Library. Assessment of the use of the startvac vaccine on a dairy farm affected by environmental mastitis

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1 5th Startvac Library Assessment of the use of the startvac vaccine on a dairy farm affected by environmental mastitis Carlos Ribeiro, Veterinary Doctor; Dália Castro, Veterinary Doctor cr.cveta@netvisao.com Centro Veterinario de Aveiro, Portugal 1. Introduction Mastitis represents very high costs for a dairy farm. The cost of mastitis may mean a reduction of 20% to 25% in both milk production and the proportion of fat in it (Sharma et al., 2009). We must add to these economic losses the immediate costs associated with the treatment of mastitis, the value of discarded milk and penalties on the milk price as a result of an increased somatic cell count (SCC) and total bacterial count. To all this, additionally we know many cases of mastitis lead to the loss of one or more udder quarters and early culling of some cows. Mastitis can never be eradicated, because the various types are the result of multiple factors: animal, environment, handling, milking routine and microorganisms. High milk production is also Taxa Rate de of novas new infections infecções Parto Calving Drying Secagem Parto Calving Período Lactação Lactation Drying Off seco Graphic 1. Rate of new intramammary infections during lactation and the dry period (Adapted from Naztke) one of the predisposing factors for the occurrence of mastitis because it increases the sensitivity of the udder to infections. Several studies have shown

2 510 th Vacas Cows Chronic mastitis (10) Mamite crónica Damaged udder Lig. ubere (1) partdo Teat lesions (1) Lesão dos tetos Infertilidade Infertility (3) Disorder Doen. of unknown causa desconhecida cause (2) Ketosis Cetose (1) Drop in Baixa production na produção (3) Graphic 2. Reasons for rejection in the period from 15/3/2009 to 16/3/2010 Abortion Aborto (2) Sindrome Fat cow syndrome da vaca gorda (1) Hoof Problemas problems podais (1) Prob. Musculoskeletal musculoesqueléticos problems (1) Problemas Digestive problems (1) digestivos iforms with protection against S. aureus and coagulase negative staphylococcus (CNS), to reduce the severity and duration of the clinical status of mastitis and prevent new infections. In order to evaluate the efficacy and cost-effectiveness of this vaccine, we used STARTVAC on a dairy farm with high economic losses due to mastitis by coliform agents, without altering the established preventive control. This dairy farm was chosen because of the high total cell count and high costs associated with the use of intramammary antibiotics (Graphic 3). During 2009, this farm had costs of approximately 11,000 euros on intramammary antibiotics used for treating clinical mastitis in lactating cows, which corresponds to 72% of the total cost for drugs. Assessment of the use of the startvac vaccine on a dairy farm affected by environmental mastitis Carlos Ribeiro, Veterinary Doctor; Dália Castro, Veterinary Doctor cr.cveta@netvisao.com Centro Veterinario de Aveiro, Portugal that nearly half of the cases of environmental mastitis developed in early lactation were related to infections acquired during the dry period (Bradley et al., 2000) (Graphic 1). In the field of mastitis control, apart from antibiotics, environmental management, hygienic measures and milking routine, a prophylactic vaccinal treatment is now emerging in Europe. The J5 type vaccines (E. coli) have been available for several Percentagem % Antibióticos AB mamite mastitis Antibióticos AB Vacinas Vaccines Suplementos Supplements Graphic 3. Medication costs (as a percentage) in 2009 Outros Others years in the United States. They are used in preventing mastitis caused by coliform bacteria such as E. coli, Klebsiella spp, Citrobacter spp and Enterobacter spp. According to several studies, administration before calving in adult cows and heifers is a solid investment with a significant economic benefit Laboratorios HIPRA S.A. have now registered STARTVAC, which combines immune protection against E. coli and col-. Hormonas Hormones Desparasitantes Anti-parasitics Corticosteroids Anti-inflammatories Anti-infl. Antibióticos AB secagem dry 2. Methodology The protocol used was according to the manufacturer (Hipra) and consisted of two 2ml intramamuscular injections of STARTVAC at 45 and 15 days prior to calving and 50 days after calving in pregnant cows and heifers. This immunisation protocol was implemented for a period of six months, after which the incidence of new infections in all vaccinated animals was assessed, as was the evolution of cell counts in the herd and the costs associated with treatment of mastitis. The study in question was carried out on a group of 65 lactating cows. The trial began on the 27 th of July 2009 and during that time, 10 heifers and 16 adult cows were vaccinated. The first cow vaccinated was scheduled to calve on the 7 th of September 2009, so it was not until that date when data collection started for the analysis of the efficacy of the vaccinated group in comparison to the previous 6 months. 3. Analysis of the results To rule out the possibility that our results are masked by possible culling of chronic cows, we compared the number

3 Assessment of the use of the startvac vaccine on a dairy farm affected by environmental mastitis of animals culled due to mastitis in the six months preceding the trial and during it. Analysing this data, we note that the number of cows rejected due to mastitis was unchanged in both periods (Table1); so we consider that this criteria has no influence on the results. In the six-month trial (from September to February), six cows were rejected for mastitis, which was what had occured in the previous milking period. Considering that from 250,000 cells/ml upwards, an animal demonstrates both lower milk quality and production losses, we followed this criteria in our trial (Brito et al, 1997). As a result, we anlaysed the number of vaccinated cows with SCCs above 250,000 cells (SCCs above 300,000 imply the loss of one premium point in the payment for milk). (Table 2). Of the 26 vaccinated animals (heifers and multiparous cows), 5 are marked on the list of animals with SCCs higher than 250,000 cells/ml. This represents 25% of total new cases in adult cows and 10% of heifers vaccinated during this period of time. The bulk milk tank cell count decreased (on average per milking period) from 449,000 cells / ml to 239,000 cells/ml. (Graphic 4). We can verify after analysing the treatment costs that there was a significant reduction in relation to intramammary medication for mastitis (Graphic 5). The mean monthly expense for monthly intramammary treatments was in excess of 1,000 euros - by September, this expense was reduced to less than half of this. Immunisation during the dry period can be considered as a possible explanation of the immediate reduction in treatment costs, as a lower incidence of mastitis is recorded postpartum and clinical cases respond more quickly to common antibiotics. Given the hypothesis of the typical seasonal character of coliform infections, we looked to see whether if at the same time as in the previous year, this cost reduction in intramammary antibiotics had been verified. In analysing the 2008 data table, which only has figures available from April onwards, there is no Table 1. Cows rejected in the period from to Date Cow ID Cause of Rejection Calving No Days in Lactation 24/02/ CLINICAL MASTITIS 1-24/02/ SUDDEN DEATH 3-24/02/ POST CALVING DISORDERS 2-24/02/ DAMAGED UDDER 1-24/02/ ABORTION 3-24/02/ FATTY LIVER SYNDROME 2-24/02/ FATTY LIVER SYNDROME 3-18/03/ CHRONIC MASTITIS 4-18/03/ ABORTION 3-18/03/ CHRONIC MASTITIS 2-22/03/ DROP IN MILK PRODUCTION 5-19/05/ DAMAGED UDDER 3-20/06/ CHRONIC MASTITIS 3-20/06/ INFERTILITY 2-20/06/ UNKNOWN DISEASE 3-23/06/ UNKNOWN DISEASE 3-25/08/ DIGESTIVE DISORDERS 1-25/08/ CHRONIC MASTITIS 2-19/09/ KETOSIS /09/ ABORTION /09/ FATTY LIVER SYNDROME /09/ MUSCULOSKELETAL DISORDERS /12/ CHRONIC MASTITIS /12/ CHRONIC MASTITIS /12/ CHRONIC MASTITIS /12/ CHRONIC MASTITIS /12/ CHRONIC MASTITIS /12/ CHRONIC MASTITIS /01/ INFERTILITY /01/ DROP IN MILK PRODUCTION /01/ DROP IN MILK PRODUCTION /02/ INFERTILITY /02/ HOOF DISORDERS /02/ DAMAGED UDDER Table 2. List of cows with SCC values above 250,000 somatic cells. Cow ID CCS x Calving No 0 April 05 Jan May 04 Feb 04 Mar June 04 May 03 Jun July 03 Jul August 03 Oct 03 Nov September Abr Mai Jun Jul Ago Set Out Nov Dez Jan Fev Mar Graphic 4. Evolution of BSCC throughout the last year. Bulk tank milk cell count decreased (average per period) from 449,000 cells/ml to 239,000 cells/ml. 03 Dec 04 Jan October 03 Feb 03 Mar November Days in Lactation December Total milk production January February Average milk production /04/09/2/G , /05/09/2/G , /05/09/2/G , /05/09/1/G , /06/06/2/S , /07/09/2/G , /09/09/1/I , /11/09/2/I , /12/09/2/C , /01/10/2/I , /03/10/2/P /04/10/2/P March

4 5 seasonality, and costs remain constant th Assessment of the use of the startvac vaccine on a dairy farm affected by environmental mastitis Carlos Ribeiro, Veterinary Doctor; Dália Castro, Veterinary Doctor cr.cveta@netvisao.com Centro Veterinario de Aveiro, Portugal throughout the year at an average of 1,250 euros (Graphic 6). 4. Conclusions During the period we vaccinated with STARTVAC, we achieved very positive results with decreased cell counts in the herd (average SCC reduction of 210,000) and a remarkable cost reduction in intramammary antibiotics (a decrease in the monthly average, which was above 1000 euros, to less than half of that). There was also a decrease in the severity of both clinical and subclinical mastitis (SCC > 250,000). With regard to the incidence of new cases, we cannot say what their evolution is; we only know that the incidence in all of the vaccinated animals was 20%. To reinforce these findings, it would be interesting to undertake further studies to assess the cost-effectiveness of the vaccine, with a larger number of animals and for a longer time January April February March May April June May Jan Fev Mar Abr Mai Jun Jul Ago Set Out Nov Dez Jan Fev Graphic 5. Intramammary medication costs in 2009 June July July August August ai Jun Jul Ago Set Out Nov Dez Graphic average monthly costs of intramammary medications used September September October October November. December November January December February Bibliography 1. Auldist, M.J.; Hubble, I.B Effects of mastitis on raw milk and dairy products. The Australian Journal of Dairy Technology, 53: p Bradley, A.J.; Green, M.J A study of the incidence and significance of intramammary enterobacterial infection acquired during the dry period. J. Dairy Sci.; 83: Brito, J. R. F.; Caldeira, G. A. V.; Verneque, R. S. et al.; Sensitivity and specificity of Californian Mastitis Test as a diagnostic tool for subclinical mastitis. Regarding somatic cell count. Brazilian Veterinary Research, v.17 (2): p.49-53, abr./jun. 4. Erskine, R. J., J. H. Kirk, J. W. Tyler, and F. J. DeGraves Advances in the therapy for mastitis. Vet. Clin. N. Am. Food Anim. Pract. 9: Green, M. J. ; Green, L. E. ; Medley, G. F.; Schukken, Y. H.; Bradley, A. J Influence of Dry Period Bacterial Intramammary Infection on Clinical Mastitis in Dairy Cows; J Dairy Sci, October 1; 85(10): Larry Smith, K.; Hogan, J. S.; Milk quality A worldwide perspective. In: National Mastitis Council, 37, Madison. Proceedings... Madison: NMC, p. 3-9, Natzke, R. P Elements of mastitis control. J. Dairy Sci. 64: Oliver, S. P.; Calvinho, L. F Influence of inflammation on mammary gland metabolism and milk composition. In: 2nd International Workshop on the Biology of Lactation in Farm Animals, J. Animal Sci. 73: Sharma, M Recent trends in mastitis management. Publication date 2/01/2009 in Venturini1, T.; Api1, I.; Restelatto, R.; Paixão, S.J.; Ziech, M.F.; Montagner, M.M.; Cases of subclinical mastitis in Holstein and Jersey breeds. 3th Symposium. Farming Production Systems - Veterinary Medicine. Laboratorios Hipra, S.A. Avd. la Selva, Amer (Girona) Spain Tel.: (34) Fax: (34) hipra@hipra.com

