Veiga,I Ana Duarte
email: anaduarte@fmv.utl.pt

L Tavares

Genetic Diversity and Phylogenetic Analysis of Feline Coronavirus Sequences from Portugal

Keywords: FCoV, genetic diversity, Heteroduplex Mobility Assay

The distribution of FCoV Type I and II in a Portuguese cat population was studied by a RT-PCR assay targeting the 3' end of the viral RNA. For a period of three years, 120 samples were collected and 57 were found positive for FCoV RNA. Within the positive samples the presence of FCoV Type I was found in 79%. Type II was only detected in 3,5% in animals with Feline Infectious Peritonitis. The remaining 17,5% could not be differentiated. These viral sequences, comprising a region within gene S were further subjected to a Heteroduplex Mobility Assay (HMA) detecting the presence of viral quasispecies in 17% of the samples. Phylogenetic analysis for FCoV Type I revealed high genetic diversity between the Portuguese sequences and other previously characterized strains, while Type II tree showed a higher genetic homogeneity. This study confirmed the presence of FCoV Type I and II circulating in Portugal and detected high genetic diversity between circulating strains suggesting that the virus persists within the host as mixed viral populations.

Introduction

Feline Coronaviruses (FCoV) are members of genus Coronavirus, Family Coronaviridae. Two variants of FCoV have been described, Feline Enteric Coronavirus responsible for an enteric disease and Feline Infectious Peritonitis Virus (FIPV) the etiological agent of FIP, a fatal disease of domestic and wild cats. FIP development in a small percentage of animals infected with FCoV is related to the occurrence of genomic mutations supported by the close genetic relationship between the higher virulent virus and the parental FCoV [START_REF] Herrewegh | Persistence and evolution of feline coronavirus in a closed catbreeding colony[END_REF][START_REF] Vennema | Feline infectious peritonitis viruses arise by mutation from endemic feline enteric coronaviruses[END_REF][START_REF] Vennema | Genetic drift and genetic shift during feline coronavirus evolution[END_REF].

Additionally, FCoVs were classified as Type I and II according to their serological and genomic features. Type I is strictly feline. Type II FCoV, although sharing with Type I a high degree of similarity in the 3' end of the genome, is antigenic and genetically more related to Canine Coronavirus (CCoV) [START_REF] Hohdatsu | Antigenic analysis of feline coronaviruses with monoclonal antibodies (MAbs): preparation of MAbs which discriminate between FIPV strain 79-1146 and FECV strain 79-1683[END_REF], suggesting that Type II resulted from a homologous and independent recombination event within the S and the M genes of Type I FCoV and CCoV [START_REF] Herrewegh | Feline coronavirus type II strains 79-1683 and 79-1146 originate from a double recombination between feline coronavirus type I and canine coronavirus[END_REF]. The current information on the geographical distribution of FCoV indicates a high incidence of Type I in Europe, Japan and USA [START_REF] Addie | Persistence and transmission of natural type I feline coronavirus infection[END_REF][START_REF] Hohdatsu | The prevalence of types I and II feline coronavirus infections in cats[END_REF]. However in Japan and Taiwan detection of FIP was associated with Type II FCoV [START_REF] Hohdatsu | The prevalence of types I and II feline coronavirus infections in cats[END_REF], Lin et al., 2008). Presently there is no available information about the molecular epidemiology of FCoV in Portugal. Due to the pathogenic impact of this virus in the feline health it was our purpose to investigate the presence of FCoV Types in the Portuguese cat population using a RTPCR-nested A c c e p t e d M a n u s c r i p t 3 PCR assay previously reported [START_REF] Addie | Persistence and transmission of natural type I feline coronavirus infection[END_REF] and to assess its genetic diversity by Heteroduplex mobility assay (HMA) and phylogenetic analysis.

Material and Methods

Sampling collection

The biological material included rectal swabs, stool, plasma, thoracic and ascite fluids collected from animals attending the Veterinary Hospital of the Faculty of Veterinary Medicine, Lisbon.

Detection of FCoV RNA

FCoV RNA was extracted using the QIAmp Viral RNA or Qiagen RNeasy mini kits (Qiagen), following the manufacturer's instructions. A fragment of 177bp located in the 3'-UTR was amplified by RT-PCR followed by a nested PCR using two primer pairs described by [START_REF] Herrewegh | Detection of feline coronavirus RNA in feces, tissues, and body fluids of naturally infected cats by reverse transcriptase PCR[END_REF] The 25 µL reactions were amplified with one step RT-PCR Fidelity Taq 2x Master Mix (GE Healthsciences), 50pmol of each primer and 10µL of RNA sample. The nPCR was performed with PCR Fidelity Taq 2x

Master Mix (GE Healhsciences), 50pmol of each primer and 5 µL of the previous amplification in a 25 µL reaction.

Differentiation between Type I and Type II FCoV:

Positive FCoV RNA samples were subjected to a RT-PCR assay to amplify a 376-283bp fragment located in the 3'region of the spike gene (S), followed by a nPCR to amplify a 360-218 bp internal fragment, for differentiation of Type I from Type II FCoV according to the fragment length [START_REF] Addie | Persistence and transmission of natural type I feline coronavirus infection[END_REF]. The amplification was performed with one step RT-PCR Fidelity Taq 2x Master Mix (GE Healthsciences), 5µL of RNA sample and primers Iffs, Icfs, Iubs in a 25 µL reaction, followed by a nested PCR with PCR Fidelity Taq 2x Master Mix (GE Healthsciences), primers nIffles, nIcfs, nIubs [START_REF] Addie | Persistence and transmission of natural type I feline coronavirus infection[END_REF] and 2,5 µL of the previous reaction in 25 µL final volume. The amplicons were analysed in a 2,5% agarose gel stained with 0.5 µg/mL of Ethidium Bromide in 1x TBE.

Heteroduplex Mobility Assay (HMA) and Cloning

The amplicons were subjected to a HMA before cloning [START_REF] Delwart | Genetic relationships determined by a DNA heteroduplex mobility assay: analysis of HIV-1 env genes[END_REF]. Briefly, 10 μL of the second round S amplicons were combined with 1 μL of 10x annealing buffer (1M NaCl, 100mM Tris pH 7.8, 20mM EDTA), heated to 94ºC/5min, cooled on ice, added of 1 μL of loading buffer (50% glycerol, 0,01% of xyleno cyanol) and loaded in a 5% polyacrylamide gel (29,2:0,8 acrylamide: bisacrylamide) poured into plates of 15 x 15 cm. The electrophoresis was carried at a constant 150V/3 h in a vertical gel apparatus (Biorad) in 1x TBE. After electrophoresis the gel was stained with 0.5 µg/mL of Ethidium Bromide in 1x TBE and observed in a UV transiluminator. The visualized Type I and II assignment based on the amplicons length were in accordance with the nucleotide sequence of each sample.

Phylogenetic analysis

The nucleotide sequences were aligned (PILEUP from GCG Wisconsin Package v. 10.0), including sequences of FCoV Type I (KU_2 AN-D32044; UCD1 AN-AB088222; Black AN-AB088223; C2_0301UK AN-AY159737; J8_0196UK AN-AY159758; FQ1_1099UK AN-AY159754; H9_1100UK AN-AY159770; FUI_1191UK AN-AY159735) and FCoV Type II (79_1683 AN-X80799; 79_1146 AN-X06170; G6_1095 AN-AY159784). Due to its recombinant status FCoV Type II sequences were aligned and submitted to phylogenetic analysis separately from Type I sequences.

The phylogenetic relationship between sequences was inferred by genetic distances between pairs of sequences using Kimura's two parameter method (DNADIST, Phylip Package).The values of transition/transversion ratio and nucleotide frequencies were calculated from the data sets (TREE-PUZZLE) [START_REF] Strimmer | Likelihood-mapping: A simple method to visualize phylogenetic content of a sequence alignment[END_REF]. The resulting trees were constructed by Neighbor Joining (NEIGHBOR, Phylip Package) and the branching order reliability was evaluated by bootstrap analysis of 1000 replicates (SEQBOOT, Phylip Package). To maximize the phylogenetic information of the nucleotide alignment closely related sequences were excluded but were indicated between brackets following the sequence chosen to represent each set of viruses as summarized; 65LVI_05

[74LVI_05, 81LVI_05, 86LVI_05, 87LVI_05]; 14LVI_05 [10LVI_05a]; 19LVI_04a [19LVI_04b]; 58LVI_05 [59LVI_05a/b]; 1Lg_06 [1Fg_06a].

3-Results

Detection of Type I and II FCoV

Detection of viral quasispecies

The presence of viral quasispecies within the animal, was evaluated by HMA for each sample (Fig. 1). This assay detects the presence of fully (homoduplexes) and partially (heteroduplexes) complementary DNA chains formed between highly related sequences during the process of denaturing and annealing. Homoduplexes molecules tend to migrate faster, while heteroduplexes show a reduced mobility in a non-denaturing PAGE. The observation of two or more homoduplexes bands, plus the corresponding heteroduplexes bands indicates the presence of different DNA populations within the sample, named viral quasispecies and mirror the in vivo genetic diversity of the virus [START_REF] Delwart | Genetic relationships determined by a DNA heteroduplex mobility assay: analysis of HIV-1 env genes[END_REF].

Within the 47 S gene amplicons 37 samples (78,7%) showed no genetic diversity whereas in 10 samples (21,3%) two homoduplexes bands were observed and individually amplified. Comparison of the nucleotide sequences of the heteroduplexes positive samples revealed similarities of 90% to 99% along the 320nt in Type I and 170nt in Type II FCoV. Although very similar, six sequences (13LVI_04b; 19LVI_04b; 10LVI_05a; 11LV_05a; 33LVI_05b; 99LVI_05b) showed a 5'end variation length of 28 to 56 nucleotides justifying the homo/heteroduplexes formation. Sequences 33LVI_05a/b, 25LVI_04a/b, 10LVI_05a/b and 11LVI_05a/b were amplified from animals with multi-cat environment (table 2).

