Bibliografische Informationen der Deutschen Bibliothek

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4 Bibliografische Informationen der Deutschen Bibliothek Die Deutsche Bibliothek verzeichnet diese Publikation in der Deutschen Nationalbibliografie; Detaillierte bibliografische Daten sind im Internet über abrufbar. 1. Auflage by Verlag: Deutsche Veterinarmedizinische Gesellschaft Service GmbH, Giessen Printed in Germany ISBN Verlag: DVG Service GmbH Friedrichstraße Gießen 0641/

5 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

6 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:

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

8 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

9 Contents Contents 1 Introduction Literature Overview Bovine udder Anatomy of bovine udder and natural defense systems The teat canal barrier The streak canal Teat cistern (Sinus papillaris) Fürstenberg's rosette Cricoid rings (Annular folds) Drying off period Mammary epithelial cells Mastitis Causative factors of mastitis Pathogenic agents of mastitis Staphylococal mastitis Streptococcal mastitis Arcanobacterium pyogenes Coliform mastitis Mycoplasmal mastitis Mastitis and somatic cell counts Mastitis in organic dairy herds The main environmental pathogens of mastitis Clinical and subclinical mastitis Treatment of mastitis Parenteral treatment Udder infusions Treatment of dry cows The aim of dry cow therapy Dry cow preparations Systemic dry cow therapy..22 I

10 Contents Drying-off chronically affected quarters Supportive therapy Control of mastitis Magnesium Biochemistry of magnesium Physiology of magnesium Silver Mode of action Cytotoxicity of silver Antibacterial activity of silver Uses of silver In health and medicine Non medical uses of silver.36 3 Materials and Methods Experimental setting Materials Cell culture Culture media Cell cultures Cell culture reagents Cell culture disposable materials Cell culture counting and viability Cell culture equipments Magnesium-silver 1 % (MgAg1%) sticks and silver nitrate Materials and reagents Materials and reagents for Bio-Rad assay Materials and reagents for measuring cytokines (interlekin-1beta (IL-1beta), interleukin- 6 (IL- 6), tumour necrotic factor (TNF-alpha) Equipments used for measuring cytokines Materials and reagents used for measuring silver concentrations II

11 Contents Materials and reagents used for measuring calcium and Magnesium concentrations Materials and reagents used for the brilliant black reduction test (BRT MRL screening test) Equipments used for BRT MRL screening test Materials and reagents used for measuring Prostaglandin E Equipments used for measuring PGE Materials and reagents used for the bouillion dilution test Materials and reagents used for histology and histochemistry Equipments used for histology and histochemistry Materials used for succinate dehydrogenase test (SDH) Materials used for pyruvate kinase test (PK) Materials used for isolated perfused bovine udder Equipments used for isolated perfused bovine udder Materials and reagents used for the lactate dehydrogenase assay Materials and reagents used for the glucose assay Materials and reagents used for the lactate assay Analytical equipments Solutions and buffers Methods Bovine udders Cell culture experiments Primary mammary epithelial cell cultures Separation of primary mammary epithelial cells contaminated with mammary fibroblasts Culturing primary mammary fibroblasts Cell counting Immunocytochemistry Treating of the cells AgNO 3 treatments Incubation with MgAg1% sticks III

12 Contents Degradations process of MgAg1% sticks The isolated perfused udder Preparation of the udder Measuring parameters of viability of bovine udder The degedradation experiments Histological examination Degradation in dry off period secretions Determination of the silver concentrations in the degradation medium Determination of the magnesium and calcium concentrations in the degradation medium Biocompatibility tests Measurements of cell viability and proliferation MTS assay (3 - (4, 5-dimethylthiazol -2-yl) -5- (3-carboxymethoxyphenyl)- 2- (4-sulfophenyl) - 2H-tetrazolium]) Neutral red assay Measurements of metabolic activity Measurement of the succinate dehydrogenase (SDH) activity in the supernatant Measurement of the pyruvate kinase (PK) activity in the supernatant Determination of the protein content Histochemistry analysis Measurements of biomarkers of inflammatory reactions Preparations of the udder tissue for PGE 2 measurements Measurements of PGE 2 in the udder tissue supernatants Measurements of bovine and mouse TNF-alpha in the culture medium supernatant Measurements of IL-6 in the culture medium supernatant Measurements of IL-1 in the culture medium supernatant Detection of antibacterial activity Bouillion dilution testand cultivation of bacteria in petri dishes IV