5 4 th Startvac Library General aspects of biofilm and its implication in ruminant mastitis Antoni Prenafeta R&D Dept., HIPRA. Avda. La Selva, 135. Amer (Girona) - Spain 1. Biofilm as a survival mechanism Biofilm can be defined broadly as a dynamic and well structured microbial community, attached to a solid surface and aggregated by an extracellular matrix. The ability to form biofilm is a widespread feature among prokaryotes (both in the archaeal and bacterial domain) and it has been found in fossil formations dating back 3.2 billion years. From an evolutionary standpoint, the formation of biofilm probably conferred an adaptive advantage by providing homeostasis against extreme conditions and fluctuations of the primitive earth (temperature, ph and exposure to UV radiation). In addition to offering protection from physical and chemical environmental factors, biofilm facilitates extracellular catalytic functions (because cells remain close to each other) and promotes the concentration of nutrients on the surface (Hall-Stoodley et al., 2004). Biofilm resistance to antimicrobial agents (e.g. antibiotics) may be due to difficulty in penetration of the antimicrobial agent through the extracellular matrix, to the decreased growth rate of biofilm cells (β-lactam antibiotics are effective in Grampositive cells that are actively dividing) or the existence of resistant phenotypes among a genetically heterogeneous population. 2. Biofilm in natural environments and its implication in infections Biofilm formation is ubiquitous in natural environments. These types of biological structures are found at the bottom of rivers or on the surface of stagnant water; in extreme environments, from hot springs to glaciers in the Antarctic; in showers or baths favoured by the warm moist environment; inside water ducts or industrial gas and oil pipes; in symbiosis with plants, etc. Biofilm formation is also implicated in the pathogenesis of many human infections. The adhesion of Staphylococcus or Streptococcus to the proteins of the basal membrane of the damaged heart epithelium is a cause of endocarditis. In the case of cystic fibrosis patients, decreased ciliary activity of the respiratory mucosa and mucus hyperviscosity promote colonisation and biofilm formation by Staphylococcus aureus, Haemophilus influenzae and Pseudomonas aeruginosa. Another well known example of biofilm is the subgingival plaque of Streptococcus mutans. Biofilm formation has also been described in uropathogenic strains of Escherichia 1 coli. Finally, biofilm is an important virulence factor involved in the development of implant-related infections of intravenous catheters, heart valves, prostheses, peritoneal dialysis catheters, endotracheal tubes, etc., which are mainly caused by the adhesion of S. aureus and Staphylococcus epidermidis to the surface of these implants (Hall-Stoodley et al., 2004). The contribution of biofilm to pathogenesis is attributed to its resistance to antibiotics and phagocytosis, thereby facilitating chronic infections. On the other hand, detachment of biofilm bacteria cells is a cause of septicaemia and new colonisations, while the production of endotoxins and exotoxins produce inflammation and tissue damage. One of the difficulties in eliminating infections associated with biofilm production is resistance to antibiotics. Table 1 shows that bacterial death within biofilm requires a greater amount of antibiotic than the concentration of bactericidal activity in planktonic bacterial cells (free or in suspension).

6 4th Startvac Library General aspects of biofilm and its implication in ruminant mastitis Antoni Prenafeta R&D Dept., HIPRA. Avda. La Selva, 135. Amer (Girona) - Spain Table 1. Sensitivity to antibiotics of different bacterial genera growing in planktonic form (free or in suspension) or in biofilm (Donlan and Costerton, 2002). Microorganism S. aureus NCTC Pseudomonas aeruginosa ATCC E. coli ATCC P. pseudomallei Streptococcus sanguls 804 Reference Organism Antibiotic Concentration effective MIC or MBC of Antibiotic against biofilm planktonic phenotype (µg/ml) phenotype (µg/ml) Vancomycin 2 (MBC) 20 Imipenem 1 (MIC) 1024 Ampicillin 2 (MIC) 512 Ceftazidime 8 (MBC) 800 Doxycycline (MIC) 3.15 MIC: Minimum Inhibitory Concentration MBC: Minimum Bactericidal Concentration 3. Development of S. aureus biofilm Biofilm formation by S. aureus is a well characterised process that occurs in two steps. First, bacterial cells adhere to a surface specifically or by physicochemical interactions. In the former case, specific S. aureus adhesion proteins bind to the extracellular matrix factors of the host, such as fibronectin binding proteins (FnBPA, FnBPB) (O Neill et al., 2008), fibrinogen binding proteins (ClfA, ClfB) (McDevitt et al., 1994), collagen binding protein (Can) (Patti et al., 1992) or bone sialoprotein binding protein (BBP) (Tung et al., 2000). In a second step, adherent bacterial cells multiply, interact with each other and accumulate in layers, embedded by an extracellular matrix secreted by the bacterial cell itself (Figure 1). The main constituent of the S. aureus and S. epidermidis extracellular matrix, responsible for the intercellular interactions, is the exopolysaccharide poly-n-acetyl-β -1,6-glucosamine (PNAG), synthesized by enzymes encoded in the icaadbc operon (Cramton et al., 1999). Likewise, some S. aureus proteins have been described that can be involved in intercellular interaction and biofilm formation: Bap (Cucarella et al., 2001), protein A (Merino et al., 2009), SasC (Schroeder et al., 2009) and SasG (Corrigan et al., 2007). Figure 1. micrograph by scanning electron microscopy of a biofilm of S. aureus growing in vitro ( Implication of biofilm 5. Vaccines against the biofilm of in ruminant mastitis caused by S. aureus In bovine and ovine mastitis caused by staphylococci, bacterial cells attach to the epithelial cells of the mammary gland and grow into colonies surrounded by an extracellular matrix, thereby forming the biofilm. Because of its size, biofilm is not capable of being phagocytised by polymorphonuclear neutrophils or macrophages and, moreover, it confers resistance to antibiotics, thereby promoting the chronicity of infection. Various studies demonstrate the presence of the icaadbc operon, which encodes the enzymes responsible for the biosynthesis of the PNAG exopolysaccharide, the main component of the extracellular matrix of the biofilm, in 94.36% (Cucarella et al., 2004) or 100% (Vasudevan et al., 2003) of S. aureus isolated from bovine mastitis. Apart from this genetic capacity, a number of studies have also demonstrated the ability of bovine mastitis isolates to form biofilm in vitro. In this regard, Vasudevan et al. (2003) found that 91% of isolates of S. aureus from bovine mastitis had the ability to form biofilm in vitro by determination of colonial morphology on agar plates with Congo red (Figure 2), whereas 69% showed adhesion in a microplate (Figure 3). In another study, Oliveira et al. (2007) characterised 80.8% of isolates of S. aureus and 75.9% of isolates of S. epidermidis in bovine mastitis as in vitro producers of biofilm. Dhanawade et al. (2010) found that 48.03% of the strains of S. aureus isolated from bovine mastitis had the ability to form biofilm in vitro by the culture test on agar plates with Congo red. While the genetic capacity and in vitro biofilm production in S. aureus isolates from bovine mastitis seems clear, is there any evidence of biofilm production of S. aureus in the mammary gland? Watson et al. (1989) observed by electron microscopy the production of a polysaccharide extracellular matrix (called pseudocapsule by the authors) in S. aureus cells isolated directly from the milk of sheep and cows with clinical mastitis. Shortly afterwards, Baselga et al. (1993) demonstrated the production lular component has been determined for all isolates of S. aureus characterised as slime producers in agar plates with Congo red (Figure 4) and its production is directly related to the in vitro biofilm formation (Table 2). The chemical characterisation showed that the SAAC is comprised of 58% (w/v) polysaccharide and 42% (w/v) protein. Contents of 6.1% of glucosamine and galactosamine were found in the polysaccharidic fraction, suggesting that these sugars may correspond to deacetylated forms of PNAG. It is noteworthy that antibodies to deacetylated forms of PNAG are those with the greatest capacity for opsonization (specific antibody binding to antigen) and protection against infection by S. aureus (Cerca et al., 2007). S. aureus to combat mastitis in ruminants Figure 3. Analysis of biofilm production in microplate (adhesion test). After an incubation period of the S. aureus isolate in the wells of the microplate, the plate is emptied, the wells washed, and the cells that have adhered fixed, stained and the optical density (OD) of the wells determined by means of an ELISA plate reader. If the isolate formed biofilm during growth, the OD of the wells is high (rows A, B and C), whereas the OD is not significant if the isolate did not produce biofilm (rows D, E and F) compared with the uninoculated negative control wells (rows G and H). of an exopolysaccharide matrix in S. aureus cells by immunohistochemical analysis of mammary gland parenchymal tissue samples from sheep experimentally infected with S. aureus by intramammary route. The in vivo exopolysaccharide expression has also been shown indirectly by observing the production of specific antibodies against PNAG (Perez et al., 2009) and against SAAC (Slime Associated Antigenic Complex; Prenafeta et al., 2010) in sheep and cows, respectively, experimentally infected with S. aureus by intramammary route. Given that biofilm formation is an important virulence factor of S. aureus in the pathogenesis of mastitis in sheep and cows, the efficacy of different experimental vaccines has been tested, showing different levels of protection. Watson et al. (1993) and Nordhaug et al. (1994) used vaccines based on whole S. aureus inactivated cells embedded in their own extracellular matrix called pseudocapsule. The experimental vaccines in a study by Amorena et al. (1994) consisted of a mixture of slime (biofilm exopolysaccharide matrix) in liposomes, toxoid and various inactivated S. aureus isolates. More recently, knowing that the PNAG exopolysaccharide is the major component of the extracellular matrix of the S. aureus biofilm, Perez et al. (2009) conducted an efficacy trial against an intrammamary challenge with a virulent S. aureus strain in sheep, using bacterins (whole and inactivated bacterial cells), crude extract, or purified PNAG, with different adjuvants, as vaccines. The results of this study showed that bacterins from strong biofilm-producing bacteria induced the highest titres of specific antibodies to PNAG and conferred the greatest protection against an intramammary challenge, compared to vaccines containing bacterins from weak biofilm-producing bacteria, crude extract or purified PNAG. The study by Prenafeta et al. (2010) clarifies the role of SAAC-specific antibodies in protecting against the mastitis caused by S. aureus in an experimental infection in cows. SAAC is an isolated cell fraction from S. aureus strains that produce biofilm. The presence of this extracel- Table 2. Determination of a slime producing phenotype (/-), biofilm formation capacity in microplate (OD of the biofilm in the test) and production of SAAC (mg SAAC/mg total protein) in isolates of S. aureus. The correlation between the production of SAAC and the ability to form biofilm in microplate is significant (R = 0.882). Isolate S. aureus SA1H SA2H SA3H SA4H SA5H SA6H SA7H SA8H SA9H SA10H SA11H SA12H SA13H Slime producing phenotype OD in the biofilm test (SD1) (0.04) (0.02) (0.03) (0.04) (0.03) (0.01) (0.02) (0.06) (0.02) (0.01) (0.01) (0.01) (0.02) Production of SAAC (SD1) 54.0 (0.012) 63.3 (0.015) 20.8 (0.011) 60.5 (0.012) 27.6 (0.015) 2.2 (0.011) 26.5 (0.012) 0.1 (0.010) 6.9 (0.013) 1 SD: standard deviation of the mean. 2 Nd. : Not detected. The main defence mechanism of the mammary gland against infections is antibody-mediated opsonization and subsequent phagocytosis by polymorphonuclear neutrophils. However, we must not exclude that the biofilm-specific antibodies also act in a direct manner in protection, binding to cells and preventing bacterial adherence to epithelium and intercellular interaction that leads to the formation of biofilm. In this regard, in an in vitro study, it was shown that antibodies to SAAC are capable of inhibiting biofilm formation without the presence of neutrophils (Figure 5 shows the results of a study conducted by the author). One of the advantages of using PNAG or the SAAC component as vaccine antigens, unlike capsular antigens, is that no serotypes have been reported among isolates of S. aureus. Therefore, the antibodies induced by vaccination with these antigens confer crossprotection regardless of the capsular type of S. aureus. STARTVAC (HIPRA) is the first vaccine against bovine mastitis registered throughout the European Union via the EMEA (European Medicines Agency). This vaccine contains inactivated cells of a high biofilm-producing S. aureus strain with a high content of cellassociated SAAC. Moreover, the vaccine contains the inactivated E. coli J5 strain and a suitable adjuvant to boost the immune response. Figure 2. Determination of the ability to form biofilm by characterising colonial morphology in agar plates with Congo red. Slime positive or biofilm-producing isolates form colonies that have rough and irregular outlines in Congo red plates (A), whereas slime negative or non-biofilm producing isolates form colonies with shiny, smooth and well-defined boundaries (B). 3 Figure 4. Analysis by immunoelectrophoresis in agarose gel for bacterial extracts from a strain of an S. aureus biofilm producer (A: wells 1 and 3) and non-producing strain (B: wells 4 and 6), using a polyclonal serum against whole bacteria (lines 2 and 5). The arrow shows the line of immunoprecipitation for the SAAC antigen, which is present only in strains characterised as exopolysaccharide producers in agar plates with Congo red. In both preclinical testing and in clinical trials conducted during the development of the vaccine, immunisation with STARTVAC induced high and long-lasting titres of anti-saac antibodies in blood and milk. Clinical trials carried out with STARTVAC on six farms (198 cows vaccinated with STARTVAC and 188 unvaccinated control cows) showed that vaccination significantly reduced the incidence of clinical and subclinical mastitis and severity of clinical symptoms of mastitis caused by S. aureus, coliforms and coagulase negative staphylococci (CNS) (reduction in somatic cell counts, decreased clinical signs and reduction of antibiotic treatment in infected animals). Additionally, vaccination with STARTVAC increased the spontaneous cure rate in 4

