Dr Bjoern Jacobsen
email: bjoern.jacobsen@tiho-hannover.de

Lars Krueger

Frank Seeliger

Michael Bruegmann

Joaquim Segalés

Wolfgang Baumgaertner

Retrospective study on the occurrence of porcine circovirus 2 infection and associated entities in Northern Germany

Keywords: Porcine circovirus type 2, in situ hybridisation, retrospective study, postweaning multisystemic wasting syndrome, porcine dermatitis and nephropathy syndrome

Porcine circovirus type 2 (PCV 2) represents a widespread, globally occurring pathogen with an increasing number of associated entities. To further elucidate the origin, spread and pathogenesis of PCV2 and associated changes archived material of pigs originating from Northern Germany and submitted for necropsy between 1961 to 1998 were investigated by using in situ hybridisation and polymerase chain reaction. PCV2 was first detected in a pig from 1962. However, incidence of detectable viral DNA and occurrence of PCV2-associated lesions varied substantially in the following years. The overall incidence of PCV2 infection was low between 1961 and 1984 (0 to 11.7 %) and increased between 1985 and 1998 (14.3 to 53.3 %). PCV2-associated pathological changes including postweaning multisystemic wasting syndrome (PMWS) and most likely porcine dermatitis and nephropathy syndrome (PDNS) were first observed in 1985. Selected sequence analyses of PCV2 DNA segments revealed high homology with current virus strains. In summary, findings showed that PCV2 has been present in the pig population in Northern Germany since 1962. This represents worldwide the earliest report about the detection of the PCV2 genome in pigs. Associated lesions such as PMWS and PDNS were not observed before 1985, indicating that virus infection alone does not seem to be sufficient enough to trigger the development of associated entities. Limited sequence analysis revealed no changes in the viral genome thus suggesting that other factors including environmental changes or co-infections with other agents might play a contributing role in the altered virulence of this pathogen and the occurrence of PCV2-associated lesions.

Introduction

Porcine circoviruses (PCV), members of the family Circoviridae, are composed of nonenveloped viruses with a single-stranded circular DNA (ICTVdB Management, 2006). Two genetically and antigenically different viruses, PCV1 and PCV2, can be distinguished. PCV1 was first described 1974 as a contaminant of a porcine kidney cell line (PK 15) and is regarded as being nonpathogenic [START_REF] Tischer | Studies on epidemiology and pathogenicity of porcine Circovirus[END_REF]. In 1991 a "postweaning multisystemic wasting syndrome" (PMWS) was reported in Canadian pigs [START_REF] Harding | Recognizing and diagnosing postweaning multisystemic wasting syndrome (PMWS)[END_REF]. PCV2 was isolated in association with PMWS and the Koch's postulates were fulfilled [START_REF] Ellis | Isolation of circovirus from lesions of pigs with postweaning multisystemic wasting syndrome[END_REF][START_REF] Ellis | Reproduction of lesions of postweaning multisystemic wasting syndrome in gnotobiotic piglets[END_REF]. In the late 1990s, reports of similar disease outbreaks in several countries including France and Spain followed and both diseases as well as PCV2 infection are now found worldwide [START_REF] Segalés | Porcine circovirus diseases[END_REF]. The first isolation of PCV2 in Germany in association with PMWS was reported in 1998 [START_REF] Hinrichs | First detection of porcine circovirus type 2 infection in Germany[END_REF]. PCV2 is nowadays highly prevalent in swine herds worldwide and infection is often subclinical [START_REF] Harding | The clinical expression and emergence of porcine circovirus 2[END_REF]. Recent studies indicate the presence of at least two PCV2 genotypes associated with different pathogenic potential [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]. However, PCV2 has also been associated with various clinical disease syndromes in swine including PMWS, porcine dermatitis and nephropathy syndrome (PDNS) as well as enteritis, proliferating and necrotising pneumonia (PNP), encephalopathy, congenital tremor and reproductive disorders [START_REF] Ellis | Reproduction of lesions of postweaning multisystemic wasting syndrome in gnotobiotic piglets[END_REF][START_REF] Harding | The clinical expression and emergence of porcine circovirus 2[END_REF][START_REF] Segalés | Pathological findings associated with naturally acquired porcine circovirus type 2 associated disease[END_REF]. PMWS is characterised clinically by wasting, dyspnoea, and lymphadenopathy and maybe associated with diarrhoea, pallor, and jaundice [START_REF] Harding | The clinical expression and emergence of porcine circovirus 2[END_REF][START_REF] Rosell | Pathological, immunohistochemical, and in-situ hybridization studies of natural cases of postweaning multisystemic wasting syndrome (PMWS) in pigs[END_REF]. The most prominent histological lesions in PMWS occur in lymphoid organs and consist of extensive lymphocytic depletion, infiltration of macrophages, few multinucleated giant cells, and botryoid basophilic cytoplasmic inclusion bodies. PCV2 antigen and DNA has been demonstrated by immunohistochemistry and/or in situ hybridisation (ISH) in various organs in the cytoplasm of macrophages, multinucleated giant cells, dendritic cells in lymphoid tissues, alveolar A c c e p t e d M a n u s c r i p t 4 macrophages, Kupffer cells, and, to a lesser extent, epithelial cells in the lungs, liver, pancreas, and kidneys. Endothelial cells in several organs, including lymph nodes and spleen, are rarely positive for PCV2 [START_REF] Opriessnig | Cardiovascular lesions in pigs naturally or experimentally infected with porcine circovirus type 2[END_REF][START_REF] Rosell | Pathological, immunohistochemical, and in-situ hybridization studies of natural cases of postweaning multisystemic wasting syndrome (PMWS) in pigs[END_REF]. Although vascular lesions are not prominent in PMWS, vasculitis is the hallmark of PDNS [START_REF] Segalés | Pathological findings associated with naturally acquired porcine circovirus type 2 associated disease[END_REF].

Lesions in PDNS are characterised primarily by a cutaneous and subcutaneous necrotising vasculitis as well as glomerulonephritis and are likely to be mediated by immune complex depositions [START_REF] Sierra | Porcine immune complex glomerulonephritis dermatitis (PIGD) syndrome[END_REF]. The responsible antigen or antigens triggering this immune complex-mediated disorder is currently unknown. A wide spectrum of factors including drugs, chemicals, food allergens, endogenous antigens, and infectious agents must be considered [START_REF] Drolet | Porcine dermatitis and nephropathy syndrome (PDNS): an overview of the disease[END_REF]. At present, the role of infectious agents has been suspected more than other factors; among them, PCV2 and porcine respiratory and reproductive syndrome virus (PRRSV) are considered the most likely causative agents [START_REF] Rovira | Experimental Inoculation of Conventional Pigs with Porcine Reproductive and Respiratory Syndrome Virus and Porcine Circovirus 2[END_REF][START_REF] Segalés | Pathological findings associated with naturally acquired porcine circovirus type 2 associated disease[END_REF][START_REF] Thibault | Cutaneous and systemic necrotizing vasculitis in swine[END_REF]. However, to date PCV2 antigen has not been detected within PDNS vascular lesions [START_REF] Segalés | Pathological findings associated with naturally acquired porcine circovirus type 2 associated disease[END_REF].

The increasing reports of new PCV2 associated lesions and syndromes including enteritis, PNP, encephalopathy, cardiovascular lesions, reproductive disorders, and congenital tremor raised the question, whether these findings represent truly new emerging entities directly or indirectly associated with PCV2 infection [START_REF] Opriessnig | Cardiovascular lesions in pigs naturally or experimentally infected with porcine circovirus type 2[END_REF][START_REF] Seeliger | Porcine Circovirus Type 2-Associated Cerebellar Vasculitis in Postweaning Multisystemic Wasting Syndrome (PMWS)-Affected Pigs[END_REF][START_REF] Segalés | Pathological findings associated with naturally acquired porcine circovirus type 2 associated disease[END_REF].

The aim of this study was to determine the presence of PCV2 and associated lesions in archived paraffin-embedded material prior to the first report from 1998 in Northern Germany.

Material and methods

Case selection

Out of 18,855 necropsied pigs originating from Northern Germany between 1998, the year of the first description of PMWS in Germany, and 1961Germany, and (excluding the years 1987Germany, and -1990 A c c e p t e d M a n u s c r i p t 5 and 1992-1995), 445 animals, at least 10 pigs per year, were randomly selected for the present study. Material earlier than 1961 was unfortunately not available in the department archive.

The criteria for selection included age (between 4 and 16 weeks), weight (under 50 kg), clinical signs, as well as gross and histological findings such as lymph node enlargement, lymphocytic depletion in lymphoid tissues, interstitial pneumonia, and interstitial nephritis. In addition, the availability of formalin-fixed and paraffin-embedded tissues of the lymph nodes, spleen, ileum, lung or tonsil was a further requirement. However, not all tissues were available in each case.

Results of previous virological investigations including classical swine fever virus (CSFV) and porcine herpesvirus 1 (PHV-1, Aujeszky's disease) were included. Haematoxylin and eosin (HE) stained sections from all available organs were investigated for histopathological changes.

In situ hybridisation

DNA in situ hybridisation (ISH) to detect PCV2 and PCV1 DNA was carried out as described [START_REF] Rosell | Pathological, immunohistochemical, and in-situ hybridization studies of natural cases of postweaning multisystemic wasting syndrome (PMWS) in pigs[END_REF][START_REF] Seeliger | Porcine Circovirus Type 2-Associated Cerebellar Vasculitis in Postweaning Multisystemic Wasting Syndrome (PMWS)-Affected Pigs[END_REF]. Digoxigenin-labeled, oligonucleotide DNA-probes of 41 basepairs (bp) was designed based on the sequence of the PCV2 and PCV1 open reading frame 1 (ORF1; GenBank accession number AF027217 and NC001792, respectively; Tab. 1). Briefly, tissue sections were dewaxed in xylene, hydrated in graded ethanol and washed in ultrapure, pyrogen-free, DEPC-treated water. After proteolytic digestion, postfixation, acetylation, and prehybridisation, hybridisation was performed overnight in a moist chamber at 56°C with a probe concentration of 100 ng/ml. The detection system consisted of an anti-DIG antibody conjugated with alkaline phosphatase and the substrates nitroblue tetrazoliumchloride (NBT) and 5-Bromo-4-chloro-3-indolyl phosphate (BCIP, X-phosphate), which yielded a bluish precipitate. Formalin-fixed and paraffinembedded pellets from PK15 cells either infected or non-infected with PCV2 or PCV1 as well A c c e p t e d M a n u s c r i p t 6 as a PCV2 positive and non-infected juvenile pig served as positive and negative controls [START_REF] Seeliger | Porcine Circovirus Type 2-Associated Cerebellar Vasculitis in Postweaning Multisystemic Wasting Syndrome (PMWS)-Affected Pigs[END_REF].

