Bibiana Peralta
email: bibiana.peralta@cresa.uab.es

Maribel Casas

Nilsa De Deus

Marga Martín

Anna Ortuño

Eva Pérez-Martín

Sonia Pina

Enric Mateu

Anti-HEV antibodies in domestic animal species and rodents from Spain using a genotype 3-based ELISA

Keywords: HEV, domestic animals, rodents, ELISA, genotype

A truncated ORF2 capsid HEV antigen derived from a genotype 3 strain was developed in insect cells and insect larvae, and compared with the Sar55 antigen and a commercial ELISA.

The antigen expressed in insect cells showed a better correlation with Sar55 (kappa value (k)=0.84) than the insect larvae antigen (k=0.69), and a better reproducibility as indicated by the intra and interplate variation coefficients. Commercial ELISA designed for human diagnosis but adapted to animal use using specific secondary antibodies demonstrated to have a very low sensitivity. The insect cell expressed antigen was used to develop an ELISA to detect antiHEV-IgG in serum samples of different domestic animal and rodents. Seropositivity in the studied animal populations was of 71.4% for pigs, 0.60% for goats, 1.92% for sheep, and 11.11% for cats. None of the 1170 cattle samples or 166 rodent samples analyzed was positive.

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

INTRODUCTION

Hepatitis E virus (HEV) is a small non-enveloped virus belonging to the Genus Hepevirus (Emerson et al., 2004), proposed as the Hepeviridae family. The virus is the causative agent of hepatitis E in humans. Four HEV genotypes have been described so far [START_REF] Lu | Phylogenetic analysis of global hepatitis E virus sequences: genetic diversity, subtypes and zoonosis[END_REF] with several sub-genotypes that seem to have a geographical distribution. However, only one serotype has been identified. Genotype 1 is common in Asia, particularly in the Indian subcontinent [START_REF] Arankalle | Phylogenetic analysis of hepatitis E virus isolates from India (1976-1993)[END_REF]. Genotype 2 was originally detected in Mexico but later on has been described in Africa [START_REF] Huang | Molecular cloning and sequencing of the Mexico isolate of hepatitis E virus (HEV)[END_REF][START_REF] Buisson | Identification of a novel hepatitis E virus in Nigeria[END_REF]. Genotype 3 strains are present in Europe, America, Asia and Oceania and genotype 4 strains seem to be autochthonous of Asia [START_REF] Nishizawa | Characterization of Japanese swine and human hepatitis E virus isolates of genotype IV with 99 % identity over the entire genome[END_REF]. Human hepatitis E is endemic in many developing countries where epidemics caused by genotypes 1 and 2 are common and usually associated with contaminated drinking water. In industrialized countries, hepatitis E appears most often as sporadic cases either related to travelling to endemic areas or caused by autochthonous strains (Péron et al., 2006). Several studies have suggested that HEV might be a zoonotic agent. Thus, it has been shown that exposure to domestic pigs could be a risk factor for seropositivity in humans [START_REF] Meng | Prevalence of antibodies to hepatitis E virus in veterinarians working with swine and in normal blood donors in the United States and other countries[END_REF], human and swine strains of genotypes 3 and 4 seem to be closely related [START_REF] Herremans | Swine-like hepatitis E viruses are a cause of unexplained hepatitis in the Netherlands[END_REF][START_REF] Wibawa | Identification of genotype 4 hepatitis E virus strains from a patient with acute hepatitis E and farm pigs in Bali, Indonesia[END_REF] and some documented sporadic cases of hepatitis E in humans have been related to the consumption of uncooked or undercooked meat or viscera of wild boars or deers [START_REF] Takahashi | Complete or near-complete nucleotide sequences of hepatitis E virus genome recovered from a wild boar, a deer, and four patients who ate the deer[END_REF][START_REF] Tei | Consumption of uncooked deer meat as a risk factor for hepatitis E virus infection: an age-and sex-matched case-control study[END_REF]. In those cases, genotype 3 was involved. In Europe sporadic cases of human hepatitis E are mostly related to genotype 3 of the virus. Recent studies showed that up to 97% of the pig herds studied in some European countries including Spain [START_REF] Rutjes | Increased hepatitis E virus prevalence on Dutch pig farms from 33 to 55% by using appropriate internal quality controls for RT-PCR[END_REF][START_REF] Seminati | Distribution of hepatitis E virus infection and its prevalence in pigs on commercial farms in Spain[END_REF] have HEV seropositive animals and porcine HEV isolates in European pigs belong to genotype 3 (Van der [START_REF] Van Der Poel | Hepatitis E virus sequences in swine related to sequences in humans, The Netherlands[END_REF][START_REF] Clemente-Casares | Hepatitis E virus epidemiology in industrialized countries[END_REF][START_REF] De Deus | Detection of hepatitis E virus in liver, mesenteric lymph node, serum, bile and faeces of naturally infected pigs affected by different pathological conditions[END_REF].

In addition to pigs and deers, so far HEV has been detected only in horses [START_REF] Saad | Hepatitis E virus infection in work horses in Egypt[END_REF]; mongooses [START_REF] Nakamura | Hepatitis E virus infection in wild mongooses of Okinawa, Japan: Demonstration of anti-HEV antibodies and a full-genome nucleotide sequence[END_REF] and chickens [START_REF] Haqshenas | Genetic identification and characterization of a novel virus related to human hepatitis E virus from chickens with hepatitis-splenomegaly syndrome in the United States[END_REF]; in the last case, the avian strains seem to belong to a different HEV species or, at least, to a different genotype.

A c c e p t e d M a n u s c r i p t 4 Antibodies against HEV have been also demonstrated in species other than the abovementioned such as cows, sheeps, goats, dogs, cats and rodents [START_REF] Tien | Detection of immunoglobulin G to the hepatitis E virus among several animal species in Vietnam[END_REF][START_REF] Arankalle | Prevalence of anti-hepatitis E virus antibodies in different Indian animal species[END_REF][START_REF] Okamoto | Presence of antibodies to hepatitis e virus in Japanese pet cats[END_REF][START_REF] Vitral | Serological evidence of hepatitis E virus infection in different animal species from the Southeast of Brazil[END_REF][START_REF] Mochizuki | Epidemiological study of hepatitis E virus infection of dogs and cats in Japan[END_REF][START_REF] Zhang | Hepatitis E Virus Infection among Domestic Animals in Eastern China[END_REF] but the epidemiological role of those domestic species is uncertain. Most of these serological studies in domestic animals other than pigs have been carried out in Asia and information about Europe is still lacking.

In the current study, we developed and applied an ELISA test based on the truncated ORF2 capsid protein from a genotype 3 strain for the screening of serological evidence of HEV infection among various domestic animal species.

MATERIALS AND METHODS

Expression of a truncated HEV ORF2

Virus sample and ORF2 amplification

One HEV RT-PCR positive bile sample collected from a 14-weeks-old pig was selected and stored at -80ºC until used. Total viral RNA was extracted from 150 µl of bile with Nucleospin ® RNA virus kit (Macherey-Nagel Gmbh & Co., Düren, Germany) and the full length HEV ORF2 was amplified by means of a two round PCR. Primers were primers F5000 (5'-AATGTKGCKCAGGTYTGTG-3') and R7260

(5'-TTTTTTTTTTTTTCCKGGGRGCGCG-3') for the first round of amplification, and primers F5160 (5'-MGGSTRGAATGAATAACATG-3') and R7260 were used for the seminested amplification [START_REF] Peralta | Genetic characterization of the complete coding regions of genotype 3 hepatitis E virus isolated from Spanish swine herds[END_REF]. The second round PCR product was cloned into pCR®II-Blunt-TOPO® (Invitrogen).

Generation of HEV ORF2 recombinant baculovirus

Generation of the recombinant baculovirus containing the truncated HEV ORF2 was done using the Bac-to-Bac Baculovirus Expression System (Invitrogen) following manufacturers's instructions. Briefly, a set of primers was designed to amplify a fragment of 1,491 bp: the DH10Bac™ were transformed with pFBAC-ORF2H 6+ for production of a recombinant bacmid BAC-ORF2H 6+ . Then, BAC-ORF2H + was extracted following standard methods and transfected into Sf9 cells using Lipofectamine™ (Invitrogen). Cells were cultured in Grace's insect media (Gibco BRL) supplemented with 10% foetal bovine serum (FBS), 3% nonessential amino acids, 100 µg/ml of streptomycin and 100 U/ml of penicillin and 20 μg/ml gentamycin and grown at 27ºC. At 96 hours post-transfection the supernatant containing the recombinant baculovirus was recovered and stored at 4ºC.

Truncated ORF2 expression in insect cells and insect larvae

The recombinant baculovirus was used to infect Sf9 cells using a multiplicity of infection (MOI) of 0.05. Infected cells were incubated for 6 days (the optimal conditions for recovery of the HEV protein as determined in previous assays). Total protein was extracted by collecting the infected cells and incubating them in milliQ water for 15 min in ice followed by a centrifugation at 2500xg for 10 min. The recombinant protein ORF2H 6+ contained in the Alternatively, Trichoplusia ni larvae were grown and inoculated as previously described [START_REF] Medin | Expression of foreign proteins in Trichoplusia ni larvae[END_REF][START_REF] Pérez-Filgueira | Optimization and validation of recombinant serological tests for African Swine Fever diagnosis based on detection of the p30 protein produced in Trichoplusia ni larvae[END_REF]. The inocula consisted in 5x10 5 recombinant virus particles (BAC-ORF2H 6+ ) or wtBAC (control baculovirus). Three days post infection, the larvae were frozen and homogenized in an extraction buffer to obtain total soluble proteins (TSP), including our recombinant ORF2H 6+ protein, as previously described [START_REF] Pérez-Filgueira | Optimization and validation of recombinant serological tests for African Swine Fever diagnosis based on detection of the p30 protein produced in Trichoplusia ni larvae[END_REF]. The amount of TSP contained in the supernatant was quantified by Bradford assay [START_REF] Bradford | A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding[END_REF], and lyophilised for long time storage [START_REF] Pérez-Filgueira | Optimization and validation of recombinant serological tests for African Swine Fever diagnosis based on detection of the p30 protein produced in Trichoplusia ni larvae[END_REF].The same protocol was followed for the control protein extract (Ni), obtained from the Bac-Ni infected larvae. [START_REF] Pérez-Filgueira | Optimization and validation of recombinant serological tests for African Swine Fever diagnosis based on detection of the p30 protein produced in Trichoplusia ni larvae[END_REF].

Analysis and quantification of recombinant protein production

The purified ORF2H 6+ and the protein extract obtained from T. ni larvae were run in duplicate in NuPAGE® Novex 4-12% Bis-Tris gels (Invitrogen). One of the gels was stained with Coomasie blue and after confirmation of the presence of the protein, the other was used for Western Blot analysis (WB). After transfer, nitrocellulose membranes were blocked for 1h at room temperature with PBS added with 0.02% Tween-20 and 2% skim milk. A swine anti-HEV hyperinmune sera obtained from an experimentally infected pig was used (provided by Dr. X.J.