7 4th Startvac Library General aspects of biofilm and its implication in ruminant mastitis Antoni Prenafeta R&D Dept., HIPRA. Avda. La Selva, 135. Amer (Girona) - Spain Table 1. Sensitivity to antibiotics of different bacterial genera growing in planktonic form (free or in suspension) or in biofilm (Donlan and Costerton, 2002). Microorganism S. aureus NCTC Pseudomonas aeruginosa ATCC E. coli ATCC P. pseudomallei Streptococcus sanguls 804 Reference Organism Antibiotic Concentration effective MIC or MBC of Antibiotic against biofilm planktonic phenotype (µg/ml) phenotype (µg/ml) Vancomycin 2 (MBC) 20 Imipenem 1 (MIC) 1024 Ampicillin 2 (MIC) 512 Ceftazidime 8 (MBC) 800 Doxycycline (MIC) 3.15 MIC: Minimum Inhibitory Concentration MBC: Minimum Bactericidal Concentration 3. Development of S. aureus biofilm Biofilm formation by S. aureus is a well characterised process that occurs in two steps. First, bacterial cells adhere to a surface specifically or by physicochemical interactions. In the former case, specific S. aureus adhesion proteins bind to the extracellular matrix factors of the host, such as fibronectin binding proteins (FnBPA, FnBPB) (O Neill et al., 2008), fibrinogen binding proteins (ClfA, ClfB) (McDevitt et al., 1994), collagen binding protein (Can) (Patti et al., 1992) or bone sialoprotein binding protein (BBP) (Tung et al., 2000). In a second step, adherent bacterial cells multiply, interact with each other and accumulate in layers, embedded by an extracellular matrix secreted by the bacterial cell itself (Figure 1). The main constituent of the S. aureus and S. epidermidis extracellular matrix, responsible for the intercellular interactions, is the exopolysaccharide poly-n-acetyl-β -1,6-glucosamine (PNAG), synthesized by enzymes encoded in the icaadbc operon (Cramton et al., 1999). Likewise, some S. aureus proteins have been described that can be involved in intercellular interaction and biofilm formation: Bap (Cucarella et al., 2001), protein A (Merino et al., 2009), SasC (Schroeder et al., 2009) and SasG (Corrigan et al., 2007). Figure 1. micrograph by scanning electron microscopy of a biofilm of S. aureus growing in vitro ( Implication of biofilm 5. Vaccines against the biofilm of in ruminant mastitis caused by S. aureus In bovine and ovine mastitis caused by staphylococci, bacterial cells attach to the epithelial cells of the mammary gland and grow into colonies surrounded by an extracellular matrix, thereby forming the biofilm. Because of its size, biofilm is not capable of being phagocytised by polymorphonuclear neutrophils or macrophages and, moreover, it confers resistance to antibiotics, thereby promoting the chronicity of infection. Various studies demonstrate the presence of the icaadbc operon, which encodes the enzymes responsible for the biosynthesis of the PNAG exopolysaccharide, the main component of the extracellular matrix of the biofilm, in 94.36% (Cucarella et al., 2004) or 100% (Vasudevan et al., 2003) of S. aureus isolated from bovine mastitis. Apart from this genetic capacity, a number of studies have also demonstrated the ability of bovine mastitis isolates to form biofilm in vitro. In this regard, Vasudevan et al. (2003) found that 91% of isolates of S. aureus from bovine mastitis had the ability to form biofilm in vitro by determination of colonial morphology on agar plates with Congo red (Figure 2), whereas 69% showed adhesion in a microplate (Figure 3). In another study, Oliveira et al. (2007) characterised 80.8% of isolates of S. aureus and 75.9% of isolates of S. epidermidis in bovine mastitis as in vitro producers of biofilm. Dhanawade et al. (2010) found that 48.03% of the strains of S. aureus isolated from bovine mastitis had the ability to form biofilm in vitro by the culture test on agar plates with Congo red. While the genetic capacity and in vitro biofilm production in S. aureus isolates from bovine mastitis seems clear, is there any evidence of biofilm production of S. aureus in the mammary gland? Watson et al. (1989) observed by electron microscopy the production of a polysaccharide extracellular matrix (called pseudocapsule by the authors) in S. aureus cells isolated directly from the milk of sheep and cows with clinical mastitis. Shortly afterwards, Baselga et al. (1993) demonstrated the production lular component has been determined for all isolates of S. aureus characterised as slime producers in agar plates with Congo red (Figure 4) and its production is directly related to the in vitro biofilm formation (Table 2). The chemical characterisation showed that the SAAC is comprised of 58% (w/v) polysaccharide and 42% (w/v) protein. Contents of 6.1% of glucosamine and galactosamine were found in the polysaccharidic fraction, suggesting that these sugars may correspond to deacetylated forms of PNAG. It is noteworthy that antibodies to deacetylated forms of PNAG are those with the greatest capacity for opsonization (specific antibody binding to antigen) and protection against infection by S. aureus (Cerca et al., 2007). S. aureus to combat mastitis in ruminants Figure 3. Analysis of biofilm production in microplate (adhesion test). After an incubation period of the S. aureus isolate in the wells of the microplate, the plate is emptied, the wells washed, and the cells that have adhered fixed, stained and the optical density (OD) of the wells determined by means of an ELISA plate reader. If the isolate formed biofilm during growth, the OD of the wells is high (rows A, B and C), whereas the OD is not significant if the isolate did not produce biofilm (rows D, E and F) compared with the uninoculated negative control wells (rows G and H). of an exopolysaccharide matrix in S. aureus cells by immunohistochemical analysis of mammary gland parenchymal tissue samples from sheep experimentally infected with S. aureus by intramammary route. The in vivo exopolysaccharide expression has also been shown indirectly by observing the production of specific antibodies against PNAG (Perez et al., 2009) and against SAAC (Slime Associated Antigenic Complex; Prenafeta et al., 2010) in sheep and cows, respectively, experimentally infected with S. aureus by intramammary route. Given that biofilm formation is an important virulence factor of S. aureus in the pathogenesis of mastitis in sheep and cows, the efficacy of different experimental vaccines has been tested, showing different levels of protection. Watson et al. (1993) and Nordhaug et al. (1994) used vaccines based on whole S. aureus inactivated cells embedded in their own extracellular matrix called pseudocapsule. The experimental vaccines in a study by Amorena et al. (1994) consisted of a mixture of slime (biofilm exopolysaccharide matrix) in liposomes, toxoid and various inactivated S. aureus isolates. More recently, knowing that the PNAG exopolysaccharide is the major component of the extracellular matrix of the S. aureus biofilm, Perez et al. (2009) conducted an efficacy trial against an intrammamary challenge with a virulent S. aureus strain in sheep, using bacterins (whole and inactivated bacterial cells), crude extract, or purified PNAG, with different adjuvants, as vaccines. The results of this study showed that bacterins from strong biofilm-producing bacteria induced the highest titres of specific antibodies to PNAG and conferred the greatest protection against an intramammary challenge, compared to vaccines containing bacterins from weak biofilm-producing bacteria, crude extract or purified PNAG. The study by Prenafeta et al. (2010) clarifies the role of SAAC-specific antibodies in protecting against the mastitis caused by S. aureus in an experimental infection in cows. SAAC is an isolated cell fraction from S. aureus strains that produce biofilm. The presence of this extracel- Table 2. Determination of a slime producing phenotype (/-), biofilm formation capacity in microplate (OD of the biofilm in the test) and production of SAAC (mg SAAC/mg total protein) in isolates of S. aureus. The correlation between the production of SAAC and the ability to form biofilm in microplate is significant (R = 0.882). Isolate S. aureus SA1H SA2H SA3H SA4H SA5H SA6H SA7H SA8H SA9H SA10H SA11H SA12H SA13H Slime producing phenotype OD in the biofilm test (SD1) (0.04) (0.02) (0.03) (0.04) (0.03) (0.01) (0.02) (0.06) (0.02) (0.01) (0.01) (0.01) (0.02) Production of SAAC (SD1) 54.0 (0.012) 63.3 (0.015) 20.8 (0.011) 60.5 (0.012) 27.6 (0.015) 2.2 (0.011) 26.5 (0.012) 0.1 (0.010) 6.9 (0.013) 1 SD: standard deviation of the mean. 2 Nd. : Not detected. The main defence mechanism of the mammary gland against infections is antibody-mediated opsonization and subsequent phagocytosis by polymorphonuclear neutrophils. However, we must not exclude that the biofilm-specific antibodies also act in a direct manner in protection, binding to cells and preventing bacterial adherence to epithelium and intercellular interaction that leads to the formation of biofilm. In this regard, in an in vitro study, it was shown that antibodies to SAAC are capable of inhibiting biofilm formation without the presence of neutrophils (Figure 5 shows the results of a study conducted by the author). One of the advantages of using PNAG or the SAAC component as vaccine antigens, unlike capsular antigens, is that no serotypes have been reported among isolates of S. aureus. Therefore, the antibodies induced by vaccination with these antigens confer crossprotection regardless of the capsular type of S. aureus. STARTVAC (HIPRA) is the first vaccine against bovine mastitis registered throughout the European Union via the EMEA (European Medicines Agency). This vaccine contains inactivated cells of a high biofilm-producing S. aureus strain with a high content of cellassociated SAAC. Moreover, the vaccine contains the inactivated E. coli J5 strain and a suitable adjuvant to boost the immune response. Figure 2. Determination of the ability to form biofilm by characterising colonial morphology in agar plates with Congo red. Slime positive or biofilm-producing isolates form colonies that have rough and irregular outlines in Congo red plates (A), whereas slime negative or non-biofilm producing isolates form colonies with shiny, smooth and well-defined boundaries (B). 3 Figure 4. Analysis by immunoelectrophoresis in agarose gel for bacterial extracts from a strain of an S. aureus biofilm producer (A: wells 1 and 3) and non-producing strain (B: wells 4 and 6), using a polyclonal serum against whole bacteria (lines 2 and 5). The arrow shows the line of immunoprecipitation for the SAAC antigen, which is present only in strains characterised as exopolysaccharide producers in agar plates with Congo red. In both preclinical testing and in clinical trials conducted during the development of the vaccine, immunisation with STARTVAC induced high and long-lasting titres of anti-saac antibodies in blood and milk. Clinical trials carried out with STARTVAC on six farms (198 cows vaccinated with STARTVAC and 188 unvaccinated control cows) showed that vaccination significantly reduced the incidence of clinical and subclinical mastitis and severity of clinical symptoms of mastitis caused by S. aureus, coliforms and coagulase negative staphylococci (CNS) (reduction in somatic cell counts, decreased clinical signs and reduction of antibiotic treatment in infected animals). Additionally, vaccination with STARTVAC increased the spontaneous cure rate in 4