Phylogenetic analysis

The genetic relationships between the Portuguese sequences of FCoV and previously assigned sequences from isolates of Type revealed a heterogeneous clustering of sequences with several tree branches supported by high bootstrap values (Fig. 2). Sequences from distinct viral quasispecies obtained from the same animal as well as sequences from cats sharing the same household were 170b), a likelihood mapping analysis was preformed (TREE-PUZZLE) to assess its phylogenetic content, revealing 62.86% of fully resolved, 0% of partially resolved and 37.14% of unresolved quartets demonstrating therefore its reliable use to estimate phylogenetic relationships.

Discussion

Until now, no previous study on the presence and prevalence of FCoV Type I and II in domestic cats had been done in Portugal. In our analysis 47,5% of the samples were positive in the RTPCR assay (57/120) and were obtained either from healthy animals or from cats with FIP. Moreover, the vast majority of the amplicons was classified as Type I. Type II was found only in two samples both from cats with FIP. The values found for FCoV Type I and Type II were in accordance with the distribution reported in Europe and USA [START_REF] Addie | Persistence and transmission of natural type I feline coronavirus infection[END_REF][START_REF] Benetka | Prevalence of feline coronavirus types I and II in cats with histopathologically verified feline infectious peritonitis[END_REF] although in Japan and Taiwan a higher prevalence of FCoV Type II was observed in animals with FIP [START_REF] Hohdatsu | The prevalence of types I and II feline coronavirus infections in cats[END_REF], Lin et al., 2008). In our study even though the two FCoV type II sequences were obtained from sick cats, the low incidence of this viral biotype does not allow any relationship between FCoV II and symptomatic disease.

The evaluation of FCoV genetic diversity revealed 21% of the amplicons positive for viral quasispecies. These viral variants were found within the same animal but also in samples collected from animals sharing a multicat environment. This finding supports the viral internal mutation theory [START_REF] Dye | Genomic RNA sequence of feline coronavirus strain FCoV C1Je[END_REF] and confirmes the animal exposure to divergent viral strains.

The significance of FCoV quasispecies had already been studied [START_REF] Battilani | Quasispecies composition and phylogenetic analysis of feline coronaviruses (FCoVs) in naturally infected cats[END_REF] suggesting a correlation between high viral genetic diversity within the host and development of disease. However Interestingly, FCoV Type II phylogenetic tree although with a limited number of Portuguese and previously characterised Type II sequences, showed a homogeneous distribution of sequences as also reported by Lin et al., 2008 in a similar study, indicative of high similarity between distantly related sequences (UK; Netherlands; Portugal). The genetic diversity of the Portuguese FCoV sequences confirmed and strengthens the evidence that this virus persists within the host as mingled viral populations whose interaction with the animal may or not result in severe disease.

In conclusion this study provided information on the FCoV molecular epidemiology in Southern Europe which was unavailable until now and revealed high genetic diversity between the Portuguese and previously characterized FCoV sequences.

  cuted, eluted at 56ºC/5min in 200 µL H 2 O of which 1 µL was re-amplified using the nPCR primers set described above. The resulting amplicons were cloned in pGEM-T Easy Vector System (Promega) according to the manufacture's conditions and sequenced by STAB Vida Lda, Portugal. The nucleotide sequences were submitted to Genbank and were assigned the Acession numbers EU327694-EU327752. Sample identification was included in table 1. FCoV.

For

  three years 120 samples were collected from domestic cats to detect FCoV RNA due to clinical suspicion of FIP and to confirm viral excretion from healthy animals. Fifty seven samples were found FCoV positive (47,5%) and afterwards processed for Type I and II differentiation. Out of those 57 positive samples, 45 (79%) were classified as Type I FCoV and were obtained from healthy A c c e p t e d M a n u s c r i p t 5 animals or with FIP. Only two samples (3,5%) from animals with FIP, were classified as Type II. No amplification was obtained from ten samples (17,5%).

  included in branch A indicating the presence of different viral strains within each confined cat population and within the same individual host. Sequences 83 LVI_05, 88LVI_05, 90LVI_05, 94LVI_05 and 96LVI_05 came from cats sharing the same household than the source animal of sequence 65LVI_05 [74LVI_05, 81LVI_05, 86LVI_05, 87LVI_05]. However sequence 65LVI_05 was positioned outside group A, in a separate branch with 80% of bootstrap value. The above mentioned sequences were obtained from a multi-cat household where a clinical FIP episode (65LVI_05) had been reported. Although all the other animals were FCoV positive, none developed FIP. Sequence 11LVI_05b was also included in branch A but sequence 11LVI_05a, obtained from the same cat A c c e p t e d M a n u s c r i p t was positioned in branch B. Sequence 25LVI_04a was grouped in branch A though it was obtained from the same animal as sequence 25LVI_04b placed in a different branch. These two viral quasispecies (25LVI_04a/b) came from the same household of the source cats of sequences 22LVI_04a/b and 23LVI_04, which in this analysis were found distributed in outlier branches supported by 100% of bootstrap value. Sequences 20LVI_06Z (stool), 20LVI_06PBMC (blood) and 20LVI_06L (ascites) amplified from the same animal, were placed in two separate branches, one with 100% bootstrap value (20LVI_06PBMC and 20LVI_06L). Type II FCoV phylogenetic tree included previously assigned FCoV Type II sequences and two Portuguese sequences obtained during this study, one positive to viral quasispecies (32LVI_06; 32LVI_06a/b). All the sequences clustered in one branch, although FIPV 79-1146 and 79-1683 grouped together with 100% of bootstrap value. Due to the low number of nucleotides considered in the alignment (

  it was not possible to correlate viral diversity with virus subtype or disease therefore a more extensive study should be performed to assess this connection.The phylogenetic analysis of FCoV Type I and II sequences revealed an assorted distribution of sequences in Type I tree, showing high genetic diversity of the FCoV Portuguese sequences, its divergence toward European and Japonese sequences (Fq1_1099UK, H9_1100UK, and strains KU2, Black and UCD1) confirming the previous findings.

Figure 1 -

 1 Figure captions

Figure 2 .

 2 Figure 2. Phylogenetic analysis of FCoV type I and II sequences based on the 3' end of the S gene. The trees were constructed by Neighbor Joining based on the genetic distances between the nucleotide sequences, calculated by Kimura two-parameter using a transition/transversion rate of 3.23 for Type I sequences and 9.99 for Type II sequences. A boostrap analysis of 1000 replicates was performed and values above 80% are indicated on the branches. Branches A, B and C include FCoV sequences with the lowest value of nucleotide substitutions within each branch. Sequences marked with an asterisk (*) were amplified from animals with FIP.

  Figure 1

Table 2 -

 2 : Genetic diversity between amplicons subjected to HMA. The length and the percentages of genetic similarity between homoduplexes are shown.

	FeCoV Type II
	102LVI_05*
	32LVI_06ª *
	32LVI_06b *
	G6_1095UK

Acknowledgments

This work was sponsored by the Center for Interdisciplinary Research in Animal Health (CIISA), FMV, UTL, Portugal. We are grateful to our colleagues from the Veterinary Hospital (FMV) for their collaboration in sampling collection.

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


Szilvia L Farkas
email: szilvia@vmri.hu

János Gál

Adenovirus and mycoplasma infection in an ornate box turtle (Terrapene ornata ornata) in Hungary

Keywords: Ornate box turtle, Terrapene ornata ornata, Adenovirus, Mycoplasma

A female, adult ornate box turtle (Terrapene ornata ornata) with fatty liver was submitted for virologic examination in Hungary. Signs of an adenovirus infection including degeneration of the liver cells, enlarged nuclei and intranuclear inclusion bodies were detected by light microscopic examination. The presence of an adenovirus was later confirmed by obtaining partial sequence data from the adenoviral DNA-dependent DNA-polymerase. Phylogenetic analyses revealed that this novel chelonian adenovirus was distinct from previously described reptilian adenoviruses, not belonging to any of the recognized genera of the family Adenoviridae. As a part of the routine diagnostic procedure for chelonians the detection of herpes-, rana-and iridoviruses together with Mycoplasma spp. was attempted. Amplicons were generated by a general mycoplasma polymerase chain reaction (PCR) targeting the 16S/23S ribosomal RNA (rRNA) intergenic spacer region, as well as, a specific M. agassizii

Introduction

Adenoviruses (AdVs) are double stranded non-enveloped viruses with a diameter of 80-110 nm. They are present in a wild range of vertebrate species, including mammals, birds, reptiles, amphibians and fish. They belong to the family Adenoviridae which presently contains four accepted genera (Mastadenovirus, Aviadenovirus, Atadenovirus, Siadenovirus) and a fifth proposed genus (Ichtadenovirus) containing the white sturgeon AdV 1, the only known fish AdV so far (Benkő et al., 2005;Kovács et al., 2003).

Adenovirus-like viruses have already been detected in various reptilian hosts, including snakes and lizards, as well as in Nile crocodiles (Crocodilus niloticus) (Fry et al., 1994;Ramis et al., 2000;[START_REF] Kim | An outbreak of adenoviral infection in inland bearded dragons (Pogona vitticeps) coinfected with dependovirus and coccidial protozoa (Isospora sp.)[END_REF]Wellehan and Johnson, 2005;Kübber-Heiss et al., 2006), a leopard tortoise (Geochelone pardalis) (Wilkinson, 2004) and Sulawesi tortoise (Indotestudo forstenii) (Rivera et al., 2008). Based on available sequence data, most of the snake and lizard AdVs examined have been shown to belong to the genus Atadenovirus (Wellehan et al., 2004, Farkas et al., 2008;Papp et al., 2009). From chelonians molecular data is currently available only from the Sulawesi tortoise AdV (Rivera et al., 2008). Interestingly, the recently detected Sulawesi tortoise AdV appears to be a member of the genus Siadenovirus (Rivera et al., 2008).