Polymerase chain reaction and sequencing

For detecting the PCV2 genome, DNA was isolated from formalin-fixed and paraffinembedded tissue using E.Z.N.A® Tissue DNA Mini Kit (PeqLab Biotechnology, Germany) according to the manufacturer's instructions. Additionally, polymerase chain reaction (PCR) using three different primer pairs was performed as described [START_REF] Ellis | Reproduction of lesions of postweaning multisystemic wasting syndrome in gnotobiotic piglets[END_REF][START_REF] Kim | Differentiation of porcine circovirus 1 and 2 in formalin-fixed, paraffin-wax-embedded tissues from pigs with postweaning multisystemic wasting syndrome by in-situ hybridization[END_REF]. The first two primer pairs targeted amplicons of 481 bp and 225 bp length of the ORF2 and the third pair generated an amplicon of an expected length of 66 bp localised within the ORF1 (Tab. 1). For the latter an optimised protocol with more stringent conditions was applied to exclude PCV1 detection. Hereby, an initial denaturation step for 4 minutes at 94°C, followed by 39 cycles denaturation at 94°C for 1 minute, annealing at 56°C for 50 seconds, and elongation at 72°C for 30 seconds was used. The reaction ended with a final extension step at 72°C for 10 minutes. Amplicons were visualised by standard gel electrophoresis on a 2% agarose gel. Positive DNA fragments of specific size were cut out from the gel, purified by using a commercial kit (Nucleobond, Macherey & Nagel, Germany) and submitted for sequencing. The latter was performed according to the method of Sanger with fluorescent-labelled BigDye Terminator sequencing kit using automatic sequencers (Applied Biosystems, USA) by an external laboratory (SEQLAB, Germany). Sequences were analysed with LAGLIN online version (http://www.ch.embnet.org) to confirm the identity of the obtained sequences. The CLUSTAL W online version (http://www.ebi.ac.uk/clustalw/index.html) for comparative alignments was used and data were submitted to DDBJ/EMBL/GenBank database (GenBank accession numbers EU158773, EU158774, EU158775). Formalin-fixed and paraffin-embedded pellets from PK15 cells A c c e p t e d M a n u s c r i p t 7 either infected or non-infected with PCV2 or PCV1 as well as a PCV2 positive and noninfected juvenile pig served as positive and negative controls [START_REF] Seeliger | Porcine Circovirus Type 2-Associated Cerebellar Vasculitis in Postweaning Multisystemic Wasting Syndrome (PMWS)-Affected Pigs[END_REF].

Statistical analysis for a correlation between detection of PCV2 DNA and histological lesions was performed using Fisher's exact test with significance at p≤ 0.05.

Results

Case selection

The selection criteria, met by 445 randomly selected animals, resulted in the investigation of about 10 to 21 pigs per year (on average 15) with a mean body weight of 21.9 kg (ranging from 2.5 to 50 kg) and a mean age of 9.7 weeks (ranging from 4 to 16 weeks). However, not all organs were available for each case (lung: n = 305; liver: n = 157; lymph node: n = 138; spleen: n = 103; intestine: n = 102; kidney: n = 89; brain: n = 49; heart: n = 29; tonsils: n = 2).

In situ hybridisation

Among these 445 pigs, 37 revealed positive hybridisation signals for PCV2 in at least one organ. The earliest detection of PCV2 in tissue was in the year 1962 with a few positive cells being detected in the spleen and lung tissue (Fig. 1) followed by a positive animal from the year 1964. Only 2.77 % (ranging from 0 to 11.7 %) of the animals were positive for PCV2 (10 of 361 pigs) between 1961 and 1984. Surprisingly, in the years 1985 to 1998 the percentage of infected animals increased (27 out of 84 pigs, 32.14 %, ranging from 14.3 % to 53.3 %) (Fig. 2). No animal showed positive hybridisation signals with the PCV1-specific probe. PK15 cells infected with PCV1 and PCV2, non-infected cells and various tissues from control pigs showed positive and negative results as expected.

Hybridisation signals were most frequently found within the lung (24 out of 305; 7.87 %), followed by the intestine (13 out of 102; 12.75 %), lymph node (12 out of 138; 3.82 %), heart (3 out of 29; 10.34 %), brain (3 out of 49; 6.12 %) and tonsil (1 out of 2; 50 %). PCV2 DNA was found most frequently in the lymph node (12 out of 12; 100 %), heart (3 out of 3; 100 %), lung (24 out of 29; 82.7 %), intestine (11 out of 14; 78.6 %) and spleen (8 out of 11; 72.7 %). Overall, fewer PCV2 positive organs were found between 1961 and 1984 compared to 1985 to 1998 (Fig. 3). The signals were found predominantly within macrophages varying from single to numerous positive cells. Other cells with positive hybridisation signals include alveolar macrophages, endothelial cells of blood vessels, fibroblasts and -cytes, Kupffer cells, hepatocytes, and tubular epithelial cells of the kidney.

Histology

Microscopically, PCV2 positive as well as PCV2 negative animals exhibited a variety of pathological lesions including lymphohistiocytic interstitial pneumonia, suppurative bronchopneumonia, lymphocytic depletion in the spleen and lymph nodes, infiltration of lymph nodes with histiocytes, lympho-histiocytic interstitial hepatitis and nephritis as well as necrotising and fibrinous glomerulonephritis and necrotising dermatitis.

Fisher's exact test revealed that interstitial pneumonia (rightsided-pvalues (p) = 0.0037), hepatitis (p < 0.0001) and nephritis (p < 0.0001), infiltration of lymph nodes with histiocytes (p = 0.0128), and necrotising and fibrinous glomerulonephritis (p = 0.007) were found significantly more frequently in PCV2-infected pigs (p ≤ 0.05 = significant). Only interstitial hepatitis and nephritis were significantly associated with PCV2 infection prior to 1985 (Fig. 4). However, no virus was detectable in the lesions within these kidneys, though some animals exhibited PCV2 DNA intralesionally in the liver.

Other PCV2 associated lesions as described for PDNS or PMWS were not found in pigs positive for PCV2 prior to 1985. In contrast, lymphocytic depletion, histiocytic infiltration, cytoplasmic inclusion bodies, multinucleated giant cells and interstitial pneumonia were Additionally, three animals exhibited lesions resembling PDNS showing ulcerative dermatitis with vasculitis, and fibrinous and necrotising glomerulonephritis (Fig. 6). Though only few or no cells were positive for PCV2 within the lesions, viral DNA was detected within the lymph nodes, lungs and spleen of these animals. The first animal displaying such alterations also originated from 1985 and the other two from the year 1998.

Polymerase chain reaction and sequencing

In order to substantiate the ISH findings, PCR for PCV2 and PCV1 with different primer pairs was performed. The PCR specific for PCV2 revealed 481 and 225 bp long amplicons in pigs from the year 1985 onwards. Unfortunately, this PCR design failed to confirm the ISH results prior to the year 1985 probably due to storage-related DNA damage.

However, using a third primer pair generating a short 66 bp long amplicon the ISH results could be confirmed including the case from 1962. Sequence analysis of the amplicons of the isolates from 1962 (GenBank accession number EU158774) and 1967 (GenBank accession number EU158775) revealed 98 % and 100 % homology to current PCV2 isolates (GenBank accession number AF027217), respectively. Additionally, sequence analysis of the amplicon of the isolate from 1985 (GenBank accession number EU 158773) revealed 100 % identity to current PCV2 isolates (GenBank accession number AY325515).

Additional results

Two of the 37 PCV2 positive pigs exhibited a concurrent infection with the classical swine fever virus (CSFV). In one animal from the year 1967 the diagnosis was confirmed by immunofluorescence testing and in the other animal from the year 1985 by virus isolation.

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

The animal from the year 1967 exhibited only mild lesions characterised by infiltration of plasma cells and eosinophilic granulocytes in the lamina propria of the intestine, a mild lympho-histiocytic periportal infiltration in the liver and a mild infiltration with neutrophilic granulocytes in the lymph node. In the latter a mild to moderate amount of PCV2 positive macrophages was detected. The animal from the year 1985 showed a mild vasculitis in the leptomeninx and the spleen, severe lymphocytic depletion in the spleen and lymph node, mild interstitial nephritis and severe necrotising lymphadenitis with PCV2 characteristic cytoplasmic basophilic inclusion bodies. PCV2 was detectable in all organs predominantly in macrophages with numerous positive cells in spleen and lymph node (Fig. 5). This animal represented the first pig with an unusual high PCV2 DNA load at that time point. Similar amounts were only found in pigs of the late 1990s. No animal was infected with PHV-1.