13 Contents Brilliant black reduction (BRT-MRL screening test) Statistical analysis Results Establishment of mammary cell culture Isolation of primary mammary cells Culturing of primary mammary cells Verification of primary mammary cells using immunocytochemistry Degradation process of MgAg1 % sticks Isolated perfused udder Measuring lactate production in the perfused udder Measuring glucose consumption in the perfused udder Measuring lactate dehydrogenase enzyme (LDH) amounts in the perfused udder Degradation of MgAg1% sticks in bovine udder Degradation of MgAg1% sticks in secretion samples from cows at dry off Period Histological parameters Udder tissue incubated with MgAg1% sticks Silver concentrations in the degradation medium Magnesium and calcium concentrations in the degradation medium Biocompatibility tests Cell viability and proliferation MTS assay Neutral red assay Measuring the amount of protein in the supernatant Measurement of metabolic activities Measurement of SDH activity in the supernatant Measurement of PK activity in the supernatant Succinate staining of tissue and cells Teat tissues Primmary mammary epithelial cells V

14 Contents Biomarkers of inflammatory reactions Measurement of IL-1 beta in the culture medium supernatant Measurement of IL- 6 in the culture medium supernatant Measurement of bovine TNF-alpha in the culture medium supernatant Measurement of mouse TNF-alpha in the culture medium supernatant PGE 2 concentrations in udder tissue Antibacterial activity Bouillion dilution test and cultivation of bacteria in petri dishes Brilliant black reduction (BRT- MRL screening test) Discussion Biocompatibility of silver ions and silver containing alloy Effect of AgNO 3 and MgAg1% sticks on the viability of different cells Effect of AgNO 3 and MgAg1% sticks on the metabolic activities Effect of AgNO 3 and MgAg1% sticks on the biomarkers of inflammatory reactions Detection of the degradation of MgAg1% sticks Antibacterial activity of MgAg1% sticks Outlook Summary Zusammenfassung Appendix References Abbreviations Acknowledgements..179 VI

15 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

16 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

17 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

18 Literature review 2 Literature review 2.1 Bovine udder 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)] 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

19 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

20 Literature review 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) 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 ( 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

21 Literature review 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

22 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

23 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

24 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

25 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

26 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) 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 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

27 Literature review housed or confined cattle principally in the northern hemisphere (RADOSTITS et al., 2000) 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

28 Literature review 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) 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) 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

29 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) 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) 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) 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

30 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 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) 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 (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

31 Literature review Tab. 2: Mastitis incidence (cow cases/100 cow years) O herds C herds Overall Lactation Dry period O herds = Organic managed dairy herds; C herds = conventionally managed dairy herds Average individual cow SCC levels were significantly higher in O herds ( cells/ml) than in C herds ( cells/ml; P<0.001), resulting in high subclinical mastitis levels in O herds (individual cow SCC> 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 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) 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

32 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) 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 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

33 Literature review 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 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) 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

34 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 Nil In slow release units base, 2 infusions (48 h) interval Cloxacillin 500 mg Up to In long acting Cloxacillin + Ampicillin 200 mg 75 mg base,1 infusion 3 infusions, once daily for 3 days (NEWBOULD, 1977) Spiramycin 500 mg infusions (24 h) interval Rifamycin 250 mg infusions (24 h) interval Streptomycin + Pencillin 1mg units infusions (24 h) interval Tetracyclines Up to 100 Poor Daily 2-3 days 400 mg Chloramphenicol 200 mg Daily for 24 days Neomycin 500 mg Daily (or 48 h intervals) for 2 infusions 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

35 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) 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) 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

36 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) 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