Theo A Niewold
email: theo.niewold@biw.kuleuven.

Jan Van Der Meulen

Hindrik H D Kerstens

Mari A Smits

Marcel M Hulst

Transcriptomics of enterotoxigenic E. coli infection. Individual variation in intestinal gene expression correlates with intestinal function

Keywords: enterotoxigenic E. coli, host-pathogen interaction, gene expression, micro array, PAP cytochrome P450 3A29, ETEC: enterotoxigenic E. coli, MMP1: matrix metalloproteinase-1, PAP: pancreatitis associated protein, STAT3: signal

Acute secretory diarrhea is a major cause of morbidity and mortality in young animals and humans. Deaths result from excessive fluid and electrolyte losses. The disease is caused by non-invasive bacteria such as Vibrio cholerae and E. coli which produce enterotoxins, however, much less is known about the role of individual host responses.

Here we report the response of intact porcine small intestinal mucosa to infection with enterotoxigenic E. coli (ETEC). Jejunal segments in four piglets were infused with or without ETEC, and perfused for eight hours, and net absorption measured. Micro array analysis at 8h post-infection showed significant differential regulation of on average fifteen transcripts in mucosa, with considerable individual variation. Differential net absorption varied between animals, and correlated negatively with the number of up regulated genes, and with one individual gene (THO complex 4). This shows that quantitative differences in gene regulation can be functionally linked to the physiological response in these four animals.

Introduction

Enterotoxigenic Escherichia coli (ETEC) is an important cause of secretory diarrhoea in man and animals. Upon colonization ETEC can produce several toxins, the most important of which is heat labile toxin (LT). LT is very similar to cholera toxin (CT).

ETEC strains are responsible for morbidity and mortality in neonates and infants in man.

Although oral rehydration therapy has reduced mortality significantly during the past decades, agents which could directly inhibit the intestinal secretory machinery would be a welcome addition [START_REF] Farthing | Antisecretory drugs for diarrheal disease[END_REF]. However, our understanding of the exact mechanisms involved is still relatively poor. A variety of mechanisms have been implied in the pathogenesis of secretory diarrhea. Enterotoxins play an important role, but also the enteric nervous system, and inflammatory cells such as polymorphonucleocytes are involved. Furthermore, intestinal cells respond to the infection by switching on innate defense mechanisms [START_REF] Flach | Broad up-regulation of innate defense factors during acute cholera[END_REF], which will eventually determine the outcome of the disease.

A c c e p t e d M a n u s c r i p t 3 Individual variation exists in susceptibility to the disease. This is, among others associated with the presence of adhesion factors for the bacteria and receptors for bacterial toxins [START_REF] Harris | Blood group, immunity, and risk of infection with Vibrio cholerae in an area of endemicity[END_REF]. Furthermore, individual variation can be caused by environmental, genetic and epigenetic factors determining the immunological response [START_REF] Radich | Individual-specific variation of gene expression in peripheral blood leukocytes[END_REF][START_REF] Shai | Microarray tools for deciphering complex diseases[END_REF].

ETEC strains cause a disease in piglets very similar to that in man. In a previous study, we have described the preliminary results of a micro array analysis of an ETEC infection in a pig model [START_REF] Niewold | Development of a porcine small intestinal cDNA microarray: characterization, and functional analysis of the response to enterotoxic E. coli[END_REF]. Here we give a more detailed analysis of the gene expression, with particular attention to individual variation. Furthermore, we relate ETEC associated individual gene expression differences with intestinal function.

Material and Methods

The material used derived from a previous study in which we have described some preliminary results of an comparative micro array analysis of an ETEC infection in a pig model [START_REF] Niewold | Development of a porcine small intestinal cDNA microarray: characterization, and functional analysis of the response to enterotoxic E. coli[END_REF]. We compared mucosal cDNA from normal uninfected with ETEC-infected perfused small intestinal segments of four F4 receptor positive pigs (6-7week-old) under anesthesia [START_REF] Niewold | Development of a porcine small intestinal cDNA microarray: characterization, and functional analysis of the response to enterotoxic E. coli[END_REF]. Briefly, of a pair of segments located around 25% of the length of the small intestine (anterior jejunum), before perfusion, one was mock-infected with PBS only, the other was infected with 5 ml of 10 9 CFU/ml PBS of ETEC (CVI-1000; typed as E. coli O149:K91, F4 (K88ac), LT, STb). Segments were perfused for 8 h with in total 64 ml of perfusion fluid. After the experiment, the surface area of each segment was measured. Net absorption was calculated as the difference between inflow and outflow in l/cm 2 intestinal wall, and the difference with the absorption of the control segment given as differential absorption. Mucosal scrapings were taken from the same segments, and frozen in liquid nitrogen, and stored at -70 o C for later transcript profiling. The animal study was approved by the local Animal Ethics Commission in accordance with the Dutch Law on Animal Experimentation.

Microarray analysis

A c c e p t e d M a n u s c r i p t 4 Microarray analysis was performed as described before [START_REF] Niewold | Development of a porcine small intestinal cDNA microarray: characterization, and functional analysis of the response to enterotoxic E. coli[END_REF], using slides spotted with the same collection of cDNA fragments (3486 clones in quadruplicate from a home made porcine jejunal EST library. For a complete description of the ESTs spotted on the Porcine intestinal cDNA array, see supplementary material to [START_REF] Hulst | Early transcriptional response in the jejunum of germ-free piglets after oral infection with virulent rotavirus[END_REF]. Hybridising probes were selected for further characterization when their mean value of M (n ≥ 5 or ≤ 8) was > 1.58 or < -1.58 (corresponding with a ratio greater than three-fold), and with a q-value of < 2%. Unknown probes were sequenced and compared with the NCBI mRNA reference sequence database (refseq_rna) and/or the non redundant (nr) database using the blastn option. Validation of differential expression of selected genes was performed using Northern blot analysis (NB).

Northern blot analysis.

In addition to PAP and IFABP already analyzed in our earlier publication [START_REF] Niewold | Development of a porcine small intestinal cDNA microarray: characterization, and functional analysis of the response to enterotoxic E. coli[END_REF], the expression of a further 3 genes was analyzed using the same technique.

Plasmid DNA was isolated from the EST library clones homologue to human mRNA coding for Fyn-related kinase (FRK, gi:31657133), poly (ADP-ribose) polymerase family, member 15 (PARP 15, gi:22749258), and signal transducer and activator of transcription 3 (STAT3, gi:47080105). Their inserts were sequenced to verify their authenticity. Blots were hybridized and scanned as described before [START_REF] Niewold | Development of a porcine small intestinal cDNA microarray: characterization, and functional analysis of the response to enterotoxic E. coli[END_REF]. The significance of the relationship between the values of the microarray and NB was calculated by linear regression using GraphPad Prism Version 5.00 software.

Gene expression vs differential absorption.

Differential net (negative) absorption in l/cm 2 was plotted against the differential expression of separate genes, the number of genes regulated, and the number of genes up and down regulated. The significance of the relationship was calculated by linear regression using GraphPad Prism Version 5.00 software.

Results

A c c e p t e d M a n u s c r i p t 5 Microarray analysis

The results of microarray comparisons of gene expression in segments perfused with ETEC for 8h vs. control are shown in Table 1. Animals showed variation in the number of genes significantly regulated ranging from 4 in animal 5 to 24 in animal 8. Also, the magnitude of gene expression differences varied between animals, being smallest in animal 5, and largest in animal 8. Only MMP1 and STAT3 were found to be similarly (up) regulated in all four animals. Animal 5 appears to be different from the other 3 animals. In the latter, common regulation was seen for PAP, MMP1, STAT3, GCNT3, GCLM, RIF1, THOC4, and PARP13. Other genes were found to be expressed in one or two animals only.

Northern Blot analysis

Linear regression analysis of the values of the microarray and NB for PAP, IFABP [START_REF] Niewold | Development of a porcine small intestinal cDNA microarray: characterization, and functional analysis of the response to enterotoxic E. coli[END_REF], PARP, FRK and STAT3 showed a significant relationship ( p < 0.0031, r 2 0.39). Only animal 8 showed striking differences between the values obtained in both techniques. Without animal 8, a highly significant correlation was found (p < 0.0001, r 2 0.91).

Gene expression vs differential net absorption.

The relationship between the level of gene expression of all individual genes, the number of regulated genes, and differential net absorption of individual animals was determined by linear regression. No significant correlation was found for separate genes except for THOC4 (p < 0.03, r 2 0.95). A near significant correlation was found for the total number of genes regulated (p > 0.07, r 2 0.85), and a significant relationship with the number of up regulated genes (p < 0.03, r 2 0.95) (Fig. 1). [START_REF] Shai | Microarray tools for deciphering complex diseases[END_REF], including intestinal disease like secretory diarrhea. The intestines are a complex and dynamic ecosystem [START_REF] Xu | Honor thy symbionts[END_REF], with epithelial cells influenced by intestinal content (food and microflora) in terms of differentiation and functionality. Furthermore, inborn (epi-) genetic factors and the immune system (and the other components of the intestinal mucosa) are essential in the defense against pathogens [START_REF] Diekgraefe | Analysis of mucosal gene expression in inflammatory bowel disease by parallel oligonucleotide arrays[END_REF][START_REF] Hooper | Commensal host-bacterial relationships in the gut[END_REF]. Individuals do vary in the reaction to pathogens, and thus vary in the physiological outcome. Individual variation is on the one hand cumbersome because it makes interpretation statistically difficult, on the other hand, if results obtained can be linked to functional read out, it can yield valuable information [START_REF] Shai | Microarray tools for deciphering complex diseases[END_REF][START_REF] Oleksiak | Natural variation in cardiac metabolism and gene expression in Fundulus heteroclitus[END_REF]. Furthermore, genes commonly active in all or most individuals, are most likely to be essential ones.

Discussion

Variation between animals could possibly be caused by loss of epithelial cells, but the control for epithelial content I-FABP [START_REF] Niewold | Development of a porcine small intestinal cDNA microarray: characterization, and functional analysis of the response to enterotoxic E. coli[END_REF][START_REF] Niewold | The early transcriptional response of pig small intestinal mucosa to invasion by Salmonella enterica serovar Typhimurium DT104 analyzed by cDNA microarray[END_REF] did not show differential expression.

In the present experiment, three out of four animals (nr. 6, 7, and 8) reacted in a similar way, whereas one was clearly different in response to a small intestinal ETEC infection.

Animal 5 had only 4 regulated genes, whereas all others had more than 10. Furthermore, the same animal (5) was aberrant (very low) in net fluid loss. On the other extreme is animal 8, showing 24 differentially regulated genes with the largest expression differences and the highest net fluid loss. Although the number of animals is limited for statistical analysis, it is interesting to observe that differential net absorption as the parameter for intestinal function was correlated with the number of differentially expressed genes, but most significantly with the number of up regulated genes. Apart from animal 5, animals were very similar in the number of up regulated genes (9, 10 and 11), and had 80% of those in common. As described before [START_REF] Niewold | Development of a porcine small intestinal cDNA microarray: characterization, and functional analysis of the response to enterotoxic E. coli[END_REF], the most prominent among those is PAP. PAP has been described as an antibacterial protein, and specific for gram positive bacteria in a mouse monoassociated system [START_REF] Cash | Symbiotic bacteria direct expression of an intestinal bactericidal lectin[END_REF]. Such a system is of course very different from the one used here with normal intestinal microbiota, and in contrast to [START_REF] Cash | Symbiotic bacteria direct expression of an intestinal bactericidal lectin[END_REF] we find no PAP expression A c c e p t e d M a n u s c r i p t 7 previous to infection, except in animal 5. We speculate that pre-ETEC PAP expression in the latter could reflect a pre-existing infection with another agent, since we have never been able to demonstrate PAP expression in the jejunum of normal, non-infected pigs (data not shown). In pigs PAP is induced by gram positive organisms such as Lactobacillus plantarum [START_REF] Gross | Mannose-specific interaction of Lactobacillus plantarum with porcine jejunal epithelium[END_REF] as well as by the gram negative ETEC (this paper), and by Salmonella [START_REF] Niewold | The early transcriptional response of pig small intestinal mucosa to invasion by Salmonella enterica serovar Typhimurium DT104 analyzed by cDNA microarray[END_REF]. This could suggest an antibacterial spectrum not limited to gram positives as suggested by Cash et al., also because the preexisting PAP in animal 5 appears to attenuate the diarrhea.