8 4th Startvac Library General aspects of biofilm and its implication in ruminant mastitis Antoni Prenafeta R&D Dept., HIPRA. Avda. La Selva, 135. Amer (Girona) - Spain Table 1. Sensitivity to antibiotics of different bacterial genera growing in planktonic form (free or in suspension) or in biofilm (Donlan and Costerton, 2002). Microorganism S. aureus NCTC Pseudomonas aeruginosa ATCC E. coli ATCC P. pseudomallei Streptococcus sanguls 804 Reference Organism Antibiotic Concentration effective MIC or MBC of Antibiotic against biofilm planktonic phenotype (µg/ml) phenotype (µg/ml) Vancomycin 2 (MBC) 20 Imipenem 1 (MIC) 1024 Ampicillin 2 (MIC) 512 Ceftazidime 8 (MBC) 800 Doxycycline (MIC) 3.15 MIC: Minimum Inhibitory Concentration MBC: Minimum Bactericidal Concentration 3. Development of S. aureus biofilm Biofilm formation by S. aureus is a well characterised process that occurs in two steps. First, bacterial cells adhere to a surface specifically or by physicochemical interactions. In the former case, specific S. aureus adhesion proteins bind to the extracellular matrix factors of the host, such as fibronectin binding proteins (FnBPA, FnBPB) (O Neill et al., 2008), fibrinogen binding proteins (ClfA, ClfB) (McDevitt et al., 1994), collagen binding protein (Can) (Patti et al., 1992) or bone sialoprotein binding protein (BBP) (Tung et al., 2000). In a second step, adherent bacterial cells multiply, interact with each other and accumulate in layers, embedded by an extracellular matrix secreted by the bacterial cell itself (Figure 1). The main constituent of the S. aureus and S. epidermidis extracellular matrix, responsible for the intercellular interactions, is the exopolysaccharide poly-n-acetyl-β -1,6-glucosamine (PNAG), synthesized by enzymes encoded in the icaadbc operon (Cramton et al., 1999). Likewise, some S. aureus proteins have been described that can be involved in intercellular interaction and biofilm formation: Bap (Cucarella et al., 2001), protein A (Merino et al., 2009), SasC (Schroeder et al., 2009) and SasG (Corrigan et al., 2007). Figure 1. micrograph by scanning electron microscopy of a biofilm of S. aureus growing in vitro ( Implication of biofilm 5. Vaccines against the biofilm of in ruminant mastitis caused by S. aureus In bovine and ovine mastitis caused by staphylococci, bacterial cells attach to the epithelial cells of the mammary gland and grow into colonies surrounded by an extracellular matrix, thereby forming the biofilm. Because of its size, biofilm is not capable of being phagocytised by polymorphonuclear neutrophils or macrophages and, moreover, it confers resistance to antibiotics, thereby promoting the chronicity of infection. Various studies demonstrate the presence of the icaadbc operon, which encodes the enzymes responsible for the biosynthesis of the PNAG exopolysaccharide, the main component of the extracellular matrix of the biofilm, in 94.36% (Cucarella et al., 2004) or 100% (Vasudevan et al., 2003) of S. aureus isolated from bovine mastitis. Apart from this genetic capacity, a number of studies have also demonstrated the ability of bovine mastitis isolates to form biofilm in vitro. In this regard, Vasudevan et al. (2003) found that 91% of isolates of S. aureus from bovine mastitis had the ability to form biofilm in vitro by determination of colonial morphology on agar plates with Congo red (Figure 2), whereas 69% showed adhesion in a microplate (Figure 3). In another study, Oliveira et al. (2007) characterised 80.8% of isolates of S. aureus and 75.9% of isolates of S. epidermidis in bovine mastitis as in vitro producers of biofilm. Dhanawade et al. (2010) found that 48.03% of the strains of S. aureus isolated from bovine mastitis had the ability to form biofilm in vitro by the culture test on agar plates with Congo red. While the genetic capacity and in vitro biofilm production in S. aureus isolates from bovine mastitis seems clear, is there any evidence of biofilm production of S. aureus in the mammary gland? Watson et al. (1989) observed by electron microscopy the production of a polysaccharide extracellular matrix (called pseudocapsule by the authors) in S. aureus cells isolated directly from the milk of sheep and cows with clinical mastitis. Shortly afterwards, Baselga et al. (1993) demonstrated the production lular component has been determined for all isolates of S. aureus characterised as slime producers in agar plates with Congo red (Figure 4) and its production is directly related to the in vitro biofilm formation (Table 2). The chemical characterisation showed that the SAAC is comprised of 58% (w/v) polysaccharide and 42% (w/v) protein. Contents of 6.1% of glucosamine and galactosamine were found in the polysaccharidic fraction, suggesting that these sugars may correspond to deacetylated forms of PNAG. It is noteworthy that antibodies to deacetylated forms of PNAG are those with the greatest capacity for opsonization (specific antibody binding to antigen) and protection against infection by S. aureus (Cerca et al., 2007). S. aureus to combat mastitis in ruminants Figure 3. Analysis of biofilm production in microplate (adhesion test). After an incubation period of the S. aureus isolate in the wells of the microplate, the plate is emptied, the wells washed, and the cells that have adhered fixed, stained and the optical density (OD) of the wells determined by means of an ELISA plate reader. If the isolate formed biofilm during growth, the OD of the wells is high (rows A, B and C), whereas the OD is not significant if the isolate did not produce biofilm (rows D, E and F) compared with the uninoculated negative control wells (rows G and H). of an exopolysaccharide matrix in S. aureus cells by immunohistochemical analysis of mammary gland parenchymal tissue samples from sheep experimentally infected with S. aureus by intramammary route. The in vivo exopolysaccharide expression has also been shown indirectly by observing the production of specific antibodies against PNAG (Perez et al., 2009) and against SAAC (Slime Associated Antigenic Complex; Prenafeta et al., 2010) in sheep and cows, respectively, experimentally infected with S. aureus by intramammary route. Given that biofilm formation is an important virulence factor of S. aureus in the pathogenesis of mastitis in sheep and cows, the efficacy of different experimental vaccines has been tested, showing different levels of protection. Watson et al. (1993) and Nordhaug et al. (1994) used vaccines based on whole S. aureus inactivated cells embedded in their own extracellular matrix called pseudocapsule. The experimental vaccines in a study by Amorena et al. (1994) consisted of a mixture of slime (biofilm exopolysaccharide matrix) in liposomes, toxoid and various inactivated S. aureus isolates. More recently, knowing that the PNAG exopolysaccharide is the major component of the extracellular matrix of the S. aureus biofilm, Perez et al. (2009) conducted an efficacy trial against an intrammamary challenge with a virulent S. aureus strain in sheep, using bacterins (whole and inactivated bacterial cells), crude extract, or purified PNAG, with different adjuvants, as vaccines. The results of this study showed that bacterins from strong biofilm-producing bacteria induced the highest titres of specific antibodies to PNAG and conferred the greatest protection against an intramammary challenge, compared to vaccines containing bacterins from weak biofilm-producing bacteria, crude extract or purified PNAG. The study by Prenafeta et al. (2010) clarifies the role of SAAC-specific antibodies in protecting against the mastitis caused by S. aureus in an experimental infection in cows. SAAC is an isolated cell fraction from S. aureus strains that produce biofilm. The presence of this extracel- Table 2. Determination of a slime producing phenotype (/-), biofilm formation capacity in microplate (OD of the biofilm in the test) and production of SAAC (mg SAAC/mg total protein) in isolates of S. aureus. The correlation between the production of SAAC and the ability to form biofilm in microplate is significant (R = 0.882). Isolate S. aureus SA1H SA2H SA3H SA4H SA5H SA6H SA7H SA8H SA9H SA10H SA11H SA12H SA13H Slime producing phenotype OD in the biofilm test (SD1) (0.04) (0.02) (0.03) (0.04) (0.03) (0.01) (0.02) (0.06) (0.02) (0.01) (0.01) (0.01) (0.02) Production of SAAC (SD1) 54.0 (0.012) 63.3 (0.015) 20.8 (0.011) 60.5 (0.012) 27.6 (0.015) 2.2 (0.011) 26.5 (0.012) 0.1 (0.010) 6.9 (0.013) 1 SD: standard deviation of the mean. 2 Nd. : Not detected. The main defence mechanism of the mammary gland against infections is antibody-mediated opsonization and subsequent phagocytosis by polymorphonuclear neutrophils. However, we must not exclude that the biofilm-specific antibodies also act in a direct manner in protection, binding to cells and preventing bacterial adherence to epithelium and intercellular interaction that leads to the formation of biofilm. In this regard, in an in vitro study, it was shown that antibodies to SAAC are capable of inhibiting biofilm formation without the presence of neutrophils (Figure 5 shows the results of a study conducted by the author). One of the advantages of using PNAG or the SAAC component as vaccine antigens, unlike capsular antigens, is that no serotypes have been reported among isolates of S. aureus. Therefore, the antibodies induced by vaccination with these antigens confer crossprotection regardless of the capsular type of S. aureus. STARTVAC (HIPRA) is the first vaccine against bovine mastitis registered throughout the European Union via the EMEA (European Medicines Agency). This vaccine contains inactivated cells of a high biofilm-producing S. aureus strain with a high content of cellassociated SAAC. Moreover, the vaccine contains the inactivated E. coli J5 strain and a suitable adjuvant to boost the immune response. Figure 2. Determination of the ability to form biofilm by characterising colonial morphology in agar plates with Congo red. Slime positive or biofilm-producing isolates form colonies that have rough and irregular outlines in Congo red plates (A), whereas slime negative or non-biofilm producing isolates form colonies with shiny, smooth and well-defined boundaries (B). 3 Figure 4. Analysis by immunoelectrophoresis in agarose gel for bacterial extracts from a strain of an S. aureus biofilm producer (A: wells 1 and 3) and non-producing strain (B: wells 4 and 6), using a polyclonal serum against whole bacteria (lines 2 and 5). The arrow shows the line of immunoprecipitation for the SAAC antigen, which is present only in strains characterised as exopolysaccharide producers in agar plates with Congo red. In both preclinical testing and in clinical trials conducted during the development of the vaccine, immunisation with STARTVAC induced high and long-lasting titres of anti-saac antibodies in blood and milk. Clinical trials carried out with STARTVAC on six farms (198 cows vaccinated with STARTVAC and 188 unvaccinated control cows) showed that vaccination significantly reduced the incidence of clinical and subclinical mastitis and severity of clinical symptoms of mastitis caused by S. aureus, coliforms and coagulase negative staphylococci (CNS) (reduction in somatic cell counts, decreased clinical signs and reduction of antibiotic treatment in infected animals). Additionally, vaccination with STARTVAC increased the spontaneous cure rate in 4

