type. Shamoon Naseem

Jochen Meens

Joerg Jores

Martin Heller

Stefan Dübel

Michael Hust

Gerald-F Gerlach
email: gfgerlach@gmx.de

Phage display-based identification and potential diagnostic application of novel antigens from Mycoplasma mycoides subsp. mycoides small colony

Keywords: Mycoplasma mycoides subsp. mycoides small colony type, phage display, diagnostic antigens

Contagious Bovine Pleuropneumonia caused by Mycoplasma mycoides subsp. mycoides small colony type is a respiratory disease of considerable economic importance in sub-Saharan Africa; control of the disease in Africa is hampered by diagnostic tests which are suited for herd-level but not for individual animal diagnostics. In the work presented we identified 22 potential immunogenic antigens of the Kenyan outbreak strain B237 by using phage display technology.

We determined the relative strength of immunogenicity, the discriminatory capacity between bovine positive and negative sera, and the cross reactivity with rabbit hyperimmune sera directed against 15 different mycoplasmal species. The three best-performing antigens, a conserved hypothetical protein (MSC_0636), a glycosyl transferase (MSC_0108), and an acyl carrier protein phosphodiesterase (MSC_0029) were considered candidate diagnostic proteins. They were expressed as GST-fusion proteins in E. coli, purified, and used in an ELISA as solid phase antigens. The diagnostic potential of the recombinant antigens was tested using the sera of ten experimentally infected animals and six control animals. This prototype test resulted in 100 % diagnostic sensitivity and specificity. In comparison, the complement fixation test and the competitive ELISA performed with a diagnostic sensitivity of 70 % and 60 %, respectively.

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

Introduction

Contagious Bovine Pleuropneumonia (CBPP) is a highly infectious respiratory disease in cattle caused by Mycoplasma mycoides subsp. mycoides small colony type (MmmSC) and solely transmitted by animal to animal contact. The disease is of major economic importance in subsaharan Africa (FAO, 2003) with a constant risk of reintroduction into other parts of the world. Diagnosis is primarily based on serology currently done by complement fixation test (CFT), competitive ELISA (cELISA) (Le [START_REF] Goff | A competitive ELISA for the specific diagnosis of contagious bovine pleuropneumonia (CBPP)[END_REF] and a lipoprotein Q-based ELISA [START_REF] Bruderer | Serodiagnosis and monitoring of contagious bovine pleuropneumonia (CBPP) with an indirect ELISA based on the specific lipoprotein LppQ of Mycoplasma mycoides subsp. mycoides SC[END_REF]. Both ELISA tests closely correlate with the CFT reported to have a diagnostic sensitivity of 64 to 70 % and a diagnostic specificity of 98 % [START_REF] Amanfu | Comparison between c-ELISA and CFT in detecting antibodies to Mycoplasma mycoides mycoides biotype SC in cattle affected by CBPP in Botswana[END_REF][START_REF] Marobela-Raborokgwe | Comparison of complement fixation test, immunoblotting, indirect ELISA, and competitive ELISA for detecting antibodies to Mycoplasma mycoides subspecies mycoides small colony (SC) in naturally infected cattle from the 1995 outbreak in Botswana[END_REF]. For a reliable diagnosis of disease on the individual animal level the diagnostic sensitivity needs to be improved without losing specificity. To achieve this goal, novel diagnostic antigens need to be identified.

Recently it has been shown that this goal can be achieved effectively using a nonlytic phage display approach based on phage M13 and designated as pHORFsystem [START_REF] Kügler | Identification of immunogenic polypeptides from a Mycoplasma hyopneumoniae genome library by phage display[END_REF]; it improves the panning and screening process compared to the lytic lambda-based phage display system [START_REF] Beghetto | Discovery of new Mycoplasma pneumoniae antigens by use of a wholegenome lambda display library[END_REF][START_REF] March | Phage library screening for the rapid identification and in vivo testing of candidate genes for a DNA vaccine against Mycoplasma mycoides subsp. mycoides small colony biotype[END_REF][START_REF] Kügler | Identification of immunogenic polypeptides from a Mycoplasma hyopneumoniae genome library by phage display[END_REF]. Furthermore, the included open reading frame selection and the packaging into "Hyperphage", a helperphage not encoding pIII protein on its genome, improve the subsequent panning process [START_REF] Hust | Enrichment of open reading frames presented on bacteriophage M13 using hyperphage[END_REF][START_REF] Kügler | Identification of immunogenic polypeptides from a Mycoplasma hyopneumoniae genome library by phage display[END_REF][START_REF] Soltes | On the influence of vector design on antibody phage display[END_REF][START_REF] Rondot | A helper phage to improve single-chain antibody presentation in phage display[END_REF].

In the work presented, the phage M13-based pHORF system was used to identify novel immunogenic epitopes of MmmSC which are potentially suitable not only for herd-level but also for individual animal diagnostics.

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

Materials and Methods

Bacterial strains, phages, growth conditions, plasmids and sera

The strains, plasmids, primers and sera used in this work are listed in Table 1.

Escherichia coli strains were cultured in Luria-Bertani medium supplemented with the appropriate antibiotics (ampicillin, 100 µg/l, kanamycin, 30 µg/l). The MmmSC field isolate B237 was grown as stationary culture for three days in modified PH medium [START_REF] Kirchhoff | Isolation of a motile mycoplasma from fish[END_REF], supplemented with 20 % equine serum (WDT, Garbsen, Germany) at 37 °C in a 5 % CO 2 atmosphere.

The 10 bovine anti-MmmSC sera used in this study were derived from experimentally infected cattle [START_REF] Jores | Assessment of in vitro interferon-gamma responses from peripheral blood mononuclear cells of cattle infected with Mycoplasma mycoides ssp. mycoides small colony type[END_REF]. A positive control serum was obtained by pooling equal volumes of these sera. The six negative sera used were obtained from uninfected cattle from a CBPP free region within Kenya. A negative control serum was generated by pooling equal volumes of these sera. The rabbit sera directed against different mycoplasmal species had been raised previously against the respective whole cell bacterins.

Construction of an MmmSC genomic phage display-library and enrichment of open reading frames

Site-specific mutation of the phagemid vector pHORF3 [START_REF] Kügler | Identification of immunogenic polypeptides from a Mycoplasma hyopneumoniae genome library by phage display[END_REF] was performed using primers oMHLacZPro_f, oMHORF3_pme_rX1 and oMHORF3_pme_rXX2 (Table 1) such that the unique PmeI site used for insertion of foreign DNA is shifted by 1 and 2 bp resulting in plasmids pHORF3X and A c c e p t e d M a n u s c r i p t 5 pHORF3XX, respectively (Fig. 1). Genomic DNA of the MmmSC African field strain B237 was isolated as described previously [START_REF] Meens | Identification and immunological characterization of conserved Mycoplasma hyopneumoniae lipoproteins Mhp378 and Mhp651[END_REF] and digested using five different blunt end-cutting restriction endonucleases (AluI,PsiI,SspI,HpyCH4V,RsaI). DNA fragments with a size of 100-1000 bp were isolated after separation in an agarose gel using the Qiagen PCR DNA and gel band purification kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions, and ligated into PmeI-restricted vectors pHORF3, pHORF3X and pHORF3XX (Fig. 1). The genomic MmmSC phage library was constructed as described previously [START_REF] Kügler | Identification of immunogenic polypeptides from a Mycoplasma hyopneumoniae genome library by phage display[END_REF].

Selection of phage encoding immunogenic oligopeptides of MmmSC and production of individual phage clones for screening

The panning of phage presenting immunogenic peptides on the surface was performed using the bovine positive control serum pre-absorbed to Hyperphage in order to prevent unspecific reactivity with antibodies directed against phagesurface epitopes. The preabsorbed serum was coupled to MaxiSorb ® 96 well microtitre plates (Nunc, Wiesbaden, Germany) via rabbit anti-bovine IgG (Sigma, Deisenhofen, Germany), and four successive panning rounds were performed as previously described [START_REF] Kügler | Identification of immunogenic polypeptides from a Mycoplasma hyopneumoniae genome library by phage display[END_REF]. Individual clones were picked from the titration plates of the last panning round.

For production of individual phage clones, polypropylene 96-well U-bottom plates (Greiner bio-one, Frickenhausen, Germany) containing 175 µl of 2 x TY-GA medium (1 % Bacto-Yeast Extract, 1.6 % Bacto Tryptone, 0.05 % NaCl; 100 mM glucose, 100 µg/ml ampicillin) per well were inoculated with single clones A c c e p t e d M a n u s c r i p t 6 and incubated at 37 °C with constant shaking at 250 rpm overnight. Cultures were expanded to 1.5 ml of 2 x TY-GA medium, infected with 5 x 10 9 cfu Hyperphage per tube and incubated at 37 °C without shaking for 30 min, followed by 30 min incubation with shaking at 250 rpm. Cells were centrifuged at 3,220 x g for 10 min and the supernatants were discarded. The bacterial pellets were resuspended in 1.5 ml of 2 x TY medium containing ampicillin (100 µg/ml) and kanamycin (30 µg/ml) and incubated at 30 °C at 250 rpm overnight for phage production.

In order to confirm the antigenicity of individual phage clones PolySorb ® 96 well microtitre plates (Nunc) were coated overnight with 10 µl (1 x 10 10 pfu/ml) of individual phage in 100 µl of carbonate buffer (100mM NaHCO 3 , 46 mM Na 2 CO 3 , pH 9.6). The ELISA was developed using the positive control serum (diluted 1:100), goat anti-bovine IgG conjugated with horseradish peroxidase (HRP; Jackson ImmunoResearch Laboratories, Dianova, Hamburg, Germany) as a conjugate and 2,2-azino-di-[3-ethylbenzithiazoline sulfonate] (ABTS; Roche Diagnostics, Mannheim, Germany) as a substrate. The staining reaction was stopped by adding 100 µl of 50% (v/v) methanol. The absorbance at 405 nm and scattered light at 492 nm were measured using a Sunrise ® microtiter plate reader (Tecan, Crailsheim, Germany).

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and

Western blot SDS-PAGE was performed following standard procedures using a Protean II A c c e p t e d M a n u s c r i p t 7 Minigel system (BioRad Inc, Munich, Germany). Western blotting of the SDS-PAGE minigels was done using the Mini Trans-Blot ® system (BioRad). The bovine positive and negative control serum and a rabbit anti-GST serum were used as primary antibodies; blots were developed using alkaline phosphataseconjugated goat anti-bovine or goat anti-rabbit IgG antibody (Jackson ImmunoResearch Laboratories) and BCIP (5-bromo-4-chloro-3-indolyl phosphate) and NBT (nitroblue tetrazolium) as substrate.

Preparation of recombinant fusion proteins and ELISA set-up

The MmmSC genes encoding immunogenic epitopes as assessed by ELISA of individual phage clones were identified by database analysis. The respective parts of the genes located between two TGA codons were amplified by PCR using primer pairs with an EcoRI-(upstream primer) or NotI-restriction endonuclease site (downstream primer) at their 5´ends (Table 1). The corresponding PCR fragments were digested with EcoRI and NotI and ligated into pGEX-5x-2, resulting in plasmids pMSC0029-500, pMSC0108-500 and pMSC0636-500.

Recombinant fusion proteins were prepared as inclusion bodies as described previously [START_REF] Gerlach | Cloning and expression of a transferrin-binding protein from Actinobacillus pleuropneumoniae[END_REF] and dissolved in guanidine hydrochloride (4 M).