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

Mycoplasmas can infect different reptile species, but they are most often detected from chelonians. Some of them are members of the normal flora living in association with the host, others may cause diseases leading even to the death of the animal (Brown et al., 2002). M. testudinis is a non-pathogenic agent of the excretory tract in Greek tortoises (Testudo graeca), M. agassizii (Brown et al., 1999) or M. testudineum (Brown et al., 2004) is causing the upper respiratory tract disease in wild and captive tortoises in the USA and Europe. In the eastern box turtles (Terrapene carolina carolina) the detected Mycoplasma sp. was associated with clinical signs of upper respiratory tract disease-like syndrome [START_REF] Feldman | A novel mycoplasma detected in association with upper respiratory disease syndrome in free-ranging eastern box turtles (Terrapene carolina carolina) in Virginia[END_REF]. Due to its high incidence and clinical importance mycoplasma detection is a part of the routine diagnostic procedures for chelonians in many laboratories. This is the first description of a novel AdV and a box turtle Mycoplasma sp. infection in an ornate box turtle (Terrapene ornata ornata) in Europe characterized by fatty liver degeneration and inclusion body hepatitis.

Material and methods

Case history

On February 12, 2008 the carcass of a female ornate box turtle (Terrapene ornata ornata) was delivered to Szent István University, Faculty of Veterinary Science, Department of Pathology and Forensic Veterinary Medicine for postmortem evaluation. The turtle might have been dead for several days before finding it. Prior to its death the turtle was hibernated at 6-12 o C beginning in November and monitored weekly. Before hibernation the turtle was fed daily with insects (crickets: Acheta domesticus, Gryllus bimaculatus, Gryllus assimilis; Zophobas morio larvae) purchased in a pet shop. Drinking water was constantly available in a shallow dish.

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

Gross examination and microbiological investigations

A routine pathological dissection was carried out with macroscopic examination. Various organs were examined and samples fixed in 4% buffered formalin for later histological investigations. These tissues were then embedded in paraffin, cut in ultra-thin sections and stained with hematoxilin-eosin. Bacterial culture was carried out from the liver of the turtle on blood agar and Drigalski agar at 24°C for 48 hours. Samples of the liver, lung and intestines were collected for virus isolation and polymerase chain reaction (PCR). Small pieces of the organs were homogenized in Dulbecco's modified Eagle's medium (DMEM; Invitrogen, Carlsbad, USA), diluted, passed through a 0.2 µm filter and used for inoculation of turtle heart (ATCC CCL-50) cell monolayer supplemented with DMEM and 2% fetal bovine serum.

The cultures were incubated at 28°C in 5% v/v CO 2 and observed daily for cytopathic effects.

DNA extraction and PCR

Small tissue specimens of the liver, lung and intestine were used for DNA extraction using the DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. A nested PCR targeting a portion of the adenoviral DNA-dependent DNApolymerase gene (Wellehan et al., 2004) was carried out. The sample was also screened for chelonian herpesviruses (VanDevanter et al., 1996), ranaviruses (Marschang et al., 1999) and invertebrate iridoviruses [START_REF] Jakob | Comparative analysis of the genome and host range characteristics of two insect iridoviruses: Chilo iridescent virus and a cricket iridovirus isolate[END_REF], Weinmann et al., 2007) by PCR according to the protocols described by the authors. For detection of mycoplasma infection a general mycoplasma PCR (Lauerman et al., 1995), amplifying a fragment from the end of the 16S ribosomal RNA (rRNA) gene through the intergenic spacer region (ISR) to the beginning of the 23S rRNA gene, was used. After obtaining and sequencing the amplicon of the general PCR, a specific M. agassizii PCR (Brown et al., 1999) was carried out targeting the 16S rRNA gene to gain more sequence data. Phylogenetic analyses were carried out with several programs. Bayesian analysis was performed using MrBayes 3.1 (Ronquist and Huelsenbeck, 2003) with gamma-distributed rate variation, a proportion of invariant sites and Rtrev aminoacid substitution model. Maximum likelihood analysis was carried out by using the PhyML online web server [START_REF] Guindon | PHYML Online -a web server for fast maximum likelihood-based phylogenetic inference[END_REF] with Rtrev aminoacid substitution model. Distance matrix analysis was performed with programs included in the PHYLIP (Phylogeny Inference Package, version 3.68) program package [START_REF] Felsenstein | PHYLIP -Phylogeny Inference Package (Version 3.2)[END_REF]. The trees were visualized using the TREEVIEW 1.6.6 program (Page, 1996).

Results and discussion

Adenoviral infections are very common in different reptile species, but have only been described in chelonians in two cases -in leopard tortoise (Wilkinson, 2004) and in Sulawesi tortoises (Rivera et al., 2008) -so far. There is only limited information published about the AdV of the leopard tortoise which was isolated together with a herpesvirus. The tortoise Other pathologic changes included myeloid necrosis in the bone marrow and severe necrotizing enterocolitis.

Pathologic findings observed during macroscopic and microscopic examination of the box turtle were typical of an adenovirus infection. Upon opening its body cavity the liver appeared clay yellow in color. A small amount of transparent fluid was observed in the stomach, the mucosa of which was intact and wrinkled. Transparent phlegm was seen in the intestines and its mucosa had a slightly reddish color. The microscopic examination of the liver revealed degeneration of liver cells, a pronounced vacuolization of the cytoplasm and pyknosis of nuclei (Fig. 1). Large vacuoles were observed in the cytoplasm of some liver cells, in some cases leading to marginization of the nuclei, whereas only small, fine vacuoles were present in other cells. A light-bordered bright purplish blue, intensely staining inclusion body was also seen in some nuclei. These nuclei were substantially enlarged, with chromatin marginalization. Lymphoid cell infiltration was seen along the blood-vessels of the liver. The submucosa of the small intestine also showed lymphoid cell infiltration.

Although attempts at virus isolation were unsuccessful, the sample was PCR positive for adenoviruses supporting our observations described above. Chelonian herpesviruses, as well as ranaviruses and invertebrate iridoviruses were not detected by PCR. Nucleotide sequencing confirmed our PCR results. The sequence of the 275 bp long fragment of the partial DNApolymerase (Accesion number: EU828750) gene indicated a novel AdV, with a balanced GC content (49.45%). Comparison of the Box turtle AdV sequence with the corresponding region of other AdVs showed that it was unique and distinct from previously known adenoviral A c c e p t e d M a n u s c r i p t sequences. Amino acid sequence alignment of the DNA-polymerase gene showed that motifs found in all known AdVs were less conserved in the Box turtle AdV (see Supplementary material). Phylogenetic analyses also gave an interesting result (Fig. 2; see Supplementary material). While the Sulawesi tortoise AdV seems to be related to siadenoviruses, the Box turtle AdV did not cluster to any known genera. Altough there were differences between the branching topologies obtained by the different programs, all calculations confirmed the distinctness of the Box turtle AdV. The analyses were based on a relatively short nested PCR product (91 amino acid), therefore final conclusions cannot be drawn; further investigations are needed to reveal its genome organization, further genes (hexon, protease) should be included into phylogenetic calculations to establish the exact taxonomic position of this exceptional virus. Characterization of other turtle and tortoise AdVs would also give more information about the evolution and origin of chelonian AdVs.

Mass mortality in reptiles due to an adenoviral infection was demonstrated previously only in a few cases [START_REF] Kim | An outbreak of adenoviral infection in inland bearded dragons (Pogona vitticeps) coinfected with dependovirus and coccidial protozoa (Isospora sp.)[END_REF]Rivera et al., 2008), usually single animals died, therefore the pathogenicity of reptilian adenoviruses and their ability to cause primary disease remains uncertain. Factors leading to immune suppression have been postulated to play a role in the development of adenoviral disease, including stress (transportation, overcrowding), inadequate housing, hibernation, co-infection with other pathogens (viruses, bacteria and/or parasites), etc. In our instance the death of the turtle was also a single case. Additional research is necessary to determine the prevalence of AdVs in chelonians and its clinical importance.

Bacterial culture of the liver resulted in no growth; however, mycplasma DNA was detected by PCR. Sequences of the ISR (Accesion number: FJ159565) and from the 16S rRNA (Accesion number: FJ159564) were obtained from the detected Mycoplasma sp. The analysis of the amplified partial 16S rRNA sequence showed it to be identical with the formerly Kübber-Heiss, A., Benetka, V., Filip, T., Benyr, G., Schilcher, F., Pallan, C., Möstl, K., 2006. First detection of an adenovirus infection in a bearded dragon (Pogona vitticeps) in Austria Wiener Tierärztliche Monatsschrift 93, 68-72. Labarga, A., Valentin, F., Anderson, M., Lopez R., 2007. Web services at the European bioinformatics institute. Nucleic Acids Res., 35, W6-11. Lauerman, L.H., Chilina, A.R., Closser, J.A., Johansen, D. 1995. Avian mycoplasma identification using polymerase chain reaction amplicon and restriction fragment length polymorphism analysis. Avian Dis. 39, 804-811.