9 General aspects of biofilm and its implication in ruminant mastitis Startvac Library Antoni Prenafeta R&D Dept., HIPRA. Avda. La Selva, 135. Amer (Girona) - Spain infected cows. In this regard, STARTVAC is the first vaccine registered in the world that confers protection against mastitis caused by coagulase negative staphylococci. Antibodies to the PNAG exopolysaccharide of the SAAC, induced by immunisation with STARTVAC, could be one of the factors responsible for conferring cross-protection against infections from CNS. 6. Perspectives of biofilm in mastitis The ability to form biofilm is an important virulence factor of S. aureus involved in bovine and ovine mastitis. Although other virulence factors may be involved in the pathogenesis of mastitis, the PNAG or SAAC-specific antibodies may prevent the establishment of infection of S. aureus in the mammary gland by binding to the exopolysaccharide extracellular matrix (before the establishment of the biofilm), thereby facilitating polymorphonuclear neutrophilmediated phagocytosis and elimination of infection. From this point of view, vaccination with STARTVAC offers an option for reducing intramammary infections from S. aureus and CNS. n 1,400 1,200 a b a 1,000 0,800 a a 0,600 0,400 0,200 b c c 0,000 S. aureus Ac - Ac Control- Culture Biofilm Figure 5. Inhibition of biofilm formation of S. aureus in microplate mediated by anti-saac antibodies. The graph plots the OD of bacterial growth at the end of the microplate incubation (in blue) and the OD of the biofilm after staining of adherent cells (in light blue). In this study, a biofilm producing strain of S. aureus was incubated without antibodies ( S. aureus columns), in the presence of serum without anti-saac antibodies ( Ac- columns ) or in the presence of a hyperimmune serum with anti-saac antibodies ( Ac columns ). The Control columns correspond to the wells with uninoculated culture medium. The columns with the same letter do not differ significantly between each other (P <0.05). Error bars indicate the standard deviation of the mean. 5

10 General aspects of biofilm and its implication in ruminant mastitis Antoni Prenafeta R&D Dept., HIPRA. Avda. La Selva, 135. Amer (Girona) - Spain 4 nº Bibliographic references 1. Amorena, B., Baselga, R. and Albizu, I., Use of liposome-immunopotentiated exopolysaccharide as a component of an ovine mastitis staphylococcal vaccine. Vaccine. 2: Baselga, R., Albizu, I., De La Cruz, M., Del Cacho, E., Barberan, M. and Amorena, B., Phase variation of slime production in Staphylococcus aureus: implications in colonization and virulence. 3. Cerca, N., Jefferson, K.K., Maira-Litrán, T., Pier, D.B., Kelly-Quintos, C., Goldmann, D.A., Azeredo, J. and Pier, G.B., Molecular basis for preferential protective efficacy of antibodies directed to the poorly acetylated form of staphylococcal poly-n-acetyl-β-(1-6)-glucosamine. Infect. Immun. 75: Startvac Library 13. Oliveira, M., Nunes, S.F., Carneiro, C., Bexiga, R., Bernardo, F. and Vilela, C.L., Time course of biofilm formation by Staphylococcus aureus and Staphylococcus epidermidis mastitis isolates. Vet. Microbiol. 124: O Neill, E., C. Pozzi, P. Houston, D. Smyth, H. Humphreys, D.A. Robinson, A. Loughman, T. J. Foster and J.P. O Gara., A novel Staphylococcus aureus biofilm phenotype mediated by the fibronectin-binding proteins, FnBPA and FnBPB. J. Bacteriol. 190: Patty, J.M., Jonsson, H., Guss, B., Switalski, L.M., Wiberg, K. et al Molecular characterization and expression of a gene encoding a Staphylococcus aureus collagen adhesion. J. Biol. Chem. 267: Corrigan, R.M., Rigby, D., Handley, P. and Foster, T.J., The role of Staphylococcus aureus surface protein SasG in adherence and biofilm formation. Microbiology. 153: Cramton, S.E., Gerke, C., Schnell, N.F., Nichols, W.W. and Götz, F., The intercellular adhesion (ica) locus is present in Staphylococcus aureus and is required for biofilm formation. Infect. Immun. 67: Cucarella, C., Tormo, M.A., Úbeda, C., Trotonda, M.P., Monzón, M., Peris, C., Amorena, B., Lasa, I. and Penadés, J.R., Role of biofilm-associated protein Bap in the pathogenesis of bovine Staphylococcus aureus. Infect. Immun. 72: Dhanawade, N.B., Kalorey, D.R., Srinivasan, R., Barbuddhe, S.B. and Kurkure, N.V., Detection of intercellular adhesion genes and biofilm production in Staphylococcus aureus isolated from bovine subclinical mastitis. Vet. Res. Commun. 34: Donlan, R.M. and Costerton, J.W., Biofilms: survival mechanisms of clinically relevant microorganisms. Clinical Microbiology Reviews. 15: Hall-Stoodley, L., Costerton, J.W. and Stoodley, P., Bacterial biofilms: from the natural environment to infectious diseases. Nature Reviews Microbiology. 2: Merino, N., Toledo-Arana, A., Vergara-Irigaray, M., Valle, J., Solano, C., Calvo, E., López, A.J., Foster, T.J., Penadés, J.R. and Lasa, I Protein A-mediated multicellular behaviour in Staphylococcus aureus. J. Bacteriol. 191: McDevitt, D., Francois, P., Vaudaux, P. and Foster, T.J., Molecular characterization of the clumping factor (fibrinogen receptor) of Staphylococcus aureus. Mol. Microbiol. 11: Nordhaug, M.L., Nesse, L.L., Norcross, N.L. and Gudding, R., A field trial with an experimental vaccine against Staphylococcus aureus mastitis in cattle. 1. Clinical parameters. J. Dairy Sci. 77: Pérez, M.M., Prenafeta, A., Valle, J., Penadés, J., Rota, C., Solano, C., Marco, J., Grilló, M.J., Lasa, I., Irache, J.M., Maira-Litran, T., Jiménez-Barbero, J., Costa, L., Pier, G.B., de Andrés, D., Amorena, B., Protection from Staphylococcus aureus mastitis associated with poly-n-acetyl β-1,6 glucosamine specific antibody production using biofilm-embedded bacteria. Vaccine. 27, Prenafeta, A., March, R., Foix, A., Casals, I. and Costa, LL., Study of the humoral immunological response after vaccination with a Staphylococcus aureus biofilm-embedded bacterin in dairy cows: possible role of the exopolysaccharide specific antibody production in the protection from Staphylococcus aureus induced mastitis. Vet. Immun. Immunopathol. 134: Schroeder, K., Jularic, M., Horsburgh, S.M., Hirschhausen, N., Neumann, C., Bertling, A., Schulte, Foster, S., Kehrel, B.E., Peters, G. and Heilmann, C., Molecular characterization of a novel Staphylococcus aureus surface protein (SasC) involved in cell aggregation and biofilm accumulation. PLoS ONE. 4(10): Tung, H., Guss, B., Hellman, U., Persson, L., Rubin, K. et al., A bone sialoprotein-binding protein from Staphylococcus aureus: a member of the staphylococcal Sdr family. Biochem J. 345(3): Vasudevan, P., Nair, M.K.M., Annamalai, T. and Venkitanarayanan, K.S., Phenotypic and genotypic characterization of bovine mastitis isolates of Staphylococcus aureus for biofilm formation. Vet. Microbiol. 92: Watson, D.L. and Watson, N.A., Expression of a pseudocapsule by Staphylococcus aureus: influence of cultural conditions and relevance to mastitis. Research in Veterinary Science. 47: Watson, D.L. and Davies, H.I., Influence of adjuvants on the immune response of sheep to a novel Staphylococcus aureus vaccine. Vet. Microbiol. 34: Laboratorios Hipra, S.A. Avd. la Selva, Amer (Girona) Spain Tel.: (34) Fax: (34) hipra@hipra.com

