Microbes and Infection 7 (2005) 560568 www.elsevier.

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Review

The pathogenesis and control of Staphylococcus aureusinduced mastitis: study models in the mouse
Eric Brouillette *, Franois Malouin
Centre dtude et de valorisation de la diversit microbienne (CEVDM), Dpartement de biologie, Facult des sciences, Universit de Sherbrooke, 2500 Boulevard Universit, Sherbrooke, Que., Canada, J1K 2R1 Available online 14 March 2005

Abstract The intramammary colonisation by Staphylococcus aureus provokes mastitis in the cow. Once established, the infection is difficult to eradicate with available therapies and may become chronic. The present article focuses on the use of the experimental mouse model of S. aureus-induced mastitis as a practical approach for the study of bovine mastitis. Results obtained regarding the pathogenesis of S. aureus and the development of new therapeutic approaches are discussed. 2005 Elsevier SAS. All rights reserved.
Keywords: Staphylococcus aureus; Mastitis; Intramammary infection; Cow; Bovine

1. Introduction Bovine mastitis is a costly disease for dairy producers [1]. This pathology is typically caused by microbial intramam- mary infection (IMI) that induces inflammation of the mam- mary gland (MG). During the course of the pathology, the production of milk is reduced and is of a lower quality. The milk contains less casein proteins and an increased amount of serum proteins and host cells. When acute inflammation takes place and persists, milk-secreting alveolar cells are dam- aged, and necrosis of tissue leads to a permanently reduced milk yield production. Staphylococcus aureus can provoke clinical mastitis but more frequently causes subclinical infections that tend to become chronic and difficult to eradicate by conventional anti- microbial therapies [2]. The frequent incapacity of both the immune response and antibiotics to prevent infection and destroy the pathogen in the intramammary environment

explains why S. aureus bovine mastitis constitutes a major challenge to dairy producers. More research needs to be per- formed in order to develop new prophylactic and therapeutic approaches that counteract S. aureus IMI pathogenesis. The costs associated with experimental IMI in the cow are prohibitive. Even with the use of smaller animals like goats, the cost is still a major obstacle when conducting experi- ments that require a minimal number of animals to get valid statistics. Consequently, since only a few hypotheses can be investigated in big animals, alternatives are needed. Here, we discuss the relevance of the mouse model of S. aureus mas- titis (MMSAM) as such an alternative. 2. The mouse model of S. aureus mastitis The MMSAM was first described in 1970 by Chandler [3]. Henceforth, until the middle of the 1980s, Chandler and Anderson further characterised this model and published numerous articles. In addition to staphylococci, different genera of microbial pathogens have been used to provoke mastitis in the mouse: Escherichia [4], Campylobacter [5], Can- dida [6], Corynebacterium [7], Mycoplasma [8], Pseudomonas [9] and Streptococcus [10]. Nevertheless, since the year 2000, a renewed interest in the MMSAM has been observed in the scientific community and, overall, S. aureus was employed in more than 50 studies that were

Abbreviations: CFU, colony-forming unit; FnBP, fibronectin-binding protein; IMI, intramammary infection; MG, mammary gland; MMSAM, mouse model of S. aureus mastitis; PMN, polymorphonuclear neutrophil; SCC, somatic cell count; SCV, small-colony variant; STM, signature-tagged muta- genesis. * Corresponding author. Tel.: +1 819 821 8000 ext. 2629; fax: +1 819 821 8049. E-mail address: eric.brouillette@sympatico.ca (E. Brouillette).

carried out
1286-4579/$ - see front matter 2005 Elsevier SAS. All rights reserved. doi:10.1016/j.micinf.2004.11.008