Marschang, R.E., Becher, P., Posthaus, H., Wild, P., Thiel, H.J., Müller-Doblies, U., Kalet, E.F., Bacciarini, L.N., 1999. Isolation and characterization of an iridovirus from Hermann's tortoises (Testudo hermanni). Arch. Virol. 144, 1909-1922. Nicholas, K.B., Nicholas, H.J.B., 1997. Genedoc

  Sequencing and sequence analyses PCR products were directly sequenced by using the Big Dye Terminator V3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, USA) according to the manufacturer instructions. The sequencing reaction was analyzed on an ABI PRISM® 3100 Genetic Analyzer. New sequences were compared with sequences deposited into GenBank (National Center for Biotechnology Information, Bethesda, USA) using BLASTX and BLASTN(Atschul et al., 1997). Pairwise and multiple alignments of nucleotide and amino acid sequences were made with the EMBOSS Pairwise Alignment Algorithms(Labarga et al., 2007) and theMultalin computer program (Corpet, 1988)l, respectively. The aligned sequences were edited with the GeneDoc program (Nicholas and Nicholas, 1997).

  s c r i p t exhibited biliverdinuria, wasting and episodes of hemorrhages were observed. The Sulawesi tortoise AdV caused severe systemic disease with very high mortality rate, 92 of 105 animals died due to the infection. Multifocal hepatic necrosis with amphophilic to basophilic intranuclear inclusions and diffuse hepatic lipidosis were observed in the Sulawesi tortoises.

identified

  Mycoplasma sp. of the eastern box turtle[START_REF] Feldman | A novel mycoplasma detected in association with upper respiratory disease syndrome in free-ranging eastern box turtles (Terrapene carolina carolina) in Virginia[END_REF]. There was only 1 nucleotide difference between the overlapping sequences of the 16S rRNA gene. Pairwise alignment of the amplified fragment containing the less conserved ISR sequence and the corresponding region of the other two Mycoplasma spp. using testudinian hosts, M. agassizii (AY780801) and M. testudineum (AY973570) demonstrated 87.9% (5.1% gaps) and 75.2% (11.3% gaps) sequence identity, respectively. Unfortunately the homologous sequence of the eastern box turtle Mycoplasma sp. was not available. Comparison of the chelonian mycoplasma ISR sequences also confirmed that the box turtle Mycoplasma sp. is a distinct species, since it differs from the previously characterized Mycoplasma spp. described in chelonians.Although we could detect the presence of the Mycoplasma sp. by PCR in our case, there were no pathological lesions consistent with disease caused by mycoplasma. Successful isolation of the detected infectious agents and experimental transmission studies should be carried out in order to clarify the role the Box turtle AdV and the Mycoplasma sp. plays in the death of the turtle.

  ., Harrach, B., Both, G.W., Russell, W.C., Adair, B.M., Adam, E., Dejong, J.C., Hess, M., Johnson, M.,Kajon, A., Kidd, A.H., Lehmkuhl, H.D., Li, Q.G., Mautner, V., Pring- Akerblom, P., and Wadell G., 2005. Family Adenoviridae. In: Fauquet, C.M., Mayo, M.A., Maniloff, J., Desselberger, U., Ball, L.A. (Eds.), Virus Taxonomy. Eighth Report of the International Committee on Taxonomy of Viruses. Academic Press, San Diego, CA, pp. 213-228. Brown, D.R., 2002. Mycoplasmosis and immunity of fish and reptiles. Front. Biosci. 7, 1338-46. Brown, M.B., McLaughlin, G.S., Klein, P.A., Crenshaw, B.C., Schumacher, I.M., Brown, D.R., Jacobson, E.R., 1999. Upper respiratory tract disease in the gopher tortoise is caused by Mycoplasma agassizii. J. Clin. Microbiol. 37, 2262-2269. Brown D.R., Merritt J.L., Jacobson E.R., Klein P.A., Tully J.G., Brown M. B., 2004. Mycoplasma testudineum sp. nov., from a desert tortoise (Gopherus agassizii) with upper respiratory tract disease. Int. J. Syst. Evol. Microbiol. 54, 1527-1529. Corpet, F., 1988. Multiple sequence alignment with hierarchical clustering. Nucleic Acids Res., 16, 10881-10890. Farkas, S.L., Harrach, B., Benko, M., 2008. Completion of the genome analysis of snake adenovirus type 1, a representative of the reptilian lineage within the novel genus Atadenovirus. Virus Res. 132, 132-139.

  Kovács, G.M.,LaPatra, S.E., D'Halluin, J.C., Benkő, M., 2003. Phylogenetic analysis of the hexon and protease genes of a fish adenovirus isolated from white sturgeon (Acipenser transmontanus) supports the proposal for a new adenovirus genus. Virus Res. 98: 27-34.

  : a tool for editing and annotating multiple sequence alignments. Distributed by the author. Page, R.D., 1996. TREEVIEW: An application to display phylogenetic trees on personal computers.Comput. Appl. Biosci. 12,[357][358]

  ., Fledelius, B., Schmidt, V., Kaján, G., Marschang, R.E., 2009. PCR-sequence characterisation of new adenoviruses found in reptiles, and the first successful isolation of a lizard adenovirus.Vet. Microbiol. 134, 233-240. Ramis, A., Fernandez-Bellon, H., Majo, N., Martinez-Silvestre, A., Latimer, K., Campagnoli, R., Harmon, B.G., Gregory, C.R., Steffens, W.L., Clubb, S., Crane, M., 2000. Adenovirus hepatitis in a boa constrictor (Boa constrictor). J. Vet. Diagn. Invest. 12, 573-576. Rivera, S., Wellehan,, J.F.X., McManamon, R., Innis, C.J., Garner, M.M., Raphael, B.L., Gregory, C.R., Latimer, K.S., Rodriguez, C.E., Diaz-Figueroa, O., Marlar, A.B., Nyaoke, A., Gates, A.E., Gilbert, K., Childress, A.L., Risatti, G.R., Frasca S., 2008. Systemic adenovirus infection in Sulawesi Tortoises (Indotestudo fortseni) caused by a novel siadenovirus. (Submitted for publication) Ronquist, F., Huelsenbeck, J.P., 2003. MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19, 1572-1574.VanDevanter D.R., Warrener, P., Bennett, L., Schultz, E.R., Coulter, S., Garber, R.L., Rose, T.M. 1996. Detection and analysis of diverse herpesviral species by consensus primer PCR. J.Clin. Microbiol. 34, 1666-1671. Weinmann, N., Papp, T., Alves de Matos, A.P., Teifke, J.P., Marschang, R.E., 2007.Experimental infection of crickets (Gryllus bimaculatus) with an invertebrate iridovirus isolated from a high-casqued chameleon (Chamaeleo hoehnelii). J.Vet. Diagn. Invest. 19,[674][675][676][677][678][679]

  Wellehan, J.F.X.,Johnson, A.J., 2005. Reptile virology. Vet. Clin. Exot. Anim. 8, 27-52. Wellehan, J.F.X., Johnson, A.J., Harrach, B., Benkő, M., Pessier, A.P. Johnson, C.M., Garner, M.M., Childress, A., Jacobson, E.R., 2004. Detection and analysis of six lizard adenoviruses by consensus primer PCR provides further evidence of a reptilian origin for the atadenoviruses. J.Virol. 78, 13366-13369. Wilkinson R., 2004. Clinical pathology. In: McArthur, S., Wilkinson, R., Meyer, J. (Eds.) Medicine and surgery of tortoises and turtles. Blackwell Press, Oxford, UK, pp. 141-186.

Fig. 1 .

 1 Figure captions:

Fig. 2 .

 2 Fig. 2. Unrooted phylogenetic tree based on maximum likelihood analysis of partial

  B1 NC_006324; B4 AF036092; C1 U55001; Chameleonid AY576679; D1 AC_000004; Eublepharid AY576677; F1 AC_000014; F9 AC_000013; Fr1 AF224336; Gekkonid AY576681; H2 J01917; H4 AY487947; H40 L19443; Helodermatid AY576680; M1 AC_000012; O7 U40839; P3 AB026117; P5 AF289262; Parakeet EU056825; Meyer AY644731; S21 AC_000010; Scincid AY576682; Snake DQ106414; Sulawesi EU056826; T3 AF074946; TS NC_004453.

Figure

  Figure

  Figure 2.

Acknowledgements

We would like to thank to Rachel E. Marschang, Petra Stumpf and Tibor Papp at the Institut für Umwelt-und Tierhygiene, Hohenheim University, Stuttgart, Germany for their help and advices. This work was supported by the OTKA F67847 grant of the National Research Fund of Hungary and the János Bolyai Research Scholarship of the Hungarian Academy of Sciences.


Marion Bernardeau

Micheline Gueguen

David G E Smith

Enrique Corona-Barrera

Jean Paul Vernoux

Jean-Paul Vernoux
email: jean-paul.vernoux@unicaen.fr

IN VITRO ANTAGONISTIC ACTIVITIES OF LACTOBACILLUS SPP. AGAINST BRACHYSPIRA HYODYSENTERIAE AND B. PILOSICOLI

Keywords: Lactobacillus, Brachyspira, probiotic, in vitro antagonism, coaggregation

The sensitivity of Brachyspira hyodysenteriae and B. pilosicoli, respectively the causative agents of Swine Dysentery and Porcine Intestinal Spirochaetosis to two probiotic Lactobacillus strains, Lb. rhamnosus CNCM-I-3698 and Lb. farciminis CNCM-I-3699 was studied through viability, motility and coaggregation assays. The cell-free supernatant of these lactobacilli contains lactic acid, that is stressful for Brachyspira (leading to the formation of spherical bodies), and lethal. It was demonstrated for the first time the in vitro coaggregation properties of two probiotic Lactobacillus strains (active or heat-treated) with two pathogenic strains of Brachyspira, leading to (1) trapping of spirochaetal cells in a physical network as demonstrated by SEM; (2) inhibition of the motility of Brachyspira. Such in vitro studies should encourage in vivo studies in animal model to evaluate the potential of the use of probiotic lactobacilli through a feeding strategy for the prevention of B. hyodysenteriae and B. Pilosicoli.

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

Introduction

Brachyspira hyodysenteriae and B. pilosicoli are important pathogenic intestinal spirochaetes that cause costly diarrhoeal diseases for the pig industry [START_REF] Smith | Colonic spirochetosis in animals and humans[END_REF], respectively Swine Dysentery (SD) and Porcine Intestinal Spirochaetosis (PIS). Economical losses (poor gain weight performance, high medication and death) have been recognized worldwide for decades. Recently, B. pilosicoli was considered as a zoonotic agent [START_REF] Hampson | Potential for zoonotic transmission of Brachyspira pilosicoli[END_REF].