To remove small-sized contaminating material, the solution was washed twice in guanidine hydrochloride (4M) using a 10 kDa cut-off (MicroCentricon 10 system, Amicon, Beverly, Mass., U.S.A.). Using BSA as reference the concentration of the purified protein (desalted by precipitation with trichloroacetic acid [TCA]) was then determined by SDS PAGE and staining with Coomassie blue. The structure A c c e p t e d M a n u s c r i p t and immunogenicity of the fusion proteins was confirmed by Western blot analysis using positive and negative control sera as well as an anti-GST serum.

For use in an ELISA, fusion proteins in guanidine hydrochloride were pipetted in a petri dish, diluted by adding the appropriate volume of carbonate coating buffer with a 10 ml pipette, and used as solid phase antigens on a PolySorb 96 well microtiter plate (Nunc) coating 100 µl volumes per well overnight at 4 °C. The coating concentration was optimized by checkerboard titration of the antigen, development of the ELISA using 2-fold serial dilutions of the positive and negative control sera, goat-anti-bovine peroxidase, and ABTS, and analysis of the log-log-transformed data. Prior to use in the ELISA sera were absorbed with whole-cell lysate of E. coli-pGEX-5x-2 transformants for 1 h at room temperature in order to remove antibodies directed against contaminating E. coli components in the semi-purified antigen preparation. 2).

Results

Identification of immunogenic peptides

Selection of clones with diagnostic potential

The individual clones were amplified and the relative strength of serum reactivity was determined by phage ELISA. Eight clones showing a reactivity of more than 30 OD% with respect to MmmSC whole cell lysate (Table 2) were considered to be of potential diagnostic value and were subjected to further investigation. The 3) and, therefore, this clone was excluded from further analyses. The three remaining clones (MSC_0029, MSC_0108 and MSC_0636) were expressed as GST fusion proteins.

Potential of recombinant GST-fusion proteins as diagnostic antigens

All fusion proteins had a molecular mass corresponding to the calculated masses of 32.5, 32.0, and 45.0 kDa, respectively. Approximately 90% of the fusion protein preparations consisted of recombinant antigen (Fig. 2A). The proteins reacted strongly positive with both the anti-GST and the positive control serum but did not show any reaction with the negative control serum (Fig. 2B-D).

The purified fusion proteins, upon checkerboard titration performed to determine the suitable coating concentration of 2.5 µg/ml, were used as solid-phase antigens.

In a first ELISA, using rabbit hyperimmune sera previously raised against MmmSC, a 32-times higher titre of the positive versus the negative serum was observed (data not shown) thereby showing suitability of the antigens in principal.

In a second ELISA, serial two-fold dilutions of the bovine positive and negative control sera were used after absorption against a lysate of E. coli pGEX-5x-2 transformants. The slope of the regression curve of the log-log transformed data obtained with the positive control serum was determined to be 0.72 thereby confirming the suitability of the coating concentration; in addition, an at least three-fold difference in titre and a 2.5-fold difference of OD%-values was observed between positive and negative control serum thus confirming the discriminatory power of the antigens. The intra-assay variation for all three ELISAs was determined to be below 5 OD% and the inter-assay variation below 20 OD%. Applying a cut-off range of 50 OD% all three ELISAs resulted in an analytical sensitivity and specificity of 100 % as compared to a 70 % sensitivity for the complement fixation test and 60 % for the competitive ELISA commercially available (Fig. 3).

Discussion

The control of the continuous spread of CBPP in sub-Saharan Africa is severely limited by the lack of sufficiently sensitive and specific diagnostic tests. In an attempt to improve the diagnostic possibilities we set out to identify novel MmmSC-derived antigens using a non-lytic M13-based phage display system [START_REF] Kügler | Identification of immunogenic polypeptides from a Mycoplasma hyopneumoniae genome library by phage display[END_REF]. The construction of phage display libraries in three different reading frames using five different restriction endonucleases with a 4mer recognition sequence was considered to result in a reasonable coverage of the MmmSC genome although the three vectors facilitate the generation of a functional pIII fusion protein only for one of the three possible reading frames (Fig. 1). Thus, in silico analysis of the MmmSC PG1 genome revealed that the restriction enzyme digests resulted in a total of 9,178 restriction endonuclease fragments of 100 to 1000 bp in length. In total these fragments cover 3,413,598 bp which corresponds to 2.8 times the MmmSC genome size. The finding that each peptide was present at least twice in the pool of 147 clones analyzed implied that the number of panning rounds was sufficient to eliminate non-specific binders A c c e p t e d M a n u s c r i p t 12 [START_REF] Eshaghi | Identification of epitopes in the nucleocapsid protein of Nipah virus using a linear phage-displayed random peptide library[END_REF][START_REF] Kügler | Identification of immunogenic polypeptides from a Mycoplasma hyopneumoniae genome library by phage display[END_REF]. When comparing the open reading frames (ORFs) identified with the results of a previous study by March et al. [START_REF] March | Phage library screening for the rapid identification and in vivo testing of candidate genes for a DNA vaccine against Mycoplasma mycoides subsp. mycoides small colony biotype[END_REF] only ORF MSC_0266 encoding a pyruvate dehydrogenase was identified in both studies. This is likely due to the different methodologies used; thus, March et al. [START_REF] March | Phage library screening for the rapid identification and in vivo testing of candidate genes for a DNA vaccine against Mycoplasma mycoides subsp. mycoides small colony biotype[END_REF]) used a lytic bacteriophage Lambda ZAP Express vector with insert sizes of 6 to 8 kb and screened using rabbit IgG antibodies. Some of the initially identified antigens are intracellular proteins. This is in accordance with many other studies, where intracellur proteins were found to be immunogenic [START_REF] Kügler | Identification of immunogenic polypeptides from a Mycoplasma hyopneumoniae genome library by phage display[END_REF][START_REF] March | Phage library screening for the rapid identification and in vivo testing of candidate genes for a DNA vaccine against Mycoplasma mycoides subsp. mycoides small colony biotype[END_REF][START_REF] Delvecchio | Proteomic profiling and identification of immunodominant spore antigens of Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis[END_REF][START_REF] Cho | Immunogenic proteins in the cell envelope and cytoplasm of vancomycin-resistant enterococci[END_REF][START_REF] Kodzius | Rapid identification of allergen-encoding cDNA clones by phage display and high-density arrays[END_REF]. A possible explanation for this phenomenon could be the release of intracelluar proteins by cell lysis or by programmed cell death [START_REF] Lewis | Programmed cell death in bacteria[END_REF].

Four of 22 identified antigens were consistently recognized by all positive sera and showed sufficient discrimination of all negative sera. Using rabbit hyperimmune sera directed against 15 other bovine mycoplasmal species these four antigens were tested for serological cross-reactivity which might limit diagnostic specificity. The cross-reactivity of all four antigens with M. capricolum subsp. capricolum appeared acceptable since sequence analysis of the 16S rRNA [START_REF] Pettersson | Sequence analysis of 16S rRNA from mycoplasmas by direct solid-phase DNA sequencing[END_REF][START_REF] Pettersson | Phylogeny of the Mycoplasma mycoides cluster as determined by sequence analysis of the 16S rRNA genes from the two rRNA operons[END_REF] and of five conserved protein-coding sequences [START_REF] Manso-Silván | Phylogeny of the Mycoplasma mycoides cluster based on analysis of five conserved protein-coding sequences and possible implications for the taxonomy of the group[END_REF] showed a high sequence similarity and, consequently, a close phylogenetic relationship between MmmSC, M. mycoides subsp. capri and M. capricolum subsp. capricolum, and the other members of the "mycoidescluster". In addition, it has been reported that M. capricolum subsp. capricolum can cause CBPP-like disease in cattle [START_REF] Ajuwape | Pathogenicity of Mycoplasma capricolum subspecies capricolum for cattle immunosuppressed with[END_REF]. Acholeplasma modicum and M. bovis, however, are not members of the "mycoides-cluster";

A c c e p t e d M a n u s c r i p t 13 since serological cross-reactivity particularly with the frequently occurring bovine pathogen M. bovis is unacceptable for a diagnostic test, ORF MSC_1062 was excluded from further analyses. Immunoblot analysis of the three remaining antigen-GST fusion proteins showed a strong reaction particularly of antigens MSC_0108 and MSC_0636 (Fig. 2) thereby confirming the initial panning results where these two peptides had elicited the highest reactivity.

The testing of the cohort sera (16 infected and six non-infected) resulted in a diagnostic sensitivity and specificity of 100 % each in comparison to a diagnostic sensitivity of 70 % for the CFT and 60 % for the cELISA (Fig. 3). This result implies that all three antigens are potentially suitable to improve the reliability of current serological testing which is primarily hampered by low diagnostic sensitivity [START_REF] Marobela-Raborokgwe | Comparison of complement fixation test, immunoblotting, indirect ELISA, and competitive ELISA for detecting antibodies to Mycoplasma mycoides subspecies mycoides small colony (SC) in naturally infected cattle from the 1995 outbreak in Botswana[END_REF]. However, the cohort sera used were by no means representative; in addition, MmmSC is known to show a high antigenic variability [START_REF] Gonçalves | Antigen heterogeneity among Mycoplasma mycoides subsp. mycoides SC isolates: discrimination of major surface proteins[END_REF][START_REF] Persson | Variable surface protein Vmm of Mycoplasma mycoides subsp. mycoides small colony type[END_REF] and different breeds of cattle may show a distinct serological response to MmmSC infection. Therefore, in order to support the potential suitability of these antigens for field application, additional sera from experimentally infected cattle as well as field sera from different geographical regions need to be investigated in the future. (1:100). All three clones pMSC0029-500, pMSC0108-500, pMSC0636-500 were found to strongly react with the positive control serum and not with the negative control serum. In addition, GST showed only minor reactivity with the positive control serum. CBPP-infected animals Uninfected animals

[n]

Figure 3 A c c e p t e d M a n u s c r i p t Frequency of clones among the random sample of 134 clones sequenced.

2)

The optical density obtained with MmmSC whole cell lysate used as solid phase antigen served as positive control (100 %).

Table 2 A c c e p t e d M a n u s c r i p t Table 3

  After performing four panning rounds with the positive control serum a total of 147 clones (49 from each of the three libraries based on vectors with the cloning site in three different reading frames with respect to the 3' pIII protein-encoding ORF) were sequenced. One hundred and thirty four clones contained analyzable sequence and showed an ORF consisting of a MmmSC-derived genomic DNA fragment in frame with both the pelB signal sequence and the PIII proteinencoding ORF. Upon NCBI Blast analysis (http://www.ncbi.nlm.nih.gov/blast/ A c c e p t e d M a n u s c r i p t 9 Blast.cgi) 22 different clones were identified with each clone present at least twice in the random selection of sequenced clones (Table

  clones were individually amplified, and the discrimination between positive and negative control serum was investigated by ELISA. For six of the eight clones (MSC_0029, 0108, 0636, 0656, 1009 and 1062) at least a 3-fold difference in titre was observed between the positive and the negative control serum (data not shown). These six clones were tested with respect to their discriminatory potential using the ten individual positive sera and the six individual negative sera contained in the respective control sera. Four of the clones (MSC_029, 108, 636 and 1062) reacted with all individual positive sera; no overlap in OD%-values was seen between positive and negative sera (data not shown). These four clones were investigated for cross reactivity to other mycoplasmal species using rabbit hyperimmune sera previously raised against 15 different mycoplasmal species. All four clones (MSC_0029, MSC_0108, MSC_0636, and MSC_1062) were highly positive with the hyperimmune serum directed against MmmSC. Two clones showed clear cross-reactivity (i.e. more than 1.5-fold of the average reactivity with the negative control serum) with the serum directed against M. capricolum subsp. capricolum; for clone MSC_1062 an additionalA c c e p t e d M a n u s c r i p t 10 cross-reactivity against A. modicum and M. bovis was observed (Table

  and coating concentrations the ELISA was performed three times in duplicate for 1:500 dilutions of the individual positive and negative sera.