The largest variation in gene expression was found in the down regulated genes. Only 3 constructs in 3 out of 4 animals were in common, PARP a transcription repressor, and two cytochrome P450 (CYP) species. Mucosal up regulation of CYP3A29 can reflect influx of polymorphonucleocytes (PMN) rather than in situ up regulation in Salmonella infection [START_REF] Veldhuizen | Expression of beta-defensins pBD-1 and pBD-2 along the small intestinal tract of the pig: lack of upregulation in vivo upon Salmonella typhimurium infection[END_REF], Niewold et al., 2007). Apparent down regulation of CYP could suggest depletion of PMN, consistent with the described transmigration of PMN to the intestinal lumen upon luminal infections [START_REF] Hofman | Increased Escherichia coli phagocytosis in neutrophils that have transmigrated across a cultured intestinal epithelium[END_REF], causing loss of epithelial integrity and enhanced electrogenic chloride secretion [START_REF] Parkos | Cell adhesion and migration. I. Neutrophil adhesive interactions with intestinal epithelium[END_REF]. The absence of a relationship between fluid excretion and loss of PMN as measured by CYP3A29 here, suggests that fluid loss is primarily caused by LT.

Common regulation was seen ( in animals 6, 7, 8) for PAP, MMP1, STAT3, GCNT3, GCLM, RIF1, THOC4, and PARP13. This suggests a major role for these genes in ETEC infection. PAP, MMP1, STAT3, and THOC4 are also present in Salmonella infection (Niewold et al., 2007, and see above), and are clearly involved in the innate defense. The same can be assumed for the other genes, although it is as yet unknown in what way, with the possible exception of GCNT3. This gene is abundantly expressed by goblet cells and/or differentiating immature enterocytes, and involved in forming and maintenance of the epithelial mucus layer. GCNT3 up regulation was also found in rotavirus infection [START_REF] Hulst | Early transcriptional response in the jejunum of germ-free piglets after oral infection with virulent rotavirus[END_REF].

We conclude that the mucosal gene expression response to ETEC varies considerably between animals. Animals may differ genetically or in immunological background.

Quantitative differences in gene regulation can be functionally linked to the physiological Normalization for cDNA microarray data: a robust composite method addressing single and multiple slide systematic variation. Nucleic Acids Res 30, e15.

Legends

Table 1. Differential expression (FC, fold change) between ETEC infected and mockinfected SISP loops for segment pairs collected from pig 5, 6, 7 and 8. Upper panel, mRNAs expressed higher in ETEC infected loops (ETEC/Mock). Lower panel, mRNAs expressed lower in ETEC infected loops (Mock/ETEC). Significant differential expression (FC >3.0) are in bold. The accession (acc.) number of the mRNA sequence that scored the highest degree of homology (lowest E-value) is listed. The number (n) of additional clones aligning to an identical mRNA species is given behind the ID of the clone that scored the lowest E-value. The q-value is SAM's [START_REF] Tusher | Significance analysis of microarrays applied to the ionizing radiation response[END_REF] median false discovery rate (%).

  underlie almost all diseases

  .H., Dudoit, S., Luu, P., Lin, D.M., Peng, J., Ngai, J., Speed, T.P., 2002.

Figure 1 .

 1 Figure 1. Relationship between differential net fluid absorption and the number of regulated genes (total nr: open symbols), and the number of up regulated genes (closed symbols).

Acknowledgements

This work was funded by the Animal Sciences Group of Wageningen University and Research. The authors like to thank Arie Hoogendoorn for his assistance in the SISPtechnique, Pim Kuurman is thanked for the statistical analyses of microarray data.

A c c e p t e d M a n u s c r i p t 8 response in these four animals. Furthermore, it is clear that the common expression found for a limited number of genes (such as PAP, MMP-1, and STAT3) reflects their importance in the innate defense against enteral infections. It suggests them to be promising targets for intervention. Finally, these genes could also be very useful as parameters for the severity of intestinal diseases, especially PAP is interesting in that respect since it is known that the protein can be detected in serum [START_REF] Gironella | Anti-inflammatory effects of pancreatitis associated protein in inflammatory bowel disease[END_REF].

Conflict of interest

The authors declare no conflict of interest.


Tormod Mørk
email: tormod.mork@vetinst.no

B Kvitle

T Mathisen

H J Jørgensen

Bacteriological and molecular investigations of Staphylococcus aureus in dairy goats

Keywords: Staphylococcus aureus, dairy goats, epidemiology, typing, enterotoxin genes

In order to investigate reservoirs of Staphylococcus aureus in dairy goats, samples for bacteriological analyses were collected from 7 herds. S. aureus was detected in 353 (6.2%) of 5671 milk samples, 53 (9.9%) of 535 teat skin swabs, 392 (68.9%) of 569 nasal swabs and in 180 (31.6%) of 569 vaginal swabs. Vaginal swabs were more often S. aureus-positive after kidding (44.9%) than before drying off (19.1%), while nasal swabs were more often positive before drying off (75.6%) than after kidding (62.0%). Retrieved S. aureus isolates were compared by pulsed-field gel electrophoresis (PFGE), and selected isolates were tested for enterotoxin genes (se) by PCR. By PFGE, 505 S. aureus isolates were divided into 33 pulsotypes (PTs). The 5 most prevalent PTs included 73.3% of the isolates and were found in 3─5 herds. Pairs of S. aureus isolates from persistent intramammary infections (IMI), repeated vaginal swabs, and from milk and teat skin from the same animal were usually identical. Paired isolates from other body sites of the same animal, including from bilateral IMI, were identical in less than 50% of the situations. The majority (71.9%) of analysed S. aureus isolates were se-positive. The genes sec, sell and tst were detected almost exclusively, but no correlation was observed between persistence of IMI and the enterotoxin gene profile of the causal S. aureus strains. The frequent presence of S. aureus on the mucous membranes may contribute to dispersal of the bacteria among dairy goats, hampering effective transmission control in dairy goat herds.

A c c e p t e d M a n u s c r i p t

Introduction

Staphylococcus aureus is an important udder pathogen in small ruminants [START_REF] Bergonier | Mastitis of dairy small ruminants[END_REF]White, 2007), and infected udders represent a source of bacterial spread and contamination of raw milk and raw milk products [START_REF] Headrick | The epidemiology of raw milk-associated foodborne disease outbreaks reported in the United States, 1973 through 1992[END_REF]Jørgensen et al., 2005b;Zottola and Smith, 1993).

The infected mammary gland is often considered to be the primary reservoir of S. aureus in ruminants, but S. aureus may also be isolated from skin, especially from udder and teat skin, and from the mucous membranes of the nose and vagina of both cattle [START_REF] Davidson | The epidemiology of staphylococcal mastitis[END_REF][START_REF] Haveri | Molecular types and genetic profiles of Staphylococcus aureus strains isolated from bovine intramammary infections and extramammary sites[END_REF]Jørgensen et al., 2005b;[START_REF] Roberson | Ecology of Staphylococcus aureus isolated from various sites on dairy farms[END_REF] and small ruminants [START_REF] Bergonier | Mastitis of dairy small ruminants[END_REF]Valle et al., 1991;Vautor et al., 2005;White, 2007). However, the importance of extramammary reservoirs of S. aureus remains to be elucidated, especially for small ruminants.

The virulence of S. aureus is believed, in part, to be conferred by exotoxins, including the staphylococcal enterotoxins (SE) and SE-like proteins (SEl) [START_REF] Foster | Immune evasion by staphylococci[END_REF]. Both the SEs and SEls are superantigens (SAg) and the SEs cause emesis in primates when ingested [START_REF] Dinges | Exotoxins of Staphylococcus aureus[END_REF]. The exact role of SAgs in staphylococcal infections remains unclear, but they may contribute to the pathogenesis of S. aureus intramammary infections (IMI) in ruminants by inducing aberrant activation of T cell populations which suppresses the immune response [START_REF] Ferens | Activation of bovine lymphocyte subpopulations by staphylococcal enterotoxin C[END_REF][START_REF] Park | Unique features of bovine lymphocytes exposed to a staphylococcal enterotoxin[END_REF].

The main goal of this study was to identify reservoirs of S. aureus in dairy goats, and to compare isolates from milk, teat skin, and from the mucous membranes of the nose and vagina by pulsed-field gel electrophoresis (PFGE). The second goal was to investigate the prevalence of enterotoxin determinants among retrieved S. aureus isolates, and to determine whether or not there are differences in the SE-gene profiles of S. aureus isolates that cause persistent vs. those that cause temporary IMI in dairy goats.

A c c e p t e d M a n u s c r i p t 4

Material and Methods

Herds

Seven commercial dairy goat herds (designated A─G) from 5 different counties in Norway were included in the study. They were selected following an advertisement asking for voluntary participants. The flock sizes varied from 35 to 280 milking goats. All the herds used machine milking, and the animals were housed during the winter, and grazed on mountain pastures during the summer. Small ruminant lentiviruses had been eradicated in herd A, C, D, E and F (http://leine.no/htg/sanering/). The newborn kids were, in all 7 herds, separated from their mothers at birth or within a few hours of birth.

Samples

Samples were collected between August 2005 and September 2006. Each farm was visited 4 times in order to follow one lactation; before drying off, within 4 weeks of kidding, at the onset of the outdoor season, and again at drying off.

Milk samples from both udder halves from all lactating goats were collected at each farm visit, with the exception of herd A where all animals were sampled only at the first visit. From this herd, 100 lactating goats were randomly selected for sampling at the next 3 visits.

Body site swab samples from teat skin and the mucous membranes of the nose and vagina were collected from a random subset of the lactating goats in farms A-D within 4 weeks of kidding and at the time of drying off in 2006.

In addition, swab samples from teat skin and the mucous membranes of the nose and vagina were collected from a random selection of kids within 4 weeks after birth (n=16) and 6-8 month after birth (n=23) in farms A-D.

Samples from animals with clinical mastitis were not included in the study.

A c c e p t e d M a n u s c r i p t 5

Sample collection

All samples were collected by 7 veterinarians (one for each farm) following detailed instructions. They wore disposable latex gloves that were changed between each animal.

Udder secretions were collected aseptically in 10-ml sterile plastic vials as recommended by the International Dairy Federation (1981), frozen and kept at -20 o C until submission by express mail to the National Veterinary Institute (NVI) in Oslo.

Swab samples were collected using coal swabs (Eurotubo®, Rubi, Spain) moistened in sterile saline. Separate swabs were used for each sampling site. Teat skin samples were collected by moving a swab back and forth over the external surface of one teat. Nasal swabs were collected by inserting the same swab into both nostrils and gently rolling it against the nasal wall. The vaginal mucosa was sampled by inserting a swab 1─5 cm into the vagina and rotating it. All swabs were sent chilled in polystyrene boxes by express mail to the NVI, where microbiological analyses were started upon arrival.

