Kathryn L Smith

George P Allen

Adam J Branscum

R Frank Cook

Mary L Vickers

Peter J Timoney

Udeni B R Balasuriya
email: ubalasuriya@uky.edu

Maxwell H Gluck

The Increased Prevalence of Neuropathogenic Strains of EHV-1 in Equine Abortions

Keywords: Equine herpesvirus-1, Neuropathogenic EHV-1, Equine myeloencephalopathy, Horse

A panel of 426 archived EHV-1 isolates collected (1951 to 2006) from equine abortions was analyzed using a real-time Taq-Man ® allelic discrimination PCR. Based on previous findings, isolates possessing adenine at nucleotide position 2254 (A 2254 ) in ORF30 were classified as having a non-neuropathogenic genotype and those with guanine at 2254 (G 2254 ) were designated as the neuropathogenic genotype. The resultant data demonstrated that viruses with the neuropathogenic genotype existed in the 1950s and isolates with this genotype increased from 3.3% in the 1960s to 14.4% in the 1990s. The incidence of EHV-1 isolates from 2000 to 2006 with G at position 2254 is 19.4%, suggesting that viruses with the neuropathogenic genotype are continuing to increase in prevalence within the latent reservoir of the virus, leading to greater risks for costly outbreaks of equine herpesvirus neurologic disease. Another highly significant finding was two isolates failed to react with either probe in the allelic discrimination assay. These isolates were found to possess an adenine to cytosine substitution at position 2258 (A 2258 →C 2258 ) in ORF30, in addition to A 2254 →G 2254 . Interestingly, the nonneuropathogenic RAC-H modified live vaccine strain of EHV-1 also contains both A 2254 →G 2254 and A 2258 →C 2258 substitutions. This finding clearly suggests that additional research is required before the genetic basis of the neuropathogenic phenotype in EHV-1 is fully understood.

Introduction

Equine herpesvirus-1 (EHV-1) infections cause significant economic losses for equine industries worldwide as a result of abortion, respiratory illness, and neurologic disease in all breeds of horses. Although it is believed almost all strains of EHV-1 can induce abortion in pregnant mares, only certain strains have the potential to cause neurologic disease [START_REF] Nugent | Analysis of equid herpesvirus 1 strain variation reveals a point mutation of the DNA polymerase strongly associated with neuropathogenic versus nonneuropathogenic disease outbreaks[END_REF]. Equine disease monitoring in Central Kentucky over the past 51 years has confirmed the frequency of EHV-1 induced abortions has declined, even though the number of broodmares has increased threefold [START_REF] Powell | Equine herpesvirus abortions[END_REF].

The majority of such abortions in recent years have been single, sporadic events on individual farms among populations of mares that are routinely vaccinated against disease. In contrast, cases of EHV-1 induced neurologic disease have increased significantly in number since the year 2000 [START_REF] Allen | Prevalence of latent, neuropathogenic equine herpesvirus-1 in the Thoroughbred broodmare population of central Kentucky[END_REF][START_REF] Marenzoni | Clinical, serological and molecular investigations of EHV-1 and EHV-4 in 15 unweaned thoroughbred foals[END_REF][START_REF] Patel | Equine herpesviruses 1 (EHV-1) and 4 (EHV-4)-epidemiology, disease and immunoprophylaxis: a brief review[END_REF]. 1 Within the United States and the United Kingdom, the number of reported outbreaks has risen from one occurrence in the early 1970s to 32 during the years 2001-2005 [START_REF] Anonymous | Equine herpesvirus myeloencephalopathy: a potentially emerging disease[END_REF]. 1 The associated case-fatality rate may also be increasing within the United States, ranging from 20% in some instances, to as high as 50% in others [START_REF] Slater | Report of the equine herpesvirus-1 Havermeyer Workshop[END_REF]. Additionally, in 2005 significant outbreaks occurred in Canada, South Africa, Switzerland, Ireland and other European nations [START_REF] Goehring | Equine herpesvirus type 1-associated myeloencephalopathy in the Netherlands: a four-year retrospective study (1999-2003[END_REF][START_REF] Slater | Report of the equine herpesvirus-1 Havermeyer Workshop[END_REF].

EHV-1 is a member of the subfamily Alphaherpesvirinae with a 150kbp double-stranded DNA genome, consisting of 80 open reading frames (ORFs), 76 of which are unique [START_REF] Burton | Multiple applications for replication-defective herpes simplex virus vectors[END_REF][START_REF] Patel | Equine herpesviruses 1 (EHV-1) and 4 (EHV-4)-epidemiology, disease and immunoprophylaxis: a brief review[END_REF]Telford et al., 1992). Within open reading 1 Powell D., Personal communication to George P. Allen (2006).

A c c e p t e d M a n u s c r i p t 4 frame 30 (ORF30), which encodes the viral DNA polymerase, a single nucleotide substitution is strongly associated with the occurrence of EHV-1 neurologic disease [START_REF] Nugent | Analysis of equid herpesvirus 1 strain variation reveals a point mutation of the DNA polymerase strongly associated with neuropathogenic versus nonneuropathogenic disease outbreaks[END_REF]. The exchange of adenine for guanine at position 2254 (ORF30;

A 2254 →G 2254 ) results in an asparagine (N) to aspartic acid (D) substitution at amino acid position 752 (N 752 →D 752 ) [START_REF] Nugent | Analysis of equid herpesvirus 1 strain variation reveals a point mutation of the DNA polymerase strongly associated with neuropathogenic versus nonneuropathogenic disease outbreaks[END_REF]. This genotype was identified as the causative agent for 30 out of 32 investigated outbreaks of EHV-1 neurologic disease occurring in the United Kingdom and the United States, between the years 2001 to 2006 (Allen, 2007a). The ability of EHV-1 strains possessing G 2254 to induce neurological symptoms has also been proven through experimental infection of the horse [START_REF] Goodman | A point mutation in a herpesvirus polymerase determines neuropathogenicity[END_REF]. Neuropathogenic EHV-1 strains are also able to replicate more efficiently and achieve 10-fold higher levels of leukocyte-associated viremia than observed in horses infected with non-neuropathogenic strains of EHV-1 (Allen, 2006a;[START_REF] Goodman | A point mutation in a herpesvirus polymerase determines neuropathogenicity[END_REF][START_REF] Van De Walle | A single-nucleotide polymorphism in a herpesvirus DNA polymerase is sufficient to cause lethal neurological disease[END_REF]. The identification of this unique single nucleotide polymorphism (SNP) in ORF30 allowed for the development of a real-time PCR assay to discriminate between non-neuropathogenic and neuropathogenic strains of the virus (realtime Taq-Man ® allelic discrimination PCR) (Allen, 2007a;[START_REF] Allen | Prevalence of latent, neuropathogenic equine herpesvirus-1 in the Thoroughbred broodmare population of central Kentucky[END_REF].

EHV-1 establishes a life-long latent infection in a high percentage of animals following exposure to the virus [START_REF] Allen | Equid herpesvirus-1 (EHV-1) and -4 (EHV-4) infections[END_REF]Allen, 2006b). Reactivation of the latent virus results in virus shedding for a limited period of time and the opportunity for transmission of the pathogen to susceptible, in-contact horses. Outbreaks of neurologic disease are thought to be initiated by viral reactivation and nasal shedding of neuropathogenic strains of EHV-1 by latently infected carriers (Allen, 2007b). The latently infected host is typically asymptomatic, although mares which harbor EHV-1 can abort their foals A c c e p t e d M a n u s c r i p t 5 without any prior symptoms [START_REF] Allen | Equid herpesvirus-1 (EHV-1) and -4 (EHV-4) infections[END_REF]. While the primary site of latency is the lymph nodes associated with the respiratory tract, latent virus has also been detected in circulating lymphocytes and the sensory nerve-cell bodies of the trigeminal ganglia [START_REF] Chesters | Detection of latencyassociated transcripts of equid herpesvirus 1 in equine leukocytes but not in trigeminal ganglia[END_REF][START_REF] Edington | The prevalence of latent equid herpesviruses in the tissues of 40 abattoir horses[END_REF][START_REF] Slater | The trigeminal ganglion is a location for equine herpesvirus 1 latency and reactivation in the horse[END_REF]. Where sporadic cases of abortion have been studied, the genotype of the strain of EHV-1 isolated from the aborted fetal tissues was found to be identical to the latent virus present in the mare that aborted [START_REF] Allen | Equid herpesvirus-1 (EHV-1) and -4 (EHV-4) infections[END_REF]Allen, 2006b). In light of this observation, it was felt that archived EHV-1 isolates, derived from fetal tissues and recovered from sporadic equine abortions, would be an excellent source of material to study the distribution of both neuropathogenic and non-neuropathogenic strains of EHV-1 over an extended period of time, within the latent population of the virus. To date, no previously recorded studies have focused on identification of neuropathogenic strains of EHV-1 from sporadic cases of equine abortion. We hypothesize that the neuropathogenic strain of EHV-1 was associated with sporadic cases of equine herpesvirus abortion prior to the 1960's, and the proportion of EHV-1 abortion isolates which posses the neuropathogenic genotype has increased in recent years in comparison to previous decades in Central Kentucky. To test this hypothesis, a real-time Taq-Man ® allelic discrimination PCR assay was used to analyze archived tissue culture fluid containing EHV-1 isolates recovered from tissues of foals aborted by Central Kentucky's Thoroughbred broodmare population during the past 46 foaling seasons, from 1950 through 2006.

Materials and Methods

Cells

A c c e p t e d M a n u s c r i p t 6 Confluent monolayers of fetal equine dermis (KyED) cells were maintained in 850-cm 2 tissue culture roller bottles in Eagle's Minimal Essential Medium (EMEM; Invitrogen, Carlsbad, CA), supplemented with 10% fetal bovine serum (10% FBS) and gentamicin reagent solution (50-µg/ml; Invitrogen) as previously described [START_REF] Allen | Genetic relatedness of equine herpesvirus types 1 and 3[END_REF][START_REF] Allen | Assessment of the base sequence homology between the two subtypes of equine herpesvirus 1[END_REF][START_REF] Turtinen | Serologic and molecular comparisons of several equine herpesvirus type 1 strains[END_REF]. In the present study, the KyED cells were used between passages 7 and 10.