Meng, CMMID, Virginia Tech). The antigen-antibody reaction was revealed by using a protein A-peroxidase conjugate (Sigma) and 4-chloronaftol solution (Sigma) as a substrate. Protein quantification was performed by means of the BCA protein Assay Kit (Pierce) following manufacturer's directions. The autochthonous truncated ORF2H 6+ was compared with the Sar55 strain derived protein at nucleotide and aminoacid level and the hydrophobicity profile was predicted by means of the bioinformatic program BioEdit [START_REF] Hall | BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT[END_REF] using the Kyte and Doolittle scale. Other additional 85 samples were obtained from previous studies (de [START_REF] De Deus | Detection of hepatitis E virus in liver, mesenteric lymph node, serum, bile and faeces of naturally infected pigs affected by different pathological conditions[END_REF]Deus et al., , 2008) ) and corresponded to pigs with known HEV viremia status. Finally, other 30 samples kindly provided by Dr. X.J. Meng (CMMID, Virginia Tech) corresponded to experimentally infected pigs or negative controls.

ELISA procedure

Ninety-six well polysterene plates were coated with 100 µl of either the purified protein recovered from Sf9 cell cultures at a concentration of 0.125µg/ml (final protein amount 12.5 ng/well) or with the protein extract obtained from the infected larvae at 3.5µg/ml (final protein amount 350 ng/well) diluted in coating buffer (0.015M Na 2 CO 3 , 0.035M NaHCO 3 , pH 9.6.

Those concentrations were determined to be optimal in previous titration experiments (not shown). In order to minimize the effect of unspecific binding to the plates or to the antigen, sera were analysed in duplicate in antigen-coated and mock-coated wells. For the ELISA procedure, after coating the plates for 18h, wells were washed with PBS added with 0.02% Tween-20 (PBS -T) and blocked for 1h at 37°C with blocking buffer (BB) (140µl/well; PBS, 0.035 M NaCl plus 0.5% gelatine and 10% FBS). After washing the BB, serum samples were diluted 1:100 in BB and 100µl were dispensed per well. After 45 min of incubation at 37ºC, plates were washed 5 times with PBS-T and a HRP-conjugated goat anti-swine IgG (Serotec Ltd., Oxford, UK) was added at a dilution 1:100,000. Plates were incubated for 45 min at 37ºC and washed as described above. The reaction was revealed by adding 100µl of TMB (Sigma Chemical, St.

Louis, MO., USA) and stopped with 100 µl of H 2 SO 4 2M. Plates were read in an ELISA reader at 450nm. The specific absorbance value for each sample was calculated by subtracting the value of the mock-coated wells from the values of the plates coated with the specific protein.

A c c e p t e d M a n u s c r i p t 8 Cut-off was set at 0.3 that was about 4 standard deviations above the mean OD value of the negative control sera.

2.2.3. Validation of the genotype 3 ELISA antigens and comparison with a commercial kit A truncated ORF2 obtained from the HEV genotype 1 Sar55 strain [START_REF] Robinson | Structural characterization of recombinant hepatitis E virus ORF2 proteins in baculovirus-infected insect cells[END_REF] was used in order to validate the genotype 3 ELISA antigen. Three hundred and sixty seven swine samples were analyzed in coated and uncoated wells using the Sar55 protein at a concentration of 0.25µg/ml (25 ng of protein per well) following the procedure described above. Also thirty selected samples were tested by the test Bioelisa HEV IgG (Biokit) (intended for diagnosis in humans) following manufacturer's instructions but modified for the analysis of porcine samples by using an anti-swine IgG conjugate as explained above.

Application of genotype 3 antigens for screening HEV seroprevalence in different

domestic species and rodents.

Immunization of different animal species to obtain hyperimmune serum

In order to obtain a positive serum to be used as a positive control in the developed ELISA for different species, an immunization assay was performed in cows, sheeps, goats, and rodents. For that purpose, two 3-months-old cows, two adult goats, two adult sheep and five ICR-CD1 6months-old mice were housed in the Veterinary School facilities of the Universitat Autònoma of Barcelona. One week after their arrival animals were injected with the HEV ORF2H 6+ purified recombinant protein using the dosages, adjuvants and inoculation routes summarized in Table 1.

For each group one animal was kept as a negative control inoculated with sterile saline solution with the adjuvant. Blood samples were periodically obtained by jugular punction in the case of the ruminants and by tail incision in mice. The ELISA was done as described above , but the secondary antibodies were HRP-conjugated sheep anti-bovine IgG at a 1:5,000 dilution (Serotec Ltd., Oxford, UK) for cow samples; HRPconjugated donkey anti-sheep/goat IgG (Serotec Ltd., Oxford, UK) at 1:24,000 dilution for sheep and goats, a protein A-peroxidase conjugate for cat samples (Sigma Chemical, St. Louis, MO., USA) at 1:2,000 dilution, and a goat anti-mouse IgG (Fc specific) peroxidase conjugated antibody (Sigma Chemical, St. Louis, MO., USA) at 1:80,000 for rodents. Samples were tested in duplicate using the purified antigen expressed in cell culture and in both coated and uncoated wells as detailed for pig samples. Cut-off were set to the mean OD plus 4x standard deviations of the negative controls. Thus, the cut-offs were 0.13 for cattle, 0.24 for sheep, 0.20 for goats, 0.50 for cats and 0.40 for rodents.

Western blot and dot blot analysis

Confirmation of positive ELISA results was made by WB analysis using the truncated protein expressed in insect cells. One µg of purified protein was run in a single well protein PBS 0.02% Tween 20, sera from cats, goats ans sheeps were diluted 1:1,000 in blocking solution and added to the strips. Following 1 hour incubation, the strips were washed as mentioned and the secondary antibody was added at the dilution determined in previous assays, which resulted in 1:100,000 for goats and sheep and 1:5,000 for cats. Pig results were not confirmed by WB since the infection in Spanish swine population has been reported before.

Chemiluminescence was detected using the fluorescence imager FluorChem ® HD2 Imaging System (Alpha Innotech). Sera positive in WB were re-confirmed in dot blot using both the Sar55 and the ORF2-truncated protein expressed in insect cells adsorbed in native conformation or after denaturation. Finally, in order to warrant specificity of the assay, we only considered as positive the sera yielding positive results with both Sar55 and the ORF2H 6+ protein.

Statistical analysis

Statistical analyses were done using Epi-Info 2000 v 3.4.1. Kappa value was calculated using WinEpiscope software.

RESULTS

Standardization of an ELISA test for IgG anti-HEV based on the truncated ORF2H 6+

of genotype 3

Protein production

After six days of incubation, 1.65 mg of HEV ORF2H 6+ were recovered from 3.6x10 7 Sf9 cells.

For the insect larvae expression, the amount of total protein recovered was 13 mg per larva, of which about 10% was the specific protein (determined by OD comparison with the purified A c c e p t e d M a n u s c r i p t protein). In both cases the truncated HEV ORF2H 6+ had a molecular weight of 55kDa. The protein produced in larvae was little soluble in water making very difficult to further purify the larva extracts by the column method used in this study probably due to the formation of aggregates with other molecules present in the larvae extract. In addition, storage of this extract at 4°C or -20°C rapidly produced a loss of antigenic reactivity as revealed in ELISA (data not shown). This did not occur with the cell culture-expressed protein.

Both recombinant proteins, the ORF2H 6+ and the Sar55 protein, were located between aminoacid positions 112 and 607, so the length was of 495 aminoacids for the Sar55 truncated protein and 501 aminoacids for the ORF2H 6+ protein, since a 6x histidine tail was added to the 3' end. Although at nucleotide level the identity was of 78%, only a difference of 5% at aminoacid level was observed. The sequence of the strain used for the protein expression is available at GenBank under the accession number EU723512. Moreover, the hydrophobicity analysis performed revealed almost identical patterns for both proteins (data not shown).

Sensitivity, specificity and variability of the genotype 3 antigens

When the 30 sera from experimental pigs were analysed, the ORF2H 6+ ELISA diagnosed correctly 13/13 positive and 17/17 negative samples. Considering Sar55 ELISA results as the golden standard (100% of relative sensitivity and specificity) and using the 252 field sera, the truncated ORF2H 6+ protein-ELISA expressed in cell cultures had a relative sensitivity of 98.9% (180/182; CI 95% : 97.4-100%), whereas the relative specificity was 78.8% (55/70; CI 95% : 68.9-88.2%). The ELISA set with the protein expressed in larvae showed a sensitivity of 97.8%

(178/182, CI 95% : 95.0-99.1%) and a specificity of 61.4% (43/70, CI 95% : 50.0-72.8%) compared with the Sar55 protein ELISA. In order to know if the ELISA developed with the Spanish strain of HEV genotype 3 was similar in performance to the Sar55 ELISA in terms of global agreement of results, the kappa value was calculated. Thus, kappa was 0.82 (CI 95% 0.69-0.94%)

for the cell culture-expressed protein and 0.66 (CI 95% 0.54-0.78) for the antigen expressed in insect larvae.

A c c e p t e d M a n u s c r i p t 12

To further characterize the ELISA test developed, an analysis of intra-and interplate variability was performed. This assay was conducted using only the 252 samples from commercial farms.

Samples were analyzed twice in the same plate and in different ELISA plates as described above. For the cell culture expressed antigen, the results showed a 2.7% and 6.0% of intra-and interplate variation, respectively and these values were 6.0% and 9.0% for the antigen expressed in insect larvae.

Finally, a comparison of results with a commercially available HEV-antibody ELISA kit was done. In this case, the commercial ELISA only recognized as positive pig sera yielding ODs1.0 in our ELISA. Relative sensitivity and specificity were 31.25% (8.5-53.9%) and 100% respectively.

Serological survey of HEV in domestic species and rodents

Positive control sera obtained by hyperimmunization with the truncated ORF2H 6+ protein had the following titre sin ELISA: sheep and goat sera: 1:16,000; cow and mouse sera titers were 1:1,000 and >1:128,000, respectively (Figure 1).

Two hundred and fifty-two pig samples collected in nine farms were examined; all nine herds had HEV-seropositive pigs with an average within farm prevalence of 79.45% (19.31%). For sheeps a total of 1357 samples from 89 different herds were analysed by ELISA, being positive 36 samples belonging to 27 farms. For goats the number of samples was 1143 form 76 herds.

and the ELISA revealed 18 positive sera from 16 farms. For cows, after analysing 1170 animals from 77 herds, no positive results were observed. Twenty cats of 5 catteries were also seropositive but none of the rodents presented antibodies.

Positive samples were tested in WB. For sheep, 28/33 animals of 26 herds produced a positive result in this test; for goats, 14/17 positive sera (14 farms) in ELISA were confirmed in WB.

Regarding cat samples, due to the scarce amount of serum final confirmation was only done by dot blot. Thus, final confirmation of WB positive sera by dot blot using Sar55 and the ORF2H 6+ truncated protein either under native and denaturing conditions produced the following results:

A c c e p t e d M a n u s c r i p t 13 for sheep, 26/28 WB positive sera were confirmed (22 herds); for goats 7/14 WB positive sera were also positive with Sar55 (7 herds) and 6 cats reacted positively in WB (table 2 summarizes these results). Interestingly, reactivity of sera was decreased by denaturation of Sar55 or ORF2H 6+ protein (figure 2) indicating that antibodies recognised primarily conformational epitopes.