Discussion

This retrospective study on the occurrence of PCV2 and associated lesions revealed three significant findings: (i) PCV2 infection has been present in the swine population in Northern Germany at least since 1962, (ii) single cases of PMWS and PDNS had already been observed in 1985, and (iii) associated lesions were not detectable prior to 1985. Though PCR, using various primer pairs, confirmed PCV2 infection by demonstrating a 481 and a 225 bp long amplicon of the ORF2 in the samples collected between 1985 and 1998, the same protocol failed to confirm the results of the ISH prior to the year 1985. This may be due to a time-dependent increased DNA-degradation in formalin-fixed and paraffin-embedded tissues [START_REF] Grierson | Detection and genetic typing of type 2 porcine circoviruses in archived pig tissues from the UK[END_REF][START_REF] Lisowski | Effect of the storage period of paraffin sections on the detection of mRNAs by in situ hybridization[END_REF]. Though the PCR with the primer pair for the amplification of a 66 bp long amplicon of the ORF1 was successful, the obtained amplicon could have also originated from PCV1. The primers possess 100% and 80% identity to the PCV1 genome, respectively. The specificity of the PCR for PCV2 was ensured by changing the annealing temperature allowing the detection of PCV2 but not of PCV1 in infected cells A c c e p t e d M a n u s c r i p t 11 [START_REF] Ishii | Optimization of annealing temperature to reduce bias caused by a primer mismatch in multitemplate PCR[END_REF]. The obtained sequences displayed a 98-100 % homology to other PCV2 strains. However, obtained sequences were too short for a concluding statement whether differences in tissue distribution and pathological findings are related to different PCV 2 strains obtained in various decades. Recent studies revealed two PCV 2 genotypes based on differences in the ORF2 with PCV2 genotype 1 possibly being more pathogenic than PCV2 genotype 2 [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]. In addition, a Danish study showed a temporally genotypic shift of PCV2 within a pig population from a third PCV2 genotype, termed PCV 2 genotype 3, via the known PCV2 genotype 2 towards PCV2 genotype 1 [START_REF] Dupont | Genomic analysis of PCV2 isolates from Danish archives and a current PMWS case-control study supports a shift in genotypes with time[END_REF]. Unfortunately, in the present study used primers were not able to detect PCV2 within tissues obtained prior to 1985. Therefore, possible differences with respect to different PCV 2 genotypes could not be investigated. The PCV2 sequence from a pig from 1985 displayed a sequence similar to PCV2 genotype 2 (primer pair p285/p286; GenBank accession number EU 158773).

The year 1962 represented the earliest confirmed detection of PCV 2 worldwide.

Although tissue samples collected before 1961 were not available, it seems reasonable to assume that PCV2 was already present in the pig population prior to this time point.

In previous investigations the earliest detection of PCV2 in archived formalin-fixed material originated from the 1970s in the UK using TaqMan®-PCR and immunohistochemistry; however, no information was provided as to whether tissues displayed histological lesions [START_REF] Grierson | Detection and genetic typing of type 2 porcine circoviruses in archived pig tissues from the UK[END_REF]. In Swiss archived material the earliest PCV2 infection was found in 1986 by using immunohistochemistry with the earliest histopathological lesions typical for PMWS in 1986 [START_REF] Staebler | PMWS: an emerging disease identified in archived porcine tissues[END_REF]. Similarly, a study from Spain revealed the presence of PCV2 in archived material from 1985 onwards, and the occurrence of typical PMWS lesions as early as 1986 [START_REF] Rodriguez-Arrioja | Retrospective study on porcine circovirus type 2 infection in pigs from 1985 to 1997 in Spain[END_REF].

The first animal showing light microscopic characteristics of PMWS originated from

A c c e p t e d M a n u s c r i p t 12 Germany [START_REF] Hinrichs | First detection of porcine circovirus type 2 infection in Germany[END_REF]. In Canada the disease has been known since the early 1990s, but the association to PCV2 was first reported in 1998 [START_REF] Ellis | Isolation of circovirus from lesions of pigs with postweaning multisystemic wasting syndrome[END_REF].

Two pigs infected with PCV2 exhibited a co-infection with CSFV. One animal from 1985 revealed a high virus load of PCV2 in various organs. Lesions consisting of vasculitis, lymphadenitis with botryoid cytoplasmic inclusion bodies, and interstitial nephritis were present. These pathologic changes differed substantially from other PCV2-infected pigs during this time period. The altered type of lesions indicates that coinfection with CSFV seemed to play a substantial role in the development of increased PCV2-associated changes.

Though pathological alterations in CSF include vascular changes with lymphocytic perivascular cuffing [START_REF] Jones | Diseases caused by viruses[END_REF], it remains unclear whether observed lesions are due to the infection with CSFV or PCV2 or a combination of both. However, the high virus load of PCV2 indicates an exacerbation of the infection in combination with CSF.

The presented data show a sudden increase in the incidence of PCV2 infection from 2. 77% to 32.14% between 1962-1984 and 1985-1998, respectively. It remains unclear which factors contributed to this increased appearance of PCV2 infection and associated lesions. The first reports of PCV2 and associated lesions occurred almost simultaneously on both sides of the Atlantic [START_REF] Ellis | Isolation of circovirus from lesions of pigs with postweaning multisystemic wasting syndrome[END_REF][START_REF] Hinrichs | First detection of porcine circovirus type 2 infection in Germany[END_REF]. This suggests that PCV2 was already within the pig population for a long time, and possible similar or different changes may have contributed to the increased virulence of PCV2 on both continents.

It is well established that various risk factors contribute to exacerbating PCV2 infection and the development of associated lesions. These include infections with PPV, PRRSV, swine influenza virus, and Mycoplasma (M.) hyopneumoniae as well as vaccination against PRRS and mycoplasma [START_REF] Elbers | Risk factors for clinical signs of PMWS and PDNS in pigs in The Netherlands: a case-control study[END_REF][START_REF] Ellis | Porcine circovirus-2 and concurrent infections in the field[END_REF]. In 1984 there was an outbreak of CSF predominantly in the northern part of Germany leading to a large-scale vaccination to control the epidemic [START_REF] Pittler | Control of the European hog cholera with regard to new European Community regulations. Changes in state preventive measures relating to district vaccinations[END_REF]. Whether the epidemic infection as well as the vaccination had an impact on PCV2 incidence and pathogenesis remains speculative but must be A c c e p t e d M a n u s c r i p t 13 considered. Whether vaccination against M. hyopneumoniae, commercially available since 1992, also played a role in the increased PCV2 prevalence and lesion development remains speculative. In the same time period other nowadays important swine diseases were noticed for the first time including PRRS [START_REF] Dea | Isolation of the porcine reproductive and respiratory syndrome virus in Quebec[END_REF][START_REF] Wensvoort | Mystery swine disease in The Netherlands: the isolation of Lelystad virus[END_REF] or increased in prevalence such as proliferative enteropathy due to Lawsonia intracellularis [START_REF] Kroll | Proliferative enteropathy: a global enteric disease of pigs caused by Lawsonia intracellularis[END_REF].

In summary, the presented data revealed that PCV 2 infection could be detected as early as 1962 in pigs and associated lesions such as PDNS and PMWS were already observed in 1985. Whether increased virus spread and pathogenecity are due to environmental factors, changes in the genetic make-up of the virus or host or due to immunomodulatory effects needs to be investigated in further studies. 1987-1990 and 1992-1995).

Fig. 3:

Percentage of PCV2 in situ hybridisation positive organs of PCV2-infected animals divided into early investigated period , late investigated period (1985-1998) and total period (1961-1998).

  %), spleen (8 out of 103; 7.77 %), kidney (8 out of 89; 8.99 %), liver (6 out of 157;

  infected with PCV2 from 1985 onwards. Four of these animals displayed changes characteristic for PMWS with intralesional detection of PCV2. The first animal originated from 1985, one from the year 1996 and two from the year 1998 (Fig.5).

  Figure Caption

Fig. 2 :

 2 Fig. 2:Percentage of PCV2 positive animals between 1961 and 1998 (excluding the years

Fig. 4 :

 4 Fig. 4:Percentage of virus associated lesions in PCV2 positive pigs: White columns, time

Fig. 5 :

 5 Fig. 5:Lymph node from a pig from 1985 (co-infected with classical swine fever virus)

Fig. 6 :

 6 Fig. 6: Skin (A, B) and kidney (C) from a pig from 1985 with lesions characteristic for

  Figure 1

Table 1 : Primers and probes used for detecting PCV2 and PCV1 DNA from archived material

 1

	Designation	Reference	Sequence (5´ to 3´)	length Genome position
	p196 -PCV2 for		CGGATATTGTAGTCCTGGTCG		1094-1114 a
	p197 -PCV2 rev	Ellis et al. (1999)	ACTGTCAAGGCTACCACAGTCA	481 bp	1570-1549 a
	p285 -PCV2 for		GATTGTATGGCGGGAGGAGT		1286-1305 a
	p286 -PCV2 rev	Kim and Chae (2001)	ATTGACGACTTTGTTCCCCC	225 bp	1510-1491 a
	p325 -PCV2 for		GCACCCTGTAACGTTTGTCA		464-483 a
	p327 -PCV2 rev	(this work)	ATTTTCCCGCTCACTTTCAA	66 bp	529-510 a
	PCV2 probe	Seeliger et al. (2007) CCTTCCTCATTACCCTCCTCGCCAACAATAAAATAATCAAA 41 bp	208-168 a
	PCV1 probe	Seeliger et al. (2007) CCCTCTTCCAAACCTTCCTCTCCGCAAACAAAATAATCAAA 41 bp	195-155 b
	a GenBank accession number: AF 027217
	b GenBank accession number: NC 001792
	for = forward
	rev = reverse

Table 1 : Primers and probes used for detecting PCV2 and PCV1 DNA from archived material

 1

	Designation	Reference	Sequence (5´ to 3´)	length Genome position
	p196 -PCV2 for		CGGATATTGTAGTCCTGGTCG		1094-1114 a
	p197 -PCV2 rev	Ellis et al. (1999)	ACTGTCAAGGCTACCACAGTCA	481 bp	1570-1549 a
	p285 -PCV2 for		GATTGTATGGCGGGAGGAGT		1286-1305 a
	p286 -PCV2 rev	Kim and Chae (2001)	ATTGACGACTTTGTTCCCCC	225 bp	1510-1491 a
	p325 -PCV2 for		GCACCCTGTAACGTTTGTCA		464-483 a
	p327 -PCV2 rev	(this work)	ATTTTCCCGCTCACTTTCAA	66 bp	529-510 a
	PCV2 probe	Seeliger et al. (2007) CCTTCCTCATTACCCTCCTCGCCAACAATAAAATAATCAAA 41 bp	208-168 a
	PCV1 probe	Seeliger et al. (2007) CCCTCTTCCAAACCTTCCTCTCCGCAAACAAAATAATCAAA 41 bp	195-155 b
	a GenBank accession number: AF 027217
	b GenBank accession number: NC 001792
	for = forward
	rev = reverse

Table 1

 1 Page 23 of 31

Acknowledgments

The authors acknowledge the excellent technical assistance of Danuta Waschke, Petra Grünig and Bettina Buck. PCV2-and PCV1-infected PK15 cells were kindly provided by Gerad Wellenberg, ID Lelystad, Instituut Dierhouderij en Diergezondheid, the Netherlands.