Fig. 1 .

 1 Figure captions:

Fig. 2 .

 2 Fig. 2. Characterisation of GST fusion proteins. (A) SDS PAGE showing

Fig. 3 .

 3 Fig. 3. ELISA results using GST fusion proteins. Sera of ten individual CBPP-

Fig. 2 :

 2 Figure 1

  Figure 2

Table 1

 1 Bacterial strains, plasmids, primers and sera used in this work

	Table 1
	Strains, plasmids,	Characteristic(s)	Source or Reference
	primers or sera
	Strains
	Mmm SC	MmmSC B237, isolated from an outbreak in	Jores et al., 2008
		Thika/Kenya in 1997
	E. coli EC-TS40.6	K12 strain	AG Dübel
	E. coli DH5αF´F´ endA1 hsdR17 (r K gyrA (NaI r ) relA1 Δ(lacZYA-argF)U169 deoR -m K -) supE44 thi-1 recA1	New England Biolabs, Frankfurt,
		[Φ80dlacΔ(lacZ)M15]	Germany
	M13K07	Helperphage carrying a kanamycin resistance	Stratagene,
		determinant	Amsterdam, The
			Netherland
	"Hyperphage"	Helperphage carrying a kanamycin resistance	Rondot et al., 2001
		determinant and truncated gIII
	Plasmids
	pHORF3	pHORF3 used for insertion of foreign DNA	Kügler et al., 2008
	pHORF3X	pHORF3 was modified such that the singular	This work
		PmeI site used for insertion of foreign DNA is
		shifted by 1bp, allowing the in-frame fusion with
		gIII in an additional reading frame
	pHORF3XX	pHORF3 was modified such that the singular	This work
		PmeI site used for insertion of foreign DNA is
		shifted by 2bp, allowing the in-frame fusion with
		gIII in an additional reading frame
	pGEX5x2	E. coli expression vector carrying an bla	GE Healthcare,
		resistance determinant, for construction of GST	Munich, Germany
		fusion proteins
	pMSC0029-500	plasmid encoding the GST-MSC_0029 fusion	This work
		protein
	pMSC0108-500	plasmid encoding the GST-MSC_0108 fusion	This work
		protein
	pMSC0636-500	plasmid encoding the GST-MSC_0636 fusion	This work
		protein

Table 2

 2 Immunogenic peptides identified in Mycoplasma mycoides subsp. mycoides small colony type (MmmSC)

	No Fq 1) Genome position Product from MmmSC	Insert length (bp)	Locus tag OD% 2)
	1	3	733268 -733109	Conserved hypothetical	159	MSC_0636 56.7 %
	2	2	127879 -127937	Glycosyl transferase	59	MSC_0108 51.4 %
	3	7	696995 -697122	ATP dependent protease	128	MSC_0613 43.7 %
	4	7	1153378 -1153506	DNA directed RNA	199	MSC_1009 39.5 %
				polymerase beta subunit
	5	8	32410 -32351	Acyl carrier protein	60	MSC_0029 37.3 %
				phosphodiesterase
	6	2	1205835 -1205959	AAA family ATPase	124	MSC_1062 36.9 %
	7	6	1032859 -1032922	Amino acid permease	64	MSC_0908 34.3 %
	8	5	750456 -750516	Conserved hypothetical	60	MSC_0656 32.7 %
	9	2	304438 -304508	Pyruvate dehydrogenase	71	MSC_0266 30.0 %
	10	5	218668 -218814 Oligopeptide ABC trans-	147	MSC_0184 30.0 %
				porter, component OppA
	11	8	159499 -159643	Hypothetical	144	MSC_0135 29.0 %
				transmembrane protein
	12	4	1160317 -1160401 Glucose inhibited division	85	MSC_1017 28.3 %
				protein A
	13	2	23668 -23767	Mannitol-1-phosphate 5-	99	MSC_0017 26.8 %
				dehydrogenase
	14 26	77529 -77641	Transcriptional regulator;	184	MSC_0068 26.7 %
				NifR3 family
	15	3	940581 -940693	Conserved hypothetical	112	MSC_0826 25.2 %
	16 19 1117031 -1117172 UDP glucose 4-epimerase	141	MSC_0978 24.5 %
	17	5	44289 -44393	D-Lactate dehydrogenase	105	MSC_0034 24.5 %
	18	4	701239 -701317	Prolipoprotein	78	MSC_0617 23.8 %
	19	6	1210760 -1210841 Conserved hypothetical	82	MSC_1066 22.3 %
				transmembrane protein
	20	4	698434 -698492	Putative hydrolase of	69	MSC_0614 22.1 %
				HAD family
	21	3	385965 -386234	tRNA pseudoridine	269	MSC_0336 21.0 %
				syntase B
	22	3	97781 -98005	Prolipoprotein ; putative	224	MSC_0079 21.0 %
				phosphate ABC transport
	1)

Table 3

 3 ELISA cross-reactivity of four clones with rabbit sera directed against 16 different

	Mycoplasma species
	Serum raised aganist	culture		OD% +/-standard deviation
		collection
		number	MSC_029 MSC_0108 MSC_636 MSC_1062
	A. axanthum S 743	NCTC 10138	28 ± 8.6	26 ± 4.1	35 ± 2.3	45 ± 1.8
	A. laidlawii PG 8	NCTC 10116	39 ± 8.3	43 ± 0.2	42 ± 3.5	39 ± 1.7
	A. modicum PG 49	NCTC 10134	32 ± 6.8	26 ± 0.6	32 ± 0.4	51 ± 1.2
	M. alkalescens PG 51	ATCC 29103	17 ± 0.6	21 ± 2.1	43 ± 1.3	41 ± 0.5
	M. arginini G 230	NCTC 10129	21 ± 1.6	21 ± 0.3	26 ± 1.6	32 ± 0.3
	M. bovigenitalium PG 11	ATCC 19852	17 ± 0.3	17 ± 1.9	22 ± 1.1	25 ± 0.3
	M. bovirhinis PG 43	NCTC 10118	36 ± 4.8	40 ± 2.1	44 ± 14.9	44 ± 3.2
	M. bovis Donetta PG 45	ATCC 25523	44 ± 13.7	39 ± 2.5	41 ± 5.6	49 ± 2.3
	M. bovoculi M 165/69	NCTC 10141	44 ± 0.4	42 ± 8.8	44 ± 7.1	33 ± 2.9
	M. californicum ST-6	ATCC 33461	21 ± 2.5	22 ± 0.9	32 ± 0.6	32 ± 2.2
	M. capricolum subsp. capricolum california kid	NCTC 10154	45 ± 21.7	48 ± 0.5	45 ± 1.4	57 ± 4.9
	M. gallinarum PG 16	ATCC 19708	19 ± 1.9	28 ± 2.4	45 ± 1.1	44 ± 0.3
	M. gatae C 8	ATCC 23392	30 ± 4.2	21 ± 4.0	27 ± 1.5	31 ± 2.0
	M. leachii PG 50	NCTC 10133	46 ± 6.0	27 ± 2.0	33 ± 4.3	45 ± 7.3
	M. mycoides subsp. capri	NCTC 10706	30 ± 1.2	27 ± 2.3	30 ± 0.5	34 ± 3.4
	y-goat
	MmmSC PG 1	NCTC 10114	100 ± 4.6 100 ± 12.1 100 ± 2.5 100 ± 8.9
	Negative serum	-	27 ± 3.0	30 ± 2.6	29 ± 1.0	36 ± 0.7

Acknowledgments

SN was funded by Higher Education Commission (HEC) Pakistan and German

Academic Exchange Service (DAAD), Germany.

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


Jochen Meens
email: jochen.meens@tiho-hannover.de

V Bolotin

G Gerlach

-F

Characterization of a highly immunogenic Mycoplasma hyopneumoniae lipoprotein Mhp366 identified by peptide spot array

Keywords: Mycoplasma hyopneumoniae, immunogenic proteins, peptide-spot array, lipoprotein

Enzootic Pneumonia (EP) in pigs caused by Mycoplasma hyopneumoniae is a highly prevalent, chronic respiratory disease, which causes considerable economic losses in the swine industry. Most herds are vaccinated, but classical bacterin vaccines do not prevent colonization and it is not possible to detect flourishing M. hyopneumoniae infections in vaccinated herds since commonly used commercial ELISAs can not differentiate infected from vaccinated animals. To solve this problem, new immunogenic proteins, up-regulated or solely expressed during infection, need to be identified. For this purpose a peptide spot array was constructed which presents 105 potential linear B-cell epitopes identified by in-silico analysis in 35 putative lipoproteins encoded on the genome of M. hyopneumoniae type strain 232. Subjecting this array to immunoblotting using porcine convalescent serum revealed a single strongly immunoreactive epitope on the Mhp366 protein which did not react with serum from bacterin-immunized pigs. In addition, it was not possible to detect Mhp366 in total cell lysates of in-vitro grown M. hyopneumoniae strains, using a polyclonal rabbit serum raised against a recombinant GST-Mhp366 fusion protein. To investigate the possibility of using an Mhp366-based ELISA in the field for differentiating vaccinated herds with and without a flourishing infection it was shown that i) homologues of the corresponding mhp366 gene were present in all 17 M. hyopneumoniae strains and porcine lung samples tested from different geographic origins and ii) an ELISA based on epitope-specific synthetic peptides as solid phase antigen allowed a classification of field samples. Therefore, Mhp366 might be the first antigen identified which facilitates the detection of flourishing M. hyopneumoniae infections even in vaccinated herds.

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

Introduction

Enzootic Pneumonia (EP) in pigs caused by Mycoplasma hyopneumoniae is a highly prevalent, chronic respiratory disease affecting pigs worldwide [START_REF] Ross | Mycoplasmal diseases[END_REF]. Pure M. hyopneumoniae infections are characterised by high morbidity and low mortality, but are often complicated by secondary infections with other bacterial or viral pathogens [START_REF] Maes | Control of Mycoplasma hyopneumoniae infections in pigs[END_REF].

The control of M. hyopneumoniae infections critically requires suitable vaccines and diagnostic tools. Two commercial ELISAs are commonly used for monitoring the health status of pig herds, a monoclonal antibody-based blocking ELISA (M. hyopneumoniae ELISA, Oxoid; [START_REF] Feld | A monoclonal blocking ELISA detecting serum antibodies to Mycoplasma hyopneumoniae[END_REF]) and an indirect whole cell ELISA (IDEXX HerdChek M. hyopneumoniae, IDEXX Laboratories). Comparison of ELISA results revealed a high specificity of both assays whereas the sensitivity strongly depends on the vaccination status and the time point of sampling [START_REF] Erlandson | Evaluation ot three serum antibody enzyme-linked immunosorbent assays for Mycoplasma hyopneumoniae[END_REF][START_REF] Ameri-Mahabadi | Comparison of two swine Mycoplasma hyopneumoniae enzyme-linked immunosorbent assays for detection of antibodies from vaccinated pigs and field serum samples[END_REF]. Most importantly, none of the tests can be used to differentiate infected from vaccinated animals. The development of new diagnostic tools requires the identification of new immunogenic proteins, preferentially those which are up-regulated or solely expressed during infection.