DISCUSSION

Several tools have been developed for HEV diagnosis. Molecular biology techniques such as PCR produce the more certain results since they give proof of the presence of HEV in a tissue, fluid or excreta of an animal. The main problem for the PCR detection of HEV is that, usually, viremia or shedding in faeces is of short duration and thus, the chance to find a positive animal is limited. In animals, other samples where the virus could be found easily, such as liver or bile, are most often only available post-mortem. For this reason, serological tests such as ELISA are widely used. Commercially available HEV ELISA kits for human diagnosis are usually based on HEV genotype 1 or 2 peptides. Cross-reactivity among antigens obtained from different genotypes seems to exist [START_REF] Engle | Hepatitis E virus (HEV) capsid antigens derived from viruses of human and swine origin are equally efficient for detecting anti-HEV by enzyme immunoassay[END_REF][START_REF] Arankalle | Evaluation of human (genotype 1) and swine (genotype 4)-ORF2-based ELISAs for anti-HEV IgM and IgG detection in an endemic country and search for type 4 human HEV infections[END_REF] and based on this property, most serological studies in animals have been done using genotype 1 peptides, particularly from the Sar55 strain [START_REF] Meng | A novel virus in swine is closely related to the human hepatitis E virus[END_REF][START_REF] Arankalle | Prevalence of anti-hepatitis E virus antibodies in different Indian animal species[END_REF][START_REF] Seminati | Distribution of hepatitis E virus infection and its prevalence in pigs on commercial farms in Spain[END_REF] However, genotype 3 has demonstrated to be the most common in animals, particularly in pigs [START_REF] Huang | Detection by reverse transcription-PCR and genetic characterization of field isolates of swine hepatitis E virus from pigs in different geographic regions of the United States[END_REF][START_REF] Ning | Genotype 3 hepatitis E has been widespread in pig farms of Shanghai suburbs[END_REF]. Thus, and although differences between ORF2 proteins of genotype 1 and 3 seem not to be large, minor changes could affect sensitivity and specificity of the test when applied to animals.

One of the aims of this study was to develop an antigen based on a genotype 3 European strain.

Two different expression systems were used: insect cells (a method reported to be optimal for HEV [START_REF] Mast | Evaluation of assays for antibody to hepatitis E virus by a serum panel[END_REF] and insect larvae (a large scale production system). In our hands, protein recovery was higher in insect larvae (about 1mg/larva) compared to insect cells; however the larva protein was not possible to be purified and was little stable in cold storage.

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

Although specific experiments to clarify these facts were not conducted, the reasons for this may be the formation of protein aggregates so there is no free protein with exposed epitopes which antibodies can recognize or the presence of proteases in the larva extract that even at low temperatures can degrade the antigenic protein. These were two serious disadvantages for that protein to be used in serological tests.

Regarding the performance of the commercial kit, it was inefficient for the serodiagnosis in animals due to poor sensitivity.

The performance of the two expressed proteins (cell culture and larvae-produced) was assessed by examining sera from experimental infections and by comparing results with the Sar55-ELISA. The protein expressed in insect cells always exceeded in performance the larvaeproduced protein and thus it was the protein selected for the epidemiological study. The ELISA set with this cell culture-produced protein recognized accurately positive and negative sera from experimental pig and had sensitivity and specificity of 98.9% and 78.8% relative to the Sar55-ELISA. It is worth to comment the apparent discrepancy in specificities between the recombinant ORF2H 6+ ELISA and the Sar55-ELISA. Both proteins had 497 aminoacids located between aminoacid positions 112 and 607, with minor differences (5% at the aminoacid level).

The predicted hidrofobicity of the two proteins was quite similar as well and therefore, the causes for this discrepancy were not evident. If we take into account that antibodies recognised primarily conformational epitopes as evidenced by the Dot Blot analysis there is the possibility that the 6x histidine tail is interfering in the protein natural conformation leading to a loss of sensitivity and specificity. Experiments with a truncated protein without the histidine tag were not performed. The fact that some react differently with the different proteins looked unimportant for pigs since in this species most animals are reported to be seropositive [START_REF] Seminati | Distribution of hepatitis E virus infection and its prevalence in pigs on commercial farms in Spain[END_REF] with seroprevalences that can reach up to 90% or higher. In contrast, a minor lack of specificity could lead to an overestimation of the prevalence in other species. Thus a stringent strategy for diagnosis was adopted: only sera reacting positively in ELISA, western blot and finally the dot blot using both Sar55 and our protein would be considered to be truly positive.

With this approach, none of the cows was positive but 1.92% (1.29-2.84%) and 0.60% (0.26-A c c e p t e d M a n u s c r i p t 15 1.28%) of sheep and goat, respectively were found to be positive. In the case of cows, other authors reported seroprevalences ranging from 1.42% to 6.9% [START_REF] Arankalle | Prevalence of anti-hepatitis E virus antibodies in different Indian animal species[END_REF][START_REF] Wang | Prevalence, isolation, and partial sequence analysis of hepatitis E virus from domestic animals in China[END_REF][START_REF] Vitral | Serological evidence of hepatitis E virus infection in different animal species from the Southeast of Brazil[END_REF]. For sheep and goats, reports are controversial. However, viral genome has never been detected in any of the domestic ruminant species. In our case, the seroprevalence in sheep and goats was very low and the fact that in most cases a single reactor per herd was found, opens the question of whether or not those could be false positive results in spite of the stringency of the conditions required in the present study to be considered seropositive. Our opinion is that extreme caution should be applied to the interpretation of serological results in species where HEV has not been detected directly, particularly when a very large number of sera are examined since this increases the chance of finding false positives in spite a high specificity. In any case, true or false positive, our results indicate that HEV is either not present or present at a very low frequency in domestic ruminants of Spain.

Six out of 54 cats analysed resulted positive (11%, 4.6-23.32%). In this case, HEV seropositive cats were present in four out of seven studied catteries with several seropositive individuals per cattery suggesting transmission among animals in the cattery. As carnivores, as well as dogs, there is the possibility that cats get infected via the food chain. It is a common practice to feed cats and dogs with raw meat, that could probably be contaminated with swine HEV. A seroprevalence study performed on dog samples collected in the same area in our lab revealed that more than 20% of the animals tested positive for anti-HEV IgG detection supporting this hypothesis [START_REF] Peralta | Detección de IgG e IgM contra el virus de la hepatitis E en la población canina de Barcelona[END_REF]. If this was true it does not necessarily mean that cats and dogs suffer the infection, the antibodies found in this species could only be the result of repeatedly contact with the virus. The percentage of positive cats found in our study is in agreement with other previous studies [START_REF] Okamoto | Presence of antibodies to hepatitis e virus in Japanese pet cats[END_REF][START_REF] Mochizuki | Epidemiological study of hepatitis E virus infection of dogs and cats in Japan[END_REF] that reported that the 33% of the tested cats had anti-HEV antibodies. Those studies were done in an area with the same epidemiological situation, sporadic cases in humans and endemic situation in pigs. This fact may have a very important role in HEV transmission, since there are evidences of direct transmission from a cat to its owner [START_REF] Kuno | Sporadic acute hepatitis E of a 47-year-old man whose pet cat was positive for antibody to hepatitis E virus[END_REF]. Finally, all examined rodents analyzed in this work were negative. Studies made in other countries reported seroprevalences varying from 0 to 90% in different rodent species depending on the country and the species examined [START_REF] Tien | Detection of immunoglobulin G to the hepatitis E virus among several animal species in Vietnam[END_REF][START_REF] Kabrane-Lazizi | Evidence for widespread infection of wild rats with hepatitis E virus in the United States[END_REF][START_REF] Favorov | Prevalence of antibody to hepatitis E virus among rodents in the United States[END_REF][START_REF] Arankalle | Prevalence of anti-hepatitis E virus antibodies in different Indian animal species[END_REF][START_REF] He | Evidence that rodents are a reservoir of hepatitis E virus for humans in Nepal[END_REF][START_REF] Withers | Antibody levels to hepatitis E virus in North Carolina swine workers, non-swine workers, swine, and murids[END_REF]. The species analyzed in this work were A.sylvaticus, A. flavicolis, M. musculus, R. norvegicus and M. glaerolus, and few samples of each were tested. Seropositive results had been previously reported in M.

musculus [START_REF] Favorov | Prevalence of antibody to hepatitis E virus among rodents in the United States[END_REF], A. sylvaticus [START_REF] Karetnyi | Possible involvement of rodents in the spread of viral hepatitis E[END_REF] and R. norvegicus [START_REF] Kabrane-Lazizi | Evidence for widespread infection of wild rats with hepatitis E virus in the United States[END_REF][START_REF] Favorov | Prevalence of antibody to hepatitis E virus among rodents in the United States[END_REF], but M. glaerolus and A. flavicolis have been never analyzed before. So far, the species that have shown to be more prevalent are R. rattus and R. norvergicus, which were not present or present in a little quantity in our sampling.

Before assuring that the infection is not present in rodents in Spain, a more accurate analysis should be made on species that have proved to be positive in other studies such as species of the genera Rattus, preferably caught in rural areas close to pig farms.

In conclusion, this study shows the performance of different HEV antigens used for the serological diagnosis in animals and the importance to adopt stringent criteria for the serodiagnosis. The seroprevalence of HEV in domestic ruminants was very low or nil while frequency of antibodies in cats of catteries was high. These results emphasize the need for developing more accurate serological tools for HEV diagnosis in animals as well as the importance of gaining understanding of the role of animals in this infection.

  RsrII restriction site and the initiation codon, and the reverse primer RevHindIIIHEVHist (5'-TATAAGCTTCTATTAGTGATGGTGATGGTGATGGGCTAAAGCAGAATGCGGGGC-3') containing the HindIII restriction site, two stop codons and a 6xHis (H 6+ ) tag. The N-terminal and C-terminal truncated ORF2 expressed corresponded to the minimum sequence necessary to allow virus like particles (VLPs) formation as described by[START_REF] Li | Essential elements of the capsid protein for self-assembly into empty virus-like particles of hepatitis E virus[END_REF], from aa 112 to aa 607. This means that the expressed product had an N'-terminus 111 aa deletion and a C'terminus 53 aa deletion. The PCR product was gel purified with Nucleospin® ExtractII (Macherey-Nagel), digested with restriction enzymes, and ligated into the pFASTBAC vector (Invitrogen) to yield the construction pFBAC-ORF2H 6+ . Then, ElectroMAX™DH5α E. coli cells (Invitrogen) were transformed, and the recombinant plasmids were extracted, and confirmed by PCR and fully sequencing of the insert. Competent cells MAX Efficiency ®

  using the kit Protino Ni-IDA packed columns (Macherey-Nagel) following manufacturer's instructions. The recovered protein was stored at 4ºC.

  of an ELISA for IgG detection using the protein obtained from insect cells and insect larvae as an antigen 2.2.1. Porcine sera samples Three hundred and sixty-seven pig sera were used in this study; of them, two hundred and fiftytwo samples were obtained from nine different fattening farms located in the North-East Spain.