Bacteriological procedures and identification of S. aureus

Microbiological analyses of udder secretions were performed essentially as recommended by the International Dairy Federation (1981). Briefly, milk samples were brought to room temperature, shaken, and 10 l were plated on blood agar (BA) (Oxoid, Basingstoke, United Kingdom) containing 5% washed bovine erythrocytes.

The swabs were placed in 10 ml Voegel-Johnson broth (Oxoid) with 0.5% agar in sterile glass test tubes, vortexed and incubated at 37°C for 4 h. One hundred microliters of the broth was then plated on Baird Parker agar with a Rabbit Plasma Fibrinogen supplement (BP+RPF; bioMérieux, Marcy-l'Etoile, France).

All agar plates were incubated at 37 C for 48 h and read after 24 and 48 h. Gram-positive cocci, that were catalase-and coagulase-positive were streaked on peptone agar (p-agar) (Difco, Sparks, Md.) supplemented with 7 mg/l of acriflavin (Sigma-Aldrich Chemie, Steinheim, Germany). Typical colonies from BP+RPF (glistening black and surrounded by an opaque halo) were considered to be coagulase-positive staphylococci and were streaked directly onto p-agar with acriflavine. Some colonies without halos on BP+RPF were tested for coagulase production as described above prior to plating of p-agar. P-agar plates were incubated at 37 C for 24 h, and bacterial growth in the full length of the streak was considered to be a positive reaction and confirmation of S. aureus [START_REF] Capurro | Identification of coagulasepositive staphylococci isolated from bovine milk[END_REF].

One or two selected colonies were purified and frozen in heart infusion broth (Difco) with 15% glycerol at -70 o C.

Isolates for PFGE

Before PFGE-results were available, infections and colonizations were predefined as "persistent" when S. aureus was detected in 2 consecutive samplings and as "temporary" when a positive sample was followed by a negative sample. These definitions were used when selecting isolates for PFGE.

A total of 505 S. aureus isolates were selected for PFGE analyses.

To investigate whether bilateral IMI are caused by the same or different S. aureus genotypes 28 pairs of isolates (n=56) from bilateral IMI were selected. Each pair included one isolate collected from each mammary gland of the same animal on the same day.

To investigate whether persistent IMI are caused by the same or different S. aureus genotypes 76 pairs of isolates (n=152) from persistent IMI were selected. Each pair included one isolate collected from 2 consecutive samplings of the same mammary gland. To investigate whether goats may carry several S. aureus genotypes at the same time 194 isolates were selected from 76 goats collected at 84 samplings. Isolates obtained from one goat collected on the same day were compared with each other.

Finally, 20 isolates from unbred kids and 53 isolates from temporary IMI were selected for PFGE.

PFGE

Preparation of chromosomal DNA and enzymatic digestion with Sma1 were performed as described by [START_REF] Bannerman | Pulsed-field gel electrophoresis as a replacement for bacteriophage typing of Staphylococcus aureus[END_REF] with previously described modifications (Mørk et al., 2005).

Differentiation of PFGE profiles

Differentiation of PFGE banding patterns was performed using BioNumerics (version 4.0; Applied Maths, Kortrijk, Belgium) and by visual inspection. DNA fragments between approximately 45 kb and 650 kb were included in the analysis, and a unique electrophoretic banding pattern was defined as a pulsotype (PT).

One representative for each PT was selected to create a dendrogram using BioNumerics.

Pairwise similarity coefficients were calculated using the Dice formula, and a dendrogram was created using the unweighted pair group method with arithmetic averages. Optimization and position tolerance settings were 1% and 1.5%, respectively. In order to investigate whether or not potentially enterotoxigenic S. aureus isolates are more likely to cause persistent infections of the caprine mammary gland than se-negative isolates, 153 PFGE-analyzed isolates from persistent (n=100) and temporary (n=53) IMI were selected for se-and sel-gene screening. All 153 isolates were tested for the genes sea-see, seg-selj by a multiplex PCR method (m-PCR) [START_REF] Lovseth | Modified multiplex PCR method for detection of pyrogenic exotoxin genes in staphylococcal isolates[END_REF]. In addition, 110 of the isolates were tested by conventional PCR for selk-selo, selq, selr and selu. Primers are listed in Table 1.

PCR for se-and sel-genes

These 110 isolates were selected as follows; all isolates from temporary IMI were included (n=53). If the 2 isolates in a pair from persistent IMI were identical by PFGE, one of the isolates was randomly selected (n=43), while if the 2 isolates of a pair were different by PFGE, both isolates were tested (n=14).

For DNA extraction, bacterial isolates were grown over night at 37ºC with shaking in heart infusion broth (Difco). Cells were pelleted and DNA was extracted using NucliSens ® easyMAG TM (bioMérieux).

The PCR program included denaturation at 95°C for 10 min followed by 35 cycles of 20 s denaturation at 95°C, 20 s annealing at 55°C, and 20 s extension at 72°C. A final extension for 10 min at 72°C completed the amplification. A set of in house S. aureus isolates that between them contained all the genes tested for in the PCR were included as positive controls for the PCR. Milli-Q water was used as negative control.

Statistical analyses

The difference between S. aureus isolates from persistent vs. temporary IMI with respect to se-gene profiles (se-positive or se-negative) were compared using contingency table analyses and the Chi squared statistic using JMP® version 7.0.1 (SAS institute Inc., Cary, NC, USA).

Results were considered significant if p < 0.05.

Isolation of S. aureus

Results from the bacteriological analyses with respect to sampling sites and herds are presented in Table 2. S. aureus was detected in 353 (6.2%) of the 5671 milk samples and in 625 (37.4%) of the 1673 swab samples from lactating goats. From kids, S. aureus was isolated from one (4%), 28 (61%) and 3 (8%) swab samples from teat skin, nose and vagina, respectively.

A noticeably higher percentage of vaginal swab samples were S. aureus-positive after kidding (44.9%) than before drying off (19.1%). Conversely, a higher percentage of nasal swab samples were S. aureus-positive before drying off (75.6%) than after kidding (62.0%).

Altogether 122 goats with S. aureus-positive nasal swabs after kidding were resampled before drying off and 90 (73.8%) of these were S. aureus-positive. Eighty two goats that had S. aureus-positive vaginal swabs after kidding were resampled before drying off and 22 (26.8%) were still S. aureus-positive.

PFGE

All the 505 analysed S. aureus isolates were typeable by PFGE, and were divided into 33 different PTs (Fig. 1). In a dendrogram with one representative for each PT, 6 different clusters (A─F) were defined visually. The minimum similarity between PTs within these clusters was 68.7% (Fig 1). Pairwise comparisons of the 33 PTs showed that with 4 exceptions all PTs within a cluster differed by <6 bands and all PTs differed by >5 bands to all PTs outside its cluster.

The most prevalent PT (C2) comprised 116 (23.0%) isolates. Two PTs, C4 and C11, each differed by 2 and 4 bands from C2 and comprised 111 (22.0%) and 67 (13.3%) of the isolates, respectively. Three hundred and seventy (73.3%) isolates belonged to the 5 most prevalent Within each herd, 4 to 15 different PTs were identified (Table 3). The greatest diversity was found in herd A with 15 PTs assigned to 4 clusters. PTs from more than one cluster were found in all herds except for herd E. Each of the 7 herds had a predominant PT which included 34 to 74% of the analysed isolates from that herd.

Comparisons of PTs of paired S. aureus isolates from bilateral IMI, persistent IMI, persistent body site colonizations and of isolates collected from IMI and extramammary sites of the same goat on the same day are shown in Table 4. The majority of isolate-pairs from persistent IMI, from repeated vaginal swabs and from milk and teat skin were identical, while remaining paired isolates were identical in less than 50% of the situations.

Out of the paired S. aureus isolates that had different PTs, the number of pairs that differed by ≤3 and >5 band differences are presented in Table 4. Noticeably, in the majority of situations where paired S. aureus isolates from bilateral IMI, from milk and the nose, and from milk and the vagina were different, the PTs differed by >5 bands. In situations where paired isolates from persistent IMI, from milk and teat skin, from the nose (repeated samples), and from the vagina (repeated samples) had different PTs, most of the pairs differed by 3 bands or less.

When S. aureus isolates (n=194) from 76 goats, collected at 84 samplings were compared, different PTs were found among isolates from 55 (65.5%) of the samplings. From 36 (65.5%) of the samplings where isolates had different PTs, >5 band differences were found. 3). The additional testing of 110 of the isolates by conventional PCR (for selk-selo, selq, selr and selu) did not increase the number of se-positive isolates.

Toxin gene detection

There was no significant difference between the se-profiles of isolates from predefined persistent vs. temporary IMI. This result remained the same also when the 7 pairs of isolates that were different by PFGE, but that had been predefined as from persistent IMI, were excluded from the analysis. By m-PCR the genes sec and tst were found in 109 (71.2%) isolates and were always codetected. Seventy eight of these isolates were also tested by conventional PCR and all were sell-positive. Three isolates from two animals were positive for seg, and sei by m-PCR. Two of these isolates, from the same animal, were also positive for sec and tst. Conventional PCR was performed on 2 of the seg-and sei-positive isolates (one from each animal), and both were positive for selm, seln, selo and selu. The genes sea, seb, sed, selk, selq and selr were not detected in any of the isolates.

The 153 isolates tested for SE-genes belonged to 22 of the PTs described above. There was a noticeable correspondence between SE-gene profile and PT and cluster (Fig. 1). With only 2 exceptions, isolates belonging to cluster A (PTs A1─A5 and A7) were all se-negative.

Conversely, most isolates (87.7%) belonging to cluster C were positive for sec and tst (and sell).

Discussion

In the present study, 6.2% of the goat milk samples were S. aureus positive, with a variation at herd level between 2.4 and 11.4%. In earlier studies S. aureus has generally been isolated from less than 3% of the goats [START_REF] Menzies | Staphylococcus aureus genotypes recovered from cases of bovine, ovine, and caprine mastitis[END_REF].

A c c e p t e d M a n u s c r i p t of the sampled goats and 61% of the kids had S. aureus-positive nasal swabs. This indicates that the nose is an important colonization site for S. aureus in goats, and that kids are colonized during or soon after birth. Previous reports have indicated a S. aureus nasal carriage rate of 30% in dairy sheep (Vautor et al., 2005) and 6% in dairy goats (Valle et al., 1991), and that 9% of dairy heifers were colonized with S. aureus in the muzzle area [START_REF] Roberson | Ecology of Staphylococcus aureus isolated from various sites on dairy farms[END_REF]. On the other hand, in 2 studies from Norway (Jørgensen et al., 2005b) and the United Kingdom [START_REF] Smith | Multilocus sequence typing of Staphylococcus aureus isolated from high-somatic-cell-count cows and the environment of an organic dairy farm in the United Kingdom[END_REF], respectively, S. aureus was not found in nasal swabs from dairy cows despite a high frequency of S. aureus IMI in both herds.

A higher S. aureus colonization rate was found in the vagina after kidding (45%) than before drying off (19%). Uterine involution and decreased secretions may explain the lower colonization rate before drying off. It is likely that presence of S. aureus on the mucous membranes of the vagina and in vaginal secretions contributes to the spread of S. aureus from goats to kids. The kids sampled in this study were removed from their mothers immediately after birth and received one feed of colostrum. They are unlikely, therefore, to have picked up S. aureus during muzzling and suckling.

Although the PFGE analyses revealed a great genetic diversity among the S. aureus isolates it was also evident that certain genotypes dominated. This was the case both when comparing isolates from individual herds, and when comparing the complete set of 505 isolates from all 7 herds. The 3 most commonly observed PTs were, with 2 exceptions, also the dominant PT in the herds where they were found. It appears that S. aureus strains belonging to certain genotypes are more successful at spreading in the Norwegian dairy goat population than others. This is in agreement with a previous study where a limited number of closely related PTs were found responsible for a great proportion of the cases of S. aureus mastitis in dairy A c c e p t e d M a n u s c r i p t 13 cows, dairy goats and meat-producing sheep (Mørk et al., 2005). Similar findings have been reported regarding S. aureus in dairy sheep (Vautor et al., 2003).

