane potential 3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2- (4-sulfophenyl)-2H-tetrazolium Macrophages number of experiments Nicotinamide adenine dinucleotide Nitroblue tetrazolium Neutrophil Chemotactic Factor N-acetyl B-D glucosaminidase Phenazine ethosulfate Pyruvate dehydrogenase kinases PGE 2 Prostaglanin E 2 PK Pyruvate kinase PMN Polymorphnuclear bodies ROS Reactive oxygen species SCC Somatic cell counts SDH Succinate dehydrogenase SEM Scanning electron microscopy SH Sulfhydryl groups SL Small lymphocytes S. aureus Staphylococcus aureus Tab. Table TMB Tetramethylbenzidine TNF Tumor necrosis factor 178

Acknowledgements This project work and my success were achieved by the help of God. Thanks for God. Secondly, this project work would not be done without the help, love and supervision of some people whom I already love and respect. First of all I want to express my gratitude to my supervisor Prof. Dr. Manfred Kietzmann for giving me the chance to work at his Institute, and trusting me to carry out this project. I appreciate your trust and I tried to work hard as much as I can. Thanks for all the help and support during the preparation and accomplishment of the present work. I owe him much for his assistance in all aspects of my Doctor-project, providing me great help on various topics, exchanging ideas on both academic and non-academic matters. Special thanks to Dr. Jessica Stahl for the daily thoughtful discussions, scientific support and constructive criticism, for your help and friendship during these years. You learned me a lot, you put me on the first steps to go on, pushing me to the front and asking me to be always optimistic. You were not only a supervisor but also a real friend who shared me good and hard times. Really, I will miss your jokes, sense of humor and your non stop advices, I owe you much. Prof. Dr. Wolfgang Bäumer for his skilled guidance in many fields, gentleness and being there any time for any question and with his great experience. I thank Ms. Ledwoch in the office of international academic affairs for her dedicated service of international students and postgraduates and the constant readiness to solve our problems, and sharing us hard time. You are one of the people whom helped me to have this scholarship. Thanks a lot. Really I love you. Viki and Caro for helping and caring about me, you teached me how to work in a lab and how to carry out my technical part, without you there will be no thesis at all and I will not be able to just stand in the lab. Besides, the nice and long talking we had about all topics of life, offereing me help without asking, too much cakes and writing lots of recepies. I will miss your nice stories, jokes and your great sense of humor. 179

Hans-Herbert for his kindness and nice time, really he is the boss of the laboratories, without him there will be no practical parts, no projects and no laboratories any more. He did a lot for us to be doctors. Thanks. Dr. Stephan S., for helping me, trying to follow up my work with care and attention, non stop advices and trying to solve technical problems, for nice time we spent with udder at least we had scientific talking to kill the boring time. I sincerely thank my roommates Maren F., Thomas. B., for nice time we spent together, your care about me and my family, your non stop help and share and always beside me in every occasion. We shared the room, the cable, life and feelings I really found you a brother and sister I will miss you. Katrin P., Katrin S., Ruta, Sarah, Verena and Dr. Christina for their support, care, sweet feelings and maintaining a pleasant working atmosphere and understanding. Actually with your active and great sharing, the working place looks like a comfortable home with warmness of a family. Dr. Bettina Blume for her excellent support in getting the udders; the Milchtierherden- Betreungs-und Forschungsgesellschaft mbh for applying the bouillion dilution test and cultivation of the bacterial colonies. Many thanks to my parents; I am grateful for them for their tireless support and keeping them under stress for 3 years, Iam sorry. I am sure the success of this work would make them delighted, and I really did this work just to make them proud of me, i love you all. Deeply thank and love to my husband Mohamed, my Sons Hamza and Badr, for standing beside me in hard times, encouragement, patient on me and helping me to keep my spirits up and their support along the time to complete this work. Mohamed, thank you for giving me the chance to carry out this work and for all what you did for me, you did a lot and I will be always grateful for you, I owe you a lot. My success is for you and by you.i love you. 180