11 3rd Startvac Library Literature review: Bovine mastitis caused by Coagulase-Negative Staphylococci Clara Navarro Sansano Milk Quality Services 1. Introduction One of the groups of bacteria that cause mastitis is called coagulase-negative staphylococci (CNS). These bacteria are of great interest because they are currently the most commonly isolated microorganisms in cows and heifers in herds, and are currently considered emerging pathogens of bovine mastitis (Pyöräla S. et al. 2009). CNS are normally found on the healthy skin of the nipple and the hands of the milker. They are often called opportunistic microorganisms because they live in areas where it is easy to colonize the teat canal and penetrate the secretory tissue. Implementing mastitis control programmes over the past 30 years has led to a reduction in the overall incidence of clinical mastitis in most herds. In some cases, the decrease has been 90%. Whereas the clinical disease caused by major pathogens such as Staphylococcus aureus and Streptococcus agalactiae decreased significantly, less important pathogens such as CNS have been increasingly taking on greater importance. Cows and heifers can be infected with CNS before calving. In lactation, infection due to CNS is associated with an increase in somatic cell count (SCC), which causes economic losses due to the penalty in the price of milk. The prevalence of mastitis by CNS is higher in primiparous animals. They are generally mild infections and limited to floccules in milk due to local changes in the udder. Many of these infections even heal spontaneously. But sometimes animals with intramammary infections caused by CNS are observed with symptoms at a systemic level and are animals with persistent infections that can last several months if measures are not taken. There are over 50 species of coagulase-negative staphylococci and perhaps it is a mistake to observe their behaviour as a group and not as individual species. Although they are not considered to be a group of bacteria as pathogenic as the main pathogens causing mastitis, their pathogenicity and resistance to antimicrobial treatments varies depending on the species of CNS. Some investigators consider them to be secondary pathogens of the udder, but the significance of intramammary infections are still a subject of debate since, on the other hand, other works give them great important in the aetiology of sub-clinical or clinical mastitis and increased somatic cell counts of affected cows.

12 Literature review: Bovine mastitis caused by Coagulase-Negative Staphylococci Startvac Library Clara Navarro Sansano Milk Quality Services 2. Aetiology and epidemiology 3rd CNS are Gram-positive cocci that inhabit both the outside and inside of infected udders. Often they are called opportunistic flora of the skin, because they can be isolated from the skin of the teat, the teat canal, vagina, and the coat and nostrils. This group of bacteria includes over 50 species and subspecies (Pyöräla S. et al. 2009). The most common species of CNS are isolated from cases of bovine mastitis are Staphylococcus chromogenes, Staphylococcus epidermitis, Staphylococcus hyicus and Staphylococcus simulans. Species such as Staphylococcus epider-mitis, Staphylococcus saprophyticus, Staphylococcus simulans and Staphylococcus warneri belong to the normal bacterial flora of the teat skin, while other species such as Staphylococcus xylosus and Staphylococcus sciuri seem to come from the environment. Staphylococcus chromogenes may colonize the skin of the teat and other parts of an animal s body such as hair, the vagina and teat canal. It seems that there are differences in the pathogenicity of different species of CNS that are investigated by techniques of molecular diagnosis (Zadoks and Schukken, 2006). We found species with different antimicrobial susceptibility and diverse virulence factors of CNS isolated from bovine mastitis (Taponen S. et al. 2009). The incidence of new infections is highest during the cow s dry period and prior to calving; therefore, the percentage of quarters infected is high at the time of calving. The highest prevalence of CNS is in primiparous animals rather than in mature cows. Unfortunately, many producers mistakenly believe that their heifers are healthy, and the presence of mastitis is not observed until calving. Future breeders represent future lactation and care for the udder is basic for ensuring the profitability of dairy farms. Many of the intramammary infections caused by CNS heal spontaneously and the prevalence decreases as lactation progresses. Although CNS infections are usually mild or subclinical, it has also been shown that they can cause more severe and persistent processes, causing an increase in the somatic cell counts and a decrease in milk quality and production due to damage to breast tissue (Taponen S. et al. 2009; Gillespie B.E. et al. 2009). 3. Characteristics of infections by CNS Are usually mild infections and cause subclinical cases of mastitis. Increase in SCC. Can induce persistent clinical processes that do not respond to antibiotic treatment. Milk appearance is normal, but it can induce intramammary infections with alterations in milk (floccules). High prevalence in primiparous animals (especially in the time around calving). Higher incidence of new infections in the cows dry period. The general state of the animal is not usually affected, nor are there severe systemic signs. High spontaneous cure rate. 4. Diagnosis Once the quarters with high cell counts or that display clinical mastitis are detected, samples of milk should be taken aseptically and appropriately for subsequent processing in the laboratory. Microbiological testing is the most important test for the diagnosis of mastitis control programmes. The methodology includes the usual seeding in growth media specific for the major aetiological groups. They are incubated at 37 ºC, with readings at 24 and 48 hours. Baird Parker Agar is a culture medium specific for Staphylococci. It makes it possible to differentiate between CNS and Staphylococcus aureus. The identification of the different species of CNS is important to determine their pathogenicity and to develop specific management practices to prevent mastitis. The problem is that the identification of this group of organisms is difficult and costly. That is why many laboratories do not include species identification of CNS in routine procedures. 5. Treatment It is generally assumed that the spontaneous cure rate of CNS is high. The CNS respond much better to antimicrobial therapy than Staphylococcus aureus does, and most species of CNS are susceptible to antibiotics commonly used to treat mastitis. The treatment by intramammary therapy in the peripartum and drying is effective for controlling infections caused by CNS. However, treatment is not always effective. According to the National Mastitis Council (NMC), we can classify these germs as coagulasenegative Staphylococci sensitive to novobiocin and coagulase-negative Staphylococci novobiocin resistant. Treatment of clinical mastitis due to coagulasenegative Staphylococcus (CNS) sensitive to novobiocin: Penicillins and/or Penethamate or Cefalosporins (intramammary and/or parenteral route). Treatment of mastitis due to CNS resistant to novobiocin: The treatment is not necessary since spontaneous cures are seen. Antibiotic treatment in drying: Penicillins and/or Penethamate or Cefalosporins

13 Startvac Library Literature review: Bovine mastitis caused by Coagulase-Negative Staphylococci 6. Control measures Control measures must be applied in cows in lactation, in dry cows and also breeder heifers. Rebreeding can be a source of infection on a dairy farm, particularly under the current management systems, where heifers are transported and mixed several times before coming to the dairy farm where they will give birth (Oliver and Nickerson). Generally, not much attention is given to heifers on farms, or to cows during the dry period. But if we consider that the heifers are approximately one third of the herd each year, and that together with the dry cows they are the farm s investment for the future, the health of udders and proper functioning of heifers and dry cows should be a number one priority. Control measures should lower the animals contact with mastitis causing agents before calving. Handling Separate the heifers in individual pens: do not allow them to suckle each other, because this transmits bacteria and causes persistent infections that become established early in the life of the animal. Do not feed lactating heifers with infected milk: avoid transmission of infectious agents from the adult cows to young cows. Separate the heifers from the cows before calving. Provide clean areas for the cows to calve and for heifers. Environment Control of flies: flies can be vectors of pathogenic agents and also create a lesion on the teat tip, which allows bacteria such as Staphylococcus aureus or CNS to become established on the skin of the teat and enter its orifice. Ensure a clean dry environment. Also for rebreeding. Vaccinal schedule We cannot ensure proper prevention and control of the health of the udder of heifers and cows on the farm without taking into account a good vaccination programme. It is important to protect against the incidence and severity of mastitis caused by environmental microorganisms. The placement on the market of the first vaccine registered worldwide against CNS is a good tool to increase immunization of farm operations. The protocol is based on two applications before and one after calving to help decrease the incidence and severity at the time of greatest losses on the farm Antibiotic therapy during drying and general measures Administer a proper antibiotic therapy during drying based on the farm operation s bacteriological profile. Correct the milking routine. Carry out proper disinfection of the teat tip using disinfectant dips at both pre-milking and post-milking. Eliminate chronically ill animals. Guarantee hygiene of drying period treatments. Verify the state of food and drinking water. Proper use and maintenance of the milking machine. Take special care of the state of beds and walkways during drying and lactation.