Katrijn Cobbaut

Kurt Houf

Glenn Buvens

Ihab Habib

Lieven De Zutter
email: lieven.dezutter@ugent.be

Lieven De Zutter

Occurrence of non-sorbitol fermenting, verocytotoxin-lacking Escherichia coli O157 on cattle farms

Keywords: E. coli O157, cattle, verocytotoxin-negative

Escherichia coli O157 is often associated with hemorrhagic colitis and the hemolytic uremic syndrome (HUS). The verocytotoxins are considered to be the major virulence determinants.

However, vt-negative E. coli O157 were recently isolated from patients with HUS. Several transmission routes to humans are described, but cattle feces are the primary source from which both the food supply and the environment become contaminated with E. coli O157.

In a prevalence study performed on dairy, beef, mixed dairy/beef and veal farms in the summer of 2007, vt-negative isolates were detected on 11.7% (8/68) of the positive farms. From these eight farms, a total of 43 sorbitol-negative E. coli O157:H7 were collected. On five farms, only strains negative for the vt genes were present whereas both vt-negative and vt-positive strains could be detected on three other farms. Further characterization revealed that all isolates carried the eaeA and hlyA genes. Pulsed-field gel electrophoresis (PFGE) of all isolates resulted in nine different PFGE types and within the vt-negative strains, four different genotypes were identified, indicating that certain genetic clones are widespread over the cattle population.

Introduction

Escherichia coli O157:H7/-belongs to the group referred to as enterohemorrhagic E. coli (EHEC) and was identified in 1982 as the cause of human illness associated with the consumption of contaminated hamburgers [START_REF] Riley | Hemorrhagic colitis associated with a rare Escherichia coli serotype[END_REF]. Ruminants, particularly cattle, have been identified as the major reservoirs of E. coli O157:H7, and infections have been traced mostly to consumption of raw or undercooked beef ( [START_REF] Ørskov | Cattle as a reservoir of verotoxin-producing Escherichia coli O157:H7[END_REF][START_REF] Chapman | Cattle as a possible source of verocytotoxin-producing Escherichia coli O157 infections in man[END_REF]. However, humans can also be infected by consumption of fecal contaminated vegetables [START_REF] Cieslak | Escherichia coli O157:H7 infection from a manured garden, Lancet 342, 367. establish the Escherichia coli O157 status on beef cattle farms[END_REF], raw milk [START_REF] Lahti | Use of phenotyping and genotyping to verify transmission of Escherichia coli O157:H7 from dairy farms[END_REF], dairy products [START_REF] Morgan | Verotoxin-producing Escherichia coli O157 infections associated with the consumption of yoghurt[END_REF] and drinking water [START_REF] Akashi | A severe outbreak of haemorrhagic colitis and haemolytic uraemic syndrome associated with Escherichia coli O157:H7 in Japan[END_REF].

A c c e p t e d M a n u s c r i p t 3 Other transmission routes include animal-to-person [START_REF] Crump | An outbreak of Escherichia coli O157:H7 infections among visitors to a dairy farm[END_REF] and person-to-person contact [START_REF] Reida | An outbreak due to enterohaemorrhagic Escherichia coli O157:H7 in a children day care centre characterized by person-to-person transmission and environmental contamination[END_REF]. Clinical symptoms in humans range from mild diarrhoea to hemolytic uremic syndrome (HUS), and can even lead to death [START_REF] Griffin | The epidemiology of infections caused by Escherichia coli O157:H7, other enterohemorrhagic E. coli and the associated hemolytic uremic syndrome[END_REF][START_REF] Nataro | Diarrheagenic Escherichia coli[END_REF].

The pathogenicity of E. coli O157 is linked to the presence of several virulence factors such as the phage-encoded verocytotoxins, intimin encoded by the chromosomal gene eaeA and enterohemolysin encoded by the plasmid gene hlyA. The verocytotoxins VT1 and VT2 are recognized as the most important virulence factors [START_REF] Boerlin | Associations between virulence factors of Shiga-toxin-producing Escherichia coli and disease in humans[END_REF][START_REF] Sandvig | Shiga Toxins[END_REF]. The toxins bind to the eukaryotic cell surface by the globotriosylceramide receptor [START_REF] Lingwood | Verotoxins and their glycolipid receptors[END_REF]. After internalization and activation, the biological active part of the toxin inhibits the protein synthesis leading to death of the host cell [START_REF] Sandvig | Shiga Toxins[END_REF]. Although verocytotoxins are considered as the essential virulence factors of EHEC, strains lacking those genes have occasionally been isolated from patients with HUS (Mellman et al., 2005;, Bielaszewska et al., 2007, Friedrich et al., 2007). It is unclear whether these strains are either inherently vt-negative or whether they have lost their vt gene during the course of infection [START_REF] Friedrich | Prevalence, virulence profiles, and clinical significance of Shiga-toxinnegative variants of enterohemorrhagic Escherichia coli O157 infection in humans[END_REF].

Recently, non-sorbitol fermenting, vt-negative E. coli O157:H7 strains were also isolated from cattle at the farm level [START_REF] Nielsen | Characterisation of Escherichia coli O157 isolates from Danish cattle and human patients by genotyping and presence and variants of virulence genes[END_REF][START_REF] Wetzel | Isolation of Escherichia coli O157:H7 strains that do not produce Shiga toxin from bovine, avian and environmental sources[END_REF]. The role of these vt-negative EHEC can not be neglected because the possibility exists that vt-negative isolates acquire the vt2 gene again along the farm to fork production chain and that they emerge as a new verocytotoxin-producing E. coli strain [START_REF] Wetzel | Isolation of Escherichia coli O157:H7 strains that do not produce Shiga toxin from bovine, avian and environmental sources[END_REF].

This study reports the isolation of sorbitol-negative, verocytotoxin lacking E. coli O157 in the cattle population in the Northern part of Belgium. (Cobbaut et al., 2008) with a maximum of 10 samples per farm. When possible, three different age categories were sampled: <8 months, 8-30 months, and >30 months.

Materials and methods

Bacterial isolates

To isolate E. coli O157 from the overshoes, 250 ml modified tryptone soya broth (Oxoid, Basingstoke, United Kingdom) (mTSB) supplemented with 20 mg/L novobiocin (Sigma, Aldrich, St-Louis, MO, USA) (mTSBn) was added to each pair of overshoes. After incubation in a warm water bath at 42°C for 6 h, immunomagnetic separation (Dynal, Oslo, Norway) was performed according to the manufacturer's recommendations. One hundred microliter was spread onto sorbitol-MacConkey agar (Oxoid) supplemented with cefixime (0.05 mg/l) (Dynal) and potassium tellurite (2.5 mg/l) (Dynal) and incubated for 24 h at 42°C. Following incubation, up to three non-sorbitol fermenting colonies with a typical morphology per plate were transferred to plate count agar (Oxoid), incubated for 18-24 h at 37°C, and identified serologically with the O157 antigen latex agglutination assay (Oxoid). From the isolates positive for agglutination, a maximum of nine isolates per farm were selected (three of each sampled age category) and examined by a multiplex PCR. The somatic rfb O157 and the flagellar fliC H7 gene sequences were amplified using previously developed primer pairs by [START_REF] Maurer | Development of primers to O-antigen biosynthesis genes for specific detection of Escherichia coli O157 by PCR[END_REF] and [START_REF] Wang | Sequence diversity of the Escherichia coli fliC genes: implication for a DNA-based typing scheme for E. coli O157:H7[END_REF], respectively. The PCR assays were carried out in a 25 µl volume containing 1 µl of the lysate, 1 x Taq buffer (20 mM Tris-HCl, pH 8.0 and 50 mM KCl), 0.75 U Taq DNA polymerase (Invitrogen, Carlsbad, California, USA), 500 µM dNTPs, 3 mM of MgCl 2 , and 1.7 µM of each primer.

Samples were subjected to an initial denaturation of 1 min by 95°C, 30 PCR cycles each consisting of 15 s denaturation at 95°C, 15 s of annealing at 50°C and 30 s of elongation at 72°C. The final cycle was followed by an elongation for 8 min at 72°C and hold at 4°C. The PCR amplification

Detection of virulence genes

For each isolate identified as E. coli O157, a multiplex virulence PCR was performed using the primers for vt1, eaeA and hlyA as described by [START_REF] Fagan | Detection of Shiga-like toxin (stx1 and stx2), intimin (eaeA), and enterohemorrhagic Escherichia coli (EHEC) hemolysin (EHEC hlyA) genes in animal feces by multiplex PCR[END_REF] and for vt2 as described by [START_REF] Paton | Detection and characterization of shiga toxigenic Escherichia coli by using multiplex PCR assays for stx1, stx2, eaeA, enterohemorrhagic E. coli hlyA, rfb O111 , and rfb O157[END_REF]. PCR assays were carried out in a 25 µl volume containing 1 µl of the lysates, 1 X Taq buffer (20 mM Tris-HCl, pH 8.0 and 50 mM KCl), 0.75 U Taq DNA polymerase, 500 µM dNTPs, 3 mM of MgCl 2 , 1.5 µM of the vt1, eaeA, hlyA and 0,6 µM of the vt2 primers.

Temperature conditions consisted of an initial 95°C denaturation step for 3 min followed by 30 cycles at 95°C for 20 s, 58°C for 40 s, and 72°C for 90 s. The final cycle was followed by a 8 min hold at 72°C and a final hold at 4°C. The PCR amplification products were analyzed as described above.

For confirmation, strains testing negative for the vt genes were retested for vt1 and vt2 using the primer set described by [START_REF] Karch | Single primer pair for amplifying segments of distinct Shiga-liketoxin genes by polymerase chain reaction[END_REF]. Strains were considered as vt-negative if both PCRs generated no amplicons for the vt genes.