Viral Isolates and Generation of Virus Stock

Archived EHV-1 field isolates from Central Kentucky (Bourbon, Clark, Fayette, Franklin, Jessamine, Madison, Scott and Woodford counties), stored either as lyophilized or wet frozen (-70°C) tissue culture fluid (TCF) stocks, were available for analysis. To ascertain if there was a time-related genetic shift in the latent EHV-1 reservoir, it was considered necessary to examine EHV-1 isolates from different decades, starting with the year 1950. Only isolates derived from fetal tissues of sporadic cases of abortion from Thoroughbred broodmares were included in this study. According to their case histories, none of the mares exhibited any symptoms of respiratory or neurologic disease prior to aborting. The number of EHV-1 isolates that met these criteria for each decade were as follows: 5 for the 1950s, 90 for the 1960s, 90 for the 1970s, 120 for the 1980s, 90 for the 1990s, and 31 for the 2000s (n=426). In addition to these isolates, TCF containing 14 confirmed EHV-1 isolates from known equine herpesvirus outbreaks were included in the study: 7 neuropathogenic herpesvirus strains (G 2254 ; T313, T954, T955, T956, T964, T967, and T970) and 7 non-neuropathogenic herpesvirus strains (A 2254 ; T61, T75, T220, T493, T547, T572, and T812). The genetic identity of these 14 isolates was previously [START_REF] Bell | Isolation of equine herpesvirus-5 from blood mononuclear cells of a gelding[END_REF].

To generate working virus stocks, confluent monolayers of KyED cells in 850-cm 2 tissue culture roller bottles were inoculated with individual isolates and each bottle incubated at 37ºC until 100% cytopathic effect (CPE) was evident. The TCF from each roller bottle was harvested and centrifuged at 12,000 g for 15 min at 4ºC. The supernatant was harvested and filtered using a 0.45 µm membrane filter to remove any remaining cellular debris. The filtered supernatant was subjected to a further cycle of centrifugation for one hour at 48,000 g at 4ºC to pellet the virus. Supernatants were decanted, and the viral pellets in each tube were resuspended in 700-µl of TE buffer (1.0 M Tris-HCl, [pH 8.0], 0.1 M EDTA: Sigma-Aldrich, St. Louis, MO) and stored at -80ºC.

Isolation of Viral DNA

The purified virus was treated with Proteinase-K (20 mg/ml in TE buffer containing 50% glycerol [pH 8.0]: Invitrogen), and the viral DNA was isolated by phenol chloroform extraction as previously described [START_REF] Allen | Genetic relatedness of equine herpesvirus types 1 and 3[END_REF]. The isolated viral DNA was resuspended in 700-µl of TE buffer (pH 8.0) and stored at -20°C. EHV-1 strains, using an ABI 7500 Fast Real-Time PCR System (Applied Biosystems, Foster City, CA). The primers and probes, as well as the reaction conditions, were identical to those described by Allen (2007a). The probe specific for nonneuropathogenic EHV-1 was tagged with a VIC ® label and the probe specific for neuropathogenic EHV-1 with a FAM ® label (Applied Biosystems). For each reaction, 2.5-µl of a viral DNA dilution (5ng per reaction) was combined with 22.5-µl of submaster mix, which consisted of the following: 12.5-µl of Taq-Man ® Universal PCR Master Mix (Applied Biosystems), 0.625-µl of primer/probe mix (40X; EHV1_ORF30_2254: Applied Biosystems), and 9.375-µl of nuclease free water. The nucleotide sequences of the real-time primers and probes used in the PCR assay are to those reported in [START_REF] Allen | Prevalence of latent, neuropathogenic equine herpesvirus-1 in the Thoroughbred broodmare population of central Kentucky[END_REF] The following thermocycling conditions were used with a 9600 emulation mode, per the manufacturer's recommendations: initial denaturation at 95°C for 10 min, followed by 35 cycles at 95°C for 15 sec and 65°C for 1 min. Each PCR run included a control without DNA (22.5-µl of submaster mix plus 2.5µl of nuclease-free water), and two additional controls with DNA from known neuropathogenic or non-neuropathogenic EHV-1 isolates. Prior to the commencement of analysis of the DNA samples from the 426 unknown viral isolates, the validity of this real-time Taq-Man ® allelic discrimination PCR assay was confirmed by the analysis of fourteen EHV-1 isolates of known neuropathogenic and non-neuropathogenic genotype.

PCR amplification and sequencing of DNA samples

EHV-1, EHV-3, and EHV-4 (alphaherpesviruses), as well as EHV-2 and EHV-5 (gammaherpesviruses), specific PCR assays were performed using a HotStartTaq ® DNA polymerase kit (Qiagen, Valencia, CA). Briefly, 50-µl of PCR mixture for each reaction contained 5-µl of 10X PCR buffer, 2-µl of 25mM MgCl 2 , 1-µl of 10mM dNTP mix, 0.3µl of HotStart DNA Polymerase, 38.3-µl of RNase free water, 0.5-µl of the forward primer, 0.5-µl of the reverse primer (200mM of each primer), and 2.5-µl of the template DNA. The DNA of each isolate was combined with seven different primer pairs, whose sequences are reported by Dyon et al., 2001, and[START_REF] Varrasso | Identification of equine herpesviruses 1 and 4 by polymerase chain reaction[END_REF]. Using an Eppendorf thermal cycler, the amplification parameters were set as follows: 95°C for 15 min, followed by 35 cycles at 94°C for 30 sec, 60°C for 30 sec, and 72°C for 1 min, and a final extension phase of 72°C for 10 min. The PCR products were analyzed on a 1% agarose gel and gel purified using a QIAquick gel extraction kit (Qiagen). Both sense and anti-sense strands were sequenced with the PRISM Ready Reaction DyeDeoxy

Statistical Analysis

Graphical and numerical descriptive summaries were generated using time series plots and contingency tables. Several inferential methods were used as statistical tests for the hypothesis of an increasing trend in the prevalence of neuropathogenic strains of EHV-1 from 1950-2006, including the Fisher's exact test and a chi-squared test for trend [START_REF] Rosner | Fundamentals of Biostatistics[END_REF]. Exact binomial confidence intervals were calculated for the prevalence of neuropathogenic strains in the five decades from the 1950s to the 1990s [START_REF] Clopper | The use of confidence or fiducial limits illustrated in the case of the binomial[END_REF]. In addition, a generalized additive logistic regression analysis was used to investigate a nonlinear trend in prevalence across this time period, along with an ordinary logistic regression analysis as a secondary test of the study hypothesis, and to estimate the temporal trend in prevalence of neuropathogenic strains [START_REF] Hastie | Monographs on statistics and applied probability #43[END_REF]. Data analysis was conducted using the R statistical software package and statistical significance was set at α=0.05.

Results

Allelic discrimination of wild type and neuropathogenic EHV-1 strains by real-time

Taq-Man ® PCR

The validity of the real-time allelic discrimination PCR assay was confirmed using EHV-1 isolates that had been characterized on the basis of ORF30 nucleotide sequence analysis as either neuropathogenic (G 2254 ) or non-neuropathogenic (A 2254 ). Seven viral isolates A c c e p t e d M a n u s c r i p t 11 from each genotype were used in these validity determination experiments. Only those viruses with A at position 2254 were found to react with the "non-neuropathogenic" probe, while reactivity with the "neuropathogenic" probe was confined to the G 2254 viruses. Following confirmation of reliability, the allelic discrimination assay was used to analyze herpes viral DNA isolated from tissue samples collected from 426 aborted equine fetuses covering a 56 year time period. Based on the results of the discrimination assay, isolates were classified as having the neuropathogenic genotype (G 2254 ), the nonneuropathogenic genotype (A 2254 ), or non-reactive, in that they were not detected by either probe. Of the 426 samples analyzed, 38 (8.9%) reacted with the "neuropathogenic" (G 2254 ) probe, 381 (89.4%) with the "non-neuropathogenic" (A 2254 ) probe and just 7

(1.6%) were non-reactive. The isolates were initially separated according to decade and the number of G 2254 isolates determined for each decade. Of the 5 samples available from the 1950s, 2 were found to contain G at position 2254. Although this clearly demonstrates that viruses with the neuropathogenic genotype were present in the equine population during this decade, the unusually high prevalence (40%) almost certainly reflects the low sample numbers available for this early time period. Support for this conclusion is provided by the analysis of a much larger, statistically significant group of samples from the following decade (1960)(1961)(1962)(1963)(1964)(1965)(1966)(1967)(1968)(1969), where just 3 of 90 isolates possessed the neuropathogenic genotype. However, one of the most significant findings from the allelic discrimination assay is that the prevalence of EHV-1 isolates containing G 2254 appears to have increased over time from 3.3% in the 1960s, 7.7% in the 1970s (7 of 90), 5.8% in the 1980s (7 of 120), to 14.4% in the 1990s (13 of 90). Furthermore, it appears

Statistical inference

For purposes of statistical analysis, the sample size was reduced from 426 to 419, excluding the seven non-reactive isolates. The data were then subdivided according to decade as follows: 5 were from 1950-1959, 90 were from 1960-1969, 88 were from 1970-1979, 119 were from 1980-1989, 86 were from 1990-1999, and 31 were from 2000-2006. For all analyses stratified by decade, isolates from the year 2000 and later were excluded so that valid comparisons could be made using a consistent time scale. A Fisher's exact test identified a statistically significant difference in the prevalence of neuropathogenic strains across the time period of the study (P=0.01). The confidence interval (CI) for the 1950s was non-informative regarding the prevalence during that decade since too few isolates (n=5) were available for testing. Also, due to the high variance associated with the 1950s, the risk estimates for this decade are unstable to the point of being unreliable and therefore this decade was removed from further consideration. Among the decades from 1960-1969 to 1990-1999, there was a statistically significant increasing linear trend in the prevalence of neuropathogenic strains of EHV-1 by decade (Chi-square test for trend P=0.01). The results from logistic regression of the data corroborated this finding; the estimated regression parameter for each decade was 0.05 with standard error of 0.02 and P=0.01. The odds of incident for neuropathogenic EHV-1 strains increased by a factor of 1.60 (95% CI for the odds ratio: 1.10, 2.34) in each 10 year period. Figure 1 presents the model-based estimated prevalences with a A c c e p t e d M a n u s c r i p t 13 point-wise 95% confidence band from the logistic regression analysis. The estimated prevalence of neuropathogenic strains of EHV-1 in the 1960s was 3.6% (95% CI: 0.1%, 6.5%), and in the 1990s, the prevalence had increased to 13.3% (95% CI: 7.0%, 19.5%).

To test for the possibility of a nonlinear temporal trend in prevalence during this time period, a generalized additive logistic regression model was constructed, using the year of occurrence as the independent variable; the p value for a nonlinear trend was 0.10, indicating no evidence to support nonlinearity.