Antimicrobial agents, mainly tylosin and tiamulin are widely used for extended periods to control SD and PIS in affected herds. However, in the light of increasing multiple drug resistance [START_REF] Karlsson | Antimicrobial susceptibility testing of porcine Brachyspira (Serpulina) species isolates[END_REF], more consideration has been given to reduce the risk of infection by applying preventive measures, and the use of antibiotic treatment should be combined with hygienic, technical and nutritional management practices. A nutritional strategy is the use of probiotics -live microorganisms which when administered in adequate amounts confer a health benefit on the host (FAO/ WHO, 2001). Supplementing animal feed with probiotic lactobacilli is well documented (reviewed by [START_REF] Bernardeau | Safety assessment and probiotic properties of two lactobacilli for use in pig farming and prevention of Brachyspira's disease. Thesis from the University of Caen, France[END_REF]. Some probiotics contribute to improve herd performance [START_REF] Torres-Rodriguez | Performance and condemnation rate analysis of commercial turkey flocks treated with a Lactobacillus spp.-based probiotic[END_REF][START_REF] Vasconcelos | Effects of increasing dose of live cultures of Lactobacillus acidophilus (Strain NP 51) combined with a single dose of Propionibacterium freudenreichii (Strain NP 24) on performance and carcass characteristics of finishing beef steers[END_REF] and health management [START_REF] Sargeant | Pre-harvest interventions to reduce the shedding of E. coli O157 in the faeces of weaned domestic ruminants: a systematic review[END_REF]. Multiple mechanisms of action for the beneficial effect of probiotics have been postulated, including preventing growth of pathogenic bacteria [START_REF] Tsai | Three Lactobacillus strains from healthy infant stool inhibit enterotoxigenic Escherichia coli grown in vitro[END_REF][START_REF] Wagner | Probiotic bacteria are antagonistic to Salmonella enterica and Campylobacter jejuni and influence host lymphocyte responses in human microbiota-associated immunodeficient and immunocompetent mice[END_REF], production of antimicrobial agents [START_REF] Gillor | The dual role of bacteriocins as anti-and probiotics[END_REF], coaggregation with pathogens [START_REF] Golowczyc | Protective action of Lactobacillus kefir carrying S-layer protein against Salmonella enterica serovar Enteritidis[END_REF] and stimulation of mucosal barrier function [START_REF] Isolauri | Modulation of the maturing gut barrier and microbiota: a novel target in allergic disease[END_REF].

To our knowledge, neither in vitro nor in vivo interactions between Brachyspira and probiotic strains have been studied to date. Thus, the aim of this study was to investigate the in vitro antagonistic activities of the cells of two Lactobacillus strains, Lactobacillus rhamnosus A c c e p t e d M a n u s c r i p t 4 CNCM-I-3698 and Lb. farciminis CNCM-I-3699, known as probiotics [START_REF] Bernardeau | Safety assessment and probiotic properties of two lactobacilli for use in pig farming and prevention of Brachyspira's disease. Thesis from the University of Caen, France[END_REF] and their cell-free supernatants against B. pilosicoli and B. hyodysenteriae.

Materials and methods

Bacterial strains and microbial cell suspensions

The two strains of lactobacilli, stored at -80°C, were cultured for 24 hours in 9.0 ml of MRS broth (MRS, De Man Rogosa Sharpe, AES, Combourg, France) or milk (Lait G, Standa Industrie, France) in anaerobic conditions (GENbox anaer, Biomérieux, Marcy l'Etoile, France) at 37°C prior to testing. The final Lactobacillus population was around 1x10 9 c.f.u. ml -1 . Lactobacillus cells were separated from cell-free supernatants by centrifugation at 10,000 x g at 4 °C for 10 min, the pellet was washed three times with phosphate-buffer saline (PBS: 0.8% NaCl, 0.02% KH 2 PO 4 , 0.115% Na 2 HPO 4 w/v, pH 7.4) and re-suspended with PBS in the initial volume. Cell-free supernatant was filtrated (Cellulose acetate membrane 0.2 µm, Sartorius, VWR, France).

Brachyspira pilosicoli strain ATCC-51139 (pig isolate originally known as P43/6/78) was obtained from the American Tissue and Cell Collection and kept at the Scottish Agricultural College, Veterinary Sciences Division, Edinburgh, which was then donated to us by Dr. Jill Thomson. Another strain of Brachyspira was obtained from LDA22 collection (Zoopôle Ploufragan, France). It was isolated from a clinical case of post-weaning piglet (15 kg) suffering from diarrhoea. It was identified by the LDA22 laboratory as B. hyodysenteriae and numbered 9828978.

To conduct experiments, spirochaetes were propagated on blood agar (BA) made of Columbia agar (AES, Combourg, France), supplemented with 5.0% sheep blood (AES, Combourg, France) and incubated in an anaerobic jar with GENbox anaer (Biomérieux, Marcy l'Etoile, A c c e p t e d M a n u s c r i p t 5 France) at 37 °C for B. pilosicoli and 42 °C for B. hyodysenteriae for 3 to 5 days. Spirochaete cells were harvested and suspended in PBS. Cells from several dishes were combined, washed 3 times and re-suspended in 1.0 ml of PBS to obtain a suspension containing 3x10 9 c.f.u. ml -1 (σ = 0.57), which was the working bacterial suspension.

Coaggregation between Lactobacillus and Brachyspira cells

The coaggregation analysis was performed according to [START_REF] Collado | Indigenous dadih lactic acid bacteria: cell-surface properties and interactions with pathogens[END_REF]. Absorbances (A 600 ) were monitored during the first 4 hours and after 18, 22 and 24 hours of incubation.

Coaggregation rate expressed as percentage was determined as [(A patho + A Lb )/2 -(A mix )/(A patho + A Lb )/2]× 100, where A patho and A Lb represent the absorbance at 600 nm in tubes containing respectively only the pathogen or each Lactobacillus strains and A mix represents the absorbance of the mixed culture after the same period of incubation.

Coaggregation was qualitatively evaluated by scanning and transmission electron microscopy.

The impact of coaggregation on motility was evaluated using an agar motility test that we developed for this work. Briefly, each strain of Lactobacillus (living cells or heat-inactivated at 120°C for 15min) and B. pilosicoli ATCC 51139 cell suspensions were pre-incubated (50/50 v/v) for 4 hours or 24 hours in an Eppendorf tube at 37°C. This test was performed in two ways: a "square test" and a "spot test", by spreading 100 µl of the mixed suspension with a loop on the surface of a blood agar plate respectively, in a 9 cm 2 defined square or by spotting a 5 µl drop. The plates were incubated for 8 days in anaerobic conditions as described above. The extent of motility was assessed every day, visually examining the hemolytic area and the spread of growth on the surface, compared to the natural spread of a Brachyspira cell suspension (control) not mixed with viable or heat-treated lactobacilli cells.

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

Materials were washed with PBS and fixed overnight at 4 °C with a cacodylate buffer (cacodylate 0.1M and saccharose 0.1M) containing 2.5% (v/v) glutaraldehyde. After washing with PBS, the cells were post-fixed for 30 min in the dark at room temperature with a mixture of cacodylate buffer containing 1% of osmium tetroxide OsO 4 . For scanning electron microscopic (SEM -JEOL Ltd 6400F, Tokyo, Japan) observations, cells were then washed three times with the same mixture and dehydrated in a gradually increased concentration series (30.0%; 50.0% and 70.0%) of ethanol solution for 10 min. The cells were then washed in 100 % ethanol for 10 min before being dried in a critical-point dryer (Balzers CPD 020) and coated with gold. For Transmission electron microscopic (TEM -JEOL 1011/micrograph MegaviewIII SIS) observations, the protocol was adapted from [START_REF] Wood | Spherical body formation in the spirochaete Brachyspira hyodysenteriae[END_REF]. Cells were washed through a cacodylate series (400, 300 and 200 mM), before being dehydrated through an ethanol gradually increased concentration series and infiltrated with resin. The resin was polymerized at 60°C for 24 hours. Ultra-thin sections (80 nm) obtained with a Leïca Microtome (Leïca Ultra Cut R) were mounted on formvar-coated copper grids. Poststaining was performed by soaking sections on 2.0% uranyl acetate for 15 min and lead citrate for 15 min.

Statistical analysis

The results for the viability, aggregation and coaggregation were expressed as the average of 3 independent experiments. Statistical analysis was performed by StatGraphicPlus software.

Data were subjected to a 1-way analysis of variance. 1). Nevertheless, the rate of the formation of coaggregates was delayed with heat-inactivated Lactobacillus strains compared to viable cells. Similar coaggregation results were obtained with B. hyodysenteriae 9828978 (data not shown). SEM was used to examine the morphological features of the interaction between the Lactobacillus and Brachyspira cells (Fig. 2). Large masses of coaggregated bacteria were evident in the scanning electron microscope preparations (Fig. 2-A) and there was typically a mesh-like between the cells found internally in these coaggregates (Fig. 2-B). Areas of close cell-to-cell contact between the two microorganisms are evident both on SEM (Fig. 2-C) and on TEM (Fig. 2-D). No preferential location of cell-to-cell interaction (e.g. tip versus middle) was found. The surface of Brachyspira had what appears to be thin filaments of exopolymers emerging from the outer membrane (Fig. 2-E). These fibrils or exopolymers are associated in many cases with other Brachyspira cells.

Effect of cell-free Lactobacillus supernatant on viability and morphology of Brachyspira

Coaggregation potential of Lactobacillus cells on Brachyspira

Inhibition of the spread of a motile Brachyspira strain

This was performed with the type strain B. pilosicoli ATCC 51139 which is very motile. The motility of spirochaetal cells on the surface of BA plates was entirely inhibited by viable Lb. rhamnosus 3698 after a 4 hour or 24h contact time contrary to viable Lb. farciminis 3699 which is only efficient after a 24h contact time (Table 2).