Genotyping by pulsed-field gel electrophoresis

Pulsed-field gel electrophoresis (PFGE) was performed with 50 U of XbaI (Invitrogen) according to the PulseNet protocol (2007). The Salmonella Braenderup standard strain H9812 was used as a molecular size marker to produce the dendrogram. After electrophoresis, the gel was stained with ethidium bromide and digitally captured under UV light. Band position differences were used to allocate PFGE profiles using GelCompar version 3.0 (Applied Maths, Sint-Martens-Latem, Belgium). The similarities between the fingerprints were calculated using the band-based Dice A c c e p t e d M a n u s c r i p t 6 coefficient, with an optimization and position tolerance of 1%. The fingerprints were grouped according to their similarities by use of the unweighted-pair group method using arithmetic averages algorithm. Isolates showing identical XbaI-PFGE patterns were compared further using 30 U of NotI. For NotI, a linearly ramped switching time from 10 to 30 s for 18 h was used. PFGE genotypes were assigned on the basis of major polymorphisms, defined as a difference in the presence of at least one band in the XbaI fingerprint. The genotypes were indicated by a capital. Some strains showed 100 % similarity after cluster analysis, but a small polymorphism, defined as a small shift of one band, was visually seen. This was indicated by one or more apostrophes after the capital.

Statistical analysis

Dependant variable of interest was the presence/absence of vt-negative E. coli O157 in a sample, and that was correlated with the categorical explanatory variables (age and farm type). Statistical analysis was carried out in Stata SE/8.0 using a logistic regression model with a logit function and a binomial error distribution.

Results

E. coli O157 was present on 68 of the 180 participating farms. All isolates harboured the eaeA and hlyA genes, but on eight of the 68 positive farms (11.8%) vt-negative E. coli O157 were detected.

On those eight farms, a total of 43 sorbitol-negative E. coli O157:H7 isolates were further examined. Twenty-nine isolates had no vt genes and the remaining 14 isolates only possessed the vt2 gene.

On five farms (6, 44, 101, 102 and 177), only strains negative for the vt genes were present whereas both vt-negative and vt-positive strains could be detected on three other farms (93, 104 and 159) (Fig. 1). The relationship between age category, cattle farm type and the presence of vt-negative A c c e p t e d M a n u s c r i p t 7 isolates is shown in Table 1. The prevalence of vt-negative E. coli O157 in animals older than 30 months was higher (75% (15/20)) compared to the 8-to-30-month old animals (45% (5/11)) and animals younger than 8 months (75% (9/12)). The statistical difference between the age groups was not convincingly significant (P-value=0.084 and 0.086, respectively) due to the limited number of recovered isolates, however this should not underestimate the biological significance of such findings. In addition, E. coli O157 lacking the vt genes were detected on five dairy farms, two beef cattle farms and one mixed cattle farm.

The restriction patterns of the 43 isolates could be categorized into nine XbaI-PFGE genotypes (Fig. 1). Restriction with NotI did not result in additional information and confirmed the results obtained with XbaI (data not shown). Most genotypes were farm specific, however one PFGE type was associated with five farms (44, 101, 102, 104, and 177). A small band shift was seen between the genotypes of farms 44 and 177 on the one hand and farms 101, 102, and 104 on the other hand, respectively assigned as subtype E' and E''. Two farms (104 and 159) harboured three different genotypes. The vt-positive and vt-negative strains did not cluster together within the same genotype, though they originate from the same farm (e.g. strain 786 and 787). A difference of at least four bands and a similarity of maximum 86% was found between the profiles of vt-negative and vtpositive strains originating from the same farm.

Discussion

The present study demonstrates that vt-negative E. coli O157:H7 are common on cattle farms. The influence of age category and cattle farm type on the presence of vt-negative isolates was borderline significant and only a large-scale study including more farms and isolates could clarify a possible relationship. Tutenel et al. (2000) conducted a prevalence study in the period 1998-1999 on Belgian cattle at the slaughterhouse level, using the same isolation method as the one that was applied in the present study. Only three E. coli O157 lacking the vt genes were detected in a total of 82 isolates [START_REF] Nielsen | Characterisation of Escherichia coli O157 isolates from Danish cattle and human patients by genotyping and presence and variants of virulence genes[END_REF]. In a longitudinal study conducted on dairy farms in the USA, vt-negative strains were isolated from four farms [START_REF] Wetzel | Isolation of Escherichia coli O157:H7 strains that do not produce Shiga toxin from bovine, avian and environmental sources[END_REF]. In Italy, [START_REF] Bonardi | Isolation of verocytotoxin-producing Escherichia coli O157:H7 from cattle at slaughter in Italy[END_REF] described the isolation of 15 vt-negative strains from feedlot cattle at the slaughterhouse.

Two hypotheses can be postulated for the absence of the vt genes in strains carrying other virulence genes such as eaeA and hlyA. Firstly, the vt-negative strains could represent strains that have lost the vt genes during culturing. Loss of vt genes during in vitro culture has been demonstrated in other verocytotoxin-producing E. coli serotypes [START_REF] Karch | Frequent loss of Shiga-like toxin genes in clinical isolates of Escherichia coli upon subcultivation[END_REF]. Secondly, the vt-negative strains may already have lost the vt genes in the natural reservoir. In support of this hypothesis, [START_REF] Mellmann | Enterohemorrhagic Escherichia coli in human infection: in vivo evolution of a bacterial pathogen[END_REF] suggested that loss of the vt genes may provide an evolutionary advantage.

Survival might be favoured by loss of the phage, because such vt-negative progeny of vt-positive progenitors are less prone to lysis. Although the loss of vt genes during isolation or subculturing in this study can not be excluded, clustering of the strains lacking the vt genes (originating from up to six samples from the same farm) supports the hypothesis that such strains are present in the natural reservoir.

All vt-negative E. coli O157:H7 isolates of this study possessed two other virulence traits: the hlyA and eaeA genes. Such strains may be considered as atypical enteropathogenic E. coli (aEPEC), since they do not harbour the bpf gene encoding for the enteropathogenic E. coli adherence factor (EAF). aEPEC have emerged in recent years as cause of diarrhea in both children and adults (Bielaszeswska, 2008). Taking into account the importance of verocytotoxins in the pathogenesis of EHEC-associated HUS [START_REF] Boerlin | Associations between virulence factors of Shiga-toxin-producing Escherichia coli and disease in humans[END_REF][START_REF] Sandvig | Shiga Toxins[END_REF], it seems unlikely that these isolates could still play a role in the development of this syndrome. Since they have unoccupied vt-

  of 2007, an E. coli O157 prevalence study on 180 randomly selected Belgian cattle farms was carried out. The farms were dairy (n=49), beef (n=75), mixed dairy/beef (n=45) or veal farms (n=11). Bedding material of different cattle pens was sampled by the "overshoe method"

  A c c e p t e d M a n u s c r i p t 5 products were determined by analyzing 8 µl of product on 1.5% agarose gels, staining with ethidium bromide and comparison with a molecular weight marker (TrackIt TM 100bp DNA ladder, Invitrogen).

  positive farms. Therefore, the present data may indicate that the prevalence of vt-negative E. coli O157:H7 has increased over the years. Previously vt-negative strains have been reported in cattle in other countries. During a 60-farm study in Denmark, vt-negative/eaeA-positive strains were isolated from five farms out of 17 E. coli O157 positive farms

Figure 1 .

 1 Figure legend

Figure 1 Figure 1

 11 Figure 1

Acknowledgements

We thank all the farmers involved in this study for their cooperation.

A c c e p t e d M a n u s c r i p t 9 bacteriophage integration sites, vt-negative O157:H7 isolates could be transduced with such phages and converted to EHEC. vt-negative attaching and effacing aEPEC presenting the same phenotype of virulence factors and belonging to the most frequent EHEC serotypes have been isolated at a low frequency from patients with diarrhea [START_REF] Bielaszewska | Shiga toxin-negative attaching and effacing Escherichia coli: distinct clinical associations with bacterial phylogeny and virulence traits and inferred in-host pathogen evolution[END_REF] and HUS [START_REF] Bielaszewska | Shiga toxin-mediated hemolytic uremic syndrome: time to change the diagnostic paradigm[END_REF]. Within serotype O157:H7, most of these aEPEC fermented sorbitol, but two did not. The authors of these papers suggest that these EHEC lost the vt gene during infection and are no simple aEPEC but a potentially highly virulent group they call EHEC-LST.

The genotypes of vt-negative and vt-positive strains differed more than described by [START_REF] Feng | Isogenic strain of Escherichia coli O157:H7 that has lost both Shiga toxin 1 and 2 genes[END_REF] who reported that when PGFE was performed, loss of vt genes resulted in a difference of two bands and the strains without the vt gene had still a similarity of 90% compared to the vtpositive strain. Possibly, the vt-negative strains in the present study do not originate from the vtpositive strains present on the same farm. The vt-negative strains from five farms displayed the same PFGE type (Fig. 1, PFGE type E). Three farms (101, 102, and 104) were situated within a distance of maximum 10 km suggesting a transmission between farms [START_REF] Wetzel | Clonal dissemination of Escherichia coli O157:H7 subtypes among dairy farms in Northeast Ohio[END_REF].

However, transmission between farms by bovine animals seems unlikely because all farms were of the closed farm type. Human contact may be the cause of transmission between farm 101 and 102 as they belonged to the same owner. Farm 44 and 177 were located at a distance of 150 km and 30 km from the other three farms, indicating the circulation of one vt-negative clone.

The high number of vt-negative E. coli O157:H7 isolates in this study stresses the importance of testing E. coli O157:H7 isolates for the presence of other virulence factors, because pathogenic strains could be overlooked by protocols that rely exclusively on vt genes or verocytotoxin detection. Further studies are necessary to determine if these strains really can acquire vt genes by transduction with vt-phages present in other E. coli strains in the intestinal flora in animal or human intestine or in food and to examine the possible relationship between vt-negative isolates from humans and cattle.