  NuPAGE 4-12% Bis-Tris Gel 1.0mm x 2D, Invitrogen) for 1 h at 170V and transferred to a nitrocellulose membrane Amersham Hybond ™ ECL ™ (GE Healthcare). Each membrane was cut into 4mm strips and stored at 4ºC until use. The Amersham ™ ECL Advance ™ Western Blotting Detection Kit (GE Healthcare) was used following manufacturer's instructions. Strips were treated with the blocking solution at 4ºC overnight. After washing with

Figure 1 .

 1 Figure legends Figure 1. Anti-HEV antibodies titre in inoculated animals measured by indirect ELISA.

Figure 2 .

 2 Figure 2. Dot Blot analysis of ELISA positive samples. Lines 1 to 7 belong to cat samples, lines 8 to 14 belong to goat samples and lines 15 to 20 are sheep samples. C-, negative control sera; C+, positive control sera. a) blank, b) denatured Sar55 protein, c) native Sar55 protein, d) denatured ORF2H 6+ , e) native ORF2H 6+ . Only samples positives to ORF2H 6+ and at least one of the two conditions of the Sar55 protein were considered to be positive.

  Sampling was adjusted to detect a 3% of infected herds. Only counties accounting for ≥2.5% of the livestock census were taken into account. At the end, serum samples were collected from 242 herds accounting for 1,170 cows, 1,357 sheeps and 1,143 goats. Cat samples (n=54) were collected from seven cat shelters located in urban areas near Barcelona. Sera from wild rodents (n=166) were collected in rural areas in central Spain and belonged to the species Apodemus sylvaticus, Apodemus flavicolis, Mus musculus, Myodes glaerolus and Rattus norvergicus. .

	2.3.2. Sampling in domestic animals and rodents
	Sera from ruminants (cows, sheeps, goats) were collected in farms of Catalonia (Spain).

The procedures were performed in accordance with the guidelines of the Good Experimental Practices (GEP), under the supervision of the Ethical Welfare Committee on Human and Animal Experimentation of the Universitat Autònoma de

A c c e p t e d M a n u s c r i p t 9 Barcelona (CEEAH), Spain. Blood samples were centrifuged for 5 min at 2,500 xg, and sera were stored at -80°C until used. 2.3.3. ELISA for anti-HEV IgG detection

Acknowledgements

This study was supported by the research grant AGL2004/06688 from the Spanish government. Bibiana Peralta, Eva Pérez-Martín and Maribel Casas have a fellowship from the Generalitat de Catalunya. Nilsa de Deus has a fellowship from CReSA. The authors are grateful to Drs. R.H. Purcell and R.E. Engle for providing the Sar55 antigen and for technical advice, Maribel Gegúndez from Universidad de Alcalá de Henares and from Red EVITAR (Fondo de Investigaciones Sanitarias, Spanish Ministry of Health, grant no. G03/059) for providing rodent

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


M Sibila

L Martínez-Guinó

E Huerta

M Mora

L Grau-Roma

T Kekarainen

J Segalés

TORQUE TENO VIRUS (TTV) INFECTION IN SOWS AND SUCKLING PIGLETS

Keywords: Torque Teno virus (TTV), piglet, sow, PCR, serum, lactation period

Torque Teno virus (TTV) is a single stranded DNA virus that has been detected in serum of primate and non-primate species including swine. Little information on swine TTV infection and transmission dynamics is nowadays available. The goal of this study was to gain insight into the potential role of the sow in transmitting TTV to piglets and the infection dynamics of both swine TTV genogroups (TTV1 and TTV2) during the lactation period. Serum samples from 44 sows at 1 week post-farrowing and 215 piglets at 1 and 3 weeks of age were tested using TTV1 and TTV2 PCR methods. Sow parity distribution and the number of delivered piglets (liveborn, stillborn and mummified) per each studied sow were recorded. TTV1 was detected in higher percentages than TTV2 in both sows (75% vs 43%, respectively) and piglets at 1 (17% vs 7%, respectively) and 3 (32% vs 12%, respectively) weeks of age. TTV1 and TTV2 co-infections were observed in higher percentages in sows (34%) than in piglets (2% and 4% at 1 and 3 weeks of age, respectively). Detection of swine TTV genogroups in sows was not associated with their detection in piglets. Moreover, there were piglets infected at 1 week of age with a swine TTV genogroup different from the one detected in their dam.

The number of sows delivering stillborns and the mean number of stillborns per sow tended to be higher in the TTV2 infected sows; this value was significantly higher when co-infected sows (TTV1 and TTV2) were compared with non-co-infected ones. Old parity sows had a higher percentage of TTV1 infected 1 week-old piglets. Results of the present study showed that the TTV infection occurs early in the production system and that these viruses may be transmitted from sow-to-piglet but also from piglet-to-piglet in farrowing facilities.

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

Introduction

Torque Teno virus (TTV) is a non-enveloped, circular, single-stranded DNA virus able to infect several vertebrate species, including human, swine, poultry, sheep, cattle, dogs and cats [START_REF] Kekarainen | Torque teno virus infection in the pig and its potential role as a model of human infection[END_REF].

In humans, several TTV genogroups have been described [START_REF] Peng | Analysis of the entire genomes of thirteen TT virus variants classifiable into the fourth and fifth genetic groups, isolated from viremic infants[END_REF]. The frequency of their detection in serum is very variable between countries and increases with age [START_REF] Saback | Age-specific prevalence and transmission of TT virus[END_REF]. Apart from serum, human TTV has been detected in saliva, nasal secretions and faeces, suggesting the faecal-oral as the main transmission route [START_REF] Maggi | TT Virus in the Nasal Secretions of Children with Acute Respiratory Diseases: Relations to Viremia and Disease Severity[END_REF][START_REF] Okamoto | Fecal excretion of a nonenveloped DNA virus (TTV) associated with posttransfusion non-A-G hepatitis[END_REF][START_REF] Deng | Higher prevalence and viral load of TT virus in saliva than in the corresponding serum: Another possible transmission route and replication site of TT virus[END_REF]. Nevertheless, detection of this virus in serum of mother-to-child pairs [START_REF] Kazi | High frequency of postnatal transmission of TT virus in infancy[END_REF], cord blood [START_REF] Matsubara | Existence of TT virus DNA and TTV-like mini virus DNA in infant cord blood: mother-to-neonatal transmission[END_REF] and in breast milk [START_REF] Schröter | Detection of TT virus DNA and GB virus type C/Hepatitis G virus RNA in serum and breast milk: determination of mother-to-child transmission[END_REF] indicates that TTV transmission may also occur vertically. To date, no definitive association between human TTV infection and a specific disease or pathology has been reported.

In swine, two TTV genogroups (TTV1 and TTV2) have been described so far [START_REF] Niel | Rolling-circle amplification of Torque teno virus (TTV) complete genomes from human and swine sera and identification of a novel swine TTV genogroup[END_REF][START_REF] Okamoto | Genomic characterization of TT viruses (TTVs) in pigs, cats and dogs and their relatedness with speciesspecific TTVs in primates and tupaias[END_REF]. Up to now, swine TTV has been detected in pigs from different countries [START_REF] Mckeown | Molecular characterization of porcine TT virus, an orphan virus, in pigs from six different countries[END_REF][START_REF] Bigarré | Pig anelloviruses are highly prevalent in swine herds in France[END_REF], ages [START_REF] Kekarainen | Detection of swine Torque teno virus genogroups 1 and 2 in boar sera and semen[END_REF][START_REF] Kekarainen | Prevalence of swine Torque teno virus in post-weaning multisystemic wasting syndrome (PMWS)-affected and non-PMWS-affected pigs in Spain[END_REF][START_REF] Martelli | Detection of Swine Torque Teno Virus in Italian Pig Herds[END_REF], sex [START_REF] Kekarainen | Prevalence of swine Torque teno virus in post-weaning multisystemic wasting syndrome (PMWS)-affected and non-PMWS-affected pigs in Spain[END_REF][START_REF] Segalés | Retrospective study on swine Torque teno virus genogroups 1 and 2 infection from 1985 to 2005 in Spain[END_REF] and production systems [START_REF] Martelli | Detection of Swine Torque Teno Virus in Italian Pig Herds[END_REF]. Like its human counterpart, swine TTV has not been clearly linked to any specific pathology. However, TTV2 has been more frequently found in animals suffering from postweaning multysistemic wasting syndrome (PMWS), a disease caused by PCV2 [START_REF] Segalés | Porcine circovirus diseases[END_REF], than in healthy pigs [START_REF] Kekarainen | Prevalence of swine Torque teno virus in post-weaning multisystemic wasting syndrome (PMWS)-affected and non-PMWS-affected pigs in Spain[END_REF].

At present, transmission routes of swine TTVs are still unknown. However, evidence of potential vertical transmission exists since both swine TTVs have been recently

A c c e p t e d M a n u s c r i p t 4 detected in colostrum samples and in sera of sows and their stillborns [START_REF] Martínez-Guinó | Evidence of Torque Teno virus (TTV) vertical transmission in swine[END_REF]. Moreover, swine TTVs have been detected in semen of boars suggesting that the sexual route may contribute to the viral dissemination [START_REF] Kekarainen | Detection of swine Torque teno virus genogroups 1 and 2 in boar sera and semen[END_REF].

The main objective of this work was to investigate the potential role of the sow in transmitting swine TTVs to piglets and the infection dynamics of TTV genogroups during the lactation period.

Materials and Methods

Animals and farm

Eleven batches from a total of 7 Spanish multi-site herds were investigated (Table 1). Four sows of different parity numbers (parities 1 to 10) per batch and 5 healthy piglets per sow were randomly selected and included in this study. A total of 44 sows and 215 piglets (from five of the sows, 4 piglet sera were available) were finally included in the study. Blood samples from cava vein (5 ml Venoject, Terumo Europe, Madrid, Spain) were taken from sows at 1 week post-farrowing and from piglets at 1 and 3 weeks of age. Data of delivered pigs (liveborn, stillborn and mummified) of the sows included in the study were recorded. Treatments, housing, and husbandry conditions conformed to the European Union Guidelines and Good Clinical Practices.

Swine TTV1 and TTV2 PCR methods

DNA was extracted from 200 µl of serum using a commercial kit (Nucleospin® Blood, Macherey-Nagel GmbH & Co KG Düren, Germany), according to manufacturer's instructions. Presence of TTV1 and TTV2 DNA was assessed with two previously described specific polymerase chain reaction (PCR) methods [START_REF] Segalés | Retrospective study on swine Torque teno virus genogroups 1 and 2 infection from 1985 to 2005 in Spain[END_REF]. To minimize the risk of contaminations, each stage of the PCR process (DNA The amplified products (305 bp for TTV1 and 250 bp for TTV2) were run in a 2% agarose gel with 0.05 mg/ml of ethidium bromide.

Statistical analyses

Sows were grouped by parity numbers into two different groups: young (from 1 st to 3 rd parity, n=21) and old (from 4 th to 10 th , n=23) sows. Bivariate analyses using contingency tables (Chi-square statistics or Fisher's exact test for 2x2 tables) were used to compare: 1) prevalence of TTV1 and/or TTV2 in serum of sows according to their parity group and the presence or absence of stillborns and 2) prevalence of both TTV genogroups in piglets at different ages and between piglets and sows. Moreover, a nonparametric statistical analyses (Kruskal-Wallis test) was used to test differences in the mean number of liveborn, stillborn and mummified piglets according to the TTV infection status of the sow. Statistical analyses were performed with the SAS system for Windows version 9.0 (SAS Institute Inc, Cary, North Carolina, USA). Statistical significance level was set at α=0.05.