E. Brouillette, F. Malouin / Microbes and Infection 7 (2005) 560568

using the mouse model of microbial mastitis. Table 1 presents the object of the majority of these reports as well as the mouse lineages and the strains of bacteria used. 2.1. The basic MMSAM: methodological aspects Most of the time, mouse IMI with S. aureus is performed as follows. The mouse possesses five pairs of MGs, three pairs on the thorax and two on the abdomen, that are identified from head to tail by a letter and a number indicating their relative position. The MGs usually infected are the R4 (fourth on the right) and L4 (fourth on the left) abdominal glands, since they are the biggest and the easiest to observe and har- vest. For S. aureus infection, a level II biological hood is required to both work in a sterile environment and protect the experimenter. One to two hours before bacterial inoculation, the 415-day-old pups are removed from their mother at her first lactation. Lactating mice are anaesthetised, put on their back under a binocular, and the teats and the surrounding area is disinfected with 70% ethanol, and looking through the bin- ocular, the very near end of the teats is then cut with small scissors. No more than a millimetre of tissue must be removed in order to avoid possible contamination of the MG by environmental microbes. One teat is then held with fine forceps, and the duct orifice is located. A volume of 50100 l is slowly injected through the orifice with a syringe, preferably with a blunt needle not bigger than 30-gauge. The needle has to be inserted for no more than about three to four millimetres deep into the canal. This procedure is repeated for the second gland to be inoculated, if required. Afterwards, the mouse is placed into a filter-cage on its back to allow drying of the skin sur- face, thus reducing possible contamination. In most studies, infection has been allowed from 24 to 48 h before MG har- vesting. 2.2. Experimental variations of the MMSAM Several modifications of the MMSAM have been done. Different combinations of S. aureus strains and mouse lineages have been utilised (see Table 1), and the duration of infection allowed was from 90 min to 21 days. To inoculate MGs, regular bevelled needles have been used without remov- ing the near end of the teat. It was reported that reproducible infection can be achieved this way even if possible intrader- mic injection may occur [3]. To reproduce natural infection, we however, believe that inoculation should be carried out with a blunt needle to minimise alterations of deep tissue along the teat canal. In this way, it is much easier to be sure that injection is into the streak canal. Besides, CF-1 mice with multiple lactations were employed instead of primiparous ani- mals [11]. Investigators have also occasionally put the off- spring back to the mother after inoculation to permit suck- ling of MGs [12,13]. Suckling reduced the level of colonisation in terms of total colony-forming unit (CFU) recovered and led to a more variable infection between ani- mals. It should be noted that removing the end of the teats did

not prevent suckling any more than did the presence of bacteria in the milk of infected mice generally. 2.3. Quantifying the level of infection and tissue damage in mammary gland In the MMSAM, the level of infection is typically estimated by determining the number of bacterial CFU per gland or per gram of gland by plating serial dilutions of MG homo- genates on agar plates. In the cow, at the microscopic level, subclinical IMIs are detectable by the presence of phagocytic cells, mainly polymorphonuclear neutrophils (PMNs), and epithelial cells in milk samples. The quantity of these cells found is referred to as the somatic cell count (SCC), and it gives information about the intensity of inflammation. In the mouse, inflammation also occurs, since PMN infiltration is observed following bacterial inoculation (Fig. 1). However, contrary to the cow, inflammation is most of the time partly and indirectly assessed by evaluating mouse mammary tis- sue damage, which results from inflammation but also possi- bly from the direct action of bacterial toxins. Specific descriptive details regarding tissue alteration and damage induced during S. aureus IMI in the mouse can be found in articles published by Chandler and Anderson [1416]. In addition, Haraldsson and Jonsson have described a well-defined and standardised scale that can be use to depict the severity of mammary tissue lesions observed by micros- copy [17,18]. To evaluate inflammation, exclusively in terms of inflammatory cell infiltration, a method that allows count- ing of many thousand PMNs in tissue has been reported by our laboratory [19]. As seen, mastitis in the mouse can be characterised by CFU counts (bacterial infection), PMN infiltration (tissue inflam- mation) and histological changes (tissue damage). Each of these methods gives specific and relevant information, as evi- denced by the fact that a given S. aureus strain may be more virulent than another in terms of tissue damage caused by the types and/or the quantities of toxins produced, and this, with a similar bacterial burden in the gland. Likewise, evaluation of PMN infiltration gives information about the level of inflammation at a specific time, whereas tissue damage is the result, at least in part, of the cumulative effect caused by that inflammation.