Winy Messens
email: winy.messens@ilvo.vlaanderen.be

Lieve Herman

Lieven De Zutter

Marc Heyndrickx

Multiple Typing for the Epidemiological Study of Contamination of Broilers with Thermotolerant Campylobacter

Keywords: Campylobacter, broilers, risk factor, molecular epidemiology, transmission

This study aims to investigate the genetic diversity of thermotolerant Campylobacter in commercial broiler flocks and in the environment of broiler farms in Belgium. Seven out of 18 investigated flocks became colonized during rearing. Fluorescent amplified fragment length polymorphism (FAFLP), pulsed field gel electrophoresis (PFGE), restriction fragment length polymorphism of the flagellin A gene (flaA-RFLP) and antimicrobial resistance profile (ARP) were used for typing of the isolates. By the combination of FAFLP and PFGE, 22 Campylobacter genotypes could be distinguished. Colonization was almost exclusively with Campylobacter jejuni and unique genotypes were found in each flock. Multiple genotypes were detected in the broilers of three flocks, either simultaneously or successively.

In five flocks, strains that were resistant to at least one antibiotic (mostly tetracycline) were found. The presence of other broiler houses on the farm did not result in a higher probability of colonization. The nipple water was contaminated with the same genotype as the broilers, illustrating its importance for transmission of Campylobacter. The same genotype was detected in a water puddle and in the broiler flock during rearing in three flocks. Once, the same genotype was isolated from the ditch water shortly before it was detected in the broilers.

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

Introduction

Campylobacteriosis was the most frequently reported zoonotic disease in humans within the European Union (EU) in 2006. Its overall incidence was 54.6 cases per 100,000 inhabitants in the EU and 64.3 per 100,000 inhabitants in Belgium [START_REF] Efsa | The community summary report on trends and sources of zoonoses, zoonotic agents, antimicrobial resistance and foodborne outbreaks in the European Union in 2006[END_REF]. Its true incidence is uncertain since many unreported infections occur (WHO 2000). Ninety-one percent of human isolates speciated in the EU were identified as Campylobacter jejuni subsp.

jejuni (hereafter C. jejuni), while Campylobacter coli accounts for most of the remaining cases [START_REF] Efsa | The community summary report on trends and sources of zoonoses, zoonotic agents, antimicrobial resistance and foodborne outbreaks in the European Union in 2006[END_REF]. The majority of infections occur sporadically, with the major vehicle of infection assumed to be contaminated food, and in particular chicken [START_REF] Friedman | Bactericidal activities of plant essential oils and some of their isolated constituents against Campylobacter jejuni, Escherichia coli, Listeria monocytogenes, and Salmonella enterica[END_REF][START_REF] Skirrow | Diseases due to Campylobacter, Helicobacter and related bacteria[END_REF]. In Belgium, in recent years 72% of the broiler flocks arriving at the slaughterhouse were colonized with campylobacters. After slaughter, 79% of the flocks yielded contaminated carcasses. Isolates were mainly identified as C. jejuni (89%) [START_REF] Rasschaert | Campylobacter contamination during poultry slaughter in Belgium[END_REF]. It has been estimated that consumption of poultry meat in Belgium causes at least 22,000 campylobacteriosis cases with a cost of illness of €10.9 million [START_REF] Gellynck | Economics of reducing Campylobacter at different levels within the Belgian poultry meat chain[END_REF]. The relative risk reduction by reducing the flock prevalence has a linear (one-to-one) relationship, i.e. 50% reduction in flock prevalence results in a 50% reduction in risk [START_REF] Messens | Quantitative risk assessment of human campylobacteriosis through the consumption of chicken meat in Belgium[END_REF]).

The most significant sources for flock colonization remain unclear. However, a number of epidemiological studies have shown that lack of hygienic barriers, use of non-disinfected drinking water and deficient hygienic measures are significant risk factors for introducing Campylobacter in broiler flocks [START_REF] Johnsen | Genetic diversity and description of transmission routes for Campylobacter on broiler farms by amplified-fragment length polymorphism[END_REF]. Other vectors such as litter beetles, house flies and wild birds have also been identified as potential transmission risks [START_REF] Chuma | Detection of thermophilic Campylobacter from sparrows by multiplex PCR: the role of sparrows as a source of contamination of broilers with Campylobacter[END_REF].

Materials and methods

Sampling

Eighteen Belgian commercial broiler flocks from 17 different farms were examined (flocks nos. 6 and 7 were successive flocks in the same broiler house). A detailed description about sample collection can be found in [START_REF] Herman | Routes for Campylobacter contamination of poultry meat: epidemiological study from hatchery to slaughterhouse[END_REF] and in the Table. Briefly, before arrival of the one-day-old chicks (day 1), the hygiene inside the broiler houses was checked. During rearing, the farms were visited three times (days 14, 28 and 42) with sampling of cecal drops (mostly 4 pools of 10 cecal drops; detecting a prevalence of 7.3% at 95% confidence level for 10,000 birds), overshoes, farmer' indoor footwear, feed and drinking water. The environment of the farms was sampled before and during rearing.

Isolation and confirmation of Campylobacter

The isolation protocol included both direct (using modified Charcoal Cefoperazone Desoxycholate Agar (mCCDA, Oxoid, Basingstoke, UK)) and enrichment culturing (using selective Preston broth (Oxoid) and mCCDA plating). Incubation was for 24 to 48 h at 42°C

A c c e p t e d M a n u s c r i p t under microaerophilic conditions (5% O 2 , 10% CO 2 , 85% N 2 in an O 2 /CO 2 incubator, Thermo Forma, Ohio, USA). Isolates were stored in brain-heart infusion broth (BHI; Oxoid) supplemented with 5% (vol/vol) lysed, defibrinated horse blood at -80°C using 15% (w/v) glycerol as cryoprotectant. A sample was determined as positive for Campylobacter as soon as at least one of the isolation methods yielded a positive result. The Campylobacter status of the flock was considered as positive if Campylobacter was detected in at least one of the pools of cecal drops.

Identification of the isolates was performed on the Campylobacter genus level and as C. jejuni or C. coli in a multiplex PCR. When no PCR identification could be achieved, the identity of the presumptive Campylobacter isolates was determined by sequence analysis of the 16S ribosomal DNA as described in [START_REF] Herman | Routes for Campylobacter contamination of poultry meat: epidemiological study from hatchery to slaughterhouse[END_REF].

Fluorescent amplified fragment length polymorphism genotyping

FAFLP was performed on 500 ng of genomic DNA digested in a total volume of 30 µl, which consisted of 5 U of HindIII and HhaI endonuclease (Promega, Leiden, The Netherlands), 3 µl of buffer C (Promega) and 0.3 µl of 100 bovine serum albumin (BSA) (Promega), for 3 h at 37°C. To the digested DNA, 25 µl of a solution containing 0.5 µl of 2 µM HindIII adaptor, 0.5 µl of 20 µM HhaI adaptor (both from Eurogentec, Seraing, Belgium), 40 U of T4 DNA-ligase, and 5 µl of 10 T4 ligase buffer (both from Amersham Pharmacia Biotech, Uppsala, Sweden) was added. Ligation was carried out overnight at room temperature. The sequence of the HindIII adaptor was 5'-CTC GTA GAC TGC GTA CC, 3'-CTG ACG CAT GGT CGA. The sequence of the HhaI adaptor was 5'-GAC GAT GAG TCC TGA TCG, 3'-G CTA CTC AGG ACT A.

A c c e p t e d M a n u s c r i p t 6 Preselective PCRs were carried out in 25-µl volumes containing 5 µl of the two-fold diluted ligation product, 2.2 µl of HindIII primer (5' GAC TGC GTA CCA GCT T-3', 1 µM, Eurogentec), 2.2 µl of HhaI primer (5'-GAT GAG TCC TGA TCG C-3', 5 µM, Eurogentec), and 15.6 µl of amplification core mix (Applied Biosystems). Amplification was performed in a programmable thermocycler (GeneAmp ® PCR System 9700; Applied Biosystems) using an initial denaturation step at 94°C (2 min), followed by 20 cycles consisting of denaturation (94°C for 20 s), annealing (56°C for 45 s) and extension (72°C for 2 min). Next, selective PCRs were carried out in 20-µl volumes containing 3 µl of the five-fold diluted PCR product, 3 µl of FAM labeled HindIII+A primer (1 µM), 3 µl of HhaI+A primer (5 µM, Eurogentec), and 11 µl of the amplification core mix. Amplification was performed in the GeneAmp ® PCR System 9700 programmable thermocycler using an initial denaturation step at 94°C (2 min), followed by 20 cycles consisting of denaturation (94°C for 20 s), annealing (66°C for 45 s) and extension (72°C for 2 min), and a final extension step (72°C for 10 min). Three microliters of the selective PCR product was mixed with 12 µl of Hi-Di formamide (Applied Biosystems), 1 µl of Gene-Scan-500 size standard labeled with the red fluorophore 6carboxy-x-rhodamine (ROX) (Applied Biosystems) and heated at 92°C for 2 min.

Subsequently the mix was chilled on ice for a few minutes.

Separation of the selective PCR products was generated in an ABI PRISM 310 genetic analyzer (Applied Biosystems). Each run was performed at 60°C for 35 min at 10 mA and 15 kV. After electrophoresis, the banding pattern data were collected with the ABI GeneScan 3.1 software (Applied Biosystems). Each gel track was then imported into the Bionumerics 4.00 software package (Applied Maths, Sint-Martens-Latem, Belgium) with the program ABICON (Applied Maths). Gels were normalized using the internal standard and the levels of genetic similarity between patterns were calculated with the Pearson product-moment correlation coefficient. For cluster analysis the unweighted pair group method using average A c c e p t e d M a n u s c r i p t 7 linkages (UPGMA) was used at an 0.06% optimisation coefficient. Fragments in the size range of ca 50-500 bp were used in the analysis. The reproducibility of the FAFLP analysis was assessed by performing the whole assay, starting with DNA isolation, three times with three C. jejuni strains as indicated in the Table . Preselective PCRs and selective PCRs were performed in one run (as for all the samples). The similarity between patterns obtained was at least 90% (93.9, 95.8 and 97.4%) and these results correlated perfectly with previously reported values [START_REF] Duim | Differentiation of Campylobacter species by AFLP fingerprinting[END_REF][START_REF] On | Identification of taxonomic and epidemiological relationships among Campylobacter species by numerical analysis of AFLP profiles[END_REF].