Results

From the 44 sows analyzed, 33 (75%) and 19 (43%) were PCR positive to TTV1 and TTV2, respectively (Fig. 1). However, this difference was not statistically significant (p>0.05). While 15 (34%) out of the 44 sows were co-infected with both TTV genogroups, 7 (16%) sows were negative to both PCR methods. The number of sows that delivered stillborn piglets tended to be higher (p=0.051) among those TTV2 PCR positive sows than negative ones (Table 2). Moreover, this parameter was significantly higher (p=0.0479) among sows co-infected with both TTV genogroups A c c e p t e d M a n u s c r i p t 6 compared to non-co-infected sows. On the other hand, TTV2 PCR positive sows tended to have higher (p=0.0502) mean number of stillborns than the negative ones. For this parameter, similar results (p=0.0582) were obtained when comparing co-infected sows versus non-co-infected ones. In regards TTV1 PCR positive and negative sows, no significant differences were observed in both parameters (number of sows delivering stillborns and mean number of stillborns) (p>0.2). On the other hand, the mean number of liveborn and mummified piglets was independent of the TTV infection status of the sow (p>0.05).

In piglets, TTV1 was globally (at 1 and/or 3 weeks of age) detected in higher percentages (92/215, 43%) than TTV2 (41/215, 19%), although this difference was not significant (p>0.05) (Table 3). From the 215 piglets studied, 36 (17%) and 69 (32%) were positive to TTV1 at 1 and 3 weeks of age, respectively. From these 36 TTV1 PCR positive piglets at 1 week of age, only 13 were also positive at 3 weeks of age. On the contrary, from the 179 piglets TTV1 PCR negative at 1 week of age, 56 (31%) were positive at 3 weeks of age. Regarding TTV2, 15 (7%) and 26 (12%) out of the 215 piglets were positive at 1 and 3 weeks of age, respectively. None of the 15 piglets TTV2 PCR positive at 1 week of age was positive at 3 weeks of age. From the 200 piglets PCR negative to TTV2 at 1 week of age, 26 (13%) became positive two weeks later.

The rate of new infections at 3 weeks of age was significantly higher for TTV1 than for TTV2 (p<0.05). Co-infections with both genogroups were sporadic since only 3 (2%) and 8 (4%) animals were positive to both TTV PCRs at 1 and 3 weeks of age, respectively. On the contrary, 96 (45%) out of 215 piglets were negative to both TTV genogroups at both sampling times.

Detection of TTV1 and/or TTV2 in sows at 1 week post-farrowing was not statistically associated with virus detection in their piglets at 1 and/or 3 weeks of age 3). The percentage of infected pigs at 1 week of age coming from TTV1 PCR positive sows (26/163, 16%) was not different from the one coming from negative sows (10/52, 19%) (p>0.05). On the other hand, no significant differences (p>0.05) were observed between the percentage of 1 week-old TTV2 PCR positive piglets coming from TTV2 PCR positive (8/94, 8.5%) and negative (7/121, 6%) sows. Moreover, there were 5 out of the 36 (14%) TTV1 PCR positive piglets at 1 week of age that came from sows PCR negative to TTV1 but PCR positive to TTV2 (data not shown). Similarly, 4 out of the 15 (27%) TTV2 PCR positive piglets at 1 week of age came from sows PCR negative to this TTV genogroup but PCR positive to TTV1.

Swine TTV genogroups were detected in higher percentages in young parity sows (18/21 [86%] and 10/21 [47%] for TTV1 and TTV2, respectively) than in old parity sows (15/23 [65%] and 9/23 [39%] for TTV1 and TTV2, respectively. No association between parity number and TTV1 and/or TTV2 infection in sows was found (p>0.05).

Old sows had higher number of 1 week-old TTV1 PCR positive piglets (25/111,23%) than young ones (11/104, 11%) (p=0.01). On the contrary, at 3 weeks of age, the proportion of TTV1 PCR positive piglets from young sows (40/104, 39%) tend to be higher (p=0.052) than old ones (29/111, 26%). TTV2 infection in piglets was independent of the sow parity.

Discussion

Results of the present study confirm that TTV1 and TTV2 are able to infect sows and their suckling piglets. Detection of swine TTVs in sows is in agreement with two studies in which these viruses were retrospectively [START_REF] Segalés | Retrospective study on swine Torque teno virus genogroups 1 and 2 infection from 1985 to 2005 in Spain[END_REF] and contemporaneously [START_REF] Martínez-Guinó | Evidence of Torque Teno virus (TTV) vertical transmission in swine[END_REF] found in sows coming from several Spanish farms. Therefore, it seems that TTV1 and TTV2 infections are widespread in A c c e p t e d M a n u s c r i p t 8 the sow population, as it has been demonstrated in boars as well [START_REF] Kekarainen | Detection of swine Torque teno virus genogroups 1 and 2 in boar sera and semen[END_REF]. This latter point reinforces the notion that pig could serve as an animal model for human TTV infection as has been suggested [START_REF] Kekarainen | Torque teno virus infection in the pig and its potential role as a model of human infection[END_REF], since the prevalence of human TTV infection increases with age, being high in adults [START_REF] Saback | Age-specific prevalence and transmission of TT virus[END_REF].

An interesting outcome of this study is the fact that the number of sows delivering stillborns and the mean number of stillborns per sow tended to be higher in the TTV2 infected sows; this value was significantly higher when co-infected sows (TTV1 and TTV2) were compared with non-co-infected ones. This result might indicate a potential role of these viruses in reproductive failure. It must be taken into account, however, that only 44 sows were analyzed. Therefore, such potential relationship with increased number of stillborns must be taken with caution and deserves further investigations, especially in regards potential co-infection with other well-known pathogens linked to reproductive failure. On the human side, although infection in pregnant women (ranging from 28 to 83%) and young babies have been described [START_REF] Kazi | High frequency of postnatal transmission of TT virus in infancy[END_REF][START_REF] Saback | Age-specific prevalence and transmission of TT virus[END_REF][START_REF] Schröter | Detection of TT virus DNA and GB virus type C/Hepatitis G virus RNA in serum and breast milk: determination of mother-to-child transmission[END_REF][START_REF] Sugiyama | Highly diverse TTV population in infants and their mothers[END_REF], no association between TTV infections and gestation disturbances has been reported so far.

Parity number of the sow was not associated with TTV1 or TTV2 infection in sows, as has been previously suggested [START_REF] Martínez-Guinó | Evidence of Torque Teno virus (TTV) vertical transmission in swine[END_REF]. Surprisingly, old sows (parity 4-10) had a higher percentage of 1 week-old TTV1 infected piglets. There is no clear explanation of this finding; it is assumed that the potential transmission from sow to piglet should be similar in both young and old sows, since their rates of infection were not significantly different. It can not be ruled out that such result might be a spurious effect due to sample.

A c c e p t e d M a n u s c r i p t 9 The higher prevalence of TTV1 than TTV2 in sows and piglets reported in this study differs from results obtained in two previous studies in which TTV2 was more prevalent than TTV1 [START_REF] Kekarainen | Prevalence of swine Torque teno virus in post-weaning multisystemic wasting syndrome (PMWS)-affected and non-PMWS-affected pigs in Spain[END_REF][START_REF] Segalés | Retrospective study on swine Torque teno virus genogroups 1 and 2 infection from 1985 to 2005 in Spain[END_REF]. These discrepancies are probably due to the variability of origin and animal ages of the samples analyzed.

Simultaneous detection of swine TTVs in sows and their newborn piglets (1 week of age) is suggestive of vertical transmission. However, swine TTV infection in piglets was independent of the TTV infection status of the sow. These apparently controversial results have been also found in their human counterpart [START_REF] Schröter | Detection of TT virus DNA and GB virus type C/Hepatitis G virus RNA in serum and breast milk: determination of mother-to-child transmission[END_REF][START_REF] Kazi | High frequency of postnatal transmission of TT virus in infancy[END_REF][START_REF] Lin | Early acquisition of TT virus in infants: possible minor role of maternal transmission[END_REF] Nevertheless, sow-to-piglet transmission is probably the most likely transmission route taking into account the existence of piglets already infected at 1 week of age. If the observed viral dissemination was related to transplacental, intrauterine, colostrum-feeding or by daily contact transmission was not elucidated in this study.

Apart from the potential sow-to-piglet transmission route, results of the present work also support the existence of piglet-to-piglet viral dissemination. This latter route of transmission could be supported by two indirect facts. Firstly, the existence of piglets negative at 1 week of age that were infected two weeks later. And secondly, the detection of piglets infected with a swine TTV genogroup different from the one detected in their mother. This latter situation has also been described in humans [START_REF] Lin | Early acquisition of TT virus in infants: possible minor role of maternal transmission[END_REF][START_REF] Sugiyama | Highly diverse TTV population in infants and their mothers[END_REF] and also in other swine ssDNA viruses such as PCV2 [START_REF] Grau-Roma | A proposal on porcine circovirus type 2 (PCV2) genotype definition and their relation with postweaning multisystemic wasting syndrome (PMWS) occurrence[END_REF]. Both facts would explain the existence of de novo infections and the apparently increasing frequency of swine TTV infection with age, as has been shown in humans [START_REF] Saback | Age-specific prevalence and transmission of TT virus[END_REF].

In conclusion, this study showed that swine TTV infection occurs at early stages of the production system and suggests that these viruses are transmitted from sow-to- A c c e p t e d M a n u s c r i p t

  amplification and electrophoresis) was carried out in separated rooms.

  horizontal transmission piglet-to-piglet in the farrowing units is also of importance.

Figure 1

 1 Figure 1

Figure

  Figure

Table 2

 2 Number of sows that delivered stillborns (percentages) and mean number of stillborns distributed according to the TTV infection status. P-values mean differences between PCR positive and negative sows for a given TTV genogroup. Letters in superscript

	means statistically significant differences (p<0.05)

Table 3

 3 TTV1 and TTV2 PCR results in piglets at 1 and 3 weeks of age distributed according TTV detection in sows. *PCR status of the piglets at 1 and 3 weeks of age (i.e., 1+3means positive PCR result at 1 week of age and negative at 3).

					Sows
				TTV1	TTV2	Total
				Positive (%) Negative (%) Positive (%) Negative (%)
				n=33	n=11	n=19	n=25	44
			1+3+	9 (5.5)	4 (7.7)	3 (3.2)	10 (8.3)	13 (6.0)
	Piglets*	TTV1 TTV2	1+3-1-3+ 1-3-Total 1+3+ 1+3-1-3+ 1-3-	17 (10.4) 48 (29.4) 89 (54.6) 163 (100) 0 (0) 12 (7.4) 23 (14.1) 128 (78.5)	6 (11.5) 8 (26.0) 34 (57.2) 52 (100) 0 (0) 3 (5.8) 3 (5.8) 46 (88.5)	11 (11.7) 25 (26.6) 55 (58.5) 94 (100) 0 (7.0) 8 (12.1) 10 (80.9) 76 (100)	12 (9.9) 31 (25.6) 68 (56.2) 123 (57.2) 23 (10.7) 56 (26.0) 121 (!00) 215 (100) 0 (0) 0 (0) 7 (5.8) 15 (7.0) 16 (13.2) 26 (12.1) 98 (81.0) 174 (80.9)
			Total	163 (100)	52 (100)	94 (100)	121 (100) 215 (100)

Acknowledgments

This work was funded by the grants AGL2006-02778/GAN, TRT2006-00018 and CONSOLIDER-PORCIVIR CSD2006-00007 from Spanish government.