To by-pass the possible effect of competition for nutrients between the two bacterial species and that of the synthesis of antimicrobial substances, similar experiments were conducted with heat-inactivated lactobacilli cells. After 4 hours contact-time with heat-inactivated Lb. rhamnosus 3698, B. pilosicoli ATCC-51139 remained viable as a spot of haemolysis was still observed on the drop. However, the spirochaetal cells were not able to spread on the surface A c c e p t e d M a n u s c r i p t 9 of the agar media (Fig. 3-B). The same experiment conducted with heat-inactivated cells of Lb. farciminis 3699 did not limit the spreading or haemolysis of the spirochaetes (Fig. 3-C).

After 24 hours contact-time, no haemolysis and no spreading were observed with either strains of Lactobacillus, heat-inactivated (Fig. 3-E &F). So results were similar to those obtained with viable lactobacilli cells (Table 2) and competition for nutrients between the two bacterial genera or effect of antimicrobial substances produced by active Lactobacillus, are not responsible for inhibiting the spirochaetes motility. The prevention of the motility of the spirochaetes was interpreted as a consequence of the coaggregative properties of Lb. rhamnosus 3698 and Lb. farciminis 3699.

Discussion

Here we demonstrated and illustrated the sensitivity of one strain of B. hyodysenteriae and of one strain of B. pilosicoli (two pathogenic agents of economical importance in the pig industry) to two probiotic Lactobacillus strains. Contact with cell-free Lactobacillus supernatant had a lethal effect on Brachyspira cells, which was characterized by broken cells walls. Sensitivity of Brachyspira to acidic compounds and disinfectants has already been shown by one of us [START_REF] Corona-Barrera | Epidemiology, detection molecular typing and control of porcine colonic spirochaetosis (Brachyspira pilosicoli)[END_REF]2004). Morphological changes due to stress have been observed here and lead to "spherical body" formation. De Ciccio (1997) has demonstrated that an accumulation of toxic products, a change in environmental conditions caused by lactic acid have conducted to the formation of spherical body in Treponema denticola (a member of the same phylum as spirochetes). Spherical bodies have also been observed with Brachyspira strains and they can revert to normal helical form if conditions improve [START_REF] Wolf | Formation of multiple treponemes[END_REF]. Wood et al., 2006 described Coaggregation -process by which genetically distinct bacteria become attached to one another via specific molecules-is of considerable importance in several ecological niches [START_REF] Jankovic | Contribution of aggregation-promoting factor to maintenance of cell shape in Lactobacillus gasseri 4B2[END_REF]. It could protect from specific diseases, as has been demonstrated for the oral cavity [START_REF] Willcox | Coaggregation of oral lactobacilli with streptococci from the oral cavity[END_REF]), urogenital tract (Mastromarino et al., 2002) and intestinal tract of humans and pigs [START_REF] Drago | Inhibition of in vitro growth of enteropathogens by new Lactobacillus isolates of human intestinal origin[END_REF][START_REF] Kmet | Aggregation promoting factor in pig intestinal Lactobacillus strains[END_REF]. We demonstrated for the first time the in vitro coaggregation properties of two probiotic Lactobacillus strains (active or heattreated) with two pathogenic strains of Brachyspira, leading to (1) trapping of spirochaetal cells in a physical network as demonstrated by SEM; (2) inhibition of the motility of Brachyspira. The effect on motility is very important result as motility (along with chemotaxis) has emerged as a major virulence factor of intestinal spirochaetes, allowing them to escape the physical effect of mucus [START_REF] Witters | Motility-regulated mucin association of Serpulina pilosicoli, the agent of colonic spirochetosis of humans and animals[END_REF]. Mucus eliminates unattached micro-organisms efficiently from the gastro-intestinal tract and, unlike other enteric pathogens, B. hyodysenteriae and B. pilosicoli are highly motile in the presence of mucus [START_REF] Nakamura | Improvement in motion efficiency of the spirochete Brachyspira pilosicoli in viscous environments[END_REF].

The coaggregation properties of some lactobacilli cells have already been observed by [START_REF] Schachtsiek | Characterization of Lactobacillus coryniformis DSM 20001T surface protein Cpf mediating co-aggregation with and aggregation among pathogens[END_REF] for Lb. coryniformis strains DSM 20001 T and LTH 1750, Lb. gasseri 4B2, on cells of pathogens involved in enteric disorders: Escherichia coli K88, Campylobacter coli and Campylobacter jejuni. The nature of the coaggregation of some Lactobacillus strains with Escherichia coli has also been determined and linked to high level of exopolysaccharides production [START_REF] Aslim | Factors influencing autoaggregation and aggregation of Lactobacillus delbrueckii subsp. bulgaricus isolated from handmade yogurt[END_REF]. The coaggregative property of Lactobacillus is neither genus-nor species-specific. Lactobacillus strains have also demonstrated coaggregative properties with yeasts of urogenital importance such as Candida albicans [START_REF] Boris | Adherence of human vaginal lactobacilli to vaginal epithelial cells and interaction with uropathogens[END_REF][START_REF] Ocana | Vaginal lactobacilli: self-and co-aggregating ability[END_REF][START_REF] Hronek | Antifungal effect in selected natural compounds and probiotics and their possible use in prophylaxis of vulvovaginitis[END_REF]. In vitro observations of antagonistic properties of two probiotic strains of Lactobacillus against two strains of Brachyspira suggest a great potential as a preharvest strategy against Brachyspira carriage. It agrees with our preliminary field observations of a reduce carriage of Brachyspira following a feeding probiotic therapy with these two lactobacilli [START_REF] Bernardeau | Diet supplementation with fermentative heat-inactivated lactobacilli based product can help to prevent swine dysentery in pigs[END_REF]. Further investigations are needed to explain the nature of coaggregative properties and to determine whether lactobacilli and Brachyspira species compete for similar specific receptors on the intestinal epithelium. Such in vitro studies should encourage in vivo studies in animal model to evaluate the potential of the use of probiotic lactobacilli through a feeding strategy for the prevention of B. hyodysenteriae and B. Pilosicoli.

  cellsLactobacillus supernatant obtained from MRS was an acidic solution (pH 3.5), and was shown to contain 14.4 g of lactic acid/l (especially the L-form: 13.7 g/l as determined by D-Lactic acid/L-Lactic acid Enzymatic kit, Boehringer Mannheim). A highly significant (P<0.001) inhibitory effect against B. hyodysenteriae 9828978 and B. pilosicoli ATCC-51139 was observed after 1 hour of contact time with Lactobacillus supernatant obtained from MRS since the initial bacterial count of 1x10 9 c.f.u. ml -1 diminished completely by this time point. Similar inhibitory effects were observed with acidic solutions 14.4 g/L of lactic acid at pH 3.5.The results of the adverse effect of the acidic Lactobacillus cell-free supernatant obtained from milk (pH 4.5), on the cell morphology of the spirochaetes are shown on Fig.1. In Fig.1(A), all spirochaetal cells from the PBS control exhibit a typical thin, smooth helical form with pointed ends. The exposure of spirochaetal cells to a stressful acidic (pH 4.5) environment for 1 hour showed besides scattered cells poles (data not shown), cells with morphological variations presumably due to a progressive degree of injury ranging from stress to lethality. For example, cells from Fig.1(B) exhibits progressive stressed stages which could be summarised as: (1) cell retracts and folds in two, (2) exopolymers are probably synthesised between the two parallel strands of the cell, (3) cell curls up, and (4) spherical body is formed. The observations were similar for both Brachyspira species.

Each

  Lactobacillus strain -either viable or heat-inactivated-showed coaggregation abilities towards B. pilosicoli ATCC-51139 (Table

  after a nutritional depletion and postulated that it is an adaptive survival response to adverse environmental conditions.

Figure caption Fig. 1 .

 caption1 Figure caption

Fig. 2 .

 2 Fig. 2. Electron micrographs of co-aggregates of heat-inactivated Lb. rhamnosus 3698 and B.

Fig. 3 .

 3 Fig. 3. Study of the motility (spot motility test) of B. pilosicoli ATCC 51139 pre-incubated

  Figure 1

  this kind of spherical body with

Table 1 :

 1 Co-aggregation of viable or heat-inactivated cells of Lb. rhamnosus 3698 or Lb.

	farciminis 3699 and B. pilosicoli ATCC-51139 as determined by the spectrophotometry
	assay; results expressed as average (n=3)  S.D.

Table 2 :

 2 Effect of a 4 or 24 hours contact-time of viable or heat-inactivated (120°C/15min) lactobacilli cells on haemolysis, motility (spot motility test) and growth of a B. pilosicoli ATCC 51139 suspension.

Acknowledgments

This study was supported by a grant from the Association Nationale de la Recherche Technique (France) and Sorbial, a French company. Authors wish to thank Atmane Asselate and Raouf Tareb for their technical support. Authors gratefully acknowledge Dr. Didier Goux from the Microscopy centre of the University of Caen (France) for his scientific and technical assistance getting the electron micrographs.

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


Wiebren Santema
email: w.j.santema@uu.nl

Marije Overdijk

Judith Barends

Jeroen Krijgsveld

Victor Rutten

Ad Koets

Searching for proteins of Mycobacterium avium subspecies paratuberculosis with diagnostic potential by comparative qualitative proteomic analysis of mycobacterial tuberculins Authors

Keywords: Tuberculins, Proteomics, Paratuberculosis, Antigen

Accurate immunodiagnosis of bovine paratuberculosis is among others hampered by the lack of specific antigens. One of the most frequently used antigen preparations is purified protein derivative (PPD), also known as tuberculin. This crude extract has limitations when used in diagnostic assays due to the presence of cross-reactive antigens. The aim of the current study was to systematically analyze the qualitative protein composition of PPD of the major mycobacterial pathogens.