Pulsed field gel electrophoresis genotyping

PFGE was performed as described previously [START_REF] Zorman | Genotyping of Campylobacter coli and C. jejuni from retail chicken meat and humans with campylobacteriosis in Slovenia and Bosnia and Herzegovina[END_REF] using SmaI as restriction enzyme (Boehringer, Mannheim, Germany). The macrorestriction fragments were separated using the Clamped Homogeneous Electric field method on a Chef Mapper (CHEF-DRII, Bio-Rad Laboratories, Richmond, USA) using pulse times from 4 s to 40 s in 22 h. The gels were stained with ethidium bromide, photographed and analyzed using a GelDoc™ 2000 system (Bio-Rad Laboratories). PFGE profiles were clustered with the BioNumerics 4.00 software package (Applied Maths) using the Dice coefficient (1% position tolerance) and UPGMA.

Restriction fragment length polymorphism genotyping

For flaA-RFLP, the consensus pair of primers for flaA gene amplification was used [START_REF] Wassenaar | Genotyping of Campylobacter spp[END_REF]. PCR reagents and conditions were from [START_REF] Nachamkin | Flagellin gene typing of Campylobacter jejuni by restriction fragment length polymorphism analysis[END_REF]. For flaA PCR amplicons of the expected 1.7-kb size RFLP analysis was performed using DdeI (New England Biolabs, Hitchin, United Kingdom) and HinfI (New England A c c e p t e d M a n u s c r i p t 8 Biolabs) as restriction enzymes. PCR amplicons (3-14 µl) were digested according to the manufacturer's instructions and then analysed by electrophoresis using 3% (w/v) agarose in 0.5 Tris-Acetate-EDTA (TAE) buffer. The gels were stained, photographed and analyzed similarly as the PFGE gels.

Definition of genotypes

For every typing experiment separately, genotypes were defined within the strain collection. FAFLP genotypes were assigned with a capital letter on the basis of an interpretation of the UPGMA-dendrogram: a cut-off similarity value of 90% was used to delineate genotypes labelled with a capital letter (e.g. A); genotypes clustering at a similarity level between 80 and 90% were classified as subtypes of a genotype, labelled with a capital letter followed by a number (e.g. A 1 , A 2 ); if visual but small band pattern differences were observed within the 90% cut-off range, this was indicated as a variant of a genotype with a quotation mark after the capital genotype letter. PFGE genotypes were assigned with a number on the basis of any difference in the presence of at least one band (above 50 kb); variants of a genotype with minor polymorphisms, defined as a small shift or density difference of a particular band, were indicated with one or more apostrophes after the PFGE genotype number. FlaA genotypes were assigned with a number on the basis of any difference in the presence of at least one band.

Overall genotypes were defined by a combination of the separately defined genotypes by FAFLP, PFGE, flaA-DdeI and flaA-HinfI in that order.

Antibiotic resistance profiling (ARP)

A c c e p t e d M a n u s c r i p t Minimal inhibitory concentrations (MICs) were obtained with the Etest (AB Biodisk, Solna, Sweden) according to the manufacturer's instructions. Isolates were classified as sensitive (S), intermediate (I) or resistant (R) applying the breakpoints of S, I and R (in µg/ml) for ampicillin (Am) 8, 16, 32, for amoxicillin-clavulanic acid (2/1) (Ac) 8, 16, 32 for ciprofloxacin (Ci) 1, 2, 4, for erythromycin (Em) 2, 4, 8, for gentamycin (Gm) 4, 8, 16, for nalidixic acid (Na) 8, 16, 32, and for tetracycline (Tc) 4, 8, 16. E. coli ATCC 25922 was used as reference strain. Mueller Hinton agar plates (BioMerieux, France) supplemented with 5% (vol/vol) sheep blood were used.

Results

Molecular typing of Campylobacter isolates

Using FAFLP, 20 genotypes could be distinguished within the 92 Campylobacter isolates (Figure). Three FAFLP-genotypes could be further divided in 2 (genotypes P and Q) or 3 (genotype N) subtypes; genotype F contained two variants. Using PFGE, 19 genotypes could be distinguished with genotype 11 showing 3 variants. FAFLP and PFGE results showed to be complementary (Figure). FAFLP-genotype F was divided in PFGE-genotypes 2 and 14 (corresponding with the variant FAFLP-genotypes F and F'), FAFLP-genotype E was divided in PFGE-genotypes 3 and 9, FAFLP-genotype O was divided in PFGE-genotypes 11' and 12 and FAFLP-genotype I was divided in PFGE-genotype variants 11 and 11''. Non-typeable strains in PFGE could be typed with FAFLP and belonged to FAFLP-genotypes M and P (P 1 and P 2 ). When FAFLP and PFGE were combined, this resulted in an increased total of 22 genotypes (+ 2 variants) for 92 isolates of 14 flocks where Campylobacter was isolated.

A c c e p t e d M a n u s c r i p t 10 Using flaA-RFLP, 13 genotypes could be distinguished using DdeI as restriction enzyme and only 9 using HinfI. FlaA-RFLP was not complementary to the FAFLP-PFGE combination, with one exception where flaA-DdeI enabled the distinction of genotypes 11 and 12 for 2 isolates belonging to PFGE genotype 11 (this also corresponded with a different antibiotic resistance profile (ARP) for these 2 isolates). PFGE-genotype 5 was divided in flaA-DdeI genotypes 5 and 13, which corresponded with the FAFLP results.

Broiler houses

Campylobacters were not isolated from the samples taken in the broiler house shortly before arrival of the 1-day-old chicks. During rearing, seven of the 18 broiler flocks became colonized with Campylobacter spp. First detection was for three flocks after 14 days (flocks 2, 3 and 11), for two flocks after day 28 (flocks 7 and 9), and at depletion for flocks 12 and 16. The Table shows the Broiler flocks were colonized by a variety of unique strains, as a strain with the same genotype was not isolated from more than one flock. The birds of flock 2 were colonized with C. jejuni strains of genotype P 1 /P 2 ,-,9,9 at day 14, but these were replaced by a C. coli A c c e p t e d M a n u s c r i p t 11 strain with genotype S,1,1,1. This latter type was also isolated from the nipple water at day 42. Colonization of flock 3 was detected from 14 days of rearing until depletion onwards with a C. jejuni strain of genotype F,2,2,2. The nipple water was also contaminated with this genotype both at 28 d and 42 d of rearing. Only at depletion, birds from flocks 12 and 16 were colonized with C. jejuni. For flock 12, the genotype C,16,7,7 was found on samples of broilers, nipple water and indoor footwear; for flock 16, the genotype M,-,-,-was found only on samples of broilers. C. jejuni strains with either genotype R 1 ,5,5,5 or R 2 ,5,5,5 were found colonizing flock 7 at 28 to 42 days of rearing and contaminating the nipple water and indoor footwear at 42 days of rearing. One of the two other poultry houses on this farm was colonized with a C. jejuni strain of another genotype (L,6,6,6). Flock 9 was colonized with two genotypes at 28 days of rearing (H,7,7,7 and N 2 ,7,7,7). Both genotypes were also isolated from the outdoor footwear at 28 days and genotypes H,7,7,7 and N 1 ,7,7,7 were also isolated from the nipple water at 42 days. At depletion, this flock was still found colonized with genotype N 1 ,7,7,7 (no more with genotype H,7,7,7), but also with another genotype B,8,8,8.

The indoor footwear was at that moment still found contaminated with genotype H,7,7,7. At depletion, the other two flocks housed at this farm were colonized with C. jejuni: one with genotype B,8,8,8 and the other with genotypes G,13,12,7 and N 3 ,7,7,7. In flock 11, a succession of different genotypes was found. C. jejuni strains with genotypes Q 1 ,15,1,7 or Q 2 ,15,1,7 were found colonizing flock 11 at 14 days of rearing, but these genotypes were not found later on. At 28 days, the flock was colonized with a strain with genotype I,11,11,7, while the nipple water and the indoor footwear were contaminated with another genotype (A,10,10,7). At depletion, the flock was still colonized with genotypes I,11,11/12,7, but also with genotypes O,11'/12,12,7; nipple water and indoor footwear were then contaminated with genotype I,11/11",11,7.

Environment of broiler house

Campylobacter was isolated from the environment surrounding the broiler house and/or from other broilers on the same farm in four of the seven colonized flocks but also in four of the eleven negative flocks. On the farms with Campylobacter-positive status, isolation was done most frequently from the faecal material of other poultry houses (2 of 2 flocks sampled or 2/2, number of samples taken N=12) and from the ditch water (1/1, N=2), but also from the water puddles (3/5, N=22), from the footwear used outside the broiler house (1/4, N=5), from the container with dead chickens (1/4, N=12) and from the dung hill (1/6, N=16). All these isolates were identified as C. jejuni and some were of identical genotype as those of the broilers (Table ). This was the case for one of the two broilers at the same farm for flock 9, but not for flock 7. All positive samples of the ditch water (flock 7), water puddles (flocks 9, 11 and 12) and footwear used outside the broiler house (flock 9) and from the container with dead chickens (flock 11) harboured the same genotype as those of the broilers. The dung hill of flock 7 harboured another genotype (D,5,13,5) as the ones that colonized the broilers (genotype R 1 /R 2 ,5,5,5). The preceding flock in this broiler house (flock 6) remained negative during rearing, but the dung hill was also found contaminated with a strain with genotype K,4,4,4. These subsequent flocks 6 and 7 on the same farm harboured the same FAFLPgenotype K (in dung hill and wild birds' faeces, respectively), but this genotype was not isolated from the broilers.

On the farms with Campylobacter-negative status, C jejuni was isolated from the drains of the broiler house (1 of 2 flocks sampled or 1/2, N=2), from the dung hill (2/5, N=14), from the faecal material from other poultry houses (2/7, N=36), from the ditch water (1/5, N=7) and from the water puddles (1/8, N=23), but not from the footwear used outside the broiler house (0/7, N=14) or from the container with dead chickens (0/7, N=21). Remarkably, in the A c c e p t e d M a n u s c r i p t 13 environment of flock 18, three genotypes were already at day 1 found contaminating either wild birds' faeces (-,11,1,7), the dung hill (-,18,1,7) or ditch water (J,19,9,9), but none of them was isolated from the broilers. The same FAFLP-genotype E was isolated from the environment of two flocks (wild birds' faeces in flock 5 and faecal material other poultry house, drains of poultry house and water puddle in flock 13); this FAFLP-genotype was divided in two PFGE-genotypes according to the flock.