Dr Jose L Blanco
email: jlblanco@vet.ucm.es

A c c e p t e d M a n u s c r i p t 2 ABSTRACT Clostridium difficile is considered to be an important causative agent of porcine neonatal diarrhoea, having taken over from classic bacterial pathogens. However, there are currently no clear data concerning the prevalence of this microorganism in piglets, or about its relative distributions among diarrhoeic and non-diarrhoeic animals. In the present study, we analyzed the presence of C. difficile in rectal swabs from 780 piglets from two age groups (newborn and 1-2-month-old pigs) by means of molecular and microbiological procedures. Furthermore DNA was isolated from the bacteria in order to identify toxin A and toxin B genes.

C. difficile was not found in any of the 239 samples taken from 1-to 2-month-old pigs.

Bacteria were, however, recovered from 140 out of 541 newborn piglets (25.9%), including both diarrhoeic and non-diarrhoeic animals, and animals from control farms (free of diarrhoeic animals). Genes for the production of both toxins A and B were identified in 132 of the 140 isolates (A + B + ). Only seven isolates, all from the same farm and from non-diarrhoeic animals, lacked both toxin genes (A -B -), while one isolate from this same group of animals was A -B + .

This study provides the first report comparing the prevalence of C. difficile in large numbers of diarrhoeic and non-diarrhoeic animals. There was no clear link between bacterial isolation and neonatal porcine diarrhoea.

Key words: Clostridium difficile, prevalence, pig, diarrhoea, toxin.

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

Introduction

Clostridium difficile is an ubiquitous bacterium in the environment and has been recognised as an important emerging pathogen in both humans and animals. C. difficile is also the most important cause of antimicrobial-associated and hospital-associated diarrhoea in humans. In recent years, an increase in the incidence of human C. difficile infections has been reported, as well as an increase in the virulence and antimicrobial resistance of the isolates [START_REF] Pepin | Clostridium difficile-associated diarrhoea in a region of Quebec from 1991 to 2003: a changing pattern of disease severity[END_REF][START_REF] Mcdonald | An epidemic, toxin genevariant Strain of Clostridium difficile[END_REF][START_REF] Kuijper | ESCMID Study Group for Clostridium difficile; EU Member States; European Centre for Disease Prevention and Control[END_REF][START_REF] Jhung | Toxinotype V Clostridium difficile in humans and food animals[END_REF].

Veterinary medicine has highlighted the role of animals as reservoirs for C. difficile, as well as its zoonotic implications and its pathogenicity in different animal species, mainly in equids and swine [START_REF] Borriello | Household pets as a potential reservoir for Clostridium difficile infection[END_REF][START_REF] Arroyo | PCR ribotyping of Clostridium difficile isolates originating from human and animal sources[END_REF]Rodriguez-Palacios et al., 2006;[START_REF] Rupnik | Is Clostridium difficile-associated infection a potentially zoonotic and foodborne disease?[END_REF]. The importance of C. difficile as an agent involved in porcine neonatal diarrhoea has increased, displacing classic bacterial pathogens [START_REF] Songer | Infection of neonatal swine with Clostridium difficile[END_REF][START_REF] Yaeger | A survey of agents associated with neonatal diarrhea in Iowa swine including Clostridium difficile and porcine reproductive and respiratory syndrome virus[END_REF][START_REF] Songer | Clostridial enteric infections in pigs[END_REF]. However, some questions remain to be answered, such as the role of toxins in the pathogenesis of the microorganism and the susceptibility of swine to C. difficile colonization [START_REF] Waters | Typhlocolitis caused by Clostridium difficile in suckling piglets[END_REF][START_REF] Songer | The emergence of Clostridium difficile as a pathogen of food animals[END_REF][START_REF] Songer | Clostridium difficile: an important pathogen of food animals[END_REF].

In spite of the importance of C. difficile as a swine pathogen, there are currently no clear data concerning the prevalence of this microorganism in pig farms in relation to age, or its relative prevalence in animals with and without diarrhoea.

The aims of this study were to analyse the prevalence of C. difficile in two age groups of diarrhoeic and non-diarrhoeic pigs (newborn pigs (1-7 days old) and 1-2-month-old pigs), and to determine the presence of C. difficile toxin A and B genes in the isolates.

Samples

Rectal swabs were obtained from 780 animals: 541 from 1-to 7-day-old piglets (287 non-diarrhoeic and 254 diarrhoeic animals) and 239 from 1-to 2-month-old pigs (187 non-diarrhoeic and 52 diarrhoeic animals). Samples came from 13 pig farms located in three densely swine populated areas in Spain, located in three different Autonomous

Communities (zone A [Aragon], zone B [Catalonia] and zone C [Castile-La Mancha]).

A total of 13 herds were sampled: Ten had experienced continuous problems with porcine neonatal diarrhoea and, in some cases, they also had 1-2-month-old diarrhoeic animals. The remaining three herds were clinically free of diarrhoea in growing piglets at the time of sampling, and were then considered as controls.

A fresh stool sample was taken from each animal by introducing a sterile swab into the rectum. Swabs without transport media were stored at -20ºC in the herds until all samples of the herd were collected. Then they were transported in frozen conditions to the laboratory to be analysed.

Culture, isolation and identification of C. difficile

In order to select for bacterial spores, the tip of each rectal swab was submerged for 30 s in a 1.5-ml microtube containing 500 l of 70% ethanol. All the microtubes were incubated at room temperature for 20 min. After incubation, an aliquot of 75 l was C. difficile isolates were identified by colony morphology, Gram stain and the typical horse-manure odour of this microorganism. Their identification was confirmed using the API rapid ID 32 A system (BioMérieux), following the manufacturer's instructions.

DNA isolation and polymerase chain reaction (PCR) detection of C. difficile toxin A and toxin B genes

In order to isolate C. difficile DNA, a single colony was taken from a pure culture of each isolate and was suspended in 1 ml of sterile milli-Q water. Microtubes were incubated at 100ºC for 20 min to lyse the bacterial cells and then centrifuged for 2 min at 8000 g. Two hundred microliters of each supernatant were transferred to a new microtube and stored at -20ºC until used in PCR assays.

A modification of the PCR method, described by [START_REF] Kato | Identification of toxin A-negative, toxin B-positive Clostridium difficile by PCR[END_REF] The presence of the C. difficile toxin B gene (tcdB) was also detected by PCR. The reaction described by Fluit et al. (1991) was adapted to our laboratory conditions.

Briefly, 2 l of DNA extract was added to a PCR mixture that contained 1.5 mM MgCl 2 , 50 mM KCl, 10 mM Tris-HCl (pH 8.3), 100 M of each dNTP, 50 pmol of each primer (5'TAATAGAAAACAGTTAGAAA3' and 5'TCCAATCCAAACAAAATGTA3') and 2.5 U of AmpliTaq Gold DNA Polymerase in a final volume of 50 l. Amplifications were carried out under the following conditions: 5 min denaturation at 94ºC followed by 40 cycles of 1 min at 94ºC, 1 min at 50ºC and 1 min at 72ºC. Specific amplification products of 252 bp for the tcdA fragment and 301 bp for tcdB were visualized under UV illumination in a 1.6% agarose gel stained with ethidium bromide (Sigma-Aldrich, Madrid, Spain).

Analysis of results

A χ 2-test was used to compare the prevalence of C. difficile among the different production areas and to study the possible association between C. difficile isolation and the presence or absence of diarrhoea. We used statistical software (Statgraphics Plus 5.0

for Windows, Statistical Graphics Corp.) and applied Yates' correction when necessary.

P-values <0.05 were considered significant.

Results

A c c e p t e d M a n u s c r i p t 7

The results of C. difficile isolation from 1-to 7-day-old piglet faecal samples are shown in Table 1. In zone B was detected a higher prevalence of C. difficile in non-diarrhoeic animals, that was a statistically significant relationship (p<0.05). However, when the 13 farms were considered individually, a significant association between the variables was only found at farm B1 (p<0.05). In the other two zones, A and C, no association was found between C. difficile isolation and the presence or absence of diarrhoea (p>0.05, in both cases).

In the 1-7-day-old non-diarrhoeic piglets, a higher prevalence of C. difficile was seen in zone B than in zones A or C, or in the control farms (p<0.01). There were no statistically significant differences between zones A and C, or between these zones and the control farms. When the data for C. difficile prevalence in the different farms within each sampled zone were analysed, significant differences were only observed among the farms located in zone C (p<0.05). There were no significant differences in prevalence among the three control farms.

When a similar analysis was performed for the 1-7-day-old diarrhoeic piglets, statistically significant differences were only found between zones B and C (p<0.01).

Both tcdA and tcdB genes were amplified by PCR (A + B + isolates) in 132 out of the 140 (94.3%) C. difficile isolates. Only seven isolates, all from non-diarrhoeic piglets from farm B3, showed no amplification of either of these genes (A -B -isolates). In one isolate, C. difficile was not isolated from any of the 239 samples taken from 1-to 2-month-old pigs, regardless of the presence or absence of diarrhoea. Of these 239 samples, 187 were from non-diarrhoeic pigs and 52 were from diarrhoeic animals.

Discussion

In the present work, C. difficile was not isolated from any 1-2-month-old pig. However, this bacterium was recovered from a considerable proportion of 1-7-day-old piglets, regardless of the presence or absence of diarrhoea. Moreover, differences in C. difficile prevalence in piglets were observed among the three production areas sampled, though significant differences were only found between zone B and other zones, or the control farms.

It has been suggested that C. difficile may be currently the most important uncontrolled cause of neonatal diarrhoea in pigs [START_REF] Songer | The emergence of Clostridium difficile as a pathogen of food animals[END_REF][START_REF] Songer | Clostridium difficile: an important pathogen of food animals[END_REF], having displaced other bacterial and viral agents as the main cause of this disease [START_REF] Yaeger | A survey of agents associated with neonatal diarrhea in Iowa swine including Clostridium difficile and porcine reproductive and respiratory syndrome virus[END_REF]. Although C. difficile-associated infection affects mainly 1-7-day-old piglets [START_REF] Songer | Infection of neonatal swine with Clostridium difficile[END_REF][START_REF] Songer | Clostridial enteric infections in pigs[END_REF]. Furthermore, outbreaks in adult pigs have also been described [START_REF] Kiss | A new periparturient disease in Eastern Europe, Clostridium difficile causes postparturient sow losses[END_REF].

The carrier rate of C. difficile seems to vary among asymptomatic individuals by species and, within the same species, depending on age and other population characteristics [START_REF] Keel | The comparative pathology of Clostridium difficileassociated disease[END_REF]. Our results confirm these observations.