PCR amplification and sequencing of real-time Taq-Man ® PCR negative DNA samples

The DNA from the seven isolates (1974D, 1974E, 1982C, 1997A, 1997B, 1997F, 1997G) that were non-reactive in the real-time Taq-Man ® allelic discrimination PCR assay were further characterized by direct PCR sequencing. DNA from these isolates was subjected to PCR amplification using primers specific for EHV-1, EHV-2, EHV-3, EHV-4 and EHV-5. Three of the seven DNA samples (1997B, 1997F, 1997G) gave a positive result with both sets of EHV-4 gB gene (encoded by ORF33) specific primer pairs: a 510bp product was produced with the first set of primers and a 324bp product was produced with the second set (Fig. 2). Sequence analysis further confirmed that these three isolates were EHV-4. DNA extracted from 1982C gave a 445bp product with the EHV-2 gB gene (ORF8) specific primers as well as a 324bp product with the EHV-4 gB gene (ORF33) specific primers (Fig. 2). Sequencing of individual gel purified PCR products further confirmed the authenticity of the PCR products, suggesting dual EHV-2 and EHV-4 infection of this particular fetus. The remaining three isolates, 1974D, 1974E and A c c e p t e d M a n u s c r i p t 14 1997A, reacted positively with both sets of EHV-1 gH gene (ORF39) primers (Fig. 2).

The sequence analysis of these PCR products further confirmed their authenticity as EHV-1.

To investigate whether the lack of reactivity in the allelic discrimination assay was due to nucleotide mismatches in the primer and/or probe binding sites of 1974D, 1974E and 1997A, we PCR amplified The PCR amplification and sequencing of the same region of the 1997A isolate revealed that it was a non-neuropathogenic EHV-1 isolate and did not contain any additional nucleotide substitutions in the primer/probe binding regions. Therefore, DNA from 1997A was retested in the allelic discrimination assay and shown to react with the "nonneuropathogenic" probe when the PCR cycle number was increased from the standard 35 to 50. In summary, the data from these three EHV-1 field isolates clearly demonstrate that the current assay conditions used in the real-time Taq-Man ® allelic discrimination assay may infrequently produce either no result, or an aberrant result.

A c c e p t e d M a n u s c r i p t 15

Discussion

Of the 426 TCF samples that underwent PCR analysis, 8.9% (38 of 426) were identified as having a neuropathogenic genotype, while the remaining 89.4% (381 of 426) were categorized as possessing a non-neuropathogenic genotype based on the possession of an A or G residue at nucleotide position 2254 in ORF30. It should be noted that the neuropathogenic genotype was present in Central Kentucky as early as 1951 and therefore, was not introduced into the population in subsequent decades. This study also demonstrated a statistically verifiable increase from the 1960s to the end of the 1990s in the occurrence of neuropathogenic strains of EHV-1 within the latent viral reservoir of Central Kentucky's Thoroughbred broodmare population. If this expansion continues, along with frequent reactivation of the latent neuropathogenic strain, it is possible that the number of neurological outbreaks attributed to EHV-1 will continue to increase and result in greater economic losses for the equine industry.

Aside from the increased occurrence of the neuropathogenic strain in the latent population of EHV-1, allelic discrimination analysis of the viral isolates yielded additional findings. No evidence of simultaneous dual infections with both A 2254 and G 2254 EHV-1 genotypes was found in the 419 fetal isolates that reacted in this assay. This is in contrast with two previous studies, where both EHV-1 genotypes were detected in submandibular lymph nodes, blood and nasopharyngeal secretions in a number of horses [START_REF] Allen | Prevalence of latent, neuropathogenic equine herpesvirus-1 in the Thoroughbred broodmare population of central Kentucky[END_REF][START_REF] Pusterla | Characterization of viral loads, strain and state of equine herpesvirus-1 using real-time PCR in horses following natural exposure at a racetrack in California[END_REF]. In addition to different tissue sources, different PCR techniques were used in each study. In the [START_REF] Allen | Prevalence of latent, neuropathogenic equine herpesvirus-1 in the Thoroughbred broodmare population of central Kentucky[END_REF] study, sequence-capture, nested PCR and sequence-capture, reverse transcription-nested PCR assays were utilized to discern the difference between the active and latent forms of A c c e p t e d M a n u s c r i p t 16 EHV-1, along with differentiating between neuropathogenic and non-neuropathogenic virus genotypes [START_REF] Allen | Prevalence of latent, neuropathogenic equine herpesvirus-1 in the Thoroughbred broodmare population of central Kentucky[END_REF]. In the present study, nested PCR assays were not used to determine whether both genotypes were present in aborted fetal tissues. [START_REF] Pusterla | Characterization of viral loads, strain and state of equine herpesvirus-1 using real-time PCR in horses following natural exposure at a racetrack in California[END_REF] study used a real-time Taq-Man PCR assay with a 7900 HTA thermocycler/fluorometer. They demonstrated that it is possible for the same horse to be infected with both strains of EHV-1 and those strains be in different stages of the virus life cycle (latent, lytic, or non-replicating), but did not address the subject of dual reactivation of EHV-1 strains. Furthermore, both previous studies extracted viral DNA directly from the primary tissue source. In the current study, aborted fetal tissues were the primary source of EHV-1 and the virus was grown in equine dermal cells first before the viral DNA was extracted.

While it is possible that both EHV-1 genotypes cannot be grown in culture at the same time, this seems unlikely given that equine dermal cells have been reported to sustain two separate types of EHV within the same culture (Diallo et al., 2008). Our study demonstrates that the occurrence of only one strain of EHV-1 within the fetal tissues could indicate that dual reactivation of EHV-1 is not possible, or at the very least, is a rare occurrence. However, further investigation is needed before any definitive conclusions can be drawn regarding dual reactivation of EHV-1 strains leading to abortion in pregnant mares. Therefore, in future studies, we intend to reanalyze fetal tissues using the PCR techniques described by [START_REF] Allen | Prevalence of latent, neuropathogenic equine herpesvirus-1 in the Thoroughbred broodmare population of central Kentucky[END_REF] to determine if dual infection with both A 2254 and G 2254 EHV-1 genotypes can be detected in these samples.

Of the seven samples that failed to react in the allelic discrimination assay, three (1997B, 1997F, 1997G) were subsequently shown by PCR sequencing to be EHV-4 and not EHV- viruses. This interesting finding warrants further investigation albeit due to the infrequent association of EHV-2 with equine abortions and the possible role of this virus in the pathogenesis of EHV-1 and EHV-4 infections by facilitating reactivation and/or transactivation [START_REF] Borchers | Distribution and relevance of equine herpesvirus type 2 (EHV-2) infections[END_REF][START_REF] Galosi | Isolation of equine herpesvirus-2 from the lung of an aborted fetus[END_REF][START_REF] Purewal | Identification and control of the cis-actinng elements of the immediate early gene of equid herpesvirus type 1[END_REF]Welch et al., 1992). In contrast to the three instances where there was likely initial misdiagnosis of the viral strain involved, the three remaining samples (1974D, 1974E, 1997A) that failed to react in the allelic discrimination assay were identified as EHV-1 by sequence analysis. Therefore, they were retested and the DNA from one sample (1997A) reacted with the "non-neuropathogenic" probe when the PCR cycle number was increased from 35 to 50. This is consistent with previous reports that the Taq-Man ® allelic discrimination PCR assay, as originally described, can produce false negative results and has a sensitivity of 96.3% when compared with alternative nucleic acid detection methods [START_REF] Allen | Equid herpesvirus-1 (EHV-1) and -4 (EHV-4) infections[END_REF]. However, the most significant finding occurred with the final two non-reactive EHV-1 isolates, 1974D and 1974E. In addition to the A 2254 →G 2254 substitution, they possessed an A to C transposition at nucleotide position 2258 in ORF30. As this is within the allelic discrimination assay probe-binding site, it introduces a base-pairing mismatch that prevents either of the assay-specific probes from annealing under the conditions of the study. Although obviously important for the diagnosis EHV-1 infections, this finding may have considerable implications for our understanding of the genetic basis of neuropathogenicity.

Since A 2258 →C 2258 transposition is non- such a replacement would be conservative. Since the transposition is located within the "palm domain" of the polymerase protein and is directly adjacent to the critical aspartic acid 752 residue implicated in neuropathogenicity, it may have a significant effect on the activity of this enzyme. Furthermore, while both amino acids contain hydroxyl residues, the R group in serine is significantly smaller and less hydrophobic than that in tyrosine, suggesting that this substitution would affect the folding of the palm domain.

Interestingly, the RAC-H modified live vaccine strain of EHV-1, used extensively in both Europe and the United States during the 1970s and 1980s, also contains both A 2254 →G 2254 and A 2258 →C 2258 substitutions [START_REF] Burki | Viraemia and abortions are not prevented by two commercial equine herpesvirus-1 vaccines after experimental challenge of horses[END_REF][START_REF] Burki | Attempts to immunoprotect adult horses, specifically pregnant mares, with commercial vaccines against clinical disease induced by equine herpesvirus-1[END_REF][START_REF] Frymus | Foetal and neonatal foal losses on equine herpesvirus type 1(EHV-1) infected farms before and after EHV-1 vaccination was introduced[END_REF]. Although this vaccine has been implicated in several equine abortions, there have been no reports of neurologic disease associated with its use [START_REF] Nugent | Analysis of equid herpesvirus 1 strain variation reveals a point mutation of the DNA polymerase strongly associated with neuropathogenic versus nonneuropathogenic disease outbreaks[END_REF]. As suggested previously, the presence of serine rather than tyrosine at amino acid position 753 in the viral polymerase might counteract the neuropathogenic effects of an aspartic acid residue at position 752 [START_REF] Nugent | Analysis of equid herpesvirus 1 strain variation reveals a point mutation of the DNA polymerase strongly associated with neuropathogenic versus nonneuropathogenic disease outbreaks[END_REF]. This possibility will be the focus of future investigations in this laboratory.

Conclusion

In conclusion, this study demonstrates EHV-1 isolates with the neurovirulent genotype are not new to Central Kentucky, but clearly were present as far back as the 1950s. The data also lend credence to the clinical observations that neuropathogenic strains of EHV-1 have recently become more prevalent, suggesting that they have a selective advantage selective advantage remains to be determined. Moreover, it will be important to ascertain the extent to which additional amino acid substitutions occur within ORF30 and how they impact the neurologic phenotype. This research will also help in redesigning the real-time allelic discrimination assay to increase its reliability in detecting all neuropathogenic phenotypes of EHV-1.

Acknowledgements

Dr. George P. Allen, who was the principal investigator of this study, passed away in

  ORF30. Additional TCFs, containing equine herpesviruses 1-5 were used as positive controls in the PCR amplification reactions. The EHV-1, 3, and 4 controls were obtained from the American Type Culture Collection (EHV-1 [Catalog number: ATCC VR-700], EHV-3 [ATCC VR-352], EHV-4 [ATCC VR-2230]; American Type Culture Collection, Manassas, VA). The EHV-2 and 5 controls were kindly provided by the late Dr. William H. McCollum (Gluck Equine Research Center, Lexington, KY) and Dr. Stephanie Bell (University of California, Davis, CA)

  -Time Taq-Man ® PCR AssayA duplexed real-time Taq-Man ® PCR assay was performed in 96-well plates for the allelic discrimination of the non-neuropathogenic (A 2254 ) and neuropathogenic(G 2254 )

  A c c e p t e d M a n u s c r i p t 9 Post-run analysis for detection and genotype identification of EHV-1 DNA present in each test sample was performed using ABI 7500 SDS allelic discrimination analysis software (version 1.3.1.). Amplification in real time of each genotype of EHV-1 DNA present in the test sample wells was represented by the software as a plot of PCR cycle number versus the accumulated level of fluorescence (Rn) from each of the Taq-Man ® reporter probes.