3. Studies involving the use of the MMSAM 3.1. Fundamental observations concerning the MMSAM Independently of the level of the initial inoculum, maximal infection of the mammary tissue is usually achieved within the first 24 h, with a resulting bacterial burden of 108 1010 CFU/g of gland (when MG are not suckled). For the mastitis isolate S. aureus Newbould 305, this bacterial burden is reachable with an inoculum of less than 100 CFU per gland; mice can thus be considered very susceptible to IMI

Table 1 Studies supported by the mouse model of S. aureus mastitis Type of study/subject of the report Basic studies on S. aureus pathogenesis Bacterial adherence in suckling mice Comparison of chronic infections in mouse versus cow Effect of teat damage and suckling on infection provoked by inoculation of the tip surface Dissemination of bacteria from the mammary glands to other body sites Intramammary administration extracellular products and whole bacterial cells Effect of suckling on mastitis Endotoxin treatment 3 days before S. aureus inoculation Endotoxin treatment 6 h before S. aureus inoculation to induce chronic mastitis Experimental bacterial mastitis (first article published about the MMSAM) Experimental S. aureus mastitis: histopathological studies Experimental S. aureus mastitis: histopathological studies Internalization of S. aureus by mammary epithelial cells Morphologic changes in tissue during infection Pre-colonization with Corynebacterium bovis prior ot S. aureus mastitis Pre-colonization with S. epidermidis prior to S. aureus mastitis Virulence of S. aureus mutants Adherence of a fibronectin-binding proteins (FnBPs) mutant under suckling pressure Increased persistence of a hemB mutant in vivo during cephapirin treatment Intracellular location of a FnBPs mutant Role of alpha- and beta-toxin on the virulence of the pathogen Virulence of hemolysin, coagulase and protein A mutants Virulence of hemolysin, coagulase and protein A mutants Effect of antibodies and vaccination against S. aureus infection Administration of an anti-staphylococcal serum Intramammary immunization with a S. aureus attenuated mutant Intramammary immunization at different times in relation to pregnancy Cell-mediated immunity conferred by intramammary immunization (no challenge) Opsonization of bacteria with an anti-clumping factor A (ClfA) serum Opsonization of bacteria with an anti-fibronectin-binding protein A (FnBP-A) serum Vaccination with the collagen-binding protein (CnBP) and alpha-toxoid Vaccination with purified fibrinogen-binding proteins (FgBPs) Vaccination with FnBP-A Antibacterial treatments and others Adaptation of the MMSAM for therapeutic compound efficacy studies Amoxillin, cloxacillin and nafcillin pretreatments on the susceptibility of S. aureus to neutrophils Ampicillin, clindamycin, cloxacillin, lincomycin, novobiocin, penicillin-G, pirlimy- cin and rifampicin Cefoperazone and cloxacillin Cefoperazone, cetiofur, cloxacillin and pirlimycin Cloxacillin action depending of S. aureus location in tissue Cloxacillin and rifampicin efficacy during acute and chronic infections Cloxacillin kinetics and therapeutic action under pathological changes Cloxacillin therapy of chronic infections Cloxacillin therapy of chronic infections with S. aureus strains from different herds Injection of a milk-derived factor to reduce the level of infection Lactoferrin and penicillin G, alone or in combination Intramammary administration of lysostaphin Lysostaphin expression in mammary glands of transgenic mice Penicillin and polymixin B Rifampin and rifamycin SV Effect of T-2 toxin administration on S. aureus colonisation TNF-alpha and antibiotic (ciprofloxacin, pirlimycin, cloxacillin) treatments Vitamin A administration Mice lineage S. aureus strain Referenc e [29] [20] [55] [24] [56] [12] [57] [58] [3] [15] [14] [22] [16] [7] [59]