A c c e p t e d M a n u s c r i p t 9 Although most C. difficile strains produce both toxin A and toxin B, some strains produce only toxin B, or no toxins at all [START_REF] Songer | Clostridial enteric infections in pigs[END_REF]. Both tcdA and tcdB genes that code for the essential virulence factors of C. difficile [START_REF] Songer | Infection of neonatal swine with Clostridium difficile[END_REF][START_REF] Keel | The comparative pathology of Clostridium difficileassociated disease[END_REF], were identified in most isolates recovered in the present study.

In contrast, A -B -and A -B + isolates were relatively rare in the sampled farms. The exception to this was farm B3, where 50% of the asymptomatic carriers of C. difficile were piglets colonized by A + B + strains, and the other 50% by A -B + or A -B -strains.

Previous studies in pigs have shown results similar to those demonstrated here. For example, the overall prevalence of C. difficile in piglets from 10 herds in North Carolina (USA) was 47.6%, and ranged from 0 to 97% across the herds [START_REF] Songer | The emergence of Clostridium difficile as a pathogen of food animals[END_REF]. Also in the United States, C. difficile toxins were detected in the colonic content of 29% of 1-7day-old piglets with neonatal diarrhoea [START_REF] Yaeger | A survey of agents associated with neonatal diarrhea in Iowa swine including Clostridium difficile and porcine reproductive and respiratory syndrome virus[END_REF]. Also, C. difficile toxins were detected in the colonic content of 23/29 (79%) apparently healthy piglets [START_REF] Yaeger | A prospective case control study evaluating the association between Clostridium difficile toxins in the colon on neonate swine and gross and microscopic lesions[END_REF].

Similar results have also been found in other animal species: for example, no association has been found between C. difficile isolation and canine diarrhoea, and no significant differences in the percentage of A + B + C. difficile isolates recovered from diarrhoeic and non-diarrhoeic dogs were detected [START_REF] Perrin | Intestinal carriage of Clostridium difficile in neonate dogs[END_REF][START_REF] Marks | Genotypic and phenotypic characterization of Clostridium perfringens and Clostridium difficile in diarrheic and healthy dogs[END_REF]. On the other hand, studies in calves detected a higher prevalence of C. difficile in faeces from healthy control animals than in faeces from calves with diarrhoea (Rodriguez-Palacios et al., 2006). Finally, C. difficile was isolated from approximately 30% of the faecal samples from healthy 0-13-day-old foals, but not from foals 1-6 months of age [START_REF] Båverud | Clostridium difficile: prevalence in horses and environment, and antimicrobial susceptibility[END_REF]. However, in this last case, only 35.7% of the isolates recovered from the 0-13-day-old foals were positive for toxin A and toxin B by PCR [START_REF] Båverud | Clostridium difficile: prevalence in horses and environment, and antimicrobial susceptibility[END_REF]. [START_REF] Bartlett | The New Clostridium difficile -What Does It Mean?[END_REF]. In domestic animals this association is less clear, as infection by this bacterium has been observed in animals not treated with antibiotics [START_REF] Waters | Typhlocolitis caused by Clostridium difficile in suckling piglets[END_REF][START_REF] Yaeger | A survey of agents associated with neonatal diarrhea in Iowa swine including Clostridium difficile and porcine reproductive and respiratory syndrome virus[END_REF]. Moreover, it has been suggested that other factors may contribute to the establishment of C. difficile infection, including diet, environmental conditions in some production farms, animal stress, and the disruption of the intestinal microenvironment by other pathogens [START_REF] Yaeger | A survey of agents associated with neonatal diarrhea in Iowa swine including Clostridium difficile and porcine reproductive and respiratory syndrome virus[END_REF][START_REF] Nagy | Neonatal piglet losses associated with Escherichia coli and Clostridium difficile in a Slovakian outdoor production unit[END_REF][START_REF] Kiss | A new periparturient disease in Eastern Europe, Clostridium difficile causes postparturient sow losses[END_REF].

Although our study did not cover the entire country, the three sampled areas account for approximately 50% of the Spanish piglet production (data from the Ministry of

1

  Prevalence of Clostridium difficile in diarrhoeic and non-diarrhoeic pigletsSergio Alvarez-Perez 1 , Jose L. Blanco 1* , Emilio Bouza 2 , Patricia Alba 1 , Xavier Gibert 3 , Jaime Maldonado 3 and Marta E. Garcia 1 1 Animal Health Department. Facultad de Veterinaria. UCM. 28040 Madrid. Spain. 2 Division of Clinical Microbiology and Infectious Diseases. Hospital General Universitario Gregorio Marañón. 28007 Madrid. Spain. 3 Laboratorios Hipra S.A. Avda de la Selva 135. 17170 Amer. Girona. Spain.

  tube and inoculated on selective medium (Clostridium difficile agar plate; BioMérieux, Marcy l'Etoile, France). The plates were incubated under anaerobic conditions for 48 h at 37ºC.

  was used to detect the non-repeating portion of the C. difficile toxin A gene (tcdA). Briefly, 3 l of DNA extract were added to a PCR mixture that contained 2.5 mM MgCl 2 , 50 mM KCl, 10 mM Tris-HCl (pH 8.3), 200 M of each dNTP, 5.5 pmol of primer NK2 (5'CCCAATAGAAGATTCAATATTAAGCTT3') (Isogen Life Science, Maarssen, GGAAGAAAAGAACTTCTGGCTCACTCAGGT3´) and 0.75 U of AmpliTaq Gold DNA Polymerase (Applied Biosystems, Madrid, Spain). The final reaction volume was 30 l. Amplifications were carried out in a GeneAmp PCR System 9700 (Applied A c c e p t e d M a n u s c r i p t 6 Biosystems, Foster City, USA) thermocycler and consisted of a denaturation step of 5 min at 95°C, followed by 35 cycles of 15 s at 95°C, 20 s at 50°C and 40 s at 72°C.

  non-diarrhoeic piglets from farm B3, tcdB but not tcdA was amplified by PCR (A -B + isolate).

  C. difficile infection is associated almost exclusively with antibiotic usage

  www.mapa.es/es/estadistica/pags/anuario/introduccion.htm), being Spain the second largest pig producer country in the European Union. The fact that zone B (Catalonia) (which is the main pig producing area in Spain) had the highest percentage of asymptomatic carrier piglets deserves further investigation. It is also necessary to clarify the relationship between the colonization and possible infection of the pig intestinal tract by C. difficile and the different factors affecting the microbial flora.In conclusion, this is the first report comparing the prevalence of C. difficile in large numbers of diarrhoeic and non-diarrhoeic pigs. No clear link between the isolation of this bacteria and neonatal porcine diarrhoea was identified, in agreement with results from other species, including humans.

  Table 1 also shows the results of the PCR detection of C. difficile tcdA and

	tcdB genes. C. difficile was recovered from 140 out of 541 (25.9%) rectal swabs taken
	from 1-to 7-day-old piglets. Moreover, C. difficile was isolated from at least one non-
	diarrhoeic piglet from this age group from all the sampled farms, including the control
	farms. Conversely, at one farm (C2), C. difficile was not isolated from any piglet with
	diarrhoea.

Table 1 :

 1 Isolation of C. difficile from 1-to 7-day-old piglet faecal samples and PCR detection of tcdA and tcdB genes

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

Table 1 :

 1 Isolation of C. difficile from 1-to 7-day-old piglet faecal samples and PCR detection of tcdA and tcdB genes

	Non-diarrhoeic animals	Diarrhoeic animals

Acknowledgements

This work was partially supported by grant FAU2006-00005-C02 INIA from the Spanish Ministry of Education and Science. This work was partially supported by LABORATORIOS HIPRA, S.A. Sergio Álvarez-Pérez acknowledges a grant from the FPU programme (ref. AP 2005-1034), Spanish Ministry of Education and Science.


Kasper A Hettinga
email: kasper.hettinga@wur.nl

H J F Van Valenberg

T J G M Lam

A C M Van Hooijdonk

ORIGIN OF METABOLITES IN MASTITIS MILK The origin of the volatile metabolites found in mastitis milk

Keywords: mastitis, pathogens, metabolites, inoculation, artificial neural networks

The possibility to detect mastitis pathogens based on their volatile metabolites was previously studied. Because the origin of the metabolites is unknown, the formation of volatile metabolites by 5 mastitis pathogens inoculated in milk of healthy cows was studied.

The volatile metabolites from inoculated samples were compared to those of mastitis milk samples from which the inoculated pathogens were isolated. Most metabolites formed in the inoculated samples were similar to the metabolites formed in mastitis samples, both in presence and in amount. Prediction by a neural network showed that the similarity between the inoculated samples and mastitis samples was sufficient for correct prediction of the pathogen in the inoculated sample. The main difference between the inoculated samples and the mastitis samples was the absence of ethyl esters of free fatty acids in inoculated samples. This could be explained by disturbance of the milk-blood barrier, allowing the transfer of esterase from the cows' blood to the milk in cows with mastitis.

Introduction

Determination of mastitis causing pathogens is of great interest, both for choice of treatment as well as for herd level management measures to prevent the spread of mastitis.

Currently, determination of mastitis pathogens is generally done by bacteriological culturing [START_REF] Nmc | Microbiological procedures for use in the diagnostics of bovine udder infection and determination of milk quality[END_REF]. Previous research (Hettinga et al., 2008b) showed that results of a method for mastitis pathogen identification based on the analysis of volatile metabolites in milk samples were comparable with bacteriological culturing. The two main advantages of this method are the total analysis time and the cost of analysis. Specific volatile metabolites detected in mastitis milk samples are formed by the pathogen itself. For example, the formation of 2-and 3-methylbutanal, as well as branched free fatty acids by S. aureus have been described by [START_REF] Ritter | The application of gas chromato-graphy in dairying. II. Detection and determination of volatile fatty acids in dairy products and cultures[END_REF]. In mastitis samples with S. aureus, other, not previously described metabolites, like ethyl esters of free fatty acids, were also detected. Whether these components are formed by S. aureus or are the result of other processes occurring during mastitis is not known.

Detected metabolites may, for example, also be formed by enzymes which travel from the blood through the blood-milk barrier to the milk, or by enzymes associated with endothelial cells of the udder tissue. To determine whether the volatile metabolites originate from the pathogens or from other sources, inoculation of milk samples from healthy cows can be used.

The goal of this study was to compare the volatile metabolites from mastitis samples with those from inoculated samples containing the same pathogen, to get insight in the mechanism A c c e p t e d M a n u s c r i p t 3 of volatile metabolite formation, and to evaluate whether inoculated samples can be used as a model for mastitis samples in studying volatile metabolites.

Materials and methods

Milk samples

Mastitis samples

The clinical mastitis samples in this study were the same as described in our previous publication (Hettinga et al., 2008b). If one of the 5 pathogens of interest (S. aureus, CNS, S. uberis, S. dysgalactiae, or E. coli) was cultured, the remainder of the milk sample (approx. 5 mL) was frozen at -20°C for later analysis of the volatile metabolites. Also, the pathogen cultured from the mastitis samples was frozen at -80°C for the inoculation procedure. Bacteriological culturing was carried out according to NMC protocols [START_REF] Nmc | Microbiological procedures for use in the diagnostics of bovine udder infection and determination of milk quality[END_REF]. Because these were milk samples from routine mastitis diagnosis, the milk samples have been incubated overnight at 37°C.