An important group of proteins found in all Mycoplasma species are surface-exposed lipoproteins which mediate different interactions with the host cells, like adherence [START_REF] Sachse | Epitope mapping of immunogenic and adhesive structures in repetitive domains of Mycoplasma bovis variable surface lipoproteins[END_REF] or damage of host tissue [START_REF] Hopfe | OppA, the ecto-ATPase of Mycoplasma hominis induces ATP release and cell death in HeLa cells[END_REF] and which are major targets of the host's immune response [START_REF] Zuo | Mycoplasma lipoproteins and Toll-like receptors[END_REF]. Since the genome sequences of three M. hyopneumoniae strains have recently been published [START_REF] Minion | The genome sequence of Mycoplasma hyopneumoniae strain 232, the agent of swine mycoplasmosis[END_REF]Vasconcelos et al., 2005), the cellular localization of all annotated proteins can be predicted to define sub-proteomes like membrane proteins or lipoproteins.

In the present study, a peptide spot array representing predicted immunogenic epitopes of putative M. hyopneumoniae lipoproteins was constructed; immunoblotting resulted in the identification of Mhp366 as an infection-related protein which might facilitate the detection of flourishing M. hyopneumoniae infections even in vaccinated herds.

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

Material and Methods

Bacterial strains and culture conditions.

All M. hyopneumoniae strains (Table 1) were cultured in modified Friis medium [START_REF] Friis | Some recommendations concerning primary isolation of Mycoplasma suipneumoniae and Mycoplasma flocculare a survey[END_REF] at 37°C and 5% CO 2 . Cells were grown to late exponential growth phase as determined by measurement of color-changing units (CCU), harvested by centrifugation at 20,000 x g for 30 min and washed three times in phosphate-buffered saline (PBS). Escherichia coli DH5 F´ [START_REF] Raleigh | Select topics from classical bacterial genetics[END_REF]) was grown at 37°C on LB agar plates or in LB medium supplemented with ampicillin (100 µg/ml), D-glucose (0.5%, w/v) or IPTG (1 mM) as required.

Synthesis and identification of immunogenic epitopes.

Putative lipoproteins were identified by analysing the genome of M. hyopneumoniae strain 232 (acc.

no. NC_006360) using the programs Lipo [START_REF] Berven | Analysing the outer membrane subproteome of Methylococcus capsulatus (Bath) using proteomics and novel biocomputing tools[END_REF] (http://www.bioinfo.no/tools/lipo) and LipoP 1.0 (www.cbs.dtu.dk/services/LipoP; [START_REF] Juncker | Prediction of lipoprotein signal peptides in Gram-negative bacteria[END_REF]. Putative lipoproteins recognized by both programs (Table 2) were used for prediction of potential B-cell epitopes using the ABCpred program (www.imtech.res.in/raghava/abcpred; (Sahaand Raghava, 2006) with a window of 20 amino acid residues as predicted epitope length and a threshold setting of 0.7.

For each putative lipoprotein, three epitopes showing the highest predictive scores were selected, and overlapping 15-mer peptides initiating at every third amino acid were synthesized as an array of spots on an aminopegylated cellulose membrane (AIMS Scientific Products, Braunschweig, Germany; [START_REF] Frank | The SPOT-synthesis technique. Synthetic peptide arrays on membrane supports-principles and applications[END_REF]). Immunoblot analyses of peptide spot arrays was essentially done as described [START_REF] Frank | SPOT synthesis. Epitope analysis with arrays of synthetic peptides prepared on cellulose membranes[END_REF]. Briefly, the ethanol-wetted membrane was washed three ) as chromogenic substrates. The reaction was stopped by washing blots twice in PBS. For repeated use, the membrane was stripped as described [START_REF] Frank | SPOT synthesis. Epitope analysis with arrays of synthetic peptides prepared on cellulose membranes[END_REF].

DNA techniques, plasmids and sequencing

Genomic DNA from culture-grown M. hyopneumoniae was isolated as described [START_REF] Caron | Diagnosis and differentiation of Mycoplasma hyopneumoniae and Mycoplasma hyorhinis infections in pigs by PCR amplification of the p36 and p46 genes[END_REF]. DNA modifying enzymes were purchased from NEB (Bad Schwalbach, Germany), preparation of plasmid DNA, DNA ligation, transformation, and agarose-gel electrophoresis were done following standard procedures [START_REF] Sambrook | Molecular Cloning: a Laboratory Manual[END_REF]. Expression vector pGEX5x3 was obtained from Amersham Biosciences (Freiburg, Germany). Taq polymerase, cloning vector pCR ® 2.1-TOPO and TopoTA cloning kits were purchased from Invitrogen (Karlsruhe, Germany).

Standard PCR reactions were performed as described earlier [START_REF] Meens | Identification and immunological characterization of conserved Mycoplasma hyopneumoniae lipoproteins Mhp378 and Mhp651[END_REF]. DNA from lung lesions was isolated from tissue pieces (20-25 mg) by digestion in 180 l of lysis buffer (20 ml 0.1 M Tris-HCl [pH 6.4]; 4.4 ml 0.2 M EDTA [pH 8.0]; 1 g/ml of guanidinium thiocyanate; 0.5 ml Triton X-100; 20 l 600 mAU/ml proteinase K) for 5 min at 65°C, followed by silica DNA extraction, essentially as described by Boom et.al. (Boom et al., 1990). In a nested PCR approach, 5 μl of the extracted DNA were first amplified using 0.2 mM of primers oMhp366A/H, followed by the second PCR, performed with primers oMhp366A/B and 1 μl of the amplification product from the first PCR as a template. Chromosomal DNA from strain M. hyopneumoniae 232 was used as positive control. Plasmid constructs were confirmed by DNA sequencing, performed by SeqLab GmbH (Goettingen, Germany). The BLAST program [START_REF] Altschul | Basic local alignment search tool[END_REF] was used for nucleotide A c c e p t e d M a n u s c r i p t 6 and protein homology search. The Clustal W program [START_REF] Thompson | CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice[END_REF] was used for multiple-sequence protein alignments.

Construction of GST-Mhp366 fusion protein expression vector

The recombinant expression of the mhp366 gene in E. coli required the replacement of 4 nonsense TGA codons at amino acid positions 279, 323, 422 and 483 of the mature protein by tryptophanencoding TGG triplets. Using chromosomal DNA of the M. hyopneumoniae type strain 232 and mutagenic primer pairs oMhp366A-H (Table 1) with class IIS restriction sites at their 5´ends [START_REF] Spring | Use of class IIS restriction enzymes for site-directed mutagenesis: variations on Phoenix mutagenesis[END_REF], four PCR fragments 366-AB (811 bp), 366-CD (152 bp), 366-EF (345 bp) and 366-GH (401 bp) were generated, restricted with BsmBI and ligated. After reamplification using primers oMhp366A and oMhp366H, the resulting PCR product was ligated into the SmaI and NotI digested plasmid pGex5x3, yielding pMhp366-501. Successful construction of the expression vector was confirmed by DNA sequencing and small-scale expression of the GST fusion protein.

Expression and purification of recombinant GST-Mhp366 fusion protein.

Protein expression using E. coli DH5 F´/pMhp366-501 was essentially done as described earlier [START_REF] Meens | Identification and immunological characterization of conserved Mycoplasma hyopneumoniae lipoproteins Mhp378 and Mhp651[END_REF]. The resulting aggregated fusion protein was isolated as previously described [START_REF] Gerlach | Cloning and expression of a transferrin-binding protein from Actinobacillus pleuropneumoniae[END_REF], followed by further purification by preparative 16 x 18 cm SDS-PAGE on a SE600 unit (Amersham Biosciences, Freiburg, Germany). After Coomassie staining, the GST-Mhp366 band was cut from the gel and isolated by electro-elution.

Protein analyses and preparation of sera.

Protein concentrations were determined using a MicroBC TM Assay (Uptima Interchim, Montlucon Cedex, France). Analysis of protein samples on 1D SDS-PAGE and Western blotting were done following standard procedures [START_REF] Sambrook | Molecular Cloning: a Laboratory Manual[END_REF]. Broad range prestained protein marker were used as molecular weight standards.

Rabbit antibodies were raised against the GST-Mhp366 protein; 100 g fusion protein dissolved in 0.5 ml PBS were emulsified with 10% (final concentration) of Emulsigen®-Plus (MVP Inc., Ralston, NE). Immunisation of a New Zealand white rabbit and preparation serum was done as described [START_REF] Meens | Identification and immunological characterization of conserved Mycoplasma hyopneumoniae lipoproteins Mhp378 and Mhp651[END_REF]. A porcine convalescent serum pool from five non-vaccinated pigs infected with M. hyopneumoniae, which was used as a positive reference serum in all experiments, and porcine hyperimmune serum have been described earlier [START_REF] Meens | Identification and immunological characterization of conserved Mycoplasma hyopneumoniae lipoproteins Mhp378 and Mhp651[END_REF]. The porcine negative control serum was pooled from 10 non-vaccinated, non-infected pigs.

ELISA

Two commercial ELISAs, an indirect ELISA (IDEXX HerdChek Mycoplasma hyopneumoniae ® , IDEXX Laboratories, Westbrook, Maine) and a blocking ELISA (Oxoid Mycoplasma hyopneumoniae ELISA, Oxoid Ltd, Cambridgeshire, UK) were used according to the manufacturers' instructions.

Putative immunogenic epitopes (nonamers) were synthesized with an amino-terminal biotinylation linked by a 2-aminohexanoic acid linker and purified by high-performance liquid chromatography.

Lyophilized peptides were resuspended in distilled water to obtain stock solutions of 10 mg/ml. For the Mhp366-specific peptide-based ELISA, Nunc Immobilizer Streptavidin F96 microtiter plates were coated with 100 l of the corresponding synthetic, biotinylated peptide at a concentration of 0,1 g/ ml in coating buffer (2.83 g/l NaHCO 3 ; 1.59 g/l Na 2 CO 3 [pH 9.6]) and incubated at 4°C overnight. Serial twofold dilutions of the sera were added and incubated for 1 h at room temperature. Goat anti-pig or goat anti-rabbit peroxidase conjugates (Dianova, Hamburg, Germany)

were each incubated for 1 h at room temperature. The ELISA was developed using 2,2-azino-di-[3ethylbenzithiazoline sulfonate] (ABTS; Roche Diagnostics, Mannheim, Germany) as a substrate.

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

Results

Construction and analysis of M. hyopneumoniae lipoprotein peptide array

The genome of the M. hyopneumoniae type strain 232 [START_REF] Minion | The genome sequence of Mycoplasma hyopneumoniae strain 232, the agent of swine mycoplasmosis[END_REF] was analysed in-silico using the programs Lipo [START_REF] Berven | Analysing the outer membrane subproteome of Methylococcus capsulatus (Bath) using proteomics and novel biocomputing tools[END_REF] and LipoP 1.0 [START_REF] Juncker | Prediction of lipoprotein signal peptides in Gram-negative bacteria[END_REF] to identify putative lipoproteins. A total of 35 ORFs were detected by both programs (Table 2) and chosen for further analysis using the ABCpred server [START_REF] Saha | Prediction of continuous B-cell epitopes in an antigen using recurrent neural network[END_REF] which is widely used to predict linear B-cell epitopes. Based on these results, three peptide sequences showing the highest predictive scores were chosen for each putative lipoprotein. If equal scores for more than three epitopes were obtained, sequences evenly distributed within the protein were selected. Since all epitope sequences were represented by three overlapping 15-mer peptides initiating at every third amino acid, a total of 315 peptides (supplemental material Table 1) were synthesized and spotted onto cellulose membranes [START_REF] Frank | The SPOT-synthesis technique. Synthetic peptide arrays on membrane supports-principles and applications[END_REF] for further analysis by immunoblotting.