  Terminator cycle sequencing kit (MWG Operon, Huntsville, AL). Sequence data were A c c e p t e d M a n u s c r i p t 10 analyzed with the CodonCode (Codon Code Corp., Dedham, MA) and Vector NTI (Invitrogen) software.

  rising trend is continuing into the twenty-first century as 19.4% (6 of 31) of the viral samples tested from 2000 to 2006 have the neuropathogenic genotype.

  and sequenced a 509bp product from ORF30, (nucleotide numbers 1901-2410; numbered according to GenBank accession #AY464052 & AY665713) which includes these target regions. Comparative nucleotide sequence analysis of ORF30 from 1974D and 1974E found an additional nucleotide substitution (A 2258 →C 2258 ) within the real-time probe binding site that was sufficient to compromise the allelic discrimination assay and give rise to a non-reactive result. The substitution at nucleotide position 2258 resulted in a tyrosine (Y) to serine (S) amino acid substitution (Y 753 →S 753 ) within the viral DNA polymerase. Therefore, both of these EHV-1 isolates from 1974 contain D 752 and S 753 amino acid substitutions in the viral DNA polymerase.

  they had been incorrectly identified during initial diagnostic testing. PCR sequencing also revealed that another sample, isolated in 1982 (1982C), did not react in the allelic discrimination assay because it contained a mixture of EHV-4 and EHV-2

  is predicted to result in the replacement of tyrosine (Y) with serine (S) at amino acid position 753 in the viral polymerase (Y 753 →S 753 ). It is highly unlikely that

  -neuropathogenic strains. The identity, or mechanism, of this

Figure 1 .

 1 Figure 1. The model-based estimated prevalences from the logistic regression analysis. The solid line in this graph represents the estimated probabilities of the

  Figure 1

Figure 2

 2 Figure 2


Astrid De Greeff
email: astrid.degreeff@wur.nl

Laurentiu Benga

Paul J Wichgers Schreur

Peter Valentin-Weigand

Johanna M J Rebel

Hilde E Smith

Involvement of NF-κB and MAP-kinases in the transcriptional response of alveolar macrophages to Streptococcus suis

Keywords: Streptococcus suis, transcriptional response, macrophages

A c c e p t e d M a n u s c r i p t 2 Abstract Interaction of Streptococcus suis with primary porcine alveolar macropahges was studied using transcriptomics. Transcriptional response of macrophages to two different S. suis strains was studied: wild-type S10 that is resistant to phagocytosis, and it non encapsulated mutant that is phagocytosed efficiently. The macrophages' transcriptional response was observed only after 60 minutes of incubation. Eleven genes were expressed significantly different between macrophages infected with streptococci and control mockinfected macrophages. These genes include IL-1-ß, MIP-2-α and TNF-α.

When gene expression was studied as function of time, transcriptional changes occurred in all macrophages independent of streptococci.

The fold induction of induced genes however, was much stronger in macrophages incubated with the non-encapsulated S. suis strain that was phagocytosed. The genes that were higher induced due to S suis suggest an innate immune response is induced in macrophages.

Pathway analysis revealed that genes that are part of the putative MAP-kinase signal transduction system are overrepresented among the regulated genes. Using an immortalized alveolar macrophage cell line it was shown that macrophages respond to interaction with S. suis by translocation of NF-κB to the nucleus, independent of phagocytosis.

This translocation subsequently induced expression of innate immune

A c c e p t e d M a n u s c r i p t 3 genes. This strongly suggests besides the MAP-kinase signaling pathway, NF-κB signaling is also induced upon interaction with S. suis.

Introduction

Streptococcus suis are gram positive cocci possessing a polysaccharide capsule that is responsible for the serotype specificity.

Worldwide S. suis serotype 2 is most frequently isolated from diseased pigs [START_REF] Staats | Streptococcus suis: past and present[END_REF]. S. suis causes severe infections in piglets, typical symptoms of the disease include meningitis, arthritis and septicemia. Animals often do not survive an infection. Recently an outbreak of human S. suis infections in China was described in which 200 people got infected, of whom 38 died [START_REF] Yu | Human Streptococcus suis outbreak, Sichuan, China[END_REF].

The severity and outcome of infections caused by S. suis are likely to depend on the ability of host innate immune mechanisms to control bacterial growth and to limit spreading of the pathogen [START_REF] Goldmann | Transcriptome analysis of murine macrophages in response to infection with Streptococcus pyogenes reveals an unusual activation program[END_REF]. Macrophages, together with the epithelial barrier, form the first line of defense against microorganisms. They identify pathogens using pattern recognition receptors (PRRs), and subsequently unfold a tightly regulated pathogen-specific immune response [START_REF] Akira | Pathogen recognition and innate immunity[END_REF]. Activation of PRRs leads to signal transduction via Myd88, and activates both NF-κB and MAP kinases, resulting in induction of genes involved in inflammatory responses. For S. suis it is known that after incubation with mononuclear cells, proinflammatory mediators are produced [START_REF] Segura | Proinflammatory cytokine and chemokine modulation by Streptococcus suis in a whole-blood culture system[END_REF]. Another study showed that S. suis can A c c e p t e d M a n u s c r i p t 5 induce an inflammatory response via the induction of prostaglandin E2 and metalloproteinase 9 production in human macrophages [START_REF] Jobin | Upregulation of prostaglandin E2 and matrix metalloproteinase 9 production by human macrophage-like cells: synergistic effect of capsular material and cell wall from Streptococcus suis[END_REF]. These reactions are part of the general 'alarm signal' for infection that can lead to a cascade of immune responses [START_REF] Jenner | Insights into host responses against pathogens from transcriptional profiling[END_REF].

In this paper we studied the 'alarm signal' of porcine alveolar macrophages in response to S. suis serotype 2 in vitro. This was achieved by comparing transcriptional profiles of alveolar macrophages after contact with either a S. suis strain that is resistant to phagocytosis or with an isogenic non-encapsulated mutant that is phagocytosed in this model [START_REF] Smith | Identification and characterization of the cps locus of Streptococcus suis serotype 2: the capsule protects against phagocytosis and is an important virulence factor[END_REF]. Our results showed that an innate immune response is induced in alveolar macrophages, that is probably induced via activation of both NF-κB and MAP-kinase pathways.

A c c e p t e d M a n u s c r i p t 6

Materials and Methods

Bacterial strains and growth conditions

Streptococcal strains were grown on Columbia agar blood base plates (Oxoid Ltd., London, United Kingdom) containing 6% (vol/vol) horse blood. Cultures were grown in Todd-Hewitt broth (Oxoid). Pathogenic S. suis strain 10 [START_REF] Smith | Mutants of Streptococcus suis types 1 and 2 impaired in expression of muramidase-released protein and extracellular protein induce disease in newborn germfree pigs[END_REF][START_REF] Vecht | Identification of two proteins associated with virulence of Streptococcus suis type 2[END_REF] and its avirulent nonencapsulated mutant strain S10cpsdelEF were used [START_REF] Smith | Identification and characterization of the cps locus of Streptococcus suis serotype 2: the capsule protects against phagocytosis and is an important virulence factor[END_REF].

For phagocytosis experiments S. suis was grown for 16 h. One ml of culture was spun down for 2 min at 20,000 x g at 4C. The pellet was washed once in cold Dulbecco's Modified Eagle's Medium (DMEM, Sigma-Aldrich, St. Louis, MO, USA) and resuspended in 1 ml of DMEM.

The inoculum size was determined by plating serial dilutions on agar plates. For stimulation of 3D4 porcine alveolar macrophages cell line, bacteria were washed once with PBS, then adjusted in PBS to an OD600 of 0.4, and subsequently diluted 1:10 in Iscove´s Modified Dulbecco´s Medium (IMDM). For NF-κB translocation studies, S. suis was grown for 16 h in IMDM and 0.5 ml of the culture was directly used for stimulation of 3D4 cells.

Phagocytosis and stimulation of primary alveolar macrophages

Isolation of alveolar macrophages

A c c e p t e d M a n u s c r i p t 7 Phagocytosis experiments were done as described before [START_REF] Smith | Identification and characterization of the cps locus of Streptococcus suis serotype 2: the capsule protects against phagocytosis and is an important virulence factor[END_REF]. Porcine lungs were collected from 6 individual specific pathogen free piglets, and used within 4 hours. As a control DMEM without bacteria was used. Animal experiments performed were approved by the ethical committee of the Central Veterinary Institute of Wageningen University and Research Center, in accordance with the Dutch law on animal experiments.

Phagocytosis

To each opsonized bacterial suspension 5 ml of macrophage suspension was added (MOI 1). The sample was mixed immediately after addition of bacteria and samples were taken at t = 0, 2, 5, 10, 15, and 60 min. One part of the sample (1.5 ml) was spun down immediately for 3 min at max speed at 4C. The supernatant was decanted and the pellet was snap frozen in liquid nitrogen for RNA isolation. The second part of the sample (0.4 ml) was added to 1,6 ml ice-cold DMEM. This sample was spun down for 10 min at 110 x g at 4C. Subsequently, the supernatant was plated in a serial dilution to determine the CFU/ml. Results of seven independent experiments were averaged. Results were analyzed using Mann Whitney testing, standard error of the mean is given for each timepoint.

Phagocytosis and stimulation of 3D4 alveolar macrophages

A c c e p t e d M a n u s c r i p t 8 3D4 porcine alveolar macrophages derived from primary porcine alveolar macrophages as previously described [START_REF] Weingartl | Continuous porcine cell lines developed from alveolar macrophages: partial characterization and virus susceptibility[END_REF] were used. Cells were maintained in Iscove´s Modified Dulbecco´s Medium (IMDM) containing 10% FCS, 5 mM glutamine, 100 units ml -1 penicillin, and 100 µg ml -1 streptomycin. For stimulation experiments, approximately 4 x 10 5 cells per well were seeded in antibiotic free media on 12 well tissue culture plates and incubated for 24 h. For NF-κB translocation, approximately 1 x 10 5 cells per well were seeded in antibiotic free media on 24 well tissue culture plates containing glass coverslips.

Phagocytosis of S. suis by adherent 3D4 alveolar macrophages cell line was determined during 90 min of co-cultivation by a gentamicin protection assay as previously described [START_REF] Valenti-Weigand | Entry and intracellular survival of group B streptococci in J774 macrophages[END_REF]. 3D4 cells in 12 well plates were stimulated with 1.5 ml S. suis suspension in IMDM for 6 h at 37°C to study the transcriptional activation.