BSVS (cows) Compton white Compton white BSVS Compton white BSVS BSVS BSVS BSVS BSVS Compton white BSVS Compton white BSVS

M60 M60 M60 M60 BB, Mexi M60 Mexi Mexi S57 BB S57, Z2 Z2, AL BB M60, M12 M60

CD-1 CD-1 CD-1 MF1 BSVS BSVS

8325-4, Du5883 Newbould hemB mutant 8325-4, Du5883 M60, 8325-4, and derived mutants SA113 and derived mutants SA113 and derived mutants

[13] [34] [23] [28] [18] [17]

CBA Swiss Swiss Swiss CD-1 BSVS BSVS BSVS BSVS

clinical strain 8325-4, 8325-4 attenuated 8325-4, 8325-4 attenuated 8325-4, 8325-4 attenuated Newman SA113 SA113 SA113 SA113

[60] [43] [44] [45] [39] [38] [42] [41] [40]

CD-1 Compton white CF1 Compton white CF1 Compton white Compton white Compton white BSVS Compton white CD-1 CD-1 MF1 BLG-lys transgenic BSVS Compton white CBA/ca CF1 B6D2F1 X SWR

Newbould Oxford, M60 U6097, UC 6899, M-60 M60, M12 UC6097 M60 NCTC 6571 and field isolates M60 M60, M63 Clinical strains Newbould 305 Field strains M60 ? S57 M60 Clinical strains B83-1 (Newbould 305 derivative) Newbould 305

[25] [61] [11] [10] [62] [63] [64] [65] [26] [27] [66] [67] [36] [37] [9] [68] [4] [69] [70]

Fig. 1. S. aureus mastitis in the mouse. Electron microscopy examination of the mammary tissue after 24 h of infection reveals that bacteria are still dividing and are predominantly found within PMNs (A, B). Also, using acridine orange staining and fluoresence microscopy, a massive infiltration of PMNs (yellowish light-green spots) can be seen in tissue of infected mice (D) compared with PBS-inoculated animals (C). Pictures were taken by Gilles Grondin (U. de Sherbrooke).

by S. aureus. Actually, the pathogens virulence in this model may be primarily modulated by the presence of milk, a good growth medium for S. aureus, and the virtual absence of phagocytic cells at the time of inoculation. For these reasons, the pathogen can efficiently colonise the MG by staying, at least for a time, a step ahead of the innate immune response. Of interest, the bovine MG contains more resident phago- cytic cells than the mouse, and chronic subclinical infections are more likely to occur [20]. The intracellular component of infection is believed to play a significant role in the persistence of S. aureus during anti- biotic therapy for different types of infections, including bovine mastitis [21]. In the mouse mammary tissue, this pathogen was found within phagocytes and sometimes also in epithelial cells [22,23]. In fact, in our hands, the vast major- ity of the bacterial cells were observed and multiplied within phagocytic cells after 1224 h of infection (Fig. 1). Never- theless, the pathogen was also found to disseminate from the MG to other organs after such a period of time [24,25]. 3.2. Induction of chronic mastitis In the MMSAM, animals typically die at 4872 h postinfection. Even if dissemination of bacteria was shown to occur, the production of bacterial toxins is thought to be the lethal factor [24]. Under these conditions, the MMSAM can be considered an acute infection model. However, the cows that are the most refractory to antibiotic treatments are those