In the present study almost all the included goats were colonized or infected with S. aureus, and individual goats could be colonised with several S. aureus genotypes. In fact in 65% of the situations, where more than one body site on the same goat was S. aureus-positive after sampling on the same day, isolates belonged to different PTs.

The majority of S. aureus IMI that were predefined as persistent were confirmed as persistent infections because two consecutively collected isolates were identical by PFGE. Isolating the same genotype on three consecutive occasions would have strengthened the definition of these infections as truly persistent, but this was not practicable in this study.

Although 18 out of the 76 compared S. aureus pairs had different PTs, 14 of these differed by only 2 bands. It could be argued that these belonged to the same infection because an infecting strain may undergo genetic change during the course of an infection [START_REF] Tenover | Interpreting chromosomal DNA restriction patterns produced by pulsed-field gel electrophoresis: criteria for bacterial strain typing[END_REF].

In contrast to the situation with persistent IMI, bilateral IMI were equally often caused by distinguishable PTs as by identical PTs. In the majority of situations where S. aureus pairs differed by PFGE, the PTs also belonged to different clusters. This indicates that S. aureus from different sources most often caused IMI in the 2 udder halves of bilateral IMI.

The nares were found to be a major S. aureus colonization site in Norwegian dairy goats. One might have expected that the nasal strain would also be the most frequent cause of IMI in individual goats. However, more than 50% of the isolate pairs, collected from the nose and an IMI of the same goat on the same day, differed by 4 bands or more by PFGE. A similar observation was made regarding pairs of isolates from the vagina and an IMI. This indicates that the mucous membranes are not the main reservoir of S. aureus IMI for individual goats.

However, the frequent presence of S. aureus on the mucous membranes probably contributes to extensive dispersal of the bacteria in the environment, hampering effective transmission A c c e p t e d M a n u s c r i p t 14 control in the herds. Out of the paired isolates from repeated nasal swabs, that were compared by PFGE, 25% of the pairs differed by >5 band. This indicates frequent strain change in the nasal membranes consistent with intermittent rather than persistent carriage [START_REF] Kluytmans | Nasal carriage of Staphylococcus aureus: epidemiology, underlying mechanisms, and associated risks[END_REF].

In agreement with previous observations (Jørgensen et al., 2005a) and investigations by others [START_REF] Da Silva | Detection of the enterotoxins A, B, and C genes in Staphylococcus aureus from goat and bovine mastitis in Brazilian dairy herds[END_REF][START_REF] Haveri | Virulence genes of bovine Staphylococcus aureus from persistent and nonpersistent intramammary infections with different clinical characteristics[END_REF][START_REF] Scherrer | Phenotypic and genotypic characteristics of Staphylococcus aureus isolates from raw bulk-tank milk samples of goats and sheep[END_REF][START_REF] Smyth | Superantigen genes encoded by the egc cluster and SaPIbov are predominant among Staphylococcus aureus isolates from cows, goats, sheep, rabbits and poultry[END_REF], the majority of analysed S. aureus isolates were SE-gene positive. The genes sec, sell and tst were detected almost exclusively, and indicates a wide distribution of the pathogenicity island Sap1bov (Fitzgerald et al., 2001b) among caprine S. aureus in Norway. It seems plausible that the toxins encoded by Sap1bov play a role in survival of S. aureus on its caprine host. However, no correlation was found between persistence of IMI and the SE-gene profile of the causal S. aureus strains. The observation that certain S. aureus PTs and PFGE clusters mostly had the same SE-gene profile indicates that it is clonal spread of toxin-encoding S. aureus strains rather than horizontal transfer of the toxin encoding genetic elements that is responsible for the wide distribution of Sap1bov.

In conclusion, S. aureus is frequently present on the mucous membranes of the nose and vagina of Norwegian dairy goats and the epidemiology is complex. The presence of S. aureus on other body sites than the mammary gland may contribute to the spread of S. aureus in dairy goat herds and thus add to mastitis control problems. The toxin genes sec, sell and tst were found to be widespread among the caprine S. aureus isolates, but the role of these toxins in colonizations and infections of goats remain to be elucidated. Vautor, E., Abadie, G., Guibert, J.M., Chevalier, N., Pepin, M., 2005. Nasal carriage of Staphylococcus aureus in dairy sheep. Vet. Microbiol. 106, 235-239. Vautor, E., Abadie, G., Guibert, J.M., Huard, C., Pepin, M., 2003. Genotyping of Staphylococcus aureus isolated from various sites on farms with dairy sheep using pulsedfield gel electrophoresis. Vet. Microbiol. 96, 69-79.

White, E. 2007, The prevalence of mastitis in small ruminants and the effect of mastitis on small ruminant production. In: Proceedings of the NMC 46th Annual Meeting. National Mastitis Council, Madison, Wis, pp. 119-127. Zottola, E.A., Smith, L.B., 1993. Growth and survival of undesirable bacteria in cheese. In: * SE-neg = no SE-genes detected; SaP1bov = detected genes belonging to the pathogenicity island SaP1bov (sec, sell and tst) (Fitzgerald et al., 2001a); egc = detected genes belonging to the enterotoxin gene cluster (egc) (sei, seg, selm, seln, selo, and selu) [START_REF] Jarraud | egc, a highly prevalent operon of enterotoxin gene, forms a putative nursery of superantigens in Staphylococcus aureus[END_REF].

A c c e p t e d M a n u s c r i p t

  suspected staphylococcal colonies were Gram-stained, tested for catalase production and for coagulase by the tube coagulase test (Becton Dickinson, Sparks, Md).

  persistent extramammary colonizations are caused by the same or different S. aureus genotypes 40 pairs of isolates (n=80) collected from persistent extramammary colonizations were selected. Each pair included one isolate collected from 2 consecutive samplings of the same body site.

  , C4, C11, A3, A1) and each of these PTs were found in 3-5 herds. With the exception of PT A1, isolates belonging to these 5 PTs were obtained from all the 4 major sampling sites. With 2 exceptions, C2, C4 and C11 were the predominant PT in each of the herds where they were found.

  153 isolates tested by the m-PCR (for sea-see, seg-selj and tst), at least one SEgene was detected in 110 (71.9%) of the isolates (Table

  ., Piriz, S., de la, F.R., Vadillo, S., 1991. Staphylococci isolated from healthy goats.Zentralbl. Veterinarmed. B 38,[81][82][83][84][85][86][87][88][89]

  Fox, P.E. (Ed.), Cheese: chemistry, physics and microbiology, Chapman & Hall, London, pp. 471-492.

  PCR primers used for detection of genes encoding staphylococcal enterotoxins SEK-SEO, SEQ, SER and SEU A c c e p t e d M a n u s c r i p t 22 Table 2: The number of different lactating goats sampled, the number of samples, and the frequency of S. aureus isolation from different sampling sites in 7 dairy goat herds A c c e p t e d M a n u s c r i p t

  Comparisons of the pulsotypes (PTs) of paired S. aureus isolates from bilateral IMI; persistent IMI; persistent body site colonizations; and of isolates from IMI and an extramammary body site on the same goat on the same day A c c e p t e d M a n u s c r i p t

Figure 1 :

 1 Figure 1: Pulsed-field gel electrophoresis of SmaI digested DNA of S. aureus isolates

Table 3 :

 3 Pulsed-field gel electrophoresis (PFGE) banding patterns (pulsotypes=PTs) and enterotoxin genes (SE-genes) among S. aureus isolates from 7 dairy goat herds

Table 1 :

 1 PCR primers used for detection of genes encoding staphylococcal enterotoxins SEK-SEO, SEQ, SER and SEU

				Produc	Accession
	Gene Primer	Sequence 5'-3'	t size	number	Reference
		sek fwd	TTA GGT GTC TCT AAT AGT GCC AGC			Smyth et al,
	sek	sek rev	AGC TGT GAC TCC GCC ATA TAT GTA	278 bp		2005
		SEL-F158	CAC CAG AAT CAC ACC GCT TA			Cremonesi et
	sel	SEL-R397	CTG TTT GAT GCT TGC CAT TG	240 bp		al., 2005
		mpSEM-1	CTA TTA ATC TTT GGG TTA ATG GAG AAC			Jarraud et al.,
	sem	mpSEM-2	TTC AGT TTC GAC AGT TTT GTT GTC AT	300 bp		2001
		sen fwd	GCT TAT GAG ATT GTT CTA CAT AGC TGC			Smyth et al,
	sen	sen rev	CAT TAA CGC CTA TAA CTT TCT CTT CAT C	448 bp		2005
		seo fwd	AAG AAG TCA AGT GTA GAC CCT ATT GCT
	seo	seo rev	AAT CGC TGA TGA GCT AAA TTC CAC	201 bp	AF285760	*
		sep fwd	ATT TAC AAA AAA AGT CTG AAT TGC AGG			Kuroda et al.,
	sep	sep rev	TGG CGG TGT CTT TTG AAC C	201 bp		2001
		seq fwd	TGG AAA ATA CAC TTT ATA TTC ACA GTT TCA
	seq	seq rev	TTT TGC TTA CCA TTG ACC CAG AG	201 bp	AF410775.	*
		ser fwd	CGG TTA GAT GTG TTT GGA ATA CCC
	ser	ser rev	ATT TGT ACT AAT TGT AAA AGA GAA CTG TTG TTT 201 bp	AB075606	*
		seu fwd	AAA ATA TGG AGT TGT TGG AAT GAA GTT			Letertre et al.,
	seu	seu rev	TTC TCT TGG GCT TTA ATG TTT GTT T	201 bp		2003

* designed in the present study.

Table A

 A

	M a n u s c r i p t
	c c e p t e d

Table 2 :

 2 The number of different lactating goats sampled, the number of samples, and the frequency of S. aureus isolation from different sampling sites in 7 dairy goat herds

	Table
	Milk samples, teat skin				Herd
	swabs, nasal swabs and vaginal swabs	A	B	C	D	E	F	G	All herds
	Milk
	No. of goats sampled	327	138	111	92	138	39	176	1039
	No. of samples	1239	916	702	701	920	245	948	5671
	No. of S. aureus (%)	130 (10.5) 27 (2.9) 17 (2.4) 41 (5.8) 44 (4.8) 40 (11.4) 54 (5.7) 353 (6.2)
	Teat skin
	No. of goats sampled	105	125	49	81
	No. of samples	122	194	91	128
	No. of S. aureus (%)	5 (4.1)	14 (7.2) 16 (17.6) 18 (14.1)				53 (9.9)
	Nasal membrane
	No. of goats sampled	119	125	48	83
	No. of goats sampled twice	27	68	43	56
	No. of samples	146	193	91	139
	No. of S. aureus (%)	102 (69.9) 118 (61.1) 68 (74.7) 104 (74.8)				392 (68.9)
	Vaginal membrane
	No. of goats sampled	119	125	48	83
	No. of goats sampled twice	27	68	43	56
	No. of samples	146	193	91	139
	No. of S. aureus (%)	47 (32.2) 60 (31.1) 28 (30.8) 45 (32.4)				180 (31.6)

Table 3 :

 3 Pulsed-field gel electrophoresis (PFGE) banding patterns (pulsotypes=PTs) and enterotoxin genes (SE-genes) among S. aureus isolates from 7 dairy goat herds ) 5, 5, 7 and 3 isolates from unbred kids included from herd A, B, C and D, respectively.