For NF-κB translocation 3D4 cells were treated with S. suis wild-type for 30 min at 37°C. Cells treated with 5 µg/ml LPS served as positive control, non-stimulated cells as negative control. After stimulation, cells were fixed with 3.7% formaldehyde for 10 min at room temperature, washed three times with PBS, and then permeabilized with 0.1% Triton-X 100 in PBS for 20 min at RT. Following, the cells were incubated for 30 min at RT with 10% FCS in PBS to block non-specific binding. Subsequently, cells A c c e p t e d M a n u s c r i p t 9 were double stained with anti-human NF-κB p65 antibodies (1:200, Cell Signalling, Frankfurt/Main, Germany) for 1h at room temperature. Cells were washed three times with PBS and then incubated with Alexa Fluor 488 goat anti-rabbit antibodies (1:500, Invitrogen, Carlsbad, CA, USA)

for 30 min at room temperature. After five washes, the cells were mounted with anti-fading solution and visualised under a fluorescence microscope.

RNA isolation

The macrophage pellet was taken up in 1 ml of Trizol and 0.2 ml of chloroform was added. The suspension was shaken vigorously for 15 sec, incubated for 3 min at RT and centrifuged for 15 min at 20,000 x g at 4C. Two phenol-chloroform-isoamylethanol extractions were performed, and 1 chloroform extraction after which the RNA was precipitated using isopropanol, washed with 70% ethanol, and dissolved in water. RNA was further purified using the Nucleospin RNA II kit (Macherey Nagel, Düren, Germany) according to manufacturer's instruction with a prolonged DNAse treatment (1hr at RT). RNA quality and quantity was checked using the NANOdrop (Thermo Fisher Scientific, Pittsburgh, PA, USA) and the BioAnalyzer (Agilent, Palo Alto, CA, USA). RNA samples were pooled per timepoint per strain.

A c c e p t e d M a n u s c r i p t 10

In experiments with 3D4 cells, RNA was harvested 6h pi. For this, cells were washed three times with ice-cold PBS then harvested and lyzed in 350 μl lysis buffer (RLT-buffer, Qiagen, Hilden, Germany) containing 1% β-mercaptoethanol. The RNA from the lysed cells was purified using the Qiagen RNeasy-Kit (Qiagen) according to the manufacturer's instructions.

Microarray analysis

Biotin-labeled cRNA was synthesized using the one-cycle target labeling and control reagents (Affymetrix, Santa Clara, CA, USA) starting with 2 μg of RNA. For hybridization to porcine whole genome Affymetrix chips, 12.5 -20 μg of cRNA was used for further fragmentation, 10 μg of this fragmented cRNA was used for hybridization. Affymetrix protocols are followed for hybridization, washing, staining and scanning of the chips. Data analysis was done using GeneSpring software (Agilent). Pathway analysis was done using a Web Based Gene Set Analysis Toolkit (Webgestalt) [START_REF] Zhang | WebGestalt: an integrated system for exploring gene sets in various biological contexts[END_REF]. All microarray data have been submitted MIAME complied to (see Table 1). Each reaction contained 12.5 pmol forward primer, 12.5 pmol reverse primer and POWR SYBR Green PCR Master Mix (Applied Biosystems) according to manufacturer's instructions. qPCR was performed using an ABI7500 (Applied Biosystems). The amount of gapdh was measured to control for variation in RNA-yield and RTreaction conditions. In each run a standard curve was incorporated consisting of a vector (pGEMT-easy) containing the cloned gene. In this way both the gene and the external control gene could be related to a standard line. The efficiency of the PCR reaction was 90-100% for all reactions (slope standard line between -3.3 and -3.6). The standard line consisted of 7 10-fold dilutions of the control vector. For each reaction negative water controls were included. Analysis was performed using the ABI7500 Software (Applied Biosystems). Porcine IL-8 and MCP-1

A c c e p t e d M a n u s c r i p t 12 transcripts of 3D4 cells were quantified by PCR using specific primers described previously [START_REF] Segura | Proinflammatory cytokine and chemokine modulation by Streptococcus suis in a whole-blood culture system[END_REF]. Porcine ICAM-1 primers were used as described previously (Stocker et al., 2000). gapdh gene was used for expression normalization, using the primers described previously [START_REF] Segura | Proinflammatory cytokine and chemokine modulation by Streptococcus suis in a whole-blood culture system[END_REF]. The PCR mixture consisted 5 μl of 10x

Taq buffer, 1.5 μl of 50 mM MgCl2, 1 μl of 10 mM dNTP, 0.5 μl of each primer (50 μM), 5 μl cDNA, 0.5 μl Taq DNA polymerase and water up to 50 μl. The PCR cycling consisted of denaturation at 94°C for 1 min, annealing at 56°C for IL-8, MCP-1 and at 58°C for ICAM-1 and E-Selectin, and elongation for 1 min at 72°C. The number of cycles used for amplification of chemokines (IL-8 and MCP-1) and ICAM-1 was 35 and 30 respectively. The amplified DNA was separated in a 1.5 % agarose gel in Tris-borate EDTA buffer (TBE), and the intensity of the bands was quantified by densitometry using the software QuantityOne.

Results were expressed as the ratio of the band intensities of the PCR products as compared to gapdh expression. Data were analyzed by analyses of variance followed by t test. A P value < 0.05 was considered significant.

A c c e p t e d M a n u s c r i p t 13

Results

Phagocytosis

Freshly isolated porcine alveolar macrophages were either incubated with wild type strain 10, or with its non-encapsulated isogenic mutant strain S10cpsdelEF, or with PBS (mock infection). Number of bacteria in the supernatant was measured by serial platings (Figure 1). The bacterial counts of wild-type bacteria remained constant in time, and even slightly increased during incubation, indicating that S. suis strain 10 was able to survive, despite the presence of macrophages. The nonencapsulated mutant strain on the contrary, was phagocytosed very efficiently; after 5 minutes of incubation 70% of the bacteria were phagocytosed. The mock-infected macrophages remained sterile during the experiment. The difference in uptake of both strains was statistically significant (P<0.05) at 5 min pi and at later timepoints. These results confirmed previous findings [START_REF] Smith | Identification and characterization of the cps locus of Streptococcus suis serotype 2: the capsule protects against phagocytosis and is an important virulence factor[END_REF].

Surprisingly, in contrast to the primary alveolar macrophages, the adherent alveolar macrophages cell line 3D4 was not able to significantly phagocytose the two S. suis strains used within 90 min of incubation as determined by a gentamycin protection assay (data not shown).

A c c e p t e d M a n u s c r i p t 14

Whole genome host response

Microarray analysis

Gene expression profiles of macrophages were studied using microarrays. Transcription profiles of macrophages incubated with wildtype S. suis, non-encapsulated mutant, or PBS (mock) were studied as a function of time.

Gene expression of macrophages incubated with either the wild type strain 10 or with the non-encapsulated mutant was compared to gene expression of macrophages that were mock-infected with PBS at all timepoints. Significant differences were only found at timepoint 60 minutes. After incubation with the wild type strain 10 that was not phagocytosed, one gene was expressed differentially compared to the mock infected macrophages (Table 2A). This gene showed homology to dynein. Ten genes were found to be differentially expressed in macrophages incubated with the non-encapsulated mutant compared to mock infected macrophages (Table 2A). Those genes include IL-1-ß, MIP-2α and TNF-α among others. The low numbers of genes that were found to be differentially regulated, suggested that either a limited response is induced in macrophages, or that mockinfected macrophages are activated as well.

To analyze this, gene expression of macrophages was studied in time by comparing expression at all timepoints to expression of timepoint t =

A c c e p t e d M a n u s c r i p t 15 0 of the same condition. This analysis showed that transcriptional changes occurred in all macrophages, independent of incubation with streptococci, and the highest number of regulated genes was found after 60 minutes. The process of phagocytosis of strain S10cpsdelEF was nearly complete by then (95% was phagocytosed). In total 377 genes were found to be regulated in this experiment, 188 in the control macrophages, 244 after incubation with wild type strain 10, and 260 genes after incubation with the non-encapsulated mutant. There is overlap among the regulated genes in the three conditions. Table 2B shows the toplist of genes that were found to be regulated. Ranking is based on macrophages incubated with S10cpsdelEF. In general it was found that the toplists of all three conditions were very similar. However, the fold-induction of gene expression was strongest in macrophages that phagocytosed S. suis strain S10cpsdelEF. This analysis indicates that the primary macrophages are activated in all three conditions. Despite this activation, genes could be identified that are differentially regulated due to interaction with S. suis (Table 2A), and S. suis could induced a stronger response in macrophage compared to the mock infected macrophages (Table 2B). A complete list of all regulated genes is given as supplementary material.

Pathway analysis

A c c e p t e d M a n u s c r i p t 16

To gain insight into the biological processes of macrophage activation, a pathway analysis was performed on genes differentially expressed in time. To increase the power of pathway analyses, the cut-off for differential expression was lowered. Genes were included for analysis when gene expression was regulated at least 1.5 fold at timepoint t = 60 minutes compared to timepoint t = 0 for (3,965 genes). Table 3 shows putative pathways found to be regulated. The MAPK-signaling pathway contained the highest number of regulated genes.

Differentially expressed genes of the MAPK-signaling pathway all directed the macrophages towards proliferation/inflammation/antiapoptosis. Expression of genes of the MAPK-signaling pathway was regulated under all three conditions, but similar to the other expression data the strongest induction/repression was found in macrophages incubated with the non-encapsulated strain s10cpsdelEF.

qPCR data

To confirm the microarray data, qPCR analysis was performed on the same samples that were used in the microarray analysis. Expression of proinflammatory cytokines like TNF-α, IL-8, IL-1-ß, IL-6 and MIP-1-ß was studied. qPCR data confirmed the data found using microarray analysis (Figure 2). Strongest induction of proinflammatory cytokines was found in macrophages incubated with the non encapsulated S.

A c c e p t e d M a n u s c r i p t 17 suis strain S10cpsdelEF, whereas much lower levels of induction were found in macrophages incubated with either S10 or PBS (mock).

Expression of Toll like receptors was also studied using qPCR. No differences in expression were found, neither in time, nor between the different conditions (data not shown).