with chronic mastitis. If required, it is possible to transform the standard MMSAM into a more chronic IMI by injecting endotoxin in MGs several hours before bacterial inoculation to stimulate PMN infiltration. The immune cells recruited this way will not prevent infection but will phagocyte bacteria more rapidly and reduce colonisation compared to control mice (which develop acute mastitis), by a factor of more or less 100 in terms of CFU [26]. Cloxacillin administrated at 48 h of infection was ineffective to reduce bacterial burden in mice with such a chronic infection, and this finding was attrib- uted to the intracellular location of S. aureus [26,27]. 3.3. Virulence of S. aureus mutants for surface proteins or secreted toxins Using the MMSAM, the roles of several putative bacterial virulence factors were investigated over the years. Haralds- son and Jonsson studied the virulence of alphahemolysin, coagulase and protein A mutants produced by chemical mutagenesis in this model of infection [17,18]. Microscopic examination of infected tissues revealed a decreased viru- lence for the first two S. aureus mutants. Similar observa- tions were made by another group with alpha-(hla or hly) and beta-hemolysin (hlb) mutants created by site-directed mu- tagenesis [28]. The adherence of bacterial cells to the bovine MG tissue is believed to be an important step to achieve S. aureus coloni- sation [21]. Anderson (1978a) stated the absence of S. aureus

parental strains. This finding needs to be confirmed in the cow, but results from the MMSAM suggest an approach for investigation. Besides, using a mouse endocarditis model, other investigators found no difference in the colonisation of tissues by an S. aureus hemB mutant under antibiotic treat- ment with oxacillin, another beta-lactam antibiotic [35]. These diverging results may imply that the MMSAM provides spe- cific in vivo conditions that are not necessarily supplied by other mouse models of infection. 3.5. Antimicrobial effcacy studies At almost the same time that Chandler published the first article about the establishment of the basic MMSAM, the capacity of penicillin to overcome IMI in this model was also assessed [9]. Since then, many antibiotics were tested for their capacity to treat infection in this animal model, as shown in Table 1. The latest development regarding the MMSAM and its use for antimicrobial efficacy studies implicated a mol- ecule that specifically acts toward S. aureus, the lysostaphin. Several years ago, Bramley and Foster [36] showed the capac- ity of lysostaphin, administrated intramammarly, to reduce or treat S. aureus IMI in the mouse. Afterwards, Kerr et al. [37] created transgenic mice expressing a modified biologi- cally active form of lysostaphin in the mouse MG and showed that protection was conferred toward S. aureus IMI. Recently, the University of Vermont and the US Department of Agriculture jointly announced the birth of transgenic cows express- ing lysostaphin in MGs. Results concerning the capacity of those animals to fight S. aureus IMI are forthcoming. Here again, the MMSAM was instrumental in establishing the proof of concept and demonstrating the value of lysostaphin trans- gene expression in MGs. The small size of the mouse permits extensive screening of promising antimicrobial compounds, given that numerous animals can be infected. Moreover, relatively small quanti- ties of compounds are necessary to carry out efficacy studies, and this is a major advantage over bigger animals, as limited supplies are available during the initial screening stages. In order to find the most appropriate experimental conditions for screening, the MMSAM was extensively characterised in our laboratory by using cephapirin, an antibiotic employed to treat infectious mastitis in the field [25]. 3.6. Opsonisation of bacteria and vaccination Sera of animals immunised against the binding region of FnBP-A [38] and clumping factor A (ClfA) [39], which are bacterial surface proteins that bind, respectively, to fibronec- tin and fibrinogen, were used to opsonise S. aureus before intramammary inoculation of mice. Antibodies contained in serum and directed toward these adhesins reduced the quan- tity of CFU recovered from MG. Of greater interest, Mamo at al. directly immunised mice with purified S. aureus sur- face adhesins and subsequently challenged these animals. More precisely, the FnBP-A protein [40], purified fibrinogen-

Fig. 2. The role of FnBPs in the virulence of S. aureus during mouse mastitis, with and without suckling pressure.