The spot membrane was blotted using a porcine convalescent serum pool and a porcine hyperimmune serum raised against a commercial M. hyopneumoniae vaccine and the signal intensity of corresponding spots was compared. Predicted epitopes showing a positive reaction in only one or two out of three peptides were not considered because the core sequence of the corresponding B-cell epitope was obviously not completely covered. As shown in Fig. 1A, the porcine convalescent serum recognized, in particular, the peptide spots 82-84 corresponding to the first epitope predicted from the mhp366 gene. In addition, epitopes of the proteins Mhp164 (spots 16-18), Mhp293 (spots 37-39), Mhp364 (spots 73-75), Mhp682 (spots 301-306) and Mhp695

(spots 310-312) were also recognized, but to a lesser extent. As shown in Fig. 1B, the Mhp366 epitope was not recognized by the hyperimmune serum, whereas Mhp164 (spots 16-18) and Mhp293 (spots 37-39) again showed a positive reaction, comparable to the convalescent serum.

None of the peptide spots was recognized by the negative control serum (data not shown). The The mhp366 gene has a length of 1,665 bp (555 amino acid residues), starting at position 432,758 in the genome of M. hyopneumoniae strain 232 (acc. no. AE017332; [START_REF] Minion | The genome sequence of Mycoplasma hyopneumoniae strain 232, the agent of swine mycoplasmosis[END_REF]). It encodes the putative lipoprotein Mhp366 (acc. no. AAV27851) with a calculated molecular mass and IP of 64,394 Da and 9.90; respectively. The predicted epitope recognized by the convalescent serum covers the amino acid positions 68 to 88 within the Mhp366 protein ( 68 QKENSQKNDVVNSQNKTEKTE 88 ), the overlapping sequence of the three peptides which should define the actual B-cell epitope, is located at position 74 to 82 ( 74 KNDVVNSQN 82 ).

Immediately following this epitope, a region of 4 tandem repeats of the sequence motif [KR]TE starts which has recently been characterized by [START_REF] De Castro | Variable number of tandem aminoacid repeats in adhesionrelated CDS products in Mycoplasma hyopneumoniae strains[END_REF].

Antigenic specificity of immunogenic Mhp366 protein.

The plasmid pMhp366-501 encodes a GST-Mhp366 fusion protein composed of the amino-terminal GST followed by the mature part of Mhp366 starting at Cys 22 . The purification of the recombinant fusion protein was controlled by SDS-PAGE analysis and Coomassie staining, which confirmed the homogeneity of the protein preparation and the apparent molecular mass of around 90 kDa (Fig. 2A), corresponding to the value calculated from the deduced amino acid sequence (89,869 Da). As expected, the GST-Mhp366 fusion protein and the GST protein were recognized by GST-specific antibodies (Fig. 2B). Using the porcine convalescent serum, the GST-Mhp366 fusion protein was clearly recognized and this reaction was solely based on the Mhp366 part of the fusion protein, since the GST protein was not recognized by the convalescent serum (Fig. 2C, lane 1). In contrast, the porcine hyperimmune serum showed only a very weak reaction with the GST-Mhp366 fusion protein (Fig. 2D), thus confirming the results obtained on the peptide spot array. In addition, a Mhp366-specific signal in the expected size of 62 kDa (mature Mhp366 protein) could not be detected in total cell lysates of M. hyopneumoniae type strains J and 232

A c c e p t e d M a n u s c r i p t 10 grown in Friis medium using a polyclonal rabbit serum raised against the GST-Mhp366 fusion protein (Fig. 3B) although a broad spectrum of immunogenic bands was visible when porcine convalescent serum was used (Fig. 3C). Two protein bands, 95 kDa and 45 kDa in size, recognized by the Mhp366-specific polyclonal rabbit serum (Fig. 3B, lanes 1 and 2) were also detected by rabbit pre-immune serum (data not shown) and, therefore, the reaction was considered as non-specific.

Sequence analysis of the immunogenic epitope and adherent VNTR regions in various M. hyopneumoniae strains and diagnostic samples

A possible future application of the immunogenic epitope identified in Mhp366 as a diagnostic marker facilitating the differentiation of infected and vaccinated animals required a more detailed examination of its distribution and sequence variations among M. hyopneumoniae isolates from different geographic origins. For this purpose, a PCR fragment (800 bp) was amplified using primers oMhp366A / oMhp366B and chromosomal template DNA prepared from a total of fourteen M. hyopneumoniae strains from Denmark (11), Belgium (2) and Germany (1) (Table 1).

In addition, three porcine lung samples from Ukraine were also analysed directly by nested PCR.

The sequencing revealed the presence of mhp366 homologous genes in all strains and samples tested. The alignment of the obtained amino acid sequences confirmed the presence of the immunogenic epitope whose core sequence is highly conserved, showing only two variations at amino acid position 75 (N/K) and 82 (N/D), respectively (Fig. 4). The VNTR region is also present in all samples. The number of repeats varies between 3 and 5, with most sequences containing 4 copies of the [KR]TE motif.

ELISA

The alignment of immunogenic epitopes (Fig. 4) revealed the presence of four different core sequences. Synthetic peptides corresponding to these sequences were synthesized (Table 1) and A total of 80 field serum samples from 9 herds were first tested using two commercial ELISA kits.

The results of IDEXX HerdChek M. hyopneumoniae were determined on the basis of the samplepositive (S/P) ratio, resulting in 17 positive (S/P > 0.4), 61 negative (S/P < 0.3) and 2 suspect samples (S/P from 0.3 to 0.4). The monoclonal antibody-based blocking ELISA (Oxoid M. hyopneumoniae ELISA) revealed 17 positive samples (OD < 50% of buffer control OD), 60 negative (OD > 65% of buffer control OD) and 3 suspect samples (50 to 65 % of buffer control OD). The overall agreement between both ELISAs was calculated to be 92.5% (data not shown), A c c e p t e d M a n u s c r i p t

Discussion

Vaccination of piglets against M. hyopneumoniae is an important prophylactic measure to control Enzootic Pneumonia. Commercial bacterin vaccines are applied in many countries in more than 70 % of the pig herds [START_REF] Maes | Control of Mycoplasma hyopneumoniae infections in pigs[END_REF]; in Germany, it is the most frequently applied vaccination in pigs with more then 85 % of the herds being vaccinated. Although the beneficial effects of vaccination are well documented [START_REF] Haesebrouck | Efficacy of vaccines against bacterial diseases in swine: what can we expect?[END_REF][START_REF] Maes | Control of Mycoplasma hyopneumoniae infections in pigs[END_REF], the failure of currently used vaccines to prevent colonization [START_REF] Sibila | Effect of sow vaccination against Mycoplasma hyopneumoniae on sow and piglet colonization and seroconversion, and pig lung lesions at slaughter[END_REF] or to reduce transmission significantly [START_REF] Meyns | Quantification of the spread of Mycoplasma hyopneumoniae in nursery pigs using transmission experiments[END_REF]) are major drawbacks. Due to the high vaccination coverage using inactivated whole cells, the detection of ongoing M. hyopneumoniae infections in vaccinated herds is strongly impeded. The construction of DIVA marker vaccines, which is based on the absence of one immunogenic protein [START_REF] Van Oirschot | Present and future of veterinary viral vaccinology: a review[END_REF] to bypass this problem, is not possible since M. hyopneumoniae is not amenable to genetic manipulation.

In order to identify new infection-specific serological markers, we used an in-silico approach to predict immunogenic epitopes within putative lipoproteins. In the first step, the genome sequence of strain 232, which is commonly used to study virulence and vaccination regimens of M. hyopneumoniae [START_REF] Minion | The genome sequence of Mycoplasma hyopneumoniae strain 232, the agent of swine mycoplasmosis[END_REF], was analysed using the programs Lipo [START_REF] Berven | Analysing the outer membrane subproteome of Methylococcus capsulatus (Bath) using proteomics and novel biocomputing tools[END_REF] and LipoP 1.0 [START_REF] Juncker | Prediction of lipoprotein signal peptides in Gram-negative bacteria[END_REF]. A total of 35 ORFs were detected by both programs (Table 2). In a second step, potential linear B-cell epitopes were predicted using the ABCpred program [START_REF] Saha | Prediction of continuous B-cell epitopes in an antigen using recurrent neural network[END_REF].

The epitope length window for predicted epitopes was set at 20 amino acid residues, corresponding to most linear B-cell epitopes which are 5 to 30 amino acid residues in length [START_REF] Saha | Prediction of continuous B-cell epitopes in an antigen using recurrent neural network[END_REF]. On the peptide array, all predicted epitopes were represented by three overlapping 15-mer peptides initiating at every third amino acid, thereby covering 21 amino acid residues. The immunoblot experiments revealed only one epitope from the Mhp366 protein which was clearly differentially recognized by convalescent and hyperimmune serum, respectively (Fig. 1). Epitopes Homology analysis of the mhp366 gene using the BLAST program revealed no significant homology to genes or proteins from other bacterial species, but within the M. hyopneumoniae genome the mhp366 gene has four paralogs (mhp367, mhp445, mhp037 and mhp414), all except mhp414 being putative lipoproteins. The sequence identity of this paralogous gene family relating to mhp366 varies between 50% (mhp367) and 29% (mhp445), but the epitope identified in our study is unique to mhp366. None of these proteins has yet been functionally characterized.

The mhp366 gene has recently been described in two publications. De Castro and colleagues analysed the distribution of variable number of amino acid repeats (VNTARs) in five M. hyopneumoniae strains [START_REF] De Castro | Variable number of tandem aminoacid repeats in adhesionrelated CDS products in Mycoplasma hyopneumoniae strains[END_REF] and found three to five copies of a conserved

[KR]TE motif in mhp366-homologous genes, located immediately downstream of the immunogenic epitope. Our analysis of 17 additional strains and diagnostic samples confirmed these results. Most strains contain four copies of the [KR]TE motif and the sequence of the immunogenic epitope is highly conserved showing only two amino acid variations (Fig. 4).

A microarray-based study to search for transcriptional changes during infection revealed a 2.43 fold up-regulation of the mhp366 gene in M. hyopneumoniae cells collected from bronchial alveolar lavage fluid of infected pigs [START_REF] Madsen | Transcriptome changes in Mycoplasma hyopneumoniae during infection[END_REF]. The values for transcript level differences observed in this study are in general comparably low, but the up-regulation of mhp366 is the thirdhighest among all genes of M. hyopneumoniae strain 232. These results are in good agreement with our findings. The Mhp366-specific epitope is strongly recognized by convalescent serum, but not by hyperimmune serum (Fig. 1), which was produced using a commercial bacterin vaccine containing inactivated M. hyopneumoniae cells. The weak reactivity might be due to low or negligible amounts of Mhp366 protein in M. hyopneumoniae cells grown under laboratory conditions as it is strongly supported by the results of our Western blotting experiments. of Mhp366-specific antibodies in both sera. For recombinant antigens, it has already been shown that higher antigen levels induce a stronger and more rapid immune response [START_REF] Himmelrich | Immune responses induced by recombinant BCG strains according to level of production of a foreign antigen: malE[END_REF] and this might also be true for native bacterial antigens. In total cell lysates of both M. hyopneumoniae strains J and 232 grown in Friis medium, it was not possible to detect a protein signal using a Mhp366-specific polyclonal rabbit serum (Fig. 3B). Nevertheless, very low amounts of Mhp366 below the detection limit of the rabbit serum might be present even in in-vitro grown cells because the hyperimmune serum still shows a weak positive reaction with GST-Mhp366.