	Herd

a

Table

Acknowledgements

The Norwegian Research Council is acknowledged for economic support of this work (grant number 164293). We also thank the veterinarians Olav Hermansen, Marianne Vinje Kilvaer,


Luisa De Martino

Gabriella Marfé

Mariangela Longo

Filomena Fiorito
email: filomena.fiorito@unina.it

Serena Montagnaro

Valentina Iovane

Nicola Decaro

Ugo Pagnini

Bid cleavage, cytochrome c release and caspase activation in canine coronavirus-induced apoptosis

Keywords: Canine coronavirus type II, apoptosis, caspases, PARP, bcl-2 family members

A c c e p t e d M a n u s c r i p t

Introduction

Apoptosis is a particular type of cell death that is characterized by distinctive changes in cellular morphology, including cell shrinkage, nuclear condensation, chromatin margination and subsequent degradation that are associated with inter-nucleosomal DNA fragmentation. Apoptosis may be initiated by a wide variety of cellular insults, including death receptor stimulation and cytotoxic compounds. Induction or inhibition of apoptosis is an important feature of many types of viral infection, both in vitro and in vivo [START_REF] Mclean | Viral manipulation of cell death[END_REF][START_REF] Hay | A time to kill: viral manipulation of the cell death program[END_REF][START_REF] Barber | Host defense, viruses and apoptosis[END_REF][START_REF] Derfuss | Herpesviral proteins regulating apoptosis[END_REF][START_REF] Benedict | To kill or be killed: viral evasion of apoptosis[END_REF][START_REF] Garnett | Accelerated degradation of FADD and procaspase 8 in cells expressing human papilloma virus 16 E6 impairs TRAIL-mediated apoptosis[END_REF]. Despite this fact, the mechanisms of virus-induced apoptosis remain largely unknown.

Canine coronavirus (CCoV), a member of antigenic group 1 of the family Coronaviridae, is a single positive-stranded RNA virus responsible for enteric disease in young puppies [START_REF] Decaro | An update on canine coronaviruses: Viral evolution and pathobiology[END_REF]. CCoV, first described in diarrhoeic dogs [START_REF] Binn | Recovery and characterization of a coronavirus from military dogs with diarrhoea[END_REF], is responsible for diarrhoea, vomiting, dehydration, loss of appetite and occasional death in puppies. Two serotypes of CCoVs were described [START_REF] Pratelli | Genetic diversity of a canine coronavirus detected in pups with diarrhoea in Italy[END_REF], CCoV-I and -II, sharing about 90% sequence identity in most of their genome. In the present study, we evaluated the processes implicated in cell death induced by CCoV, in particular of CCoV type II, which is the only CCoV that grows in cell cultures [START_REF] Pratelli | Two genotypes of canine coronavirus simultaneously detected in the fecal samples of dogs with diarrea[END_REF]).

An important regulatory event in the apoptotic process is represented by the caspases activation which regulates two major relatively distinct pathways, the extrinsic and intrinsic pathways. The extrinsic pathway is mediated by activation of caspase-8 that is initiated mainly by binding of death receptors to their ligands. The intrinsic pathway involves the release of cytochrome c from mitochondria; subsequently, cytochrome c binds to the adaptor molecule Apaf-1 (apoptotic protease activating factor-1) causing autocleavage of caspase-9 [START_REF] Adrain | Regulation of apoptotic protease activating factor-1 oligomerization and apoptosis by the WD-40 repeat region[END_REF]. This pathway known also as mitochondrial pathway involves pro and anti-apoptotic members of the proteins bcl-2 family which can be a key to trigger mitochondrial apoptosis [START_REF] Hildeman | Activated T cell death in vivo mediated by proapoptotic bcl-2 family member bim[END_REF]. Both pathways, extrinsic and intrinsic, converge at the activation of executioner caspases (caspase-3, -6 and -7), which are responsible for the characteristic morphological changes of apoptosis [START_REF] Budihardjo | Biochemical pathways of caspase activation during apoptosis[END_REF][START_REF] Elmore | Apoptosis: a review of programmed cell death[END_REF]. Generally, both death receptor and mitochondrial apoptosis signalling pathways were shown to be implicated in apoptosis induced by viruses. Induction of caspase-dependent apoptosis has been observed during infection by other coronaviruses, including transmissible gastroenteritis coronavirus [START_REF] Eleouet | Transmissible gastroenteritis coronavirus induces programmed cell death in infected cells through a caspase-dependent pathway[END_REF], avian coronavirus infectious bronchitis virus [START_REF] Liu | Induction of caspase-dependent apoptosis in cultured cells by the avian coronavirus infectious bronchitis virus[END_REF], human coronavirus strain 229E [START_REF] Collins | In vitro detection of apoptosis in monocytes/macrophages infected with human coronavirus[END_REF], and equine coronavirus (Suzuki et al., 2008).

A c c e p t e d M a n u s c r i p t

During infection of CCoV, [START_REF] Ruggieri | Canine coronavirus induces apoptosis in cultured cells[END_REF], demonstrated activation of caspase 3. The aim of this study was to provide a better characterization of the pattern of caspase activation following infection with CCoV-II. We showed that CCoV infection results in the activation of the initiator caspases, caspase-8 and -9, and of the effector caspases, caspase-3 and -6. The data demonstrated that both death receptor and mitochondrial pathways can play an essential role in CCoV-induced apoptosis. Furthermore we observed no variation of CCoV release inhibiting apoptosis by caspase inhibitors.

Materials and methods

Cell culture and virus preparation

A canine fibrosarcoma cell line (A-72 cells) was grown and maintained in complete medium consisting of Dulbecco Minimal Essential Medium (D-MEM) supplemented with 2 mM Lglutamine, 1% non essential amino acid, 5% heat-inactivated foetal calf serum (FCS), 100 IU of penicillin, and 100 g of streptomycin per ml, at 37°C in a 5% CO 2 atmosphere incubator. This cell line was maintained free of mycoplasma and of bovine viral diarrhoea virus. Cells were trypsinized once a week.

CCoV type II strain S/378 (kindly provided by Prof. C. Buonavoglia, Faculty of Veterinary Medicine, University of Bari, Italy) was used for the study. For viral infection A-72 cells at 80 to 90% confluence in complete medium as described above, were incubated with virus. One hour post-infection (p.i.) non internalized virus was removed by washing the cells three times with DMEM, and incubation continued in complete medium. Virus titers were determined by end point dilution tests using 96-well microtitre plates, and are given as 50% tissue culture infective doses (TCID 50 ) according to the method of [START_REF] Reed | A simple method of estimating 50 per cent end-points[END_REF]. Aliquots of CCoV-II were stored at -80°C until used.

Cell viability and microscopy

Cell viability after CCoV-II infection was monitored by evaluation of mitochondrial suffering through the MTT assay as previously described [START_REF] Pagnini | The involvement of oxidative stress in bovine herpesvirus type 4-mediated apoptosis[END_REF]. Data are presented as a percentage of the control, and results are the mean ± SEM of three experiments performed in triplicate.

To identify apoptotic nuclei, cells uninfected or infected with CCoV-II, were stained with acridine orange (Sigma Chemical Co, St Louis, USA). Briefly, after 1 h of virus adsorption, the A c c e p t e d M a n u s c r i p t inoculum was removed and fresh medium was added. After 24 and 48 h p.i., cells were washed with D-MEM and in each well were gently added 100 µl of phosphate-buffered saline (PBS) with acridine orange (40 µg/ml). The cells were incubated at room temperature for approximately 2 min in the dark. The preparations were washed with distilled water, covered with glass cover slips, and assessed on the same day using fluorescent microscope (Zeiss, Oberkochen, Germany) with a 460nm filter. The duration of illumination was limited to 40 seconds per field. Each experiment was repeated at least three times to confirm the reproducibility of the results. Positive controls were obtained using camptothecin as previously described [START_REF] Fiorito | 2,3,7,8-Tetrachlorodibenzo-p-dioxin increases Bovine Herpesvirus type-1 (BHV-1) replication in Madin-Darby bovine kidney (MDBK) cells in vitro[END_REF].

Caspase detection by flow cytometry

Caspase activation detection was performed by using the carboxyfluorescein FLICA Assay kits (B-Bridge International, Inc., CA, USA). Fluorochrome-labeled inhibitors of caspases (FLICA) included in kits were added during the final 1 h. Precisely, the fluorochrome-labeled inhibitors of caspase-3 (FAM-DEVD-FMK), caspase-8 (FAM-LETD-FMK) and caspase-9 (FAM-LEHD-FMK)

were dissolved in DMSO following the manufacturer's instructions and then stored at -20°C in the dark until use. Solutions of each FLICA were added to cell cultures (1x10 6 cells/ml) at different times post infection; cells were then incubated for 1h at 37°C in a 5% CO 2 atmosphere incubator.

After incubation, cells were washed twice with washing buffer, placed on ice and the cell fluorescence was measured within 15 min by a flow cytometer (Parteck Flow Cytometer). The amount of fluorescence detected was directly proportional to the amount of caspase activity. Results of all experiments are reported as mean SEM of three experiments.

Protein extraction and Western blot analysis

Mock-infected and infected cells (MOI 10) were collected at different times p.i. and washed twice in PBS, then the cell pellets were homogenized directly into lysis buffer (50 mM HEPES, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 10% glycerol, 1% NP-40, 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml aprotinin, 0.5 mM sodium orthovanadate, and 20 mM sodium pyrophosphate).

The lysates were centrifugated at 14,000 x rpm for 10 min. Protein concentrations were determined by the Bradford assay (Bio-Rad, Hercules, CA). Equivalent amounts of proteins were loaded and electrophoresed on SDS-polyacrylamide gels. Subsequently, proteins were transferred to nitrocellulose membranes (Immobilon, Millipore Corp., Bedford, MA). After blocking with Trisbuffered saline-BSA (25 mM Tris (pH 7.4), 200 mM NaCl, and 5% BSA), the membrane was incubated with the following primary antibodies: anti-caspase-6 PAb (dilution 1:1,000) (Cell Signalling), anti-caspase-3 PAb (dilution 1:1,500) (Abcam), anti-caspase-8 PAb (dilution 1:2,000)

A c c e p t e d M a n u s c r i p t (Abcam), anti-caspase-9 PAb (dilution 1:2,000) (Stressgen), anti-bax PAb (1:1,000) (Aviva Systems Biology), anti-bcl-XL PAb (1:1,000) (Abcam), anti-bcl-2 PAb (1:1,000) (Abcam), anti-bid PAb (1:1000) (Abnova Corporation), anti-cytochrome c (1:1000) (Abcam), anti-bim PAb (1:1000) (Cell Signaling) and anti-β-actin MAb (dilution 1:7,500) (Cell Signaling). Membranes were then incubated with the horseradish peroxidase-conjugated secondary antibody (dilution 1:1,000) (at room temperature), and the reaction was detected with an enhanced chemiluminescence system (Amersham Life Science). The relative amount of protein expression was quantified using Gel-Doc phosphorimanger and Quantity One software (Bio-Rad) and normalized by the band intensity of βactin.