Activation of porcine alveolar macrophages 3D4 cell line by S. suis

To study which signal transduction pathway is involved in the induction of proinflammatory cytokines, the 3D4 porcine alveolar macrophage cell line model was used [START_REF] Weingartl | Continuous porcine cell lines developed from alveolar macrophages: partial characterization and virus susceptibility[END_REF]. Although these cells did not phagocytose S. suis strains, this cell line can be used to study the effect of interaction between S. suis and alveolar macrophages strong expression of all three transcripts, whereas in non stimulated control cells no gene expression was induced (Figure 4). These data strongly suggest that after recognition of S. suis by alveolar macrophages, the signaling pathway of NF-κB is induced, as well as subsequent production of proinflammatory cytokines. We found that primary alveolar macrophages started regulating gene expression after 60 minutes of incubation with the two S. suis strains. At this timepoint, gene expression of macrophages that came into contact with the wild type strain or the non encapsulated mutant was compared to that of mock infected macrophages. Eleven genes were found to be expressed differentially due to incubation with S. suis. In macrophages that came into contact with the wild type strain, but did not phagocytose this strain, only dynein was found to be differentially expressed. Dynein is one of the microtubule associated motors.

Overexpression of this gene suggests that despite the fact that S. suis is not phagocytosed, the macrophages are rearranging their membranes upon interaction with the wild type. Macrophages that came into contact with the non encapsulated mutant, that was phagocytosed, showed differential gene expression of ten genes.

Genes involved in a proinflammatory immune response (TNF-α, MIP-2α, IL-1-ß) were found to be regulated, but genes involved in cell cycling (G0/G1 switch protein, BTG2 protein) were regulated as well. In conclusion, after contact with the wild type strain, macrophages seem to change morphologically. After interaction with the non The relatively low number of regulated genes due to incubation with S.

suis that was found can be explained by the activation of macrophages due to the isolation procedure or the incubation time.

When transcriptional responses were studied in time, 377 genes were differentially expressed compared to timepoint zero. This response was observed in macrophages incubated with S10cpsdelEF as well as in macrophages that came into contact with S10, and in control macrophages. This general transcriptional response of macrophages was described previously as the common macrophage activation program by Nau et al. [START_REF] Nau | Human macrophage activation programs induced by bacterial pathogens[END_REF]. These authors describe that macrophages respond to a broad range of bacteria with a robust, shared pattern of gene expression. The alveolar macrophages in our study were activated after contact with S. suis independent of phagocytosis. Genes that were described to be part of this 'common activation program' share a lot of homology with our gene list of differentially regulated genes at timepoint 60 min (compared to timepoint 0). These data suggest that the alveolar macrophages in our study were all activated, independent of bacterial interaction.

However, not all macrophages were activated to the same extent. Macrophages that came into contact with wild type strain S10 induced the same genes, but a much weaker induction was observed, whereas control macrophages showed an even weaker induction of gene expression. The strong transcriptional response observed in macrophages after phagocytosing the capsular mutant, will probably result in a strong local innate immune response in vivo. This will create a hostile environment for the bacteria to prevent them from multiplication and spreading through the host. Since the transcriptional response of macrophages to the wild type strain 10 is much weaker, it is expected that a weaker immune response is induced locally. In this way capsule not only protects S. suis from phagocytosis, but indirectly suppresses the immune response.

When the list of regulated genes at timepoint 60 compared to timepoint 0 is studied in more detail, it is clear that the strongest regulated genes encode proteins involved in the proinflammatory host defense like IL-1-ß, TNF-α, MIP-2-α, IL-1-α and IκB being the signal transducer. The regulation of a selected number of genes encoding A c c e p t e d M a n u s c r i p t 23 pro-inflammatory proteins was confirmed using qPCR. Our data confirm previous publications that describe induction of a proinflammatory immune response after incubation of human monocytes with S. suis [START_REF] Dominguez-Punaro | Streptococcus suis serotype 2, an important swine and human pathogen, induces strong systemic and cerebral inflammatory responses in a mouse model of infection[END_REF][START_REF] Graveline | TLR2dependent recognition of Streptococcus suis is modulated by the presence of capsular polysaccharide which modifies macrophage responsiveness[END_REF][START_REF] Segura | Heat-killed Streptococcus suis capsular type 2 strains stimulate tumor necrosis factor alpha and interleukin-6 production by murine macrophages[END_REF][START_REF] Segura | CD14-dependent and -independent cytokine and chemokine production by human THP-1 monocytes stimulated by Streptococcus suis capsular type 2[END_REF][START_REF] Segura | Proinflammatory cytokine and chemokine modulation by Streptococcus suis in a whole-blood culture system[END_REF]. CD163 is one of the genes most strongly downregulated in alveolar macrophages. CD163 is a macrophage marker that is known to be regulated by pro-and antiinflammatory stimuli [START_REF] Buechler | Regulation of scavenger receptor CD163 expression in human monocytes and macrophages by pro-and antiinflammatory stimuli[END_REF]. The low expression level observed in alveolar macrophages correlates with the strong proinflammatory response that is also observed. This indicates that our data fit the known regulation of innate immune response in macrophages, thereby strengthening our findings. Activation of the innate immune response is controlled in large part by the Toll-like receptor (TLR) family of pattern-recognition receptors. TLRs are transmembrane proteins that recognize specific pathogen-associated molecular patterns [START_REF] Akira | Pathogen recognition and innate immunity[END_REF]. It was shown that S. suis can indeed bind to TLR2, both on human macrophages, as well as in vivo in murine brain [START_REF] Dominguez-Punaro | Streptococcus suis serotype 2, an important swine and human pathogen, induces strong systemic and cerebral inflammatory responses in a mouse model of infection[END_REF][START_REF] Graveline | TLR2dependent recognition of Streptococcus suis is modulated by the presence of capsular polysaccharide which modifies macrophage responsiveness[END_REF]. In our primary alveolar macrophages no increased expression of either one of the TLRs was observed upon stimulation with S. suis. However, expression of all TLRs included in our analysis (TLR 1,2,3,4,6,8,9,10) was detected on macrophages at all timepoints. This expression A c c e p t e d M a n u s c r i p t 24 correlates very well with the ready-to-react status described previously.

It seems that alveolar macrophages are constantly armed with sensors to detect invading bacteria. Therefore, it seems very likely that S. suis binds to one or more of the TLRs, resulting in the observed proinflammatory response.

When the induced genes were clustered to functional groups, some putative pathways were strongly regulated upon contact with S. suis such as the MAPK-signaling pathway, cytokine-cytokine receptor interaction, and cell cycle. The highest number of regulated genes were involved in the MAPK-signaling pathway . MAP-kinases are key regulators for the synthesis of numerous cytokines, chemokines, and other inflammatory mediators [START_REF] Dong | MAP kinases in the immune response[END_REF]. ERK, one of the MAPkinase subfamilies is strongly upregulated, thereby inducing the inflammatory cascade. However, ERK has not yet induced its selflimiting mechanism of MKP1, since MKP1 is downregulated at 60 minutes [START_REF] Wang | Regulation of innate immune response by MAP kinase phosphatase-1[END_REF]. This means that the process that terminates cytokine production is completely off after 60 minutes, allowing the inflammatory cascade to amplify NF-κB represents another important transcriptional regulator, which also induces expression of proinflammatory cytokines [START_REF] Pahl | Activators and target genes of Rel/NF-kappaB transcription factors[END_REF]. The is also involved in the induction of expression of proinflammatory cytokines we identified with the microarray, an alveolar macrophages cell line was used (3D4) [START_REF] Weingartl | Continuous porcine cell lines developed from alveolar macrophages: partial characterization and virus susceptibility[END_REF]. Despite the fact that this cell line was unable to phagocytose either wild type S. suis or the non encapsulated mutant, the cell line offers a good model to study the transcriptional effects of interaction with S. suis. An advantage o using a cell line is that the conditions are more standardized. When using primary cells, there is always the risk the cells get activated upon isolation. When using a cell line, this 'background' effect is lacking. Taken together, we show that after interaction of S. suis with alveolar macrophages a strong proinflammatory response is induced. Although this effect occurs independent of phagocytosis, the induction of gene expression was stronger in a population of macrophages that were incubated with the non-encapsulated S. suis that was phagocytosed.

Pathway analysis and NF-κB translocation studies showed that the differential gene expression is probably induced by two major signal transduction pathways, MAP-kinase and NF-κB. This is the first time S. suis induced translocation of NF-κB is described.

A c c e p t e d M a n u s c r i p t 31

  //www.ebi.ac.uk/microarray-as/ae/). was used to synthesize cDNA in a reaction containing 25 ng/μl random primers (Promega, Madison, WI, USA), 10 mM dNTPs (Promega), 10 mM DTT (Invitrogen), 40 U RNAsin (Promega) and SuperScriptII Reverse Transcriptase (Invitrogen) according to manufacturer's instructions. qPCR cDNA was diluted 20 times for qPCR analysis. Primers were designed using PrimerExpress software (Applied Biosystems, Foster City, CA, USA)

  independent of phagocytosis. NF-κB is an important transcriptional activator of pro-inflammatory cytokine gene expression. Therefore translocation of NF-κB to the nucleus of macrophages was studied after incubation with S. suis strain 10. Imunofluorescence microscopy revealed that the p65 subunit of NF-κB was translocated to the nucleus following infection with wild-type S. suis. Translocation of p65 was seen in approximately 10% of the macrophages (Figure3). A similar picture was seen in LPS-treated cells (positive control), whereas in control cells, p65 remained in the cytoplasm. To confirm that NF-κB translocation indeed induces a proinflammatory cytokine response, S. suis-IL-8, MCP-1, and ICAM-1 was studied by RT-PCR. Both S. suis strain 10 and the non-encapsulated mutant induced a

  , gene expression of alveolar macrophages interacting with S. suis was studied using a phagocytosis model. Microarray analysis demonstrated that within the first 15 minutes of incubation of macrophages with bacteria, hardly any differences in gene expression occurred in the alveolar macrophages, whereas they did exert their biological function of clearing the bacteria within that time period.These data indicate that macrophages can phagocytose bacteria without adapting their gene regulation. Apparently, all reagents and mechanisms are ready-to-react in alveolar macrophages. It was previously shown that PMNs are capable of phagocytosing several pathogenic bacteria within 30 minutes of time, whereas regulation of gene expression started after 90 minutes[START_REF] Kobayashi | Bacterial pathogens modulate an apoptosis differentiation program in human neutrophils[END_REF]. Taken together, these data suggest that macrophages start signaling after their primary task of clearing bacteria from the host. The alveolar macrophage cell line was unable to phagocytose S. suis. Weingartl et al. described that the 3D4 cell line could phagocytose latex beads, although only a limited percentage of cells were able to exhibit this ability and only after very long incubations (3 hr -overnight)[START_REF] Weingartl | Continuous porcine cell lines developed from alveolar macrophages: partial characterization and virus susceptibility[END_REF]. Based on these results it seems likely, the 3D4 cells lost (part A c c e p t e d M a n u s c r i p t 20 of) their capacity to phagocytose efficiently during the process of immortalization.

  raised an innate immune response and changed their cell cycle. This might potentially lead to phagocytosis or proliferation of macrophages.

  microarray results and in the conformational qPCR analysis, differences in the level of induction are observed between the incubation conditions. The strongest induction of gene expression was observed in alveolar macrophages incubated with S10cpsdelEF.