Based on our initial results, synthetic peptides corresponding to the four different epitope core sequences (Fig. 4) were tested as coating antigens. A strong ability to discriminate between positive and negative reference sera from non-vaccinated infected or healthy pigs, respectively, could be demonstrated (Fig. 5a). The sequence variations observed in M. hyopneumoniae strains and diagnostic samples did not influence the recognition, since all four peptides showed a similar reactivity (Fig. 5a). The examination of 80 field serum samples supported our hypothesis. Thus, whereas 17 samples were recognized as positive by two commercial ELISA kits, only four samples showed an ELISA activity ≥ 100 OD% compared to the positive reference serum pool on our peptide based ELISA. The lower number of pigs reacting in the Mhp366 antigen ELISA most likely mirrors the absence of a prime-boost effect due to vaccination with respect to this antigen. This is supported by the gap observed in the range of ELISA results. None of the samples ranged from 70 to 100 OD%, the vast majority of samples ranged from 10 to 40 OD%. Most likely, this large group represents background levels caused by vaccination or unspecific reactions. To support this hypothesis, a more detailed examination of hyperimmune sera raised against different commercial vaccines, as well as larger numbers of field serum samples obtained from herds with a defined M. hyopneumoniae vaccination status would be necessary. These experiments should not only confirm our present results but also allow us to define a threshold value to discriminate between as listed in supplementary Table 1.

PTKESKVSPVEENTQKENSQKNDVVNSQNKTEKTEKTQGTE----NQTTDNNFWSEST J PTKESKVSPVEENT-----QKKDVVNSQNKTEKTERTE------NNQTTDSNFWSEST 7448 PTKESKVSPVEKNT-----QKKDVVNSQNKTEKTEKTEKTERTE-NQTTDNNFWSDST PMS PTKESKVSPVEENT-----QKKDVVNSQNKTEKTERTE-------NQTTDSNFWSEST 7442 PTKESKVSPVEENT-----QKKDVVNSQNKTDKTERTE-------NQTTDSNFWSEST Mp18 PTKESKVSPVEENT-----QKNDVVNSQDKTEKTEKTERTE---NNQTTDSNFWSEST Mp47 PTKESKVSPVEENT-----QKKDVVNSQNKTEKTERTE------NNQTTDSNFWSEST Mp73 PTNESKVSPVEENT-----QKNDVVNSQDKTEKTEKTERTE---NNQTTDSNFWSEST Mp88 PTKESKVSPVEENT-----QKNDVVNSQDKTEKTEKTERTE---NNQTTDSNFWSEST Mp182 PTNESKVSPVEENT-----QKNDVVNSQDKTEKTEKTERTE---NNQTTDSNFWSEST Mp195 PTKESKVSPVEENT-----QKNDVVNSQDKTEKTEKTERTE---NNQTTDSNFWSEST Mp263 PTKESKVSPVEENT-----QKNDVVNSQDKTEKTEKTERTE---NNQTTDSNFWSEST Mp473 PTNESKVSPVEENT-----QKNDVVNSQDKTEKTEKTERTE---NNQTTDSNFWSEST Mp529 PTKESKVSPVEENT-----QKNDVVNSQDKTEKTEKTERTE---NNQTTDSNFWSEST Mp612 PTKESKVSPVEENT-----QKNDVVNSQDKTEKTEKTERTE---NNQTTDSNFWSEST Mp654 PTKESKVSPVEENT-----QKNDVVNSQDKTEKTEKTERTE---NNQTTDSNFWSEST Mhyop3873 PTKESKVSPVEKNT-----QKKDVVNSQNKTEKTEKTERTE----NQTTDNNFWSDST MhyopB2V2W20 PTKESKVSPVEKNT-----QKKDVVNSQNKTEKTEKTERTE----NQTTDNNFWSDST MhyopOef91 PTKESKVSPVEKNT-----QKKDVVNSQNKTEKTEKTERTE----NQTTDNNFWSDST VB-1A PTKESKVGPVEENT-----QKNDVINSQNKTERTEKTERTE---NNQTTDSNFWSEST VB-2A PTKESKVGPVEENT-----QKNDVINSQNKTERTEKTERTE---NNQTTDSNFWSEST VB-3A PTKESKVGPVEENT-----QKNDVINSQNKTERTEKTERTE---

  times with Tris-buffered saline [pH 7.0] (TBS) for 10 min and incubated overnight at 4°C in membrane blocking solution (MBS; 80% TBS-T [TBS with 0.05% Tween 20], 20% [vol/vol] casein-based blocking buffer concentrate [Thermo Scientific 37532], 5% [w/v] sucrose [pH 7.0]).

  min with T-TBS, the membrane was incubated for 2 h at room temperature with porcine convalescent or hyperimmune serum against M. hyopneumoniae[START_REF] Meens | Identification and immunological characterization of conserved Mycoplasma hyopneumoniae lipoproteins Mhp378 and Mhp651[END_REF] diluted 1:200 in MBS. After washing three times with T-TBS, alkaline phosphatase-conjugated goat anti-swine immunoglobulin G (Dianova, Hamburg, Germany) 1:2000 diluted in MBS was applied for 1 h. Blots were washed with T-TBS and citrate-buffered saline [pH 7.0] (CBS) and developed using BCIP (5-bromo-4-chloro-3-indolylphosphate) and MTT (thiazolyl blue tetrazolium, Sigma M2128

  Frankfurt, Germany) and LMW calibration kit (Amersham Biosciences, Freiburg, Germany)

  the Mhp366 epitope by convalescent serum but not by hyperimmune serum led us to examine its properties in more detail.

  antigens. A porcine convalescent serum pool from five non-vaccinated pigs identified as infected with M. hyopneumoniae was used as a positive reference serum. Plates were coated with 0.1 g/ml of the particular peptides (as determined by checkerboard titration using the porcine convalescent serum) or with an equal mixture of all peptides, each at a concentration of 0.025 g/ml. As shown in Fig.5a, the four single peptides and the peptide mixture showed very similar reactivity with the convalescent serum pool, and a strong discrimination between positive and negative control serum (pooled serum from 10 non-vaccinated, non-infected pigs).

  which is in good agreement with recently published comparison of both tests[START_REF] Ameri-Mahabadi | Comparison of two swine Mycoplasma hyopneumoniae enzyme-linked immunosorbent assays for detection of antibodies from vaccinated pigs and field serum samples[END_REF].The relative strength of field serum samples reactivity towards the immunogenic epitope was determined on ELISA plates coated with the equal mixture of epitope-specific peptides. The results, given as OD% with respect to the positive reference serum pool are shown in Fig. 5b. The field samples could be clearly separated into two groups. The first group includes four samples showing ELISA activities ≥ 100 OD% compared to the positive reference serum pool. The second group combines the remaining 76 samples with OD% values between 67.7% and 14,7%, with a majority of 59 samples showing activities ≤ 40 OD%. The negative control serum always resulted in OD% values ≤ 5% of the positive reference serum. None of the field serum samples showed comparably low values.

  five additional proteins reacted with both sera, indicating that the selection of a limited number of B-cell epitopes to identify immunogenic proteins was successful.

  of the GST-Mhp366 fusion protein with the convalescent serum pool and the weak recognition by the hyperimmune serum (Fig. 2C/D) clearly reflects a different concentration

  positive signals. Additional experiments will also be necessary to characterise the expression pattern of the mhp366 gene in-vivo and in-vitro and the time course of Mhp366-specific antibody formation during infection to support our hypothesis, that Mhp366 might be the first antigen identified to facilitate the detection of flourishing M. hyopneumoniae infections even in vaccinated herds.

  Fig. 1

Fig. 2

 2 Fig.2

Fig. 4

 4 Fig. 4

  Figure 1

Figure 4 A

 4 Figure 4

Figure

  Figure

  binding at position 937 to 970 in mhp366; BsmBI recognition site underlined

Table 1

 1 Bacterial strains, plasmids, primers and peptides used in this study 1

	Characteristics

Acknowledgments V.B. was supported by a DAAD grant (Ref. A/06/09151). We thank Dr. D. Maes and Prof. F. Haesebrouck, Ghent, and Dr. B. Kokotovic, Kopenhagen, for providing M. hyopneumoniae strains.

We thank Susanne Daenicke for expert technical assistance in the synthesis of the peptide array.

Putative lipoproteins were predicted using the programs Lipo [START_REF] Berven | Analysing the outer membrane subproteome of Methylococcus capsulatus (Bath) using proteomics and novel biocomputing tools[END_REF] and LipoP 1.0) [START_REF] Juncker | Prediction of lipoprotein signal peptides in Gram-negative bacteria[END_REF]. Starting and endpoint as well as the number of amino acids, molecular weight, pI and functional description are taken from http://mycoplasma.genome.uab.edu/genomes.asp.


Heterogeneity among methicillin-resistant Staphylococcus aureus from Italian pig finishing holdings

Keywords: MRSA, pigs, survey, genotyping, antimicrobial resistance, zoonoses

e d M a n u s c r i p t isolates, one t4794, one t4795, and one t2922 were positive for LukE-LukD genes. All 64 antimicrobial susceptibility tested isolates were resistant to tetracyclines, with high resistance rates to trimethoprim (68.8%), erythromycin (60.9%), and ciprofloxacin (35.4%). All t127, ST1 isolates were resistant to tetracycline-ciprofloxacin-erythromycin. This survey provides the first report of MRSA ST1 and ST(CC)97 among pigs and the first report of MRSA ST9 from pigs in Europe. The presence of human-associated CA-MRSA (t127, ST1, SCCmec type V) in 6% holdings surveyed can represent an additional MRSA reservoir for infections in humans.

A survey for methicillin-resistant Staphylococcus aureus (MRSA) in finishing pig holdings was carried out in Italy in 2008. MRSA isolates were characterised by spa-, MLST-, SCCmec-and antimicrobial susceptibility typing. A prevalence of 38% (45/118, 95% CI 29.4%-46.9%) positive holdings was observed. Eleven different spa-types were found among 102 MRSA isolates, clustering in lineages associated with farm animals (ST398, ST9, ST(CC)97 in 36 holdings) and humans (ST1, 7 holdings). Nine (7.6 %) holdings were positive for two, three or four different and unrelated spa-types in various combinations. ST398 was the most prevalent lineage (33 positive holdings). The most prevalent spa-type was t899 (ST398), detected in 22 positive holdings. Three novel spa-types (t4794 of ST9; t4795 of ST97; t4838 of ST398) were detected. Ten holdings were positive for spa-type t1730, that proved to be a new single-locus variant of ST97, within the CC97.

The most prevalent SCCmec was Type V (79 isolates), while Type IVb was found in 10 isolates.