14 Literature review: Bovine mastitis caused by Coagulase-Negative Staphylococci Startvac Library Clara Navarro Sansano Milk Quality Services Vaccinal schedule STARTVAC Efficacy at post-partum thanks to its vaccinal schedule By using two applications before calving (45 and 10 days before) and one application post-partum (on day 52) the objective of reducing mastitis is achieved at the time of greatest risk of infections and economic losses. 3rd Bibliographic references 1. John R.Middleton, Christopher D.Luby, D.Scott Adams, 2009 Efficacy of vaccination against staphylococcal mastitis: A review and new data 2. Schukken, Y.H., et al., 2009 CNS mastitis: Nothing to worry about? 3. Stephen C.Nickerson, 2008 Control of heifer mastitis: Antimicrobial treatment-an overview 4. Suvi Taponen, Satu Pyörälä, 2009 Coagulase-negative staphylococci as cause of bovine mastitis-not so different from Staphylococcus aureus? 5. Satu Pyörälä, Suvi Taponen, 2009 Coagulase-negative staphylococci- Emerging mastitis pathogens 6. White, L.J., et al., 2001 A multispecies model for the transmission and control of mastitis in dairy cows 7. Zadoks, R.N., et al., 2001 Cow- and quarter-level risk factors for Streptococcus uberis and Staphylococcus aureus mastitis 8. Zadoks, R.N., Schukken, Y.H., 2006 Use of the molecular epidemiology in veterinary practice. 9. A.A. Sawant, B.E. Gillespie, S.P.Oliver, 2008 Antimicrobial susceptibility of coagulase-negative Staphylococcus species isolated from bovine milk 10. O.C.Sampimon, H.W.Barkema, I.M.G.A.Berends, J.Sol, T.J.G.M.Lam, 2008 Prevalence and herd-level risk factors for intramammary infection with coagulasa-negative staphylococci in Dutch dairy herds 11. National Mastitis Council Annual Meeting Proceedings, StephenC. Nickerson 12. Wilson, D.J., Gonzalez, R.N. and Das, H.H. Bovine Mastitis Pathogens in New York and Pennsylvania: Prevalence and Effects on Somatic Cell Count and Milk Production 13. W. Nelson Philpot, Ph.D. y Stephen C. Nickerson, Ph.D. Ganando la lucha contra la mastitis 14. Arthur Saran-Marcelo Chaffer Mastitis y Calidad de Leche 15. Boehringer Ingelheim España, S.A. Div. Veterinaria Dosier solomamitis etiologia, tratamiento y diagnótico.

15 2nd Startvac Library Colibacillar mastitis Demetrio Herrera 1. Introduction 2. Pathogeny Despite the efforts of farmers and technicians to improve the health of herds udders, colibacillary mastitis remains a major problem in many livestock farms. In farms where contagious mastitis is virtually eliminated, and that have low tank cell counts, between 20 and 40% of episodes of clinical mastitis are caused by coliforms. Escherichia coli, Klebsiella spp. and to a lesser degree Enterobacter spp. are the most commonly isolated coliforms from clinical episodes of this kind. The presentation of clinical symptoms and costs stemming from them (discarded milk, treatment costs, replacement due to the death or slaughter of animals, etc.) is highly variable and depends primarily on factors in relation to the cow, rather than the pathogenicity of the strain involved. In this paper, we will discuss the predisposing factors and preventive measures to fight against this disease. Escherichia coli, and most Gram-negative bacteria, have a characteristic and essential macromolecule in their external cell membrane called lipopolysaccharide (LPS). This LPS is the major factor of pathogenicity of the bacterium. It triggers the typical set of symptoms of hyperacute coliform mastitis. The experimental intramammary injection of LPS in healthy animals causes the same symptoms, being dose dependent, and causes the death of the animal at high doses. The bacterium only enters via the teat canal; it multiplies rapidly in the cistern of the udder, and in the process of multiplication and lysis, the LPS toxicity and potent induction of inflammatory cytokines causes generally acute symptoms in cows. After running their course, it can cause an almost total loss of production, as well as an acute inflammation of the affected quarter and often loss of appetite, fever, listlessness, shock and sometimes death. Depending on the immune status of the cow, the presentation may be less acute. Chronic infection with recurrent clinical episodes may also occur but that is less frequent. The ability of the immune system of the cow is a key factor to limit the rapid spread of E. coli in the udder and reducing the toxic action of LPS. Neutrophils are key players in the fight against intramammary infections. They are responsible for sequestering, killing and eliminating the pathogen. They are aided by opsonising antibodies, mainly IgG 2 and pro-inflammatory cytokines, which are responsible for the massive influx of neutrophils from the blood capillaries of the udder into the cistern. The rapid mobilization of neutrophils into the udder is essential in reducing the impact of clinical symptoms.

16 23. Predisposing factors nd Most coliform intramammary infections occur in the first two weeks of the dry period and especially at peripartum. Furthermore, almost half of the cases of clinical mastitis that occur within the first 100 days in milk originate in the dry and peripartum periods. The presentation of hyperacute or acute colibacillary mastitis is not exclusive to post-partum, but a high percentage is. Intramammary coliform infections in advanced lactation cause mild or moderate cases, that a cow s immune system is able to resolve and that often go unnoticed. Colibacillar mastitis Startvac Library Demetrio Herrera qlettscp@gmail.com The onset of the dry period is a phase of risk due mainly to: Increased pressure within the udder, which sometimes causes loss of milk. Antibiotic syringes used during drying, can leave the sphincter open, so bacteria can enter. Bacterial proliferation on the skin of the teat, which is the result of the cessation of milking and the practice of pre-and postdipping. Delay in the formation of the keratin plug. It takes days or even weeks for the teat of some cows to seal. Poor hygiene when applying drying intramammary cannulae, which can cause intramammary infections. The peripartum phase is also a time of risk because the immune system is compromised by several factors: Calving is stressful for the cow. Plasma levels of cortisol experience a sharp physiological increase, which is needed for the development of the delivery and colostrogenesis. Cortisol inhibits the inflammatory response and adversely affects the operation of the neutrophils. Negative Energy Balance (NEB). There are numerous studies that relate a NEB with post-partum pathologies. Increased energy needs in post-partum along with a reduced ability to intake, induces the mobilization of fat reserves, which after metabolism in the liver can cause ketosis. The ketone bodies negatively influence the ability of migration and recruitment of neutrophils into the udder, phagocytosis and the ability of oxidation and destruction by the neutrophils. Others * Loss of milk is the result of the increase in intramammary pressure at the end of the dry period. The sphincter is open for the entry of pathogens. * The majority of antibiotic formulations administered through cannulae at drying do not cover the final phase of drying, especially in standard 60-day dry periods. Furthermore, the majority of products on the market have limited activity against Gram-negative bacteria. * Postpartum milking that is often difficult due to udder oedemas, thereby facilitating air intake during milking and the subsequent entry of pathogens into the cistern. Stress. Stress factors such as heat, metabolic stress, competition, transport, etc. induce the secretion of cortisol and cause immunosuppression. Stress is a vicious circle in post-partum. Cows eat less when stressed, which lengthens or increases the NEB, and immunosuppression is enhanced.

17 Startvac Library Colibacillar mastitis 4. Treatment Treatment should be focused towards the cow, not the bacteria. E. coli rapidly multiply in the udder reaching peak concentration in less than 12 hours (Erksine et al 1989). The recognition of clinical signs of colibacillary mastitis normally occurs after the maximum bacterial concentration in the udder is reached. This idea questions the appropriateness of treating colibacillary mastitis with antibiotics. In addition, there are many studies demonstrating the poor efficacy of treatment with antibiotics against gram-negative mastitis. Therefore, we will focus on symptomatic treatment: 1. IV Hypertonic saline serum. Cows must have free access to clean fresh water. 2. NSAIDs for controlling fever and inflammation. 3. Calcium, iron and vitamins A, D, and E to enhance neutrophil function. 4. Frequent milking and oxytocin. The pain and inflammation inhibits milk drop. Oxytocin helps better emptying of the udder, thus removing more bacteria. 5. Antibiotics active against Gram-negative bacteria by parenteral route (as a preventive measure against sepsis, not for curing the infection). 5. Prevention Given the low efficiency of any treatment against the hyperacute colibacillary mastitis, prevention is the best possible treatment. Knowing the periods of greatest risk and predisposing factors, prevention strategies, are mainly focused on 2 routes: 1.Minimize exposure of the teat tip to bacteria present in the environment: Maximize hygiene in areas where cows rest, especially drying yards, during pre- and postpartum since these are the periods of maximum risk of intramammary infection by coliforms. Clean dry cubicles or beds are key to prevent the proliferation of E. coli in resting areas. Inert materials such as sand or marble dust are more suitable when compared to materials such as straw, sawdust or husks because bacteria proliferate less. Milk clean dry teats. 2. Increase the animal's resistance to infection: Minimize stress of any kind. Rations and feeding strategies that minimize the NEB and its duration. The objective is to maximize intake of dry matter. Securing the necessary intake of Vitamin E and Se in the ration; they are important for the immune system and increasing the phagocytary activity of neutrophils. A state of deficiency of these elements increases the likelihood of suffering from mastitis, as well as the severity and duration of infection. Vaccination. Vaccination against colibacillary mastitis is a commonly implemented strategy on dairy farms in the United States (between 40-65% of the farms apply vaccination). The most widely used vaccines are based on the J5 strain of E. coli. This strain is a mutant that lacks the O-polysaccharide chain of the LPS, leaving the LPS antigen core exposed to the immune system. Unlike the O-polysaccharide chain, the composition and structure of the antigen core is highly conserved among the various Gramnegative bacteria, so vaccines with J5 induce opsonising anti-core antibodies with crossimmunity against different strains of E. coli and other Gram-negative bacteria. The efficacy of vaccination in protecting against acute colibacillary mastitis has been demonstrated in several field studies. In many references, it is clear that immunization with J5 does not prevent coliform intramammary