One-dimensional gel electrophoresis followed by tandem mass spectrometry analysis of PPD from Mycobacterium avium subspecies paratuberculosis (MAP), Mycobacterium avium subspecies avium (MAA) and Mycobacterium bovis (MB) identified 156, 95 and 132 proteins, respectively. Comparative sequence analysis led to the selection of a MAP-specific protein (MAP1718c), and finally heterologous expression in E. coli of this and other diagnostic candidate proteins (MAP3515c and MAP1138c (LprG)) enabled evaluation of their immunogenicity. Lymphocyte proliferation responses did not indicate substantial diagnostic potential of the antigens tested In contrast serum antibody levels for MAP1138c in paratuberculosis infected cows (N=20) were significantly higher (p<0.05) than in control animals (N=20), despite the conserved nature of this protein.

In conclusion, this study showed that a combination of proteomics and genomics, starting from complex protein mixtures, present in tuberculins, can reveal novel proteins aiding the development of immunodiagnostics for mycobacterial diseases.

Accurate immunodiagnosis of bovine paratuberculosis, a chronic incurable infection of the small intestine caused by Mycobacterium avium subspecies paratuberculosis (MAP), is among others hampered by the lack of specific antigens [START_REF] Harris | Mycobacterium avium subsp. paratuberculosis in Veterinary Medicine[END_REF]. Purified protein derivative (PPD), also known as tuberculin, produced from heat-processed mycobacterial culture filtrates, is one of the antigen preparations most used in mycobacterial immunodiagnostics, like skin tests in animals and humans, IFNγ assays, and in serum antibody ELISA [START_REF] Monaghan | The tuberculin test[END_REF]. The PPD protein mixture likely contains many individual proteins. To limit false positive reactions to antigens shared with other pathogens or innocuous environmental micro-organisms, MAP-specific antigens are needed. Aided by the completion of the prototype mycobacterial genomes of MAP, MAA and Mycobacterium bovis (MB) / Mycobacterium tuberculosis (MT) we performed a qualitative, comparative proteomic analysis of commercially produced PPDs from MAP, MAA and MB, and evaluated the immunogenicity of three selected MAP proteins.

Material and Methods

Proteomics

The purified protein derivatives, produced according to the OIE manual for diagnostic tests from MAP strain 3+5/C (PPDP), MAA strain D4 (PPDA) and M. bovis strain AN5 (PPDB), were contracted from two producers, CVI-Lelystad (Lelystad, the Netherlands) and CZ Veterinaria (Pontevedra, Spain). Hundred microgram of each PPD was separated by onedimensional gel electrophoresis in a 13% (w/v) SDS-PAGE gel, followed by in-gel digestion with trypsin. Proteins were reduced with 6.5mM DTT (Roche Diagnostics) followed by alkylation with 54mM iodoacetamide (Sigma-Aldrich, St. Louis, USA) for one hour, and then digested with trypsin at an enzyme: substrate ratio of 1:50 (w/w). Nano-LC-MS/MS was A c c e p t e d M a n u s c r i p t 4 performed as previously described [START_REF] Van Balkom | Mass spectrometric analysis of the Schistosoma mansoni tegumental sub-proteome[END_REF]. Peptides were identified using Mascot software (MatrixScience, version 1.9) using the following settings: trypsin was used as an enzyme with one miscleavage allowed, peptide tolerance was set to 1.2 Da; carbamidomethyl and oxidation of methionine were set as fixed and variable modifications, respectively. Scaffold (version Scaffold_2_00_05, Proteome Software Inc., Portland, OR) was used to validate MS/MS based peptide and protein identifications. Peptide and protein identifications were accepted if they could be established at greater than 90.0% and 95.0% probability as specified by the Peptide/Protein Prophet algorithms, respectively. Protein identifications were accepted if they contained at least 2 identified peptides. The Geneplot program (available at http://www.ncbi.nlm.nih.gov/) was used to identify protein homologues between the sequenced genomes of Mycobacterium avium spp. paratuberculosis strain K-10, Mycobacterium avium 104 and Mycobacterium bovis AF2122/97. TBLASTN was used to calculate the homology on protein level.

Recombinant proteins

Primers were designed from the coding sequence of the MAP strain K10 genome (GenBank: AE016958) using Primer software (Version 1.0). Each forward primer contained the 5'extension CACC to facilitate directional cloning into the pET100/D TOPO vector (Invitrogen, USA). The following primers were used to clone the genes of interest: MAP1718cF, 5'CACCGTGGCGTTCTCACACATTG3'; MAP1718cR, 5'CTAGCTCTTCCAGACCACCTTG3'; MAP3515cF, 5'CACCATGTATTCCGAGTCGAAG3'; MAP3515cR, 5'TCATGTGCGGCAGCTGATTTCC3'; MAP1138cF,5'CACCATGCTGGGTATGCAGACCCG3'; MAP1138cR, 5'TCACTTGGTGACCTGGACCTGCTC3'.

A c c e p t e d M a n u s c r i p t 5 Genomic DNA from MAP strain G195 and MAA strain D4 was used as template for the PCR reactions. Amplification was performed on an I-Cycler (BioRad, USA) with Pfu Turbo Cx Hotstart DNA Polymerase (Stratagene, USA). The selected genes were cloned into the pET100/D TOPO vector (Invitrogen, USA), containing an N-terminal His-tag. Expression was performed in BL21 Star (DE3) E. coli cells (Invitrogen, USA) using 1.0 mM isopropyl ß-D-thiogalactoside induction. Recombinant proteins were affinity-purified on a Ni-column, using Chelating Sepharose Fast Flow (Amersham Bioscience, USA). Purity was checked on 13% (w/v) SDS-PAGE gels and confirmed by Western blot analysis using an anti-His monoclonal antibody (Amersham Bioscience, USA). The purified recombinant proteins were dialyzed against PBS, concentrated using a Vivaspin 10,000 MWCO PES membrane (VivaScience, Germany) and quantified by the BCA kit (Pierce, USA). All procedures were performed according to instructions provided by the manufacturers.

Immunoassays

Sera were derived from Holstein-Friesian cattle on farms which participated in paratuberculosis field surveys in the Netherlands. The sera were classified based on results from repeated fecal culture and serological responses measured in the absorbed ELISA for paratuberculosis diagnosis (Pourquier, France) and stratified in three groups: frequent shedders (N=20), intermediate shedders (N=20) and control animals (N=20). Serological responses to the recombinant proteins were measured using an ELISA technique as described previously [START_REF] Koets | Differential changes in heat shock protein-, lipoarabinomannan-, and purified protein derivative-specific immunoglobulin G1 and G2 isotype responses during bovine Mycobacterium avium subsp. paratuberculosis infection[END_REF] were performed as described previously [START_REF] Koets | Heat-shock protein-specific T-cell responses in various stages of bovine paratuberculosis[END_REF], with the antigens at a concentration of 10 μg/ml. To correct for differences in background proliferation in wells stimulated with culture medium alone, data were expressed as Δ-cpm (cpm in antigen stimulated wells minus cpm in medium control wells). Data were analyzed using SPSS 15 software. ELISA data were normalized by Log transformation and ANOVA was used for comparison of groups. Results of the proliferation assays were compared using a nonparametric Mann-Whitney test on Δ-cpm values. The level of significance was set at p<0.05.

Results

Protein identification and selection

Analysis of the three Lelystad PPDs by tandem mass spectrometry led to the generation of proteomes for PPDP, PPDA and PPDB, in which 156, 95 and 132 proteins were identified, respectively. Comparison of the generated proteomes by matching protein homologues showed that a large proportion (44-73%) of proteins was shared between the PPDs (figure 1a).

In PPDA the total number of proteins identified was markedly less than in both other PPDs, however the ratio of shared proteins with the other PPDs was the highest (73%). In total 25 proteins were shared by PPDP, PPDA and PPDB produced by one manufacturer (CVI-Lelystad). To determine the overlap in proteome within a specific PPD, we next analysed three PPDA's from different manufacturers (two independent batches from CZ Veterinaria and one from CVI-Lelystad). The comparison of the proteomes generated for the PPDA's is depicted in figure 1b. The ratio of shared proteins ranged from 67-81%. In total 39 proteins A c c e p t e d M a n u s c r i p t were shared between all PPDA preparations, representing 35-43% of the total proteins identified in the PPDA preparations.

Nineteen proteins were identified in each of the PPDs analyzed (table 1). The average homology on protein level as established with TBLASTN analysis for this group of proteins compared to PPDP was 99% (range 99-100%) for PPDA and 87% (range 67-96%) for PPDB homologues. Of these 19 proteins 8 and 5 proteins have been described as immunogenic in bovine paratuberculosis and bovine tuberculosis, respectively.

Next, we selected specific proteins for MAP (table 2). Since MAP and MAA genomes are known to be highly similar, and as a consequence very few MAP-specific proteins exist, we also selected MAP proteins differentially expressed in mycobacteria. To define MAP-specific proteins we selected MAP proteins present in PPDP that were not identified in PPDA, and had no homologues in the sequenced MB genome. Only hypothetical protein MAP1718c had no homologue identified in the sequenced MAA genome and was therefore selected as MAPspecific protein. For the remaining proteins in PPDP homologues are present in the MAA genome with on average over 98% similarity on protein level, which was established with TBLASTN analysis. The hypothetical protein MAP3515c was selected as a first protein, based on differential expression in MAP and MAA, to be screened for immunogenicity. Furthermore, MAP1138c (LprG), a conserved protein present in all PPDs, was selected for evaluation of immunogenicity in paratuberculosis infection, based on immunogenicity of the M. tuberculosis homologue. The homology on protein level of MAP1138c as compared with the M. bovis LprG protein is 67%.

Immunoassays

The selected proteins (MAP1718c, MAP3515c and MAP1138c) were successfully cloned and expressed in E. coli. The purified proteins were used to evaluate their potential as T cell antigen in a proliferation assay. The results are summarized in figure 2 (A-D). Animals A c c e p t e d M a n u s c r i p t 8 infected with paratuberculosis showed higher responses to PPDP as compared to control animals (p<0.05). No significant differences between groups were observed for the recombinant MAP1718c, MAP3515c and MAP1138c (LprG) proteins. The purified proteins were also evaluated for immunogenicity in a serum antibody ELISA. The results are summarized in figure 2 (E-H). No significant differences between groups were observed for the recombinant MAP1718c and Map3515c protein. However, high shedders showed significant higher responses than control animals (p<0.01) for the MAP1138c (LprG) protein.