None of the isolates was positive for Panton-Valentine Leukocidin, while most of t127 and t1730

Introduction

Methicillin-resistant Staphylococcus aureus (MRSA) is a leading cause of hospital-associated infections [START_REF] Johnson | Surveillance and epidemiology of MRSA bacteraemia in the UK[END_REF] and has during the last decade emerged as a significant pathogen in the community (Vandenesch et At present, there is no available information in Europe on the possibility that pigs can represent a competent host and a reservoir for STs other than CC398 MRSA and other farm-associated clones.

Lineages of human origin (spa-type t002, CC5, USA 100) have been isolated from pigs in 3/9 holdings investigated in Ontario, Canada, in a small-scale population study [START_REF] Khanna | Methicillin resistant Staphylococcus aureus colonization in pigs and pig farmers, 2008[END_REF].

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

The aim of our study was to estimate the herd prevalence of MRSA colonization in finishing pig holdings in Italy, to provide phenotypic and molecular characterisation of isolates for epidemiological purposes, and to gather information about their relationships and their possible origin.

Materials and Methods

a. Survey at slaughter and data analysis

Herds were selected by a random sampling procedure from the national registry of holdings rearing pigs for slaughter (farrow to finish, weaner to finisher, finisher holdings), at the base of the pig production pyramid. For among-herd prevalence estimates, a simple random sampling technique was used, with a minimum desired sample size (primary sample size) calculated based on a 20% expected prevalence, a 95% confidence level and 7.5% desired absolute precision [START_REF] Thrusfield | Veterinary Epidemiology, Second Edition[END_REF]. Through this procedure, 118 holdings from 29 provinces and 10 different regions (Northern Central and Southern Italy, accounting for >90% of the Italian pig population) were enrolled for the survey. The study was conducted from January to June 2008 in several major slaughterhouses in Italy, located in Northern and Central Italy that received finishers from the selected holdings. From each holding, 60 individual nasal swabs were sampled (secondary sample size), in order to allow the detection of at least one positive animal with a within-herd expected prevalence of 5% and a 95% confidence level. The swabs were put in Amies with charcoal transport medium, stored at +4°C and sent to laboratory for cultures.

Prevalence estimates and multivariate analysis of data were performed with EpiInfo version 3.5.1 (CDC, Atlanta, United States). Outcomes examined included: the positive status of the holding and the positive status from a human-associated spa-type/sequence type. Information on independent variables included: province and region of holdings sampled, production technique (e. g. farrow to finish, weaner to finisher, finisher herds), holding size, slaughterhouse of sampling. For purposes of the analysis, a variety of summary variables were created: size category of holdings (small: 500-A c c e p t e d M a n u s c r i p t 3000, medium: 3001-9000, large: 9001+), macro-regions (north-east, north-west, central-south), putative origin of spa-type/sequence type detected in the holding (human, animal, human and animal). A multivariate analysis (logistic regression) was performed with initial model containing all the selected variables followed by a backward deletion procedure, and prevalence Odds Ratios (OR) with 95% Confidence Intervals (CI) and p-values were calculated.

b. Laboratory procedures

Pooled cultures of 10 nasal swabs (6 pools from each holding) were carried out in Müller-Hinton broth containing 6.5% NaCl and incubated at 37°C for 16-20 hours (pre-enrichment); One millilitre of the pre-enrichment culture was added to 9 ml of Phenol Red Mannitol Broth + 5 mg/L oxacillin and 75 mg/L aztreonam and incubated at 37°C for 16-20 hours (selective enrichment). The selective enrichment culture was then plated on Oxacillin Resistance Screen Agar (ORSA, Oxoid) and 5% sheep blood agar and incubated at 37°C for 48 hours. Suspected colonies were identified as S. Susceptibility testing was performed on a representative collection of isolates, including at least one isolate for each spa-type (or when appropriate, for each spa-type and SCCmec combination) detected in positive holdings. Broth microdilution testing was performed in 96-well microtiter plates (Trek Diagnostic Systems, Westlake, OH) and interpreted according to the European Committee on Antibiotic Susceptibility Testing (EUCAST) epidemiological cut-offs. The following drugs were tested: erythromycin, ciprofloxacin, chloramphenicol, clindamycin, gentamicin, kanamycin, linezolid, quinopristin-dalfopristin, streptomycin, sulphonamides, tetracycline, trimethoprim, vancomycin.

Results

A total of 45 out of 118 (38.1%, 95% CI 29.4%-46.9%) holdings had at least one MRSA-positive pool. A total of 98 (14%) positive pools were detected out of the 701 cultured. Nearly 50% (22/45) of positive holdings had only one positive pool. Two, three and four positive pools were found in 18%, 13% 11% of positive holdings, respectively, while five and six positive pools were found in 4% of positive holdings. Eleven different spa-types were detected, three of which have never been described before (t4794, t4795, t4838), and belonging to five different MLST profiles, ST398, ST1, ST9, ST97, ST1476 (Table 1). An estimated 7.6% (9/118; 95% CI 2.8-12.4%) of the holdings had their animals positive for 2, 3 or 4 unrelated spa-types belonging to ST1, ST97, ST1476 or ST398 in different combinations. Four holdings (3.4%, 95% CI 0.1-6.7%) were positive for both humanand animal-associated MRSA: t127 and t011, t034, t889, t2922, t1730, t4795 (Table 2). An estimated 28% (33/118; 95% CI 19.9-36.1%) of holdings were found positive for pig-associated A c c e p t e d M a n u s c r i p t ST398 lineage and an estimated 5.9% (7/118; 95% CI 1.7-10.2%) were positive for humanassociated MRSA (t127, ST1). Fourteen holdings were positive for other lineages, one for ST9 (t4794), three for t4795, ST97 and ten for t1730. The MLST performed on t1730 isolates showed that they are a single-locus variant (in the gmk gene) of ST97, now classified as ST1476, and thus they belong to CC97. The most prevalent spa-type was t899, ST398, detected in 22/45 (49%) positive holdings, with an overall holding prevalence of 18.6% (95% CI 11.6-25.7%). Among the 102 isolates from the 45 positive holdings, the most frequent SCCmec ccr and mec combination [START_REF] Kondo | Combination of multiplex PCRs for staphylococcal cassette chromosome mec type assignment: rapid identification system for mec, ccr, and major differences in junkyard regions[END_REF]) was 5C (i. e. Type V), detected in 79 isolates. Ten isolates carried the 2B combination (i. e. Type IV) and were also positive in the IVb subtype molecular assay. Thirteen isolates showed a positive result in the PCR assay for both ccrA2-ccrB and ccrC genes in a novel 5+2B combination (i. e. genetic elements described in the Type IV and Type V SCCmec cassettes).

Among the novel spa-types, both t4794, ST9 and t4795, ST97 isolates carried the SCCmec 5C combination (Type V), and the t4838 the 5+2B combination.

None of the 102 isolates carried the Panton-Valentine Leukocidin (PVL) genes. Most of the t127 (10/12) and t1730 (11/13) along with one t4794, one t4795, and one t2922 were positive for LukE-LukD genes.

Antimicrobial susceptibility testing performed on 64 isolates (Table 3) showed that 100% were tetracycline-resistant. A high proportion of isolates were also resistant to trimethoprim (44/64, 68.8%) and to erythromycin (39/64, 60.9%). The overall resistance rate to ciprofloxacin was 35.9% (23/64). No resistance to linezolid, quinopristin-dalfopristin and vancomycin was observed.

All the t127, ST1 and most (9/10) t1730, CC97 isolates tested showed a common pattern of tetracycline, macrolide and fluoroquinolone resistance, while among the three t4795, ST97 isolates, two were resistant to ciprofloxacin and none to erythromycin. Conversely, among ST398 isolates, only 43% (12/28) of t899 and related spa-types were resistant to erythromycin (all t899 isolates), with only one isolate resistant to ciprofloxacin (t2922). Isolates of the related spa-types t011, t034

A c c e p t e d M a n u s c r i p t and t108 showed a high rates of resistance to erythromycin (12/16, 75%), but lower resistance rates to ciprofloxacin (4/16, 25%).

When analysing the data in a multivariate model for possible predictor variables, the only significant risk factor observed was holding size ("large" vs "small") for the outcome variable "positive status of the holding" with a prevalence Odds Ratio (OR) 3.67 (95% CI 1.04-112.91). The best fitting model for the predictor variable holding size "large vs "small" and the outcome variable was that adjusted for "macro-region", "production technique", and "slaughterhouse of sampling" (likelihood ratio test 11.7; 2 d. f.; p-value 0.0028), as a significant difference with the reduced model without the variable "slaughterhouse of sampling" was found: -2 Ln (likelihood M1/likelihood M2) chi square p-value < 0.05; 2 d. f.; where M1 is the reduced model and M2 is the extended model. No significant association between independent variables and the outcome "positive status of the holding from a human-associated spa-type/sequence type" were found.

Discussion

This is the first description of MRSA among pig population in Italy, the first description of three novel spa-types (t4794, ST9; t4795, ST97; t4838, ST398), the first report of MRSA of ST1 ST1476 and ST97 lineages among pigs, and the first report of MRSA ST9 in Europe.

Pigs reared in Italy may represent a relevant reservoir for both farm-associated and humanassociated MRSA clones, as animals of nearly 40% of Italian finishing holdings were found positive. The majority of holdings were positive for ST398 lineage, with two main spa-types groups detected (t899 and related t2922, t4838 spa-types, along with t011 and related t034, and t108 spatypes). Spa type t899 proved to be the most prevalent spa-type in Italian pig holdings, although it had been considered a rare type in pigs until recent description in the Netherlands (van Dujikeren et al., 2008). The presence of unrelated spa-types within the same holding (up to four spa-types), either belonging to ST398, CC97 or to ST1, suggests subsequent introduction of new strains over time as the most common pattern of colonization and diffusion among animals and holdings. Clonal Complex 97 is known at present to be one of the S. aureus lineages associated with cattle, and especially with bovine mastitis [START_REF] Sung | Staphylococcus aureus host specificity: comparative genomics of human versus animal isolates by multi-strain microarray[END_REF]Hasman et al., in press). The new spa-type t4795 differs from t1730 in a single deletion event (repeat 34) and shown to belong to ST97. Spatype 4838 is closely related to t899 and differs only by a single point mutation in the first repeat (now repeat 283 instead of 07), while spa-type t4794, ST9 has a Ridom succession sequence of only two repeats , suggesting multiple deletion events in the X-region. The MSSA phenotype of lineage ST9 is known to be present in pigs in Europe [START_REF] Kehrenberg | Methicillinresistant and -susceptible Staphylococcus aureus strains of clonal lineages ST398 and ST9 from swine carry the multidrug resistance gene cfr[END_REF]Hasman et al., in press) and MRSA isolates of this ST type, spa-type t899, were found recently in pigs in China [START_REF] Wagenaar | Methicillin-resistant Staphylococcus aureus in horses and horse personnel, 2000-2002[END_REF]. In this respect, as all the t899 isolates from this study were classified as ST398 by MLST, the same Ridom sequence (spa-type t899) both in ST9 and ST398