18 2infections, but does reduce the severity, the onset of clinical cases and economic nd losses from death or slaughter. According to economic studies conducted in the USA, a vaccine against this type of mastitis is economically profitable if more than 1% of lactations are affected by colibacillary mastitis. According to the literature, vaccination can be an important tool in preventing mastitis caused by Gram-negative bacteria on farms where there is such a problem. Considering that the post-partum period is the most critical and where there are most cases for the reasons already mentioned, the goal should be to strengthen immunity in that period by vaccinating animals in the drying period and revaccinating before calving. A booster dose Colibacillar mastitis during the first months of lactation may be appropriate to extend the duration of immunity. In hot and humid climates where the incidence can be high in the summer months, a booster dose to all the animals could also protect the herd. Bibliographic references Startvac Library 1. Bradley et al Adaptation of E. coli to the bovine mammary gland, J Clin Microbiol Mai 2001; 39(5):1845-9). 2. Passey S, Bradley A, Mellor H. Escherichia coli isolated from bovine mastitis invade mammary cells by a modified endocytic pathway. V et Microbiol. 27 Juillet 2008;130(1-2): Wilson DJ et al., Comparison of J5 Vaccinates and Controls for Incidence, Etiologic Agent, Clinical Severity, and Survival in the Herd Following Naturally Occurring Cases of Clinical Mastitis J.DairySci, Sept. 2007,90(9):4282-8) 4. Mallard BA, Burnside EB, Burton JH, Wilkie BN. Variation in serum immunoglobulins in Canadian Holstein-Friesians. J Dairy Sci 1983; 66: Burton JL, Chaiyotwittayakun A, Smith K, et al. Novel applications for coliform vaccine programs. Proceedings of the 41st Annual Meeting of the National Mastitis Council. Orlando (FL); p Preisler MT, Weber PSD, Tempelman RJ, et al. Glucocorticoid receptor down-regulation in neutrophils of periparturient cows. Am J Vet Res 2000; 61:14 9. Demetrio Herrera qlettscp@gmail.com Conclusions Colibacillary mastitis is an important pathological condition on many farms due to the economic impact it implies. Prevention is the best tool to control this problem. The management of the dry period and the peripartum phase is crucial. Cows housed in clean, dry and comfortable cubicles and yards will have reduced intramammary coliform infections. In addition, feeding strategies to minimize the NEB in the post-partum period and reducing stress on the cow will help facing hyperacute mastitis. Finally, it should be noted that a vaccination protocol in the drying period can help to prevent clinical cases of coliform on farms where there is a problem. 7. David J. Wilson, Ruben N. Gonzalez,Vaccination strategies for reducing clinical severity of coliform mastitis Vet Clin Food Anim 19 (2003) P, Ruegg. Evaluating the effectiveness of Mastitis Vaccines, Adrian Gonzalez Garrido. Manejo del posparto para el control de las enfermedades Metabólicas. Libro ponencias Congreso ANEMBE wilson DJ, Grohn YT, Bennett GJ, González RN, Schukken YH, Spatz J.Milk production change following clinical mastitis and reproductive performance compared among J5 vaccinated and control dairy cattle. J Dairy Sci. Oct. 2008; 91(10): Jeanne L. Burton,Ronald J. Erskine, Immunity and mastitis. Some new ideas for an old disease Vet Clin Food Anim 19 (2003) Erskine RJ, VanDyk EJ, Bartlett PC, Burton JL, Boyle MC. Effect of hyperimmunization with an Escherichia coli J5 bacterin in adult lactating dairy cows J Am Vet Med Assoc. Oct. 2007; 231(7): NAHMS Dairy 2007 Part III: Reference of Dairy Cattle Health and Management Practices in the United States, vs/ceah/ncahs/nahms/dairy/dairy07/dairy2007_partiii.pdf.

19 1st Startvac Library Immunity and Mastitis: Is it possible to vaccinate? Marcelo Chaffer DVM PhD Atlantic Veterinary College Prince Edward Island University Canada 1. Introduction Mastitis is an inflammation of the secretory tissues or milk ducts in the mammary gland in response to a bacterial infection. It affects the quantity and quality of milk production. The causal agents of bovine mastitis are microorganisms that live in the udder of the cow and its enviroment. They can be divided into three groups according to their epidemiology: 1) contagious: with bacteria such as Staphylococcus aureus, Streptococcus agalactiae, 2) those that are environmentally related: such as Streptococcus agalactiae and Gram-negative bacteria such as E. coli and 3) opportunistic: coagulase-negative Staphylococci. Mastitis control is based on various measures that can include: 1) Proper and hygienic milking routine; 2) Proper use and maintenance of milking equipment; 3) Appropriate dry period therapy, 4) Treatment of clinical cases during lactation; 5) Treatment of skin problems of the udder and teats; 6) Culling of cows with chronic mastitis; 7) Examination of cows that will enter the farm as replacements, 8) Recording of data and 9) Maintaining a clean environment. 2. Is it possible to vaccinate? Along with all the above-mentioned classic measures of control, we have added another measure: vaccination. Taking into account the difficulties we have when facing agents such as S. aureus or E. coli due to their poor response to antibiotic treatments, prevention through proper vaccination plus the above-mentioned measures would be of great importance. In the case of mastitis caused by Staphylococcus aureus, dairy cows are the reservoirs of the bacteria. Results for antibiotic therapy are poor when the bacteria are found in the deepest udder tissue (Ma et al., 2004). Authors such as Blowey et al (1995), conducting a literature review of treatments with Cloxacillin, showed cure rates of mastitis caused by S. aureus of 24% of clinical cases and 40% for sub-clinical cases. The highest rate of therapy was during drying (60%), and that is why the treatment of choice for this bacterium is during drying. The low cure rate could be attributed to the ability of bacteria to survive the treatment when it is found intracellularly in epithelial cells or macrophages (Hensen et al., 2000; Herbet et al 2000).

20 1 Immunity and Mastitis: is it possible to vaccinate? st With regard to E. coli, according to the study by Sandholm et al (1995), antibiotic therapy would have little effect on improving symptoms caused by the bacterium. That is because these symptoms are, more than anything, caused by the bacterium s endotoxin. Vaccination aims to improve and enhance the immune system against a specific antigen. In the case of vaccines against mastitis, what is sought is an adequate arrival of neutrophils to the place where the pathogenic agent is found and with the appropriate amount of immunoglobulins, opsonization and the subsequent phagocytosis occur. In addition, antibodies generated by vaccination, may also have an important role in neutralizing toxins, interfering with the adhesion mechanisms of bacteria and inducing the bacterial lysis. A review of the literature has shown benefits in the use of protective vaccines against S. aureus or E. coli. The effect of vaccination is seen in the next table: Protective vaccines benefits against S. aureus or E. coli. 1) Reduction in the severity and duration of symptoms of coliform mastitis 2) Decrease in the rate of infections 3) Decrease in antibiotic use and its possible occurrence as residues in milk 4) Decrease in somatic cell counts and increases in daily production of milk Nordaugh et al., (1994) used an inactivated vaccine of S. aureus, and showed its positive effect on the appearance of clinical cases in the vaccinated group of cows as opposed to 6% of cases in the non-vaccinated group. With regard to cases of subclinical mastitis caused by S. aureus, it was diagnosed in 8% of the vaccinated group and 14% of the unvaccinated group of cows. In Israel, in a field trial, (Leitner et al., 2003) a vaccine composed of fragments of S. aureus obtained by sonication was used. It showed statistically significant beneficial effects with respect to milk production and somatic cell count in the group of vaccinated cows. The important thing to note in a vaccine against S. aureus, is to vaccinate as early in the life of the cow as possible. This immunization should be performed in pre-partum heifers, thereby avoiding potential infection that would compromise the productive life of the animal. With regard to coliform mastitis, after performing a challenge with a virulent strain of E. coli in a group of cattle vaccinated with the E. coli J5 bacterin and a non-vaccinated group, Hogan et al. (1995) showed that duration of intramammary infection, as well the intensity of the symptoms were lower in the vaccinated group. Deluyker et al. (2005) found in field tests that, although vaccination against E. coli does not help in reducing the number of cases Startvac Library in the vaccinated group compared with the nonvaccinated, there were significant differences in the number of cases of systemic toxic mastitis in favour of the vaccinated group. In the case of S. aureus, various types of vaccines have been developed in the past with mixed results. These vaccines could be divided into the two major groups, 1) bacterin and 2) vaccines that include a component of the bacterium considered to be of antigenic importance. The first group, with bacterins, are vaccines prepared with all of the components of the bacterial cell and they may be dead or alive; so, mastitis tests were developed with this type of vaccine by Pankey (1985) or Leitner et al. (2003). The second group, are those vaccines that include elements of antigenic importance, these vaccines are developed from virulence factors such as: a) Protein A, a component of the cell wall of the bacterium that binds to Immunoglobulins. (Pankey et al., 1985, Carter and Kerr, 2003) b) Pseudocapsule, extracellular polysaccharide with antiphagocytic properties (Watson et al. 1992; Nordhaug et al., 1994) c) Capsular antigens, such as expolysaccharide: also called Slime Associated Antigenic Complex (Yosida et al. 1987, Calzolari et al. 1997; Giraudo et al., 1997). d) Alpha and Beta toxins (Herbelin et al., 1997) e) Fibronectin binding protein, surface molecule that acts as a factor for bacterial adherence (Shkreta et al., 2004). f) Clumping factor A, surface molecule that acts as a factor for bacterial adherence (Brouillete et al. 2002). Marcelo Chaffer DVM PhD Atlantic Veterinary College Prince Edward Island University Canada 3. Vaccination trial against mastitis conducted in Spain In a multicentre trial conducted on 6 dairy farms in Catalonia (Spain), 386 primiparous and multiparous dairy cows were divided in two groups. The first group consisted of 188 cows, and as control group was not vaccinated, while the second group of 198 cows was vaccinated. The vaccination schedule for this group consisted of a first dose of vaccine 45 days before the expected date of birth; the second dose was administered at ten days prior to delivery, and the third dose of vaccine was given at about 50 days postpartum.

21 Startvac Library Immunity and Mastitis: is it possible to vaccinate? The vaccine used contained antigens of the CP8 S. aureus strain, which is a high producer of the Slime Associated Antigenic Complex plus the E. coli J5 strain. (Laboratorios Hipra, Amer, Girona, Spain). Data collected were analyzed by logistic regression with an analysis of variance Somatic cell count, cure rate and additional pharmacological treatments during the assay The somatic cell count was measured during the field trial. It is the most accepted parameter for monitoring udder health and milk quality (Laevens, 1997; Pyorala, 2003; Schukken et al., 2003). The vaccinated group had a cell count of x 103 compared to x 103 in the control group. When compared in logarithmic form, these differences were found to be statistically significant (p = ). The cure rate for vaccinated multiparous cows was 53.33% compared to 20.45% for the unvaccinated animals. This difference was significant (p <0.05). In primiparous animals, although the cure rate was favourable in the vaccinated group, the difference was not significant. In the same trial, drug treatments were measured in both groups of cows, vaccinated and control. Twenty-four animals were treated for mastitis in the vaccinated group, 14 primiparous and 10 multiparous cows. Multiparous cows in this vaccinated group received 21 treatments, giving an average of 1.5, whereas primiparous animals received 13 treatments, and averaged 0.7. Moreover, in the control group, 40 animals received additional drug treatment, 28 of them were multiparous and 12 primiparous. The average for the non-vaccinated control group was 2.1 and 2.8 for multiparous and primiparous animals respectively. The statistical analysis of these results was that for the multiparous group this difference in necessary additional pharmacological treatments was significantly different (p = 0.003). As the number of treatments per cow was lower in the vaccinated group, the treatment time required is also less. These points are extremely interesting because they ultimately determine not only the reduced use of drugs, but mean that less milk is discarded due to the use of antibiotics. The experiment conducted in Catalonia as well as the literature provides us with data showing positive effects of vaccines against mastitis. Whilst it is an element to be considered and recommended in the fight against mastitis, it should not be forgotten that it must be combined with traditional measures to control mastitis on a farm operation.