adherence during colonisation in the MMSAM [29]. However, in vitro, a lower level of adherence on endothelial cells was observed under shear stress for a double mutant lacking both fibronectin-binding protein (FnBP) genes [30]. Similarly, in the MMSAM, we found a slight but statistically sig- nificant difference in favour of the wild-type strain, com- pared to the FnBP deficient mutant strain, in its ability to colonise mouse MGs under suckling, and this, by using inocula composed of both strains mixed together (Fig. 2) [13]. This finding can possibly translate to cattle, since a similar shear stress arises under lactation or milking. Furthermore, it has been shown that FnBPs play a major role in the capacity of S. aureus to invade cultured bovine mammary epithelial cells in vitro [31] and could thus contribute to the establish- ment of a chronic disease. On the other hand, we have showed that a FnBP mutant was still able to invade mouse MG epi- thelial cells in the MMSAM, therefore, indicating that the presence of these adhesion proteins is not an absolute require- ment for in vivo invasion of non-phagocytic cells [23]. 3.4. Small-colony variants The small-colony variant (SCV) phenotype of S. aureus has been suspected to play a role on the persistence of S. aureus in human infections during antibiotic therapies [32]. SCVs were also isolated from bovine mastitis [33], but no study assessed the importance of this phenotype in the diffi- culty to overcome bovine mastitis. In the MMSAM, we have assessed the capacity of cephapirin to eliminate a genetically defined S. aureus hemB mutant, a representative of SCVs, and its parental isogenic counterpart, during infection; the hemB mutant was clearly more difficult to eradicate from the MGs [34]. This was observed despite similar in vitro suscep- tibility to cephapirin for both the S. aureus hemB mutant and

binding proteins (FgBPs) [41] and a combination of collagenbinding protein (CnBP) and alpha-toxoid [42] were utilised as antigens. A reduced IMI was observed for vaccinated mice compared to non-vaccinated controls for all the antigens tested. In addition to vaccination with purified proteins, a group from Argentina demonstrated that attenuated S. aureus cells used as vaccine helps animals to fight infection in the MMSAM, when intramammary immunisation was performed but not when the strain was administered intraperito- neally [43]. This prophylactic effect against IMI was observed when immunisations were carried out during late pregnancy or early lactation but not before pregnancy [44]. Although the results obtained from the MMSAM may not necessarily be readily applicable to bovines, they suggest, however, that this type of immunisation is promising and that the route and time of immunisation with attenuated S. aureus could be of crucial importance. This group also showed the involvement of both CD4+ and CD8+ cell-mediated immunity in the MGs towards S. aureus after local immunisation with the attenu- ated pathogen [45].

accordingly, the mouse model of S. aureus-induced mastitis constitutes a unique alternative, which can be employed in any standard laboratory equipped with animal care facilities. As evidenced by using the MMSAM, the discovery that S. aureus can be internalised within mammary epithelial cells [22] suggested new directions for research. At the time, the authors suggested a phagocytic capacity for mammary epi- thelial alveolar cells. It was later shown in vitro that the patho- gen itself possesses the capacity to invade non-phagocytic cells and that this property is for the most part mediated by the presence of FnBPs at the bacterial surface [31]. As ini- tially predicted in the MMSAM, the capacity of S. aureus to invade mammary epithelial cells in the cow was also clearly demonstrated 20 years later! [50]. This example illustrates that despite possible differences between IMIs in the two spe- cies, results obtained in the MMSAM may bring to light important mechanisms that could also take place during bovine mastitis caused by S. aureus. 5. New technologies to study S. aureus pathogenesis and the MMSAM Many new technologies and genomic-based approaches have recently been developed to identify and study genes that are essential or expressed during an infection [51]. For example, it is now possible to follow, in real-time, bacterial growth and location in animals by using bioluminescent bac- teria and a hypersensitive camera [52]. As of today, this tech- nology is only applicable to small laboratory animals, as they need to be placed in a relatively small and perfectly sealed black box, hence the potential importance of the MMSAM to study mastitis pathology. Moreover, the relative thickness of the mouse body, compared to that of the cow, allows detec- tion of photons emitted by the pathogen located in deep mam- mary tissue (and even in kidneys). Another technology that can benefit from the MMSAM is signature-tagged mutagenesis (STM), which permits identification of in vivo-induced genes required for survival and virulence [53]. Mutants are differentially tagged, pooled and inoculated in animals. After infection, the bacteria are recov- ered and identified by their tag, thus allowing assessment of the in vivo mutants viability by comparing the original and the recovered bacterial pools. Importantly, the MMSAM per- mits the recovery of bacteria from the whole MG. This pro- vides the opportunity to detect any mutant that can be disad- vantaged in milk, MG tissue, within phagocytic cells, etc. It is important to emphasise that from all the in vivo-attenuated mutants identified thus far in a variety of mouse models of infection, only a few mutants were the same [54], indicating that specific types of infection often involve the contribution of specific bacterial genes. The DNA chip technology is also a method that can help direct future research efforts on cow mastitis. In such a method, RNA first needs to be extracted from bacteria and converted to cDNA. Since the bacterial RNA half-life is very