A c c e p t e d M a n u s c r i p t could be explained with the occurrence of a horizontal transfer of the X-region between the two pigassociated lineages. Human-and cattle-associated spa-types and CCs (t127, t1730 and t4795) were also detected recently in Italian holdings of breeding pigs during an European baseline survey (European Commission Decision 2008/55/EC), which demonstrates the diffusion of these clones also at the top of the pyramid of the Italian pig population (Battisti, unpublished data). Additionally, this study shows that in Italy mecA mediated beta-lactam resistance in MRSA from pigs is carried in Type IV, V, or a combination (5+2B, [START_REF] Kondo | Combination of multiplex PCRs for staphylococcal cassette chromosome mec type assignment: rapid identification system for mec, ccr, and major differences in junkyard regions[END_REF] of the ccr and mec elements of the SCCmec cassette. None of the isolates, irrespective of their lineage, carried PVL genes or any of the other toxin genes considered for the study except for the LukE-LukD determinants, found in most of the t127, ST1 and t1730, ST1476 isolates and in three other spa-types of lineages known to be associated with animals (ST9, ST97, ST398). The LukE-LukD are bi-component leukotoxins located in a putative staphylococcal pathogenicity island (SapIn3/SapIm3) on the chromosome of S. aureus [START_REF] Kuroda | Whole genome sequencing of methicillin-resistant Staphylococcus aureus[END_REF]) and are found in most of human CA-MRSA as well as in HA-MRSA [START_REF] Vandenesch | Community acquired methicillin-resistant Staphylococcus aureus carrying Panton-Valentine leukocidin genes: worldwide emergence[END_REF] from France and USA. Those genes were also detected in a high proportion of avian and bovine isolates, but in none of the 72 isolates from pig tonsils in a study carried out in Japan [START_REF] Yamada | Leukotoxin family genes in Staphylococcus aureus isolated from domestic animals and prevalence of lukM-lukF-PV genes by bacteriophages in bovine isolates[END_REF]. All isolates tested for antimicrobial susceptibility proved to be resistant to tetracyclines, a feature consistent with the data reported from isolates of different spa-types and lineages in other European countries [START_REF] Van Duijkeren | Transmission of methicillin-resistant Staphylococcus aureus strains between different kinds of pig farms[END_REF] where these drugs have as well been widely used for mass medication. Additionally, high resistance rates to trimethoprim, erythromycin and ciprofloxacin are evident from this survey, with a pattern of tetracycline, erythromycin and ciprofloxacin resistance that seems a common feature among isolates of the ST1 and ST(CC)97 lineages. We did not detect any significant differences in the macroregional distribution of positive holdings, in the production technique, or in spa-types and STs associated with the positive status. This is probably because in Italy MRSA are also widespread in breeding holdings with an overall estimated prevalence that exceeds 30% (Battisti, unpublished data) and they act as a reservoir when colonised breeders are sold to holdings of farrow-to-finish A c c e p t e d M a n u s c r i p t and of growing-finishing pigs. Larger farms (9000+ animals reared) were more likely to be MRSApositive than small ones. The reason for this is not known, but might be because larger farms are more likely to have several trading contacts and thereby are more likely to be exposed to contamination. In conclusion, a heterogeneity of spa-types and STs (eleven spa-types clustering in five different STs) is evident in Italian pig finishing holdings. This population study also suggests that pigs may be not only competent hosts and a possible reservoir for MRSA of the cattleassociated CC97 lineages, but also for the human-associated ST1, thus representing a possible source of exposure for the community from a zoonotic pathogen that may need little or no adaptation to humans. Concerns raise also from the presence of different spa-types and STs in the same holdings, as it may favour the exchange and the spread of virulence determinants among different MRSA lineages. The antimicrobial resistance profile observed suggests that beside the use of beta-lactams, the amount and pattern of usage of other antimicrobials should be investigated as a further important predictor in the spread of MRSA within and among Italian pig holdings.

  al., 2003; Kluytmans-Vandenbergh & Kluytmans, 2007; ). Community-acquired MRSA(CA-MRSA) clones have been increasingly detected in companion animals as dogs and horses (van Duijkeren et al., 2004; Weese et al., 2005) and also observed in livestock animals (Juhász-Kaszanyitzky et al., 2007, Khanna et al. 2008). Recently, isolates belonging to MRSA Clonal Complex (CC) 398 have emerged in pig farming, later spread to other farm animal species and it is now prevalent in farm animals in some countries of Central and Northern Europe (de Neeling et al., 2007; van Duijkeren et al., 2008) and North America (Khanna et al., 2008 ). MRSA CC398 of animal origin has also emerged as a cause of colonization and infection in humans and it is responsible for >20% of all MRSA in the Netherlands (van Loo et al., 2007). Recently, MRSA isolates of Sequence Type(ST) 9 were reported in pigs from China (Wagenaar et al., 2009).

  aureus by means of standard techniques: colony morphology (blue colonies on ORSA), Gram staining, catalase, coagulase tube test and Staphytect Plus test kit (Oxoid). The identification at species level was further confirmed by Polymerase Chain Reaction assay (PCR)[START_REF] Baron | Development of a PCR test to differentiate between Staphylococcus aureus and Staphylococcus intermedius[END_REF]. The detection of the mecA gene by PCR was performed as described byStrommenger and colleagues (2003). MRSA isolates were genotyped by DNA sequencing of the X-region of theStaphylococcusProtein A (spa-typing, Harmsen at al., 2003), with repeats and spa types determined by Ridom StaphType software (Ridom GmbH, Würzburg, Germany) and Multi-Locus Sequence Typing (MLST) (Enright et al., 2000). Typing of the Staphylococcal Cassette Chromosome mec (SCCmec) was performed by PCR as previously described (Kondo et al., 2007). Isolates carrying ccr and mec gene combinations classified as Type IV SCCmec were subtyped according to Zhang and colleagues (2005). MRSA isolates were screened for genetic determinants of the following toxins: Gamma-hemolysin components A, B, and C (hlg) and Gamma-hemolysin variant (hlg-2), Panton Valentine (PV) A c c e p t e d M a n u s c r i p t leukocidin components S and F (lukS-PV-lukF-PV) according to McClure and colleagues (2006), LukM-LukF'-PV (lukM), and LukE-LukD (lukE-lukD) leukocidins, according to Jarraud and colleagues (2002), exfoliative toxins ETA (eta), ETB (etb) and ETD (etd), according to Yamaguchi and colleagues (2002), toxin shock syndrome toxin (TSST) according to Fueyo and colleagues (2005).

A

  c c e p t e d M a n u s c r i p t More than forty percent (19/45) of positive holdings had their animals colonised either by humanassociated MRSA of the ST1 lineage, or by CC97 clones, and demonstrating that pigs may acquire and spread MRSA belonging to CCs other than CC398. ST1 belongs to CC1, a particularly successful lineage which includes MW2 CA-MRSA associated with deaths in children in the United States (Centers for Disease Control and Prevention, 1999) , and lukS-PV/luk-F-PV-positive CA-MRSA also known as USA400 (McDougal et al., 2003). Additionally, PVL-negative ST1 t127, SCCmec IVa, was among the most common CA-MRSA reported by researchers in the United Kingdom, closely associated with injecting drug-users and homeless people (Otter et al., 2009). One PVL-positive t127 CA-MRSA was recently reported in a Dutch patient in a study conducted in the Euregio Meuse-Rhine Region (Deurenberg et al., 2009). A cluster of t127, ST1 infections in humans and horses in a veterinary teaching hospital in Germany (Cuny et al., 2008) and a mastitis outbreak in cattle (Juhász-Kaszanyitzky et al., 2007) were also reported recently, suggesting the ability of this lineage to adaptation to new host species. As CC97 is concerned, one t1730 MRSA isolate had already been detected from an Italian participant at a conference of the International Veterinary Pig Society (Wulf et al., 2008), but at that time this finding was not included among the spa-types associated with risk factors related to pig-farming and thus not analyzed by MLST.

Table 1 . Spa-types and MLST profiles among holdings surveyed

 1

			n holdings where	% among	% holdings
	spa-type	ST	it was detected	positive holdings	surveyed
	t011	398	4	8.9	3.4
	t034	398	6	13.3	5.1
	t108	398	4	8.9	3.4
	t2510	398	1	2.2	0.8
	t4838	398	2	4.4	1.7
	t2922	398	4	8.9	3.4
	t899	398	22	48.9	18.6
	t4794	9	1	2.2	0.8
	t4795	97	3	6.7	2.5
	t127	1	7	15.6	5.9
	t1730	1476	10	22.2	8.5

Table 2 : Holdings with more than one spa-type or ST detected

 2

	holding	n spa-		spa-type
	code	types	spa-types	relatedness	STs
	1	2	t011, t108	related	398
	2	3	t127, t899, t1730	unrelated 1, 398, 1476
	3	3	t011, t899, t1730	unrelated 398, 1476
	4	2	t108, t899	unrelated	398
	5	2	t034, t899, t2510, t4795 unrelated	398, 97
	6	2	t108, t2922	unrelated	398
	7	3	t127, t899, t2922	unrelated	1, 398
	8	2	t127, t2922	unrelated	1, 398
	9	2	t127, t899	unrelated	1, 398
	10	2	t1730, t4795	related	1476, 97
	11	2	t1730, t034	unrelated 1476, 398

Acknowledgements:

The Authors wish to thank Oliviero Bassoli, Giuseppe Cito, Giorgio Cuoghi, Alessandro De Bassa, Elena Floriani, Vitantonio Perrone, Pietro Razzini, Gaetano Liuzzo for sample collection, and Roberta Amoruso, Carmela Buccella, Gessica Cordaro, Paola Di Matteo, Angela Ianzano and Cinzia Onorati for laboratory technical support.

This study was made possible also thanks to research grants provided by the Italian Ministry of Health (code: IZS LT 03/07 RC)

 25) 0 (0)1 ( 25) 1 ( 25) 0 (0) 4 (100) 4 (100) 0 (0) t2510 398 1 0 (0) 0 (0) 1 (100) 0 (0) 0 (0) 0 (0) 0 (0)0 (0) 0 (0) 0 (0) 1 (100) 0 (0) 0 (0) t4838 398 1 0 (0) 0 (0) 1 (100) 0 (0) 0 (0) 0 (0) 0 (0)1(100) 0 (0) 0 (0) 1 (100) 1 (100) 0 (0) t2922 398 4 1 ( 25) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0)3 (75) 1 ( 25) 0 (0) 4 (100) 3 (75) 0 (0) t899 398 23 0 (0) 6 ( 26)15 (65) 12 (52) 7 ( 30) 6 ( 26) 0 (0)7 ( 30) 11 ( 48) 0 (0)23(100)15 (65) 0 (0) t4794 9 1 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0)0 (0) 1 (100) 0 (0) 1 (100) 0 (0) 0 (0) t4795 97 3 2 (67) 0 (0) 1 ( 33) 0 (0) 0 (0) 0 (0) 0 (0)1 ( 33) 0 (0) 0 (0) 3 (100) 0 (0) 0 (0) t1730 1476 10 10(100) 2 (20) 9 (90) 9 (90) 3 (30) 3 ( 30) 0 (0)3 (30) 3 ( 30) 0 (0)10(100) 8 (80) 0 (0) t127 1 6 6 (100) 0 (0) 6 (100) 6 (100) 3 (50) 3 (50) 0 (0)3 (50) 1 ( 17) 0 (0) 6 (100) 3 (50) 0 (0) a CIP, ciprofloxacin; CHL, chloramphenicol; CLI, clindamycin; ERY, erythromycin; KAN, kanamycin; Gen, gentamicin; LIN, linezolid, STR, streptomycin, SUL, sulphonamides, SYN, quinopristin-dalfopristin; TET, tetracycline; TMP, trimethoprim; VAN, vancomycin