Extraction of PARP

Mock infected and infected cells (MOI 10) were washed twice with ice-cold PBS and then once with ice-cold buffer A (100 mM Tris-HCl [pH 7.4], 10 mM MgSO 4 , 500 mM sucrose, 10 mM PMSF, 0.5 μg of leupeptin per ml, 0.75 μg of pepstatin per ml, and 5 μg of antipain per ml). Cells were permeabilized by incubation on ice for 20 min with buffer B (100 mM Tris-HCl [pH 7.4], 10 mM MgSO 4 , 500 mM sucrose, 10 mM PMSF, 1% NP-40; 0.25 μg of leupeptin per ml, 0.35 μg of pepstatin per ml, and 50 μg of antipain per ml). PARP was extracted from permeabilized cells by incubation with cold buffer C (200 mM K 2 HPO 4 , 100 mM Tris-HCl [pH 7.4], 10 mM MgSO 4 , 500 mM sucrose, 10 mM PMSF, 0.5 μg of leupeptin per ml, 0.75 μg of pepstatin per ml, and 5 μg of antipain per ml) on ice for 20 min. The extract was centrifuged at 2,000 × g for 10 min at 4°C, and the supernatant was collected and mixed with 4 volumes of urea loading buffer (62.5 mM Tris-HCl [pH 6.8], 6 M urea, 10% glycerol, 2% SDS, 0.00125% bromophenol blue, and 5% βmercaptoethanol). The Western blot analysis of PARP was performed, as above reported, using anti-poly(ADP-ribose) polymerase (PARP) antibody with a dilution of 1:5,000 (BD Biosciences).

The relative amount of protein expression was quantified using Gel-Doc phosphorimanger and Quantity One software (Bio-Rad) and normalized by the band intensity of β-actin.

Cytosolic and mitochondrial protein extraction

Isolation of mitochondria and cytosolic fractions were carried out using a modified protocol from [START_REF] Kluck | The release of cytochrome c from mitochondria: a primary site for Bcl-2 regulation of apoptosis[END_REF]. Mock-infected and infected cells were collected at different times p.i. and washed twice in PBS. The cell pellets were resuspended in lysis buffer (50 mM Tris [pH 7.5], 150 mM NaCl 2 , 5 mM EGTA, 1 mM CaCl 2 , 1 mM MgCl 2 , 1% NP-40, 1 µg/ml leupeptin, 1 µg/ml aprotinin, 1 µM PMSF, and 100 µM Na 3 VO 4 ). Samples were then incubated on ice for 20 minutes and centrifuged at 14,000 rpm for 15 min. Equivalent amounts of proteins were loaded and A c c e p t e d M a n u s c r i p t electrophoresed on SDS-polyacrylamide gels. The Western blot analysis of cytochrome c was performed, as above reported, using anti-cytochrome c PAb with a dilution of 1:1,000 (Abcam).

The relative amount of protein expression was quantified using Gel-Doc phosphorimanger and Quantity One software (Bio-Rad) and normalized by the band intensity of β-actin.

Inhibition experiments of CCoV-II-induced apoptosis

A-72 cells in 24-well plates, at confluency, were treated with 25 μM of each of the caspase inhibitors Z-VAD-FMK (pan-caspase inhibitor), Z-IETD-FMK (caspase-8-specifc inhibitor), Z-LEDH-FMK (caspase-9-specific inhibitor), Z-DEVD-FMK (caspase-3-specifc inhibitor) (Calbiochem, EMD Biosciences), 2 h prior to infection with CCoV-II at MOI 10.

At 24 h p.i. cell viability was evaluated by MTT assay as above reported, and virus titers recovered from the culture medium were assayed by TCID50 method according to [START_REF] Reed | A simple method of estimating 50 per cent end-points[END_REF].

Statistical analysis

The results are presented as mean ± SEM of three experiments. One-way ANOVA with Turkey's post test was performed using GraphPad InStat Version 3.00 for Windows 95 (GraphPad Software, San Diego, CA, USA). P value < 0.05 was considered statistically significant.

Results

Cell viability and morphological evidence of apoptosis induced in A-72 cells by CCoV-II infection.

Infection of A-72 cells with CCoV-II resulted in cell death in a time-dependent manner, as detected by MTT assay (Fig. 1). This loss of viability was also dependent on the MOI at which the cultures were infected. Moreover, a significant reduction of mitochondrial dehydrogenases activity was already detectable at 8 h p.i. using a MOI 10 (P<0.05). A significant decrease (P<0.01 and P<0.001) in cell number was observed at all examined times post infection independently of the used MOI .

Using a MOI of 10, the typical cytopathic effects (CPE) characterized by cytoplasmic vacuolation, fusion, rounding up, and detachment of infected cells from the cultured plates were first detected in CCoV type II-infected A-72 cells at 24 h p.i. and the extent of CPE gradually increased by 48 h. (Fig. 2).

Cellular morphological changes were also investigated by acridine orange staining using fluorescence microscopy (Fig. 3). Cells at 80% confluence were mock-infected or infected with A c c e p t e d M a n u s c r i p t

CCoV-II at a MOI of 10. At various time p.i., cells were stained and observed. The results demonstrated that apoptotic morphological changes could be detected overall at 48 h p.i., when cells showed chromatin condensation. Thus, it could be seen clearly that the strong apoptotic signs were evident late in the infection. The data were in accordance with the results of flow cytometric assay (data not shown).

Activation of caspase cascade

To explore the cell death program, first we investigated caspase activation cascades during

CCoV-II-induced apoptosis using a MOI of 10 and analyzing the cell lysates at different times p.i.

(8, 12, 24 and 48 h). The activity of two initiator caspase-8 (death receptor-mediated) and caspase-9

(mitochondrial-mediated) as well as the downstream effector caspase-3 were measured using fluorochrome-labeled inhibitors of caspases (FLICA) assay kits.

Fig. 4 shows at and after 12 h p.i. a significant increase (P<0.01 and P<0.001) of caspase-3, -8 and -9 activity in a time-dependent manner in CCoV-II-infected cells. These data suggested that both extrinsic and intrinsic pathways were involved in CCoV-II-induced apoptosis.

For further evidence, we investigated the initiator caspase-8, 9 activation as well as the executioner caspase-3 and -6 by Western blot analysis. A representative blot (Fig. 5a) and the relative densitometric analysis (Fig. 5b) show the involvement of the examined cleaved caspases.

As described in Materials and Methods, β-actin was used as an internal loading control. The cleavage of caspase-8 and -9 was detected from 12 h p.i. in CCoV infected cells (Fig. 5a), and such result was in accordance with FLICA analysis. In particular, it was evident a higher expression level of cleaved caspase-8 compared to cleaved caspase-9 at 12 h p.i.. Then, whereas the cleaved caspase-8 showed a time-dependent downward trend, the cleaved caspase-9 didn't change over time.

The caspase-3 activation pattern was also well correlated with the FLICA analysis. In fact, we observed the caspase-3 proteolytic processing at and after 12 h p.i., and a significant peak at 24 and 36 h p.i. (Fig. 5a andb). By Western blot analysis, we have also observed the cleaved caspase-6 (Fig. 5a andb) and a higher activation of caspase-6 at and after 24 h p.i. than that of caspase-3.

PARP cleavage

We next assessed the cleavage of PARP in the induction of apoptosis in A-72 cells by CCoV-II. PARP is a substrate for activated caspase-3 and cleaved PARP is considered a hallmark of apoptosis [START_REF] Elmore | Apoptosis: a review of programmed cell death[END_REF]. PARP cleavage was monitored during CCoV-II infection by Western blot in order to provide additional evidence of caspase activation and apoptosis induced by CCoV-II. As shown in Fig. 6a, the full-length 116 kDa PARP was cleaved to active form of 85 kDa in

B D

A c c e p t e d M a n u s c r i p t time-dependent manner. A representative blot (Fig. 6a) and its quantification (Fig. 6b) shows the involvement of proteolytic cleavage of PARP in virus-infected cells harvested at 12, 24, 36 and 48 h p.i., when the typical DNA laddering was also evident (data not shown). Furthermore, the Fig. 6c shows that the cleaved PARP/uncleaved PARP ratio was statistically significant (P<0.01) from 24 h p.i. until the end of infection compared to that of 12 h p.i..

Bid cleavage and cytochrome c release

In normal cells, the bid protein exists as an inactive form in the cytosolic fraction and becomes activated by caspase-8 in response to apoptotic stimuli. Then, the active form of bid translocates to the mitochondria, and triggers bax activation in correlation with the translocation of bax from the cytosol to the mitochondria. These events result in the cytochrome c release from the mitochondria to the cytosol [START_REF] Korsmeyer | Proapoptotic cascade activates BID, which oligomerizes BAK or BAX into pores that result in the release of cytochrome c[END_REF][START_REF] Roucou | Bax oligomerization in mitochondrial membranes requires tBid (caspase-8-cleaved Bid) and a mitochondrial protein[END_REF]. Because CCoV-II infection, as shown above, induced caspase-8 activation in infected cells, we generated subcellular fractions to examine bid and cytochrome c cell localization. As shown in Fig. 7a

Effect of CCoV type II infection on the expression of Bcl-2 family of proteins

The Bcl-2 family of proteins, including both inhibitors and promoters of apoptosis, is involved in the controlling of mitochondrial permeability, as well as regulation of caspase activation. In this study, several Bcl-2 family proteins, including anti-apoptotic (bcl-2, bcl-xL) and pro-apoptotic members (bax, bim), were detected in response to CCoV-II infection in A-72 cells.

The densitometric analysis of the blots demonstrated an increased expression of pro-apoptotic proteins, bim and bax, which presented a peak at 12 and 24 h p.i. respectively; but while the levels of bim protein slightly decreased after 36 h p.i., bax levels didn't change (Fig. 8a andb). Whereas the expressions of the anti-apoptotic members, bcl-2 and bcl-xL, were downregulated by CCoV-II infection (Fig. 8a). The densitometric analysis of the blots demonstrated a decreased expression of A c c e p t e d M a n u s c r i p t bcl-2 and of bcl-xL after 8 h and 12 h p.i. respectively (Fig. 8b). The data suggest that bax, bim, bcl-2 and bcl-xL may play a role on arbitrating the CCoV type II-induced apoptosis.

3.2-Effects of caspase inhibitors on cell viability and viral replication

To test if the apoptotic changes observed in CCoV type II-infected A-72 cells were caspase dependent, Z-VAD-FMK (pan-caspase inhibitor), Z-DEVD-FMK (caspase-3-specific inhibitor), Z-IETD-FMK (caspase-8-specific inhibitor), Z-LEHD-FMK (caspase-9 inhibitor) were added to the culture media before the infection. As shown in Fig. 9a, in presence of caspases inhibitors was observed a significant higher cell viability (P<0.001).

The effects of apoptosis on the replication of CCoV-II were assayed by comparing virus titers in the presence of caspase inhibitors. As reported in Fig. 9b, the results showed that the virus release in presence of caspase inhibitors didn't change compared with control. It is conceivable that apoptosis is not required for the release of progeny virions. These observations therefore suggest that CCoV-II replication don't requires caspase activation to complete the virus life cycle.

Discussion

It is believed that the modulation of apoptotic cell death, also known as programmed cell death, is relevant to diseases that are caused by various viruses. One of the main advantages of apoptotic cell death for virus infectivity is to facilitate the spread of virus progeny to the neighboring cells and to minimize the inflammatory reaction evoked by virus-infected cells on the host.

Coronaviruses are known to infect host cells by receptor-mediated endocytosis and some of them to cause cell-cell fusion during the late stages of infection, resulting in synctium formation and CPE [START_REF] Lai | The molecular biology of coronaviruses[END_REF]. Apoptosis was observed in Vero E6 cells infected by SARS-CoV (Yan et al., 2004) and in a recent publication it has been reported that CCoV induces apoptosis in cultured cells [START_REF] Ruggieri | Canine coronavirus induces apoptosis in cultured cells[END_REF]. It has been shown that in other coronavirus, e.g., avian infectious bronchitis virus [START_REF] Liu | Induction of caspase-dependent apoptosis in cultured cells by the avian coronavirus infectious bronchitis virus[END_REF], swine transmissible gastroenteritis virus [START_REF] Eleouet | The viral nucleocapsid protein of transmissible gastroenteritis coronavirus (TGEV) is cleaved by caspase-6 and -7 during TGEV-induced apoptosis[END_REF] and murine coronavirus [START_REF] Liu | Role of the mitochondrial signaling pathway in murine coronavirus-induced oligodendrocyte apoptosis[END_REF][START_REF] Liu | Murine coronavirus-induced oligodendrocyte apoptosis is mediated through the activation of the Fas signalling pathway[END_REF], apoptosis is induced by a caspase-dependent mechanism.