  NF-κB is located in the cytoplasm, whereas stimulation of the NF-κB pathway leads to a degradation of the inhibitory proteins IκB, resulting in NF-κB translocation into the cell nucleus. To study if NF-κB

  Immunofluorescencent staining of the p65 subunit of NF-κB allowed us to demonstrate nuclear translocation of the transcriptional regulator in response to S. suis infection indicating activation of NF-κB. This demonstrates for the first time that S. suis is able to induce NF-κB. NF-κB activation plays an established central role in the inflammatory process[START_REF] Pahl | Activators and target genes of Rel/NF-kappaB transcription factors[END_REF]. The S. suis induced translocation of NF-κB suggest that signal transduction upon interaction with S. suis not only acts via MAPkinase, but also via NF-κB. RT-PCR analyses showed that the translocation of NF-κB indeed resulted in an upregulation of innate immune genes such as IL-8, MCP, and ICAM. This regulation of A c c e p t e d M a n u s c r i p t 26 expression was not different between stimulation with encapsulated and non-encapsulated S. suis strains.

Figure 1 .Figure 2 .

 12 Figure 1. Bacteria present in supernatant of culture medium containing

Figure 3 .

 3 Figure 3. Immunofluorescence detection of p65 translocation into the

Figure 4 .

 4 Figure 4. Contribution of S. suis to transcriptional activation of IL-8, MCP-1

  Figure 1

  Figure 3

Table 1 .

 1 Primer sequences.

	Gene
	name	Forward primer	Reverse primer
	TLR1	TGCTGGATGCTAACGGATGTC	AAGTGGTTTCAATGTTGTTCAAAGTC
	TLR2	TCACTTGTCTAACTTATCATCCTCTTG	TCAGCGAAGGTGTCATTATTGC
	TLR3	AGTAAATGAATCACCCTGCCTAGCA GCCGTTGACAAAACACATAAGGACT
	TLR4	GCCATCGCTGCTAACATCATC	CTCATACTCAAAGATACACCATCGG
	TLR6	AACCTACTGTCATAAGCCTTCATTC	GTCTACCACAAATTCACTTTCTTCAG
	TLR8	AAGACCACCACCAACTTAGCC	GACCCTCAGATTCTCATCCATCC
	TLR9	CACGACAGCCGAATAGCAC	GGGAACAGGGAGCAGAGC
	TLR10	CCTGTCCAACTGCCTCATTTG	CTAAGTGTTCTAAGGATGTGTTTCTG
	IL-1β	GGCCGCCAAGATATAACTGA	GGACCTCTGGGTATGGCTTTC
	IL-12	GGAGTATAAGAAGTACAGAGTGG	GATGTCCCTGATGAAGAAGC
	IL-8	TTCGATGCCAGTGCATAAATA	CTGTACAACCTTCTGCACCCA
	MIP-1β	CAGCACCAATGGGCTCAGA	TTCCGCACGGTGTATGTGA
	TNF-α	CGCCCACGTTGTAGCCAATGT	CAGATAGTCGGGCAGGTTGATCTC
	IL-6	GACAAAGCCACCACCCCTAA	CTCGTTCTGTGACTGCAGCTTATC
	GAPDH	TGCCAACGTGTCGGTTGT	TGTCATCATATTTGGCAGGTTTCT

Table 2A .

 2A Genelist of regulated genes in alveolar macrophages after incubation with S. suis strain 10 (S10), or its non-encapsulated mutant (S10cpsdelEF). Fold induction is expressed as expression level after incubation with S10 or S10cpsdelEF at timepoint 60 min compared to cells that were mock infected at timepoint 60 min. (-) Gene expression below cut-off of 2 fold induction

	Gene name Dynein Tumor necrosis factor (TNF-α) Chemokine ligand 4 (CCL4) Interleukin 1 beta (IL-1-ß)	Fold induction S10 S10delcpsEF 2.9 --3.9 -3.8 -3.6 a n u s c r i p t M
	Amphiregulin Put. Lymphocyte G0/G1 switch protein 2 BTG2 protein Macrophage inflammatory protein 2 alpha (MIP-2-α) Jumonji domain containing 3 Sertine/threonine proteins kinase PLK2 A c c e p t e d	------	3.0 2.9 2.2 2.2 2.1 2.0
	Pyruvate dehydrogenase kinase isozyme 4	-	-2.1
		35

Table 2B .

 2B Genelist of regulated genes in alveolar macrophages after incubation with S. suis strain 10 (S10), its non-encapsulated mutant (S10cpsdelEF) or PBS (mock). Fold induction is expressed as expression level at timepoint 60 min compared to timepoint 0 min of the same strain. (-) Gene expression below cut-off of 2 fold induction.

		Fold Induction
	Gene Name	PBS (mock)	S10	S10
				cpsdelEF
	Interleukin-1-ß	16.1	16.6	47.2
	TNF-α	9.6	13.1	43.4
	Macrophage inflammatory protein-2-α	16.0	25.5	34.3
	Mitogen-activtated kinase (MAP3K8)	13.9	16.9	23.5
	Interleukin-1-α	12.6	16.0	22.1
	IκB	14.6	19.8	21.9
	Adrenomedullin	6.2	6.9	18.0
	Immediate early protein	4.3	5.9	15.8
	Activating transcription factor 3 (ATF-3)	12.8	12.4	14.8
	FosB	10.9	13.7	12.2
	Interleukin-8	8.1	10.3	10.8
	Mitogen-inducible gene 6	6.6	5.5	9.9
	Cytochrome P450	7.1	10.1	9.5
	Small inducible cytokine like 3 A1	-	-	9.2
	Chemokine Ligand 4 like (CCL4)	2.0	2.3	8.5
	Vascular endothelial growth factor A	8.1	8.9	8.4
	Phosphatidylcholine:ceramide	-2.8	-2.7	-3.0
	cholinephosphotranferase 1
	Nuclear factor 1A	-	-	-2.8
	Muscleblind-like protein	-3.2	-3.0	-3.0
	P2Y purinoreceptor 5 (P2Y5)	-3.0	-2.9	-3.0

Acknowledgements

This project was financially supported by the Ministry of Agriculture, Nature and Food Quality (KB-08) and results were obtained through the EC funded EADgene network (contract no. FOOD- CT-2004-506416). The text represents the authors' views and does not necessarily represent a position of the Commission who will not be liable for the use made of such information.


Dr Simona Nardoni
email: snardoni@vet.unipi.it

Isolation and characterization of Malassezia spp. in healthy swine of different breeds

Keywords: Malassezia spp., swine, PCR, culture, external ear canal

Malassezia spp. genus is represented by several lipophilic yeasts, normally present on the skin of many warm-blooded vertebrates, including man. Swine are one of the less investigated animal species. The aim of the present work was to study the occurrence of Malassezia spp. in the external ear canal of 408 healthy swine of different breeds, under different breeding conditions. For this purpose N. 185 free ranging wild boars, N. 107 large size pigs and 116 Cinta Senese breed were selected. Animals were of both genders, with age ranging from 8 months to 4 years. The subjects were culturally and molecularly checked for Malassezia spp.

Ninety-two out of 408 animals scored positive for Malassezia yeasts (22.5%). M. pachydermatis, M. sympodialis and M. furfur were recognized. M. pachydermatis was the sole species isolated from wild boars (12.9%), Cinta Senese (20.7%) and juvenile large size pigs (13.6%); 88% of large size breeds adult subjects scored positive for M. sympodialis (63.6%) and M. furfur (22.7%), respectively. The study focus on scarcely investigated epidemiological aspects of Malassezia spp. in this animal species.

A c c e p t e d M a n u s c r i p t Introduction Malassezia genus encounters lipophilic yeasts, normally present on the skin of many warmblooded vertebrates. Based on morphological, ultrastructural, physiological, and genetic features, 7 species were assigned to the genus in the 90ties [START_REF] Guého | The genus Malassezia with description of four new species[END_REF][START_REF] Guillot | The diversity of Malassezia yeasts confirmed by rRNA sequence and nuclear comparisons[END_REF], Malassezia pachydermatis, the only non lipid-dependent species, and the six lipid-dependent species Malassezia furfur, Malassezia globosa, Malassezia obtusa, Malassezia restricta, Malassezia slooffiae and Malassezia sympodialis. Further lipiddependent species, namely Malassezia dermatis [START_REF] Sugita | New yeast species, Malassezia dermatis, isolated from patients with atopic dermatitis[END_REF] Malassezia japonica (Sugita et al., 2003), Malassezia nana [START_REF] Hirai | Malassezia nana sp. nov., a novel lipid-dependent yeast species isolated from animals[END_REF], Malassezia yamatoensis (Sugita et al., 2004), Malassezia equina and Malassezia caprae [START_REF] Cabañes | Two new lipid-dependent Malassezia species from domestic animals[END_REF] were then described. These fungi have a role in otitis externa and seborrhoeic dermatitis of animals, mainly carnivores, as well as in pityriasis versicolor, seborrhoeic dermatitis, atopic dermatitis and folliculitis of man. In humans, M. pachydermatis was the responsible agent of infection both in immunocompetent (Ming [START_REF] Fan | Granulomatous skin infection caused by Malassezia pachydermatis in a dog owner[END_REF], and in immunocompromised subjects [START_REF] Midgley | The lipophilic yeasts: state of the art and prospects[END_REF]; lipid-dependent species alone or variously associated among them or with M. pachydermatis are described in domestic animals (Crespo et al., 2002a;Crespo et al., 2002b;[START_REF] Nardoni | Occurrence of Malassezia species in healthy and dermatologically diseased dogs[END_REF].

Swine seem to be one of the less investigated animal species. Some reports prior to the description of the new species demonstrated the occurrence of Malassezia spp. on swine skin [START_REF] Gustafson | Lipophilic yeasts belonging to genus Pityrosporum, found in swine[END_REF][START_REF] Gustafson | The occurrence of yeasts belonging to genus Pityrosporum in different kinds of animals[END_REF][START_REF] Dufait | Présence de Malassezia pachydermatis (syn. Pityrosporum canis) sur les poils et les plumes des animaux domestiques[END_REF][START_REF] Kuttin | Mycotic otitis externa in animals[END_REF][START_REF] Guillot | Prévalence du genre Malassezia chez les mammifères[END_REF] with a marked predominance of lipid-dependent yeasts. [START_REF] Garau | Prevalence of Malassezia spp. in healthy pigs[END_REF], identified M. sympodialis and M. slooffiae from 73% of ear canals in dermatologically healthy animals. To the best of our knowledge, there are no reports about the presence of Malassezia spp., related to breed and management in this animal species. Aim of the present work was to investigate

Materials and methods

External ear canals from 408 healthy swine were culturally and molecularly checked for Malassezia spp. The animals were of both genders, with age ranging from 8 months to 4 years. Animals aged more than one year were classified as adults. On the basis of breed, the animals were divided into 3 groups: free-ranging wild boars (N. 185), large size swine, such as Landrace, Large White and their crosses (N. 107) and Cinta Senese breed (N. 116). The first group consisted of 68 juvenile and 117 adult wild boars, the second comprehended 64 juvenile and 43 adults, while the last one 42 juvenile and 74 adult subjects. Large size pigs were intensively raised in 3 farms (No. 1, 2, 3), as well as Cinta Senese subjects were managed with extensive/outdoor production system in 3 different farms (No. 4,5,6).