4. Is the MMSAM an appropriate model for the study of bovine mastitis? Mice can certainly not entirely replace cows for all research purposes. Differences most likely exist between mouse and bovine MG, and for several investigators, data obtained in the mouse must be analysed with caution. Extrapolation, from the mouse to the cow, of fine biological mechanisms observed in the host MG may be risky. However, as demonstrated by many authors, the observed consequences of bacterial inocu- lation in mouse MG are similar to those found in the cow in terms of PMN infiltration, tissue damage and S. aureus-host cell interactions. From the bacterial point of view, experimental conditions offered by the MMSAM offer the opportunity to study the bacterium itself in such an environment. It is known that spe- cific S. aureus genes can be differentially expressed in vivo or in biological fluids such as milk and serum compared to traditional laboratory media [46,47]. Moreover, when grown in milk whey, S. aureus was found to be more adherent to cultured bovine mammary epithelial cells [48]. Interestingly, the importance of the growth medium was confirmed in the MMSAM by Mamo et al. [49], who showed an increased virulence for S. aureus grown in milk whey. Based on previ- ous results, it is obvious that the growth environment and the genes expressed in vivo strongly influence S. aureus patho- genesis, and consequently, this knowledge is of crucial impor- tance for the development of new therapeutic treatments. The MMSAM provides a representative growth environment com- posed of milk and serum proteins, and it allows interaction of the pathogen with host mammary cells and with immune com- ponents present or recruited during the infection. Such a com- plex environment is impossible to recreate in vitro, and

short and experimental samples need to be processed very quickly (in a few minutes), this approach may gain from the MMSAM, in which MGs can be rapidly harvested and homogenised for isolation of bacteria and purification of RNA. Thereafter, specific hybridation of cDNAs, on microscopic spots of DNA representing selected or all bacterial genes can be detected, and the genes expressed during mastitis identi- fied. We are currently developing such an approach, combin- ing the MMSAM and DNA chip technology, to obtain a genomic-wide view of S. aureus genes expressed in vivo. We also believed that such valuable information will be useful for tackling other types of chronic infections caused by S. aureus and their SCVs in animals or humans [32]. Other methods for the assessment of important or essential in vivo-expressed genes exist and will be developed. As discussed above, the MMSAM offers the opportunity to use techniques that would benefit from the use of small-sized ani- mals and enables rapid recovery of all bacteria from MGs. It is possible to recover bacteria from milk of infected cows, although, during experimental and natural infections, bacte- ria can also be found in tissues and within cells. 6. Conclusion The MMSAM is a convenient experimental set-up for the study of acute and chronic S. aureus-induced mastitis. Despite differences between the cow and the mouse MGs, the mouse still represents a valuable model, as evidenced in this review, especially for the study of the pathogen itself in the context of an IMI. The MMSAM already suggested new research leads and possible prevention or therapeutic approaches for cow mastitis. The MMSAM could, in addition, permit exploi- tation of available new technologies developed to more pre- cisely identify bacterial genes or virulence factors expressed in vivo to further enable the rational design of new drugs or vaccines. Acknowledgements Grant no. MOP-57701 from the Canadian Institutes of Health Research to F.M. provided some support for the prepa- ration of this review and some of the studies discussed herein. References
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