The importance of caspase activation during apoptosis is well established [START_REF] Hengartner | The biochemistry of apoptosis[END_REF]. Caspases play a central role in the effector phase during apoptotic cell death. To date, 14 mammalian caspases have been described [START_REF] Earnshaw | Mammalian caspases : structure, activation, substrates, and functions during apoptosis[END_REF].

A c c e p t e d M a n u s c r i p t

In this study we show that CCoV type II infection leads to caspase-dependent apoptotic cell death in A-72 cells, in vitro. First, we examined by MTT assay and morphological evaluations the CCoV-II infection, demonstrating a dose-and time-dependent trend of the apoptotic process virusinduced. Then, in an attempt to understand the mechanism of induction of apoptosis in CCoV-IIinfected cells we have investigated the caspase pathways. The results reported herein by FLICA method [START_REF] Bedner | Activation of caspases measured in situ by binding of fluorochrome-labeled inhibitors of caspases (FLICA): correlation with DNA fragmentation[END_REF] provide an unequivocal evidence that CCoV-II induces apoptotic cell death in vitro, involving caspase-dependent pathway of caspase-8 (death receptor-mediated) and caspase-9 (mitochondrial-mediated). This suggests that both the extrinsic and intrinsic pathways in the CCoV-II-induced apoptosis were involved. Our results match those [START_REF] Eleouet | The viral nucleocapsid protein of transmissible gastroenteritis coronavirus (TGEV) is cleaved by caspase-6 and -7 during TGEV-induced apoptosis[END_REF] who demonstrated that the transmissible gastroenteritis coronavirus (TGEV), also included in group 1 coronaviruses as CCoV-II, triggers caspase activation events, involving both the extrinsic and intrinsic pathways, during infection. Further apoptosis-associated caspase activation has been documented also in other coronavirus groups such as SARS CoV (group 2) [START_REF] Mizutani | Phosphorylation of p38 MAPK and its downstream target in SARS coronavirus-infected cells[END_REF], MHV (group 2) [START_REF] Chen | Murine coronavirus-induced apoptosis in 17Cl-1 cells involves a mitochondria-mediated pathway and its downstream caspase-8 activation and bid cleavage[END_REF], and IBV (group 3) [START_REF] Liu | Induction of caspase-dependent apoptosis in cultured cells by the avian coronavirus infectious bronchitis virus[END_REF].

However, in the present study, it was evident the activation of caspase-8 and -9 at and after 12 h p.i. by FLICA and Western blot analysis. Furthermore, caspase 9 was only partially activated compared to the others investigated and not changing between times 24 -72 hours suggesting that viral proteins could interfere with such activation. A higher protein expression level of caspase-3 and -6 compared to those of caspase-8 and -9, after 12 h p.i., was observed. These higher levels of activity achieved by caspase-3 and -6 could be related to the additive effect of both caspase-8 and -9 pathways that activate these executioner caspases.

Caspase-8 has been shown to activate the intrinsic pathway following the cleavage of bid, a pro-apoptotic member of the Bcl-2 family of proteins [START_REF] Li | Cleavage of BID by caspase 8 mediates the mitochondrial damage in the Fas pathway of apoptosis[END_REF]. The data herein presented are consistent with such mechanism, showing that the full-length bid expression level in cytosolic fraction decreased in time-dependent in virus-infected cells respect to mock-infected cells. We also described the relationships with bid-induced cytochrome c release in the cytosolic fraction from 12 h p.i. until the end of infection.

Bid is part of the bcl-2 family of proteins, which includes both pro-apoptotic proteins (for example bid, bax, bak, bcl-XS, bad, bim), and anti-apoptotic proteins (for example bcl-2, bcl-XL bcl-w, mcl-1). Therefore, the bcl-2 family of proteins have been considered as pivotal players in apoptosis. Our study has shown that CCoV-II infection down-regulated bcl-2 but up-regulated bax expression. Furthermore, densitometric analysis of bim, which is one of the candidates for initial mitochondrial activation, indicated a marked increase of protein expression level up to 36 h p.i..

Bim activates bax through interactions with mcl-1 and bcl-2 (Willis et al., 2007). Here we have A c c e p t e d M a n u s c r i p t reported that bim significantly shifted the relative contributions of bax to cell death. Then, in response to apoptotic stimuli, bax has to translocate to mitochondria before it can oligomerize and form pores [START_REF] Desagher | Mitochondria as the central control point of apoptosis[END_REF], and the relative decline in bcl-2 expression permits bax to gain dominance, the downstream effects of which most likely involve both direct and indirect activation of the cell death proteases that represent the final steps in the apoptotic pathway.

We have also shown additional evidence of downstream caspase activation evaluating the PARP cleavage during CCoV-II infection. PARP is a substrate for activated caspase-3 [START_REF] Li | Cleavage of Poly(ADP-ribose) polymerase measured in situ in individual cells: relationship to DNA fragmentation and cell cycle position during apoptosis[END_REF], and here we have shown elevated expression of activated caspase-3 and cleaved PARP in the CCoV-II-infected cells in time-dependent manner. Thus, the CCoV-IIinfection significantly increased PARP cleavage levels.

Furthermore, we observed the effects of apoptosis on both the replication of CCoV type II and virus titers in the presence of caspase inhibitors. The suppression of apoptosis by these inhibitors suggested that the activation of apoptosis doesn't represent an important step in virus release. These data are in agreement with previous studies, in fact, it was demonstrated that the inhibition of apoptosis, either by caspase inhibitors or by overexpression of the bcl-2 protein, did not affect SARS-CoV replication in Vero cells [START_REF] Ren | Apoptosis induced by the SARSassociated coronavirus in Vero cells is replication-dependent and involves caspase[END_REF][START_REF] Bordi | Bcl-2 inhibits the caspase-dependent apoptosis induced by SARS-CoV without affecting virus replication kinetics[END_REF], suggesting that apoptosis does not play a role in facilitating viral release.

Collectively, the results reported here demonstrate that: (i) apoptosis developing in a

CCoV-II infection depended on the activition of caspase-3,-6 -8 and -9;

  and b, the fulllength bid expression level evaluated by Western blot analysis in cytosolic fraction, decreased in time-dependent in virus-infected cells respect to mock-infected cells. The mitochondrial localization of 15 kDa fragment truncated bid (tbid) clearly appeared at 12 h p.i. Since this fragment contains the BH3 domain which is the functional part of bid for cytochrome c release, we examined whether the bid traslocation was accompanied by a mitochondrial release of cytochrome c into the cytosol. The relationships with bid-induced cytochrome c release is evident in Fig.7c and d whichshows the presence of cytochrome c in the cytosolic fraction from 12 h p.i. until the end of infection.

  (ii) apoptosis induced during in vitro infection of A-72 cells could occur both from the extrinsic pathway and the intrinsic pathway; (iii) CPE and cell death in A-72 cells, which are triggered by CCoV-II, are due to the induction of caspase activation, given the ability of caspase inihibitors to increase viability of CCoV-II-infected cell; and (iv) there is no interference between apoptosis and viral release.These results suggest that this coordinated interplay among caspases is crucial in attaining full activation of apoptotic signal in A-72 infected cells; however, the first direct link between caspase activation and CCoV-II-induced apoptosis remains to be explored and studies are under way in order to identify targets of viral protein involved in the modulation of apoptosis during CCoV-II-infection.

  .,Shenk, T.E., 1995. Viruses and apoptosis. Curr. Opin. Genet. Dev. 5, 105-111.

Fig. 1 -

 1 Fig. 1 -Effect of various MOI of CCoV-II on A-72 cell viability. After the indicated times of post infection, cell viability was evaluated with MTT assay. Values represent the mean ( SEM) of three different experiments performed in triplicate. *p < 0.05, ** p < 0.01, *** p < 0.001 compared to mock-infected cultures.

Fig. 3 -Fig. 4 -Fig. 5 -Fig. 6 -Fig. 7 -Fig. 8 -

 345678 Fig. 3 -Photomicrographs under fluorescence microscopy showing morphological changes in CCoV-II-infected cells (MOI 10) after orange acridine staining. A, mock-infected cells; B, CCoV-II -infected cells at 24 h p.i.; C, CCoV-II -infected cells at 48 h p.i.. Magnification: 400x.

Fig. 9 -

 9 Fig. 9 -Effect of caspase inhibitors on cell viability and virus release in CCoV-II-infected cells (MOI 10). a) A-72 cells were treated with Z-VAD-FMK (pan-caspase inhibitor), Z-DEVD-FMK (caspase-3-specific inhibitor), Z-IETD-FMK (caspase-8-specific inhibitor), Z-LEHD-FMK (caspase-9 inhibitor) 1 h prior to infection with CCoV-II. The cell viability were analyzed at 24 h

  Figure 8

Figure 9

 9 Figure 9

  Suzuki, K., Matsui, Y., Miura, Y., Sentsui, H., 2008. Equine coronavirus induces apoptosis in cultured cells. Vet. Microbiol. 129, 390-395. Willis, S.N., Fletcher, J.I., Kaufmann, T., van Delft, M.F.,Chen, L., Czabotar, P.E., Ierino, H.,

	Lee, E.F., Douglas Fairlie, W., Bouillet, P., Strasser, A., Kluck, R.M., Adams, J.M.,
	Huang, D.C.S., 2007. Apoptosis initiated when BH3 ligands engage multiple Bcl-2
	homologs, not Bax or Bak. Science 315, 856-859.
	Yan, H., Xiao, G., Zhang, J., Hu, Y., Yuan, F., Cole, D.K., Zheng, C., Gao, G.F., 2004. SARS
	coronavirus induces apoptosis in Vero E6 cells. J. Med. Virol. 73, 323-331.

A c c e p t e d M a n u s c r i p t


O Couvert

I Leguerinel

Pr Mafart
email: pierremafart@univ-brest.fr

Modelling the overall effect of pH on the apparent heat resistance of Bacillus cereus spores

Introduction

Among environmental conditions that affect spore thermoresistance, pH is a major factor in microbial destruction. It has been recognised for several years that low pH values reduce spore resistance (Alderton et al., 1976;[START_REF] Townsend | Heat resistance studies on spores of putrefactive anaerobes in relation to determination of safe processes for canned foods[END_REF][START_REF] Tsuji | Thermal resistance of Clostridium sporogenes (P.A. 3679)at the intermediate pH range[END_REF]. However, available information related to the quantitative effect of pH is scarce. [START_REF] Jordan | Studies in the dynamics of desinfection. XIV. The variation of the concentration exponent for hydrogen and hydroxyl ions with the mortality level using standard cultures of Bact. coli at 51°C[END_REF] observed a linear relationship between the D-value (decimal reduction time) of Escherichia coli and the pH of the heating menstruum. [START_REF] Davey | The effect of pH on continuous high temperature/short time sterilisation of liquids[END_REF] and, more recently, [START_REF] Mafart | Modelling combined effects of temperature and pH on heat resistance of spores by a linear-Bigelow equation[END_REF], proposed a model to describe the combined effect of temperature and pH on heat resistance of spores.