Inoculated samples

Fifteen milk samples from 3 cows (5 samples/cow), without clinical signs of mastitis and with low SCC (<75,000/ml cow SCC), were supplied by the "De Ossekampen", the university farm of Wageningen University and Research Centre (Wageningen, The Netherlands). After disinfection of the teat and forestriping, 10 mL foremilk samples were taken into sterile 12 mL tubes. Directly after taking these samples, they were transported to the laboratory and inoculated, in the same tube, with one of the five pathogens cultured from the mastitis samples (3 samples per pathogen). Inoculation was performed using BHI broth (CM0225, The inoculated samples were incubated at 37°C for 8 h. After incubation, the samples were cooled on ice to approximately 0°C. Five mL of the sample was subsequently transferred to a headspace vial. The headspace vials were frozen at -20°C for later analysis of the volatile metabolites.

Analysis of volatile metabolites

The volatiles were analyzed using solid-phase microextraction (SPME) combined with gas chromatography/mass spectrometry (GC/MS) according to Hettinga et al. (2008b).

Statistical analysis

SPSS for Windows version 12.0 (SPSS Inc., Chicago, IL, USA) was used for statistical analysis. An ANOVA was performed for comparison of inoculated and mastitis samples, with a Welch test in the case of unequal variances. P<0.05 was considered statistically significant.

The obtained data were used in the prediction module of NeuralTools (Palisade, Ithaca, NY, USA) using the previously developed probabilistic neural network (PNN) to identify the mastitis causing pathogen (Hettinga et al., 2008b).

Results

Analysis of volatile metabolites

The results of the analysis of the mastitis samples have been described elsewhere (Hettinga et al., 2008b). The results of the inoculated samples were compared with the mastitis samples per pathogen.

Comparison of volatile metabolites per pathogen

Staphylococcus aureus

When comparing the data of the inoculated samples with the mastitis samples, especially the group of ethyl esters of the free fatty acids stands out. Four of the ethyl esters were not found at all in the inoculated samples, only ethyl acetate was detected in one of the samples.

Another difference between the inoculated and mastitis samples is that the samples inoculated with S. aureus contained a higher amount of dimethyl sulfide. Finally, hexanoic acid was found in the inoculated samples but not in the mastitis samples.

CNS

Just like S. aureus, samples inoculated with CNS contained hexanoic acid, whereas the mastitis samples did not. Additionally, the concentration of dimethyl sulfide and 2-butanone were significantly higher for the inoculated samples compared to the mastitis samples. The inoculated samples did not contain isopentanol, acetoin, and ethyl esters of branched fatty acids whereas some of the mastitis samples did contain these components. These differences were, however, not significant.

Streptococcus uberis

The absence of the ethyl esters and the presence of hexanoic acid in the inoculated samples compared to the mastitis samples was similar to the other pathogens. Additionally, 2methylbutanal and acetoin were absent in the inoculated samples. Furthermore, the inoculated A c c e p t e d M a n u s c r i p t 6 samples contained a higher concentration of dimethyl sulfide compared to the mastitis samples.

Streptococcus dysgalactiae

In the inoculated samples, the ethyl esters, 2-methylbutanal, isopentanol, and 3methylbutanoic acid were all absent. Hexanoic acid was present in all inoculated samples, but not in the mastitis samples. Like the other inoculated samples, samples inoculated with S. dysgalactiae contained a higher concentration of dimethyl sulfide compared to mastitis samples.

E. coli

The samples inoculated with E. coli, like the inoculated S. aureus samples, had very limited formation of ethyl esters of free fatty acids. As for other pathogens, the presence of hexanoic acid in inoculated samples was also seen in the samples inoculated with E. coli. In addition to these two differences, a 100-fold increase in dimethyl sulfide was seen for the inoculated samples. This increase was much higher than for the other pathogens.

Prediction by PNN

The results of the inoculated samples were used in the prediction module of the previously developed PNN. Of the 15 inoculated samples, 14 were correctly classified (93%). Only 1 of the E. coli samples was incorrectly classified.

Discussion

Comparing the mastitis samples to the inoculated samples, the majority of the volatile metabolites were present in both types of samples in similar amounts. The prediction of the A c c e p t e d M a n u s c r i p t 7 PNN showed that the similarity between the inoculated samples and mastitis samples was sufficient for correct prediction of the pathogen in the inoculated sample.

Looking at the 20% of metabolites which were different between the inoculated samples and mastitis samples, some differences were seen for all 5 pathogens. The main difference was that in inoculated samples, ethyl esters of free fatty acids were absent, or significantly lower. This may be explained by the activity of the esterase enzyme. Esterases are enzymes which are produced by the cow and occur in the blood [START_REF] Mcsweeney | Biochemical pathways for the production of flavour compounds in cheeses during ripening: a review[END_REF]. In case of mastitis, this enzyme can probably transfer from the blood to the milk due to disturbance of the blood-milk barrier [START_REF] Raulo | Increase in milk metalloproteinase activity and vascular permeability in bovine endotoxin-induced and naturally occurring Escherichia coli mastitis[END_REF]. These enzymes would then not be present in the milk of healthy cows, thus explaining the absence of the ethyl esters in inoculated samples.

Hexanoic acid was detected in all inoculated mastitis samples, but not in any of the 50 mastitis samples. However, in most of the mastitis samples, ethyl hexanoate was detected.

Hexanoic acid is a precursor for the formation of ethyl hexanoate. Possibly, the hexanoic acid in the mastitis samples was converted by esterase to ethyl hexanoate.

In the inoculated samples, a higher dimethyl sulfide concentration was found compared to the mastitis samples. The relative amount of dimethyl sulfide found in all inoculated samples (6×10 5 to 1×10 6 arbitrary units of area), except for the sample inoculated with E. coli, was similar to what was found in the milk of healthy cows (Hettinga et al., 2008a). The observed difference between inoculated samples and mastitis samples may be due to different sample handling, because dimethyl sulfide is easily lost due to its high volatility [START_REF] Patton | Methyl sulfide and the flavor of milk[END_REF]. This does, however, not explain the high concentration of dimethyl sulfide in the samples inoculated with E. coli. Dimethyl sulfide most likely is a metabolite formed by E. coli and was also probably lost in the mastitis samples due to handling. The formation of dimethyl sulfide by E. coli may be useful for the identification of E. coli in mastitis samples. In some of the samples, especially those inoculated with CNS and S. dysgalactiae, an increased concentration of 2-butanone was detected. However, the concentration of 2butanone still falls within the normal range of this component for fresh raw milk (Hettinga et al., 2008a).

Conclusions

When comparing the whole pattern of volatile metabolites, it can be concluded that most metabolites were present in both inoculated as well as mastitis samples, in similar concentrations. The prediction of the PNN showed that the similarity between the inoculated samples and mastitis samples was sufficient for correct prediction of the pathogen in inoculated samples (93% correct).

The absence of ethyl esters was the most striking difference between the two types of samples, and this could be explained by the absence of transfer of the esterase enzyme from the cow's blood to the milk of a healthy cow.

Therefore, it can be concluded that the metabolites found in mastitis samples are mainly formed by the pathogens and that the ethyl esters of free fatty acids are, probably, formed by the esterase enzyme from cow's blood.

Finally, because of the similarities between inoculated and mastitis samples, inoculation can be used to obtain samples for further research on the use of volatile metabolites in mastitis diagnosis.

  , UK) containing 10 9 CFU/mL, diluted to a final inoculation level of 10 5 CFU/mL in the milk sample.

  A c c e p t e d M a n u s c r i p t 5 Table5.1 contains an overview of the results of the comparison between the mastitis samples and the inoculated samples.

  Acree, T. E. and H. Arn. Website: http://www.flavornet.org/. Accessed: 20 Oct., 2008.

Table 1 :

 1 Amount (arbitrary units of area) of volatile metabolites detected in mastitis samples and inoculated samples containing S. aureus, 243 coagulase-negative staphylococci (CNS), S. uberis, S. dysgalactiae, and E. coli (n=10 for mastitis samples, n=3 for inoculated samples).

	244
		S. aureus	CNS	S. uberis	S. dysgalactiae	E. coli
	245	Metabolite Acetaldehyde Ethanol Dimethyl sulfide 2,3-Butadione 2-Butanone Eth. acetate 3-Meth.butanal 2-Meth.butanal 2-Pentanone Acetic acid Isopentanol Acetoin Eth. butyrate Butyric acid Eth. 2-meth.but. Eth. 3-meth.but. 3-Meth.but. 2-Meth.but. 2-Heptanone Hexanoic acid Eth. hexanoate a significant difference between mastitis samples and inoculated sample (p<0.05) Mastitis Inoculated Mastitis Inoculated Mastitis Inoculated Mastitis Inoculated Mastitis Inoculated 7×10 6 2×10 7 6×10 4 1×10 5 3×10 5 1×10 5 1×10 5 1×10 5 3×10 5 2×10 5 1×10 8 3×10 7 1×10 5 4×10 4 1×10 6 0 6×10 6 0 1×10 8 4×10 7 9×10 4 1×10 6 a 3×10 4 6×10 5 a 2×10 5 9×10 5 a 2×10 5 8×10 5 a 1×10 5 2×10 7 a 1×10 6 3×10 6 5×10 4 3×10 3 6×10 5 1×10 4 1×10 5 4×10 3 2×10 4 4×10 5 a 2×10 6 3×10 6 3×10 5 3×10 6 a 1×10 6 3×10 6 7×10 5 5×10 6 a 5×10 5 4×10 6 1×10 6 4×10 4 a 0 0 5×10 4 0 a 2×10 4 0 a 7×10 5 4×10 5 3×10 7 8×10 7 3×10 6 2×10 6 9×10 4 8×10 5 5×10 5 5×10 5 5×10 4 3×10 5 4×10 5 2×10 6 6×10 4 6×10 4 3×10 3 0 3×10 3 0 0 0 8×10 4 1×10 5 8×10 3 4×10 4 5×10 4 1×10 5 7×10 4 7×10 4 1×10 6 6×10 4 6×10 6 4×10 7 5×10 5 2×10 6 3×10 6 2×10 6 3×10 6 3×10 6 1×10 7 9×10 6 7×10 6 2×10 6 1×10 6 0 0 0 1×10 5 0 4×10 6 0 8×10 5 5×10 5 3×10 3 0 4×10 3 0 0 0 0 0 7×10 5 0 a 0 0 7×10 4 0 a 1×10 5 0 a 3×10 5 2×10 4 3×10 6 4×10 5 2×10 5 4×10 5 8×10 5 3×10 5 1×10 6 4×10 5 1×10 6 2×10 6 2×10 4 0 a 4×10 2 0 0 0 0 0 0 0 1×10 5 0 a 5×10 2 0 3×10 3 0 3×10 3 0 0 0 1×10 6 4×10 4 0 0 0 0 7×10 2 0 0 0 6×10 6 1×10 5 0 0 0 0 0 0 0 0 2×10 5 4×10 4 2×10 4 4×10 4 1×10 5 3×10 4 1×10 5 4×10 4 3×10 5 5×10 4 0 3×10 5 a 0 2×10 5 a 0 1×10 5 a 0 2×10 5 a 0 3×10 6 a 4×10 5 0 a 8×10 2 0 6×10 4 0 9×10 4 0 2×10 5 0 a