Samples were collected by means of sterile cotton tip swabs immediately after killing during wild board hunting season, and at the swine farm in the others.

After collection, the specimens were promptly seeded onto Sabouraud Dextrose Agar added with 0.5% of chloramphenicol and cycloheximide (Mycobiotic Agar®, DID, Milano, Italy) and mDixon Agar (3.6% malt extract, 0.6% peptone, 2% desiccated ox-bile, 1% Tween 40, 0.2% glycerol, 0.2% oleic acid, 1.2% agar, 0.5% chloramphenicol and 0.5% cycloheximide).

The plates were incubated at 30 ° C for 7 days and processed as previously reported [START_REF] Nardoni | Occurrence of Malassezia species in healthy and dermatologically diseased dogs[END_REF]. Briefly, lipid-dependent species were morphologically and biochemically identified, as described by [START_REF] Guého | The genus Malassezia with description of four new species[END_REF]. The presence of M. furfur, M. sympodialis and M. slooffiae was proven by the Tween diffusion test, on the basis of their ability to assimilate various polyoxyethylene sorbitan esters. [START_REF] Bond | Characterization of markedly lipid-dependent Malassezia pachydermatis isolates from healthy dogs[END_REF] demonstrated the possible lipid dependence of some isolates of M. pachydermatis. Also, the presence of some lipids in A c c e p t e d M a n u s c r i p t the primary culture from the specimen can allow the growth of M. furfur in lipid-free medium.

For these reasons, subsequent transfers were performed to clearly distinguish these two species. The identification of M. furfur was confirmed by using the Cremophor EL (Sigma, St Louis, MO, USA) assimilation test, as reported by [START_REF] Raabe | Demonstration of Malassezia furfur and M. sympodialis together with M. pachydermatis in veterinary specimens[END_REF]. The splitting of esculin was also performed as additional key to identify both M. furfur and M. sympodialis [START_REF] Mayser | Differentiation of Malassezia species: selectivity of cremophor EL, castor oil and ricinoleic acid for M. furfur[END_REF]. The lack of catalase activity, which is a specific feature of M. restricta, proved the presence of this species.

Morphological and biochemical identification was confirmed by means of a PCR-based technique using restriction enzyme digestion, specific for the discrimination of 11 Malassezia species, as described by [START_REF] Mirhendi | A simple PCR-RFLP method for identification and differentiation of 11 Malassezia species[END_REF]. In order to achieve pure cultures, for each positive sample 5 colonies were subcultured onto mDixon Agar and stored at -20°C until analysis. The cell walls were mechanically disrupted by freeze-thawing and genomic DNA was extracted and purified according to the DNeasy TM protocol for animal tissue (QIAGEN Inc., Valencia, Ca, USA). The primers selected for this protocol amplify the target part of 26S rDNA, providing a single PCR product of an expected size of 580 bp. The PCR products were subjected to REA using CfoI and BstF51, separately, according to the manufacturer's instructions (Fermentas International Inc. Burlington, Ontario, Canada). Digested fragments were analyzed by electrophoresis in 2% agarose gel stained with ethidium bromide.

Yeast strains with morphological, physiological and molecular characters consistent with M. sympodialis were discriminated from the newly described species M. caprae, and M. equina, amplifying by PCR the variable D1 and D2 regions of the 26S rRNA gene, and the ITS and 5.8S rRNA gene. Isolation of DNA, PCR amplification process and DNA sequencing of rRNA genes of the isolates were performed as described by [START_REF] Cabañes | Two new lipid-dependent Malassezia species from domestic animals[END_REF]. Statistic analysis to evaluate significant differences among breeds and age of pigs was performed by chi square test (P<0.01). pachydermatis isolates grew without lipid supplementation. Some of the lipid-dependent isolates grew scarcely, making difficult conventional identification. However most of the lipid-dependent species showed a strong catalase reaction, scored positive for assimilation of Cremophor EL (M. furfur isolates) and the splitting of esculin (M. sympodialis isolates) and were inhibited from Tween 20 only (M. sympodialis). PCR-RFLP results agreed with cultural findings, and resulted discriminating for isolates with a reduced growing rate, generating fragments identificative for M. pachydermatis, M. sympodialis and M. furfur, respectively.

The sequences of the isolates identified as M. sympodialis on a preliminary count, resulted completely identical to M. sympodialis CBS 7222.

M. pachydermatis was the sole species isolated from both wild boars and Cinta Senese breed, being cultured from 24 animals for each group (12.9% and 20.7%, respectively). The prevalence of recovery for Malassezia spp. in large size animals was 44.1%; M. sympodialis was cultured from 28 subjects (63.6%), M. furfur from 10 (22.7%) and M. pachydermatis from 6 (13.6%). All yeasts species were obtained as a pure culture. Animals harboring Malassezia species were present in 2 out of 3 intensive breeding farms, while all farms in which Cinta Senese were raised yielded yeasts, with different prevalences.

Lipid-dependent species among large size breeds were limited to one farm only, where 88.3% of adults scored positive. Statistical analysis showed significant differences among overall prevalence values obtained from wild boars and Cinta Senese on one hand, and large size swine, on the other hand (P> 0.01).

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

Among juvenile wild boars, M. pachydermatis was isolated from 3 subjects only (4.4%), while this same species was recovered from 23/117 adults (19.6%). Thirty-eight out of 43 (88%) large size adult animals scored positive for lipid-dependent Malassezia species, while M. pachydermatis was cultured from 6 juvenile subjects only (9.4%). This difference was statistically significant (P<0.01). Sixteen out of 74 Cinta Senese adults (21.6%) were positive for M. pachydermatis, and 8/42 juvenile pigs (19%) harbored this fungal species. For animals belonging to this last breed, different prevalences were not statistically significant.

Discussion

Epidemiology of Malassezia species in swine has been scarcely investigated. In literature there is an unique paper dealing with this topic published since the taxonomic revision in 1996, while previous reports concern the presence of both lipid-dependent species and M. pachydermatis (formerly Pityrosporum pachydermatis) in the external ear canal of healthy animals [START_REF] Garau | Prevalence of Malassezia spp. in healthy pigs[END_REF]. These authors recovered M. sympodialis and M. slooffiae from 73 out of 100 swine ears. In our paper 408 pigs were investigated, with an overall prevalence of 22.5%. The occurrence of Malassezia yeasts appeared to be significantly different depending on the productive aptitude of the animals and, in group of large size and in wild boars on the age, also. To the best of our knowledge there are no reports about the occurrence of Malassezia species in different breeds of Sus scropha.

The most striking feature was represented by the fact that lipid-dependent fungal species were cultured from adults belonging to large size breeds, only. Among these species, M. sympodialis and M. furfur were identified, in partial contrast to the data published by [START_REF] Garau | Prevalence of Malassezia spp. in healthy pigs[END_REF], who isolated M. sympodialis and M. slooffiae. The presence of lipid dependent Malassezia organisms in this group of animals could suggest that breed and lines used in commercial production as well as diet may represent sources of variation of fat quality not A c c e p t e d M a n u s c r i p t only on fatty acid profiles of muscle and adipose tissue [START_REF] Guo | Effects of dietary vitamin E and fat supplementation on pork quality[END_REF], but also on skin composition traits. M. pachydermatis was present in all the 3 groups of investigated animals, but the occurrence of isolation showed remarkable differences. It was the unique species recovered both from wild boars and Cinta Senese breed animals, although a significant higher fungal burden was present in adult wild boars, only. The presence of this yeast species in juvenile large size pigs only was striking, considering that a simultaneous recovery of mixed lipid-dependent and M. pachydermatis was not recorded neither by [START_REF] Garau | Prevalence of Malassezia spp. in healthy pigs[END_REF] nor in the present study. The colonies isolated from each positive animal were representative of an unique Malassezia species as reported by the above cited Authors.

The number animals checked for each group within different farms didn't allow to draw general conclusions from a statistic point of view. Nevertheless, different prevalence values were observed among different farms, harbouring animals with similar management systems, suggesting a circulation of fungal organisms among animals of the same piggery, whereas they are present.

PCR-RFLP performed in the present study contextually with phenotypic identification methods was chosen for its ability to discriminate among 11 yeasts species by using 2 restriction enzymes (Mihrendi et al., 2005). This identification approach, applied to cultured isolates, appeared to be relatively fast and reliable, showing both a good discriminatory power, and an unambiguous capability to differentiate newly identified species, with the exception of M. sympodialis. To distinguish this last species from related ones, the sequencing and analysis of the D1/D2 26S rRNA gene, and ITS-5.8S rRNA gene is necessary, as demonstrated by [START_REF] Cabañes | Two new lipid-dependent Malassezia species from domestic animals[END_REF].

Lipid-dependent Malassezia species have been cultured from healthy subjects as stressed by [START_REF] Garau | Prevalence of Malassezia spp. in healthy pigs[END_REF], who appreciate studies dealing with yeast prevalence in skin/ear diseased swine. It is remarkable to considerate that swine is a strictly zootechnic species, characterized

  s c r i p t the occurrence of Malassezia spp. in the external ear canal of different swine breeds, under different breeding conditions.

  out of 408 animals scored positive for Malassezia spp. (22.5%). Three different species were recognized, namely M. pachydermatis, M. sympodialis and M. furfur. All M.

  short productive life cycle, so a possible occurrence of otitis externa is often 197 neglected by breeders. 198 A c c e p t e d M a n u s c r i p t Farm No. N. of animals juvenile adults positive juvenile animals (%) M. pachydermatis positive adult animals (%) M. furfur M. pachydermatis M. sympodialis

  Table shows the prevalences of Malassezia spp. recovery from different swine breeds, based on farm and age of animals.

Table -

 - Prevalence and characterization of Malassezia species in function of farms, breed and age of examined animals.

	Table

Sugita, T., Tajima, M., Takashima, M., Amaya, M., Saito, M., Tsuboi, R., Nishikawa, A., 2004. A new yeast, Malassezia yamatoensis, isolated from a patient with seborrheic dermatitis and its distribution in patients and healthy subjects. Microbiol Immunol. 48: 579-583.