Discussion

This study describes a qualitative survey of the proteins present in mycobacterial PPDs.

Proteomics has been used to identify proteins in mycobacterial culture filtrates and cell wall extracts [START_REF] Cho | Identification of proteins of potential diagnostic value for bovine paratuberculosis[END_REF]Xiong et al., 2005). However, we were interested in the protein composition of PPD, the most widely used antigenic preparation in mycobacterial immunodiagnostics. To improve current diagnostic assays, knowledge on the composition of PPD may enable the rational design of a synthetic tuberculin with selected immunogenic noncross reactive proteins or peptides.

The proteome analysis showed that the PPDs have a high range of shared proteins most likely partly responsible for the limited specificity of PPDs. In this group well-characterized and evolutionary conserved antigens are present like the 85 A, B, C complex proteins, heat shock protein 70 and Antigen D (bacterioferritin). The protein homology for these proteins is high, especially between the closely related species MAP and MAA, and likely also non-pathogenic environmental species. However, based on the range of protein homology, we speculate that specifically when MAP/MAA and MB proteins are compared, there could be peptides or protein regions within these conserved proteins that elicit pathogen-specific immune responses.

A c c e p t e d M a n u s c r i p t 9

The majority of proteins identified in PPDB have been previously identified in culture filtrates or cell membrane analysis (Xiong et al., 2005). Tuberculosis-specific diagnostic antigens, like ESAT-6 and CFP-10, (Cockle et al., 2002) are present in PPDB. Andersen et al. described 30 antigens of M. tuberculosis recognized by T cells or effective as vaccine in animal models [START_REF] Andersen | TB subunit vaccines--putting the pieces together[END_REF]. Sixteen of these proteins were identified in PPDB in this study.

The hypothetical protein MAP1718c was identified as MAP-specific protein in PPDP, because it was absent from the sequenced genome MAA strain 104. However, in the current study a 99% identical homologue of the MAP1718c gene could also be amplified from MAA strain D4 and it was shown that a large sequence polymorphism containing MAP1718c was present in 44 of the 96 non-MAP isolates [START_REF] Semret | Genomic polymorphisms for Mycobacterium avium subsp. paratuberculosis diagnostics[END_REF]. Apparently strain variation within mycobacterial species may further complicate the search for diagnostic candidates.

The other potential MAP-specific proteins we have selected are based on differential expression of these proteins in the context of the tuberculin production process, in PPDP as compared to PPDA, rather than on the absence of the homologous gene in MAA. The immunoassay data showed that MAP1718c and MAP3515c are not immunodominant paratuberculosis antigens, unlike MAP1138c (LprG) that is recognized by antibodies during the course of paratuberculosis infection. LprG was recognized by high shedders to a larger extend than by intermediate shedders which may be related to the stage of the disease these animals are in. This phenomenon was also observed in other serodiagnostic studies using crude antigens [START_REF] Koets | Differential changes in heat shock protein-, lipoarabinomannan-, and purified protein derivative-specific immunoglobulin G1 and G2 isotype responses during bovine Mycobacterium avium subsp. paratuberculosis infection[END_REF]. Recently, MAP LprG was also identified by screening of a MAP gene fusion library and found to be immunogenic in bovine paratuberculosis [START_REF] Dupont | Identification and characterization of an immunogenic 22 kDa exported protein of Mycobacterium avium subspecies paratuberculosis[END_REF]. However, the sensitivity of the LprG ELISA appears to be much lower than the Pourquier ELISA.

  with minor modifications. Plates were coated with 1 μg protein per well, and IgG1 antibody responses were measured. For the proliferation assays, blood samples were used from Holstein-Friesian cattle on dairy farms participating in control programs in the Netherlands. Animals were classified according to their clinical history and laboratory tests as described above. Control animals (N=14) originated from a single dairy farm with no known history of paratuberculosis, participating in the Dutch paratuberculosis A c c e p t e d M a n u s c r i p t 6 control programs and having obtained the highest herd status based on repeated testing. The infected animals (N=12) originated from different dairy farms and were classified as MAP infected based on repeated positive fecal culture results. Lymphocyte stimulation tests (LST)

  .A., Brooks, B.W., Young, N.M., Watson, D.C., Nielsen, K.H., Corner, A.H., Turcotte, C., Michaelides, A., Stewart, R.B., 1991, Chromatographic purification and characterization of antigens A and D from Mycobacterium paratuberculosis and their use in enzyme-linked immunosorbent assays for diagnosis of paratuberculosis in sheep. J Clin Microbiol 29, 1659-1664. Xiong, Y., Chalmers, M.J., Gao, F.P., Cross, T.A., Marshall, A.G., 2005, Identification of Mycobacterium tuberculosis H37Rv integral membrane proteins by one-dimensional gel electrophoresis and liquid chromatography electrospray ionization tandem mass spectrometry. J Proteome Res 4, 855-861.

  Figure captions

  Figure 2

  Figure1

Table 1

 1 Proteins identified in all PPDs analyzed (N=19). The locus tag of each protein for all three mycobacterial species analyzed is depicted. 3 Antigenicity of each protein within bovine paratuberculosis and bovine tuberculosis as described in literature is shown. MW: molecular weight in 4

	5	Da, ND: not described.

Protein name Locus tag MAP Locus tag MAA Locus tag MB MW (Da) ParaTB antigen TB antigen Reference

		35kd antigen	MAP_2855c	MAV_3635	Mb2765c	29.442	ND	ND
		Aconitate hydratase 1	MAP_1201c	MAV_3303	Mb1511c	101.097	ND	ND
		Acyl carrier protein	MAP_1997	MAV_2193	Mb2268	12.467	ND	ND
		Adenosylhomocysteinase	MAP_3362c	MAV_4211	Mb3276c	54.476	ND	ND
		Antigen 85-A	MAP_0216	MAV_0214	Mb3834c	36.078	Yes	Yes	(Rhodes et al., 2000; Rosseels et al., 2006)
		Antigen 85-B	MAP_1609c	MAV_2816	Mb1918c	34.676	Yes	Yes	(Rhodes et al., 2000; Rosseels et al., 2006)
		Antigen 85-C	MAP_3531c	MAV_5183	Mb0134c	38.035	Yes	Yes	(Rhodes et al., 2000; Rosseels et al., 2006)
		ATP synthase F1, beta subunit	MAP_2451c	MAV_1527	Mb1342	53.080	ND	ND
		Bacterioferritin	MAP_1595	MAV_2833	Mb1907	18.462	Yes	ND	(Sugden et al., 1991)
		Chaperone protein DnaK	MAP_3840	MAV_4808	Mb0358	66.510	Yes	Yes	(Koets et al., 1999; Rhodes et al., 2000)
		60 kDa chaperonin 2	MAP_3936	MAV_4707	Mb0448	56.597	Yes	Yes	(Koets et al., 1999; Rhodes et al., 2000)
		LprG protein	MAP_1138c	MAV_3367	Mb1446c	24.401	Yes	ND	(Dupont et al., 2005)
		Malate dehydrogenase	MAP_2541c	MAV_1380	Mb1272	34.587	ND	ND
		ModD protein	MAP_1569	MAV_2859	Mb1891	37.512	Yes	ND	(Cho et al., 2007)
		Ribosomal protein L7/L12	MAP_4126	MAV_4507	Mb0671	13.413	ND	ND
		Translation elongation factor Tu	MAP_4143	MAV_4489	Mb0704	43.752	ND	ND
		Trypsin	MAP_3527	MAV_5187	Mb0130	36.698	No	ND	(Cho et al., 2007)
		Universal stress protein family protein MAP_1339	MAV_3137	Mb1662	13.708	ND	ND
	6	Wag31 protein	MAP_1889c	MAV_2345	Mb2169c	26.988	ND	ND
	7
	8
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Acknowledgement

The authors express their thanks to Jolanda Meijlis (CVI-Lelystad) and Eugenia Puentes (CZ Veterinaria) for the gifts of tuberculins. This work was supported by the Netherlands Proteomics Centre.

A c c e p t e d M a n u s c r i p t 10 Genomic approaches have led to the identification of MAP-specific ORFs by comparative sequence analysis followed by heterologous expression and immunogenicity evaluation [START_REF] Bannantine | Expression and immunogenicity of proteins encoded by sequences specific to Mycobacterium avium subsp. paratuberculosis[END_REF]. This has not yet resulted in novel specific antigens useful for diagnostic approaches. A disadvantage of this approach is the lack of knowledge on transcription of the identified ORFs. Proteome analysis identifies proteins which are actually transcribed under the conditions studied. Future work should also aim to identify differential expressed antigens, as this could be a key in detecting novel diagnostic antigens. If the induced proteome of MAP during infection is different from related environmental mycobacteria, resulting in a differential induction of the immune response, we propose that conserved antigens can be useful as diagnostic targets. An interesting approach in this respect is the recent study by Hughes et al., who compared the proteome of in vitro cultured MAP and MAA bacteria and selected 32 differentially expressed MAP proteins [START_REF] Hughes | Proteome-determined Mycobacterium avium subspecies paratuberculosis-specific proteins: immunogenicity in ovine paratuberculosis[END_REF].

Eight proteins were also identified in the current study, of which only MAP3627 was found to be immunogenic in ovine paratuberculosis.

In conclusion, we have used proteomics as a tool to qualitatively analyze tuberculins. Three MAP proteins (MAP1718c, MAP3515c and MAP1138c) were tested in immunodiagnostic assays. The evolutionary conserved recombinant MAP1138c protein is best recognized of these three proteins by paratuberculosis infected cows in a serum antibody ELISA. These results support the line of thought that there could be evolutionary conserved proteins or parts thereof that elicit pathogen and/or infection-specific immune responses. They warrant further study as many of these proteins are important targets for the host immune system.