These models were developed from data where D-values were estimated by recovering surviving cells at optimum conditions and, particularly, at optimal pH of the recovery medium. However, it is well known that counts of survival spores after a heat treatment are greatly influenced by the characteristcs of the recovery medium (temperature, pH, water activity, composition). At non optimum recovery conditions, both a decrease in the number of viable cells capable of producing colonies and a decrease in the estimated decimal reduction time are observed. It is generally accepted that the pH of the recovery medium exerts a great influence on the apparent heat resistance of spores: D-values decrease as pH is reduced [START_REF] Cook | Relationship between heat activation and percentage colony formation for Bacillus stearothermophilus spores: effects of storage and pH on the recovery medium[END_REF][START_REF] Yokoya | Effect of several environmental conditions on the thermal death rate of endospores of aerobic, thermophilic bacteria[END_REF][START_REF] Cook | Factors affecting the heat resistance of Bacillus stearothermophilus spores. I. The effect of recovery conditions on colony count of unheated and heated spores[END_REF][START_REF] Mallidis | Evaluation of recovery media for heated spores of Bacillus stearothermophilus[END_REF][START_REF] Santos | Evaluation of citric acid and GDL in the recovery at different pH levels of Clostridium sporogenes PA3679 spores subjected to HTST treatment conditions[END_REF]Lopez et al., 1997).

This paper aims to present a simple overall model which takes into account both the effect of the pH of the heating menstruum and that of the recovery medium upon the observed D-value.

Assuming a "multiplicative" effect of the pH on the D-value, the influence of the heating menstruum pH can be written:

D = f (pH).D* (1)
where D* is the maximum D-value at the optimum pH. Similarly, the effect of the recovery medium pH can be written:

D' = f' (pH').D (2

Materials and methods

Micro-organism and spore production

The strain of Bacillus cereus (CNRZ 110) was obtained from the Institut National de Recherche Agronomie (France). Spores were kept in distilled water at 4°C.

Cells were precultivated at 37°C during 24 h in Brain Heart Infusion (Difco ). The preculture was used to inoculate nutritive agar plates (Biokar Diagnostics BK021) added with MnSO 4 40 mg l -1 and CaCl 2 100 mgl -1 on the surface area. Plates were incubated at 37°C for 5 days. Spores were then collected by scraping the surface of the agar, suspended in sterile distilled water, and washed three times by centrifugation (10000xg for 15 min) (Bioblock Scientific, model Sigma 3K30). The pellet was then suspended againin 5 ml distilled water and 5 ml ethanol. The obtained suspension was then kept at 4°C during 12 hours in order to eliminate vegetative non sporulated bacteria, and washed again three times by centrifugation. The final suspension (about 10 10 spores ml -1 ) was finally distributed in sterile Eppendorf microtubes and kept at 4°C.

Thermal treatment of spore suspension

D values in citrate-phosphate buffer adjusted were determined at 95°C

with one replicate at each pH ranging from 5 to 7.

First, 30µl of spore suspension was diluted in 3 ml buffer. Capillary tubes of 25 µl (vitrex) were filled with 10µl of sample and submitted to a thermal treatment in a thermostated oil bath. After heating, the tubes were cooled in water/ice bath, washed in a solution of soap and rinsed with sterile distilled water.

Finally, the ends were flamed with ethanol. The capillary tubes were broken at both ends and their contents poured into a tube containing 9 ml sterile tryptone salt broth (Biokar Diagnostics) by rinsing with 1 ml tryptone salt broth contained in a needle-equipped syringe.

Recovery conditions

Viable spores were counted by duplicate plating in nutritive agar (10g tryptone, 5g meat extract, 5g sodium chloride, 15 g agar for 1000ml

water)(Biokar Diagnostic) and incubated at 25°C for 6 days. The pH medium ranging from 5 to 7 was adjusted with H 2 SO 4 1N before autoclaving. After autoclaving the pH was measured and the final pH value was kept for calculations.

For media whose pH was lower than 5.5, 10g/l of agar was added. A solution of Na 2 SO 4 was added to adjust the medium at the same ionic strength.

Data analysis

D values were based on the reciprocal of slopes obtained when the log number of survivors was plotted against time.

Parameters of the models were fitted by simple or multiple linear regressions carried out with the STAT-ITCF software (Institut Technique du Fourrage France).

The goodness of fit of the model was evaluated by using the accounted for per cent variance (Snedecor and Cochran, 1969) which is given by: R r n n N

2 2 1 1 1 1 = - - - -- ( )( ) ( )
where n is the number of observations, N the number of terms and r 2 is the multiple regression coefficient. Complementarily, mean square errors were determined.

Results

In a first set of experiments spores were heated at 95°C in media ranging from pH 5 to 7 and recovered at 25°C , at the optimum recovery pH (6.7). Observed D-values are shown in table 1. logD values were then plotted vs (pH-pH*) 2 (fig1). The pH of maximal thermal resistance related to the studied strain of B.

cereus was close to 7.5 [START_REF] Gaillard | Modelling combined effects of temperature and pH on heat resistance of spores of Bacillus cereus[END_REF]. The model was then fitted with pH* = 7.5 according to a linear regression (table 2).

In a second set of experiments, spores were heated at 95°C in a medium of pH 7

and recovered at 25°C in media ranging from pH 5 to 7. Observed apparent decimal reduction times are shown in table 3. log D' values were then plotted vs (pH'-pH'*) with pH'* = 6.7. Fitted parameters according to the linear regression are presented in table 4. The observed satisfactory goodness of fit allows to adopt the same function for the destruction factor as for the stress factor (Eqn 5 and 6 respectively).

As foods make up the heating menstruum and the recovery medium, a third set of experiments, in which spores were recovered at the same pH as those of the heating menstruum, was carried out in order to validate the overall model.

Parameters of the overall model were not fitted but parameters'values obtained from the first two independent sets of data (tables 2 and4 Fitted parameters of this simplified model are respectively D*=2.27 min and Z pH =1.69, with R 2 =0.976 and a Mean Square Error of 4.34.10 -3 . The robustness of the new overall parameter Z pH was checked through the following test: z pH and z' pH were fitted again while standard values were affected to pH* and pH'* (7 instead of 7.5 and 6.7 respectively). As expected, new values were clearly different from previous ones (z pH =2.62 instead of 3.10 and z' pH =2.04 instead of 1.75). However, when calculating the overall Z pH value from the following

relationship 1 1 1 2 2 2 Z z z pH pH pH = + ' (10)
Z pH =1.61 was found, which is close to the value determined from the last set of data.

It is clear that substituting model (7)( 5 parameters when pH* and pH'* are fitted instead of being fixed ) for the more parsimonious model (9) ( only 2 parameters) is likely to lead to some lost of goodness of fit : a R 2 value of 0.976 instead of 0.983 and a Mean Square Error of 4.34.10 -3 instead of 2.99.10 -3 are obtained. However, this drawback is largely set off by the simplification and the improvement of the model's robustness.

When a n decimal reduction ratio is aimed, at the reference temperature (121.1°C) and at pH 7, the sterilisation value must be:

F = nD* 121.1°C (11)
At 121.1°C but at a pH different from 7, the time of heat treatment leading to the same destruction ratio will be:

t = nD' 121.1°C (12)
Combining Eqn ( 11) and ( 12) gives: The function L(pH) must be regarded as a partial apparent Biological Destruction Value (partial because pH is here the only considered factor).

Obviously, the studied strain of B.cereus which is very sensitive to recovery medium pH, is not a representative strain likely to be used as an indicator strain in order to develop standard heat process calculations. From data of [START_REF] Lopez | Effect of pH heating medium on the thermal resistance of Bacillus stearothermophilus spores[END_REF], it was possible to fit z pH values of 4 strains of Bacillus stearothermophilus which were heated at 120°C. Another set of data from the same laboratory (Lopez et al., 1997) allowed to fit z' pH values related to the same strains. For each strain, overall Z pH values were deduced from Eqn (10), (see Table 6). Table 7 shows the magnitude of partial apparent Biological Destruction Values related to B. stearothermophilus with an average Z pH value of 3.4. Further works would be needed in order to validate the model more generally and to determine Z pH value related to other representative species, especially Clostridium botulinum. 2 and4.

Legends of tables

Legends of figures

  ) where D' is the apparent decimal reduction time at pH' (pH of the recovery medium). The overall model is provided by combining Eqn (1) and (2): D' = f (pH).f' (pH').D* (3) Model (3) can be linearized by the following logarithmic transformation: log D' = log D* + log f (pH) + log f' (pH'which pH* is the optimum pH of the heating menstruum corresponding to the maximum heat resistance. On account of the similitude of patterns which can be observed between curves plotting D vs pH and D' vs pH' respectively, and in order to keep the homogeneity of the overall model, we tested the following stress factor:

  ) were used in order to compute D' values. Squares on Figure 3 represent observed log D' values while the continuous line corresponds to the calculated values according to the model. The correlation between observed and calculated values gives a R 2 value of 0.968 and a Mean Square Error of 8.83.10 -3.DiscussionCurrently the D-values used to establish sterilisation process were not taken into account in the recovery phase. The overall model (Eqn 7) is proving to describe successfully the influence of the pH on the heat resistance of spores both during the heat treatment and the recovery phase. However its lack of robustness, due to overparameterization, makes it difficult for it to be used for calculating heat food processes. Lack of robustness is pointed out by the following test: if instead of calculating D' values with parameters separately estimated from the first two independent sets of data, parameters are fitted according to the actual observed D' values from the third set of data (pH* and pH'* are then held to be fitted parameters and not fixed values), quite different values are obtained from those previously determined (table5). On account of the great variability of microbial species and strains, it seems worthwhile, in the framework of standard calculations, to fix pH* and pH'* values to 7.Consequently, when pH' = pH ,

Fig. 1 .

 1 Fig. 1. Fitting of log D vs (pH-pH*) 2 with pH*=7.5

  Figure 1

Table 1 .

 1 The effect of the heating medium pH on the heat resistance of Bacillus cereus spores. D 95(min)

Table 2 .

 2 Rate coefficient related to equation 5 (pH*=7.5)

Table 3 .

 3 The effect of the recovery medium pH on the apparent heat resistance of Bacillus cereus spores. D' 95(min)

Table 4 .

 4 Rate coefficient related to equation 6 (pH'*=6.7)

Table 5 .

 5 Rate coefficient related to equation 7 Table6. z pH , z' pH and Z pH values related to Bacillus stearothermophilus , according toLopez et al.'s data (1996Lopez et al.'s data ( , 1997) )

Table 7 .

 7 Partial apparent Biological Destruction Values vs medium pH with Z pH =3.4

Table 1 .

 1

	pH	D 95°C (min)
	5	0.64
	5.25	0.79
		0.76
	5.5	0.98
		1.10
	6	1.71
		1.62
	6.5	1.91
		2.07
	7	2.52
		2.79

Table 4 .

 4

	Number of data	13
	R 2	0.942
	Mean Square Error	5.77.10 -3
	D 95°C	2.17 min
	z' pH	1.74

Table 5 .

 5

	Number of data	20
	R 2	0.983
	Mean Square Error	2.99.10 -3
	pH*	7.87
	pH'*	7.38
	D 95°C *	3.36 min
	z pH	3.59
	z' pH	2.53

Table 6 .

 6

	Strain	z pH	z' pH	Z pH
	12980	3.82	3.06	2.39
	7953	3.97	3.80	2.75
	15951	3.45	3.21	2.35
	15952	4.12	2.94	2.39

Abstract

A simple overall model was proposed to describe the effect of both pH of the heating menstruum and pH of the recovery medium on apparent spore heat resistance of Bacillus cereus. Applied to foods making up both heating and recovery media, the model can be reduced to only 2 parameters. Its goodness of fit and its robustness enable it to be applied for the optimisation of heat treatments. However, further experiments should be undertaken to validate the model for other species and to determine parameters related to reference species such as Clostridium botulinum. KEYWORDS: pH, heat resistance, recovery, Bacillus cereus.