Animals surrounding the broiler house

Both in Campylobacter positive flocks and negative flocks, Campylobacter was detected in wild bird's faeces (1 of 6 positive and 3 of 6 negative flocks sampled, N=25) and cow faeces (1 of 4 positive and 1 of 3 negative flocks sampled, N=16). The wild bird's faeces always harboured C. jejuni but the genotype was different to the one colonizing the broilers (flock 7). The cow faeces on the other hand harboured C. hyointestinales in both occasions (flocks 12 and 15).

Campylobacter was not isolated from dog faeces (N=16), horse faeces (N=16), sheep faeces (N=5), caterpillars (N=2), insects (N=33), mice or rats (N=3), snails (N=2) or worms (N=8), deer faeces (N=2) or pig faeces (N=1).

Antibiotic resistance profiles

Overall, 47 of the 87 tested strains (54%) showed resistance to at least one of the antibiotics tested. No resistance to amoxicillin and gentamycin was found. Resistance to ampicillin was found for only one strain isolated from faecal material in another house from flock 9. Ciprofloxacin resistance was found in strains isolated from cecal drops from flocks 3 A c c e p t e d M a n u s c r i p t 14 and 11. Isolates with erythromycin resistance were only found within flock 2. Resistance to nalidixic acid was found in strains isolated from cecal drops from flocks 3, 10 and 11. Also an isolate from cow faeces in flock 15 was resistant to nalidixic acid. Resistance to tetracycline was found in strains isolated from cecal drops from flocks 2, 3, 9, 10 (other house) and 11. The ARP's found were specific for strains from a certain flock. With two exceptions (sensitive to all antibiotics tested and ampicillin resistance for two strains of another poultry house), the C. jejuni isolates from flock 9 were only resistant to Tc. The profile EmTc was found occurring in C. coli isolates from flock 2. The profile CiNaTc was found in C. jejuni isolates from flock 3, while the profile NaTc was found in C. jejuni isolates from another house from flock 10. The profile CiNa was found in isolates from flock 11. In this flock, a succession of ARP's was found which correlated well with the genotypes found. At day 14, all strains isolated (belonging to genotypes Q 1 / 2 ,15,1,7) were found sensitive. At 28 days, a strain from the nipple water and belonging to genotype A,10,10,7 was found resistant according to the CiNa profile, while a strain from cecal drops belonging to the genotype I,11,11,7 was found sensitive to all antibiotics tested. At 42 days, strains belonging to genotypes I,11/11'',11,7 isolated from the nipple water and clean footwear were found resistant again according to this CiNa profile. However, at day 42, the strains from cecal drops of genotype I,11,11,7 were again sensitive, while other strains from cecal drops belonging to other genotypes were either intermediary resistant to tetracycline (genotypes -,11,12,7; O,12,12,7; O,11',12,7) or resistant to nalidixic acid (genotype -,11,11,7). in Sweden [START_REF] Berndtson | A 1-year epidemiological study of campylobacters in 18 Swedish chicken farms[END_REF] to more than 90% in the United Kingdom [START_REF] Evans | A longitudinal study of campylobacter infection of broiler flocks in Great Britain[END_REF]. Although this study was not designed to evaluate seasonal differences in flock colonization, a trend in seasonal variation as mentioned by others [START_REF] Berndtson | A 1-year epidemiological study of campylobacters in 18 Swedish chicken farms[END_REF][START_REF] Jacobs-Reitsma | Cecal carriage of Campylobacter and Salmonella in Dutch broiler flocks at slaughter: a one-year study[END_REF]) was found. The prevalence was highest when depletion occurred in the summer (75.0%, N=4) and spring (50.0%, N=4). In the autumn, the prevalence was 28.6% (N=7) and all flocks raised in the winter were considered Campylobacter free (N=3).

Discussion

In contrast to other findings [START_REF] Katsma | Campylobacter prevalence in broiler flocks in the Netherlands[END_REF], in our study the presence of other broilers on the same farm did not give rise to a more frequent colonization of the flock under investigation (P>0.05). In the absence of other flocks, 4 of 8 flocks became colonized. In the presence of two and three poultry houses on the farm, respectively 0 of 1 and 3 of 6 flocks became colonized. In the farms with each 4, 5 and 8 poultry houses, the flock under investigation was negative for Campylobacter. In the farms with 4 and 5 houses, all the other poultry houses were also sampled and were all found negative for Campylobacter. It may be that good biosecurity was practiced on these farms, as this can help to either prevent or delay flock colonization as shown before [START_REF] Gibbens | A trial of biosecurity as a means to control Campylobacter infection of broiler chickens[END_REF]. In flock 10, in total 8 poultry houses were present on the farm. At 28 days, six flocks on this farm were sampled of which two were found colonized by an indistinguishable strain of Campylobacter. At 42 days, depletion already occurred for these two flocks. The remaining six flocks tested were all negative for Campylobacter.

Multiple typing was helpful for genotyping the 92 Campylobacter isolates. Using FAFLP, 20 genotypes could be distinguished and using PFGE, 19 genotypes could be distinguished.

FAFLP and PFGE results showed to be complementary as when FAFLP and PFGE were A c c e p t e d M a n u s c r i p t 16 combined, this resulted in an increased total of 22 genotypes (+ 2 variants). FlaA-RFLP was not complementary to the FAFLP-PFGE combination, with one exception.

Multiples types of Campylobacter, as determined by FAFLP, PFGE and flaA-RFLP, were detected during rearing in three of the seven positive broiler flocks (i.e. 42.9%). This is in accordance with data presented by [START_REF] Shreeve | The carry-over of Campylobacter isolates between sequential poultry flocks[END_REF], [START_REF] Hein | Campylobacter jejuni isolated from poultry and humans in Styria, Austria: epidemiology and ciprofloxacin resistance[END_REF] and [START_REF] Bull | Sources of Campylobacter spp. colonizing housed broiler flocks during rearing[END_REF] who found that respectively 40%, 77% and 40% of flocks were colonized with more than one Campylobacter genotype. According to [START_REF] Höök | Genotype dynamics of Campylobacter jejuni in a broiler flock[END_REF], two hypotheses may explain the subsequent addition of genotypes: either subsequent introductions of Campylobacter or frequent mutations of the dominant clones. Although no firm conclusions can be drawn from our study, both processes could have taken place in some flocks.

Subsequent introduction has occurred in some flocks as genotypes of strains differed greatly for flock 2 (C. jejuni genotype P 1 /P 2 ,-,9,9 and C. coli genotype S,1,1,1), for flock 9 (C. jejuni genotypes H/N 1 /N 2 ,7,7,7 and B,8,8,8) and for flock 11 (C. jejuni genotypes Q 1 /Q 2 ,15,1,7; I,11,11,7 and O,11'/12,12,7). However, in some of these flocks related genotypes were found, supporting the mutation hypothesis: for flock 9 (C. jejuni genotypes H,7,7,7 and N 1 ,7,7,7) and for flock 11 (C. jejuni genotypes O,12,12,7 and O,11',12,7). In this study, the Campylobacter genotype that first colonized the broilers was sometimes replaced by other genotypes. At depletion, multiple types were detected in two broiler flocks. Of the seven positive flocks, in four occasions strains were resistant to at least one antibiotic tested. Mostly resistance was towards tetracycline (four flocks), but also multiresistance (EmTc, CiNaTc) was found in two flocks .

Following colonization, the nipple water of nearly all colonized flocks was contaminated with strains indistinguishable from these isolated from the broilers illustrating the importance of transmission of Campylobacter via the nipple water. This has been postulated by others [START_REF] Evans | Introduction and spread of thermophilic campylobacters in broiler flocks[END_REF][START_REF] Gregory | Epidemiological study of Campylobacter spp. in broilers: source, time of colonization, and prevalence[END_REF]), but has now been confirmed by detailed isolate A c c e p t e d M a n u s c r i p t characterization. [START_REF] Chaveerach | Effect of organic acids in drinking water for young broilers on Campylobacter infection, volatile fatty acid production, gut microflora and histological cell changes[END_REF] have shown that acidified drinking water can prevent Campylobacter spread via drinking water in broiler flocks. It should be remarked that in one flock (flock 11), the nipple water and indoor footwear were contaminated with a C. jejuni strain with genotype A,10,10,7. As this strain was not recovered from the cecal drops, it might be unable to infect the broilers.

Campylobacter was found in the environment surrounding the broiler house in four of the seven positive flocks. In three cases, isolates from water puddles were indistinguishable from isolates of the broilers. On nearly all occasions, isolation was accomplished at the same sampling time in the flock and water puddles. Once (flock 7) however, the ditch water was contaminated prior to flock colonization with a strain indistinguishable from isolates found later in the broiler flock. The study by [START_REF] Bull | Sources of Campylobacter spp. colonizing housed broiler flocks during rearing[END_REF] also confirmed that water puddles were most frequently contaminated and they explained this by protection from desiccation, a stress to which campylobacters are particularly sensitive.

Wild birds have, under good management practices, no access to the broiler houses.

However, contaminated droppings can be brought into the house, e.g. by footwear. In our study, C. jejuni was detected in wild bird's faeces of 4 of all 12 flocks sampled. Once, also the flock was positive but it harboured another genotype. However, the wild bird's faeces found at farm 5 (negative flock) harboured a Campylobacter strain with a related genotype as these isolated from the broilers at farm 10. Both farms were at a distance of about 21 km. It has been shown by [START_REF] Petersen | Comparison of genotypes and serotypes of Campylobacter jejuni isolated from Danish wild mammals and birds and from broiler flocks and humans[END_REF] that the genotypes of the isolates from wild birds and from broilers are seldom the same, suggesting that the importance of wild birds as a reservoir of infection is limited. The presence of pet animals and livestock, such as pigs, cattle and sheep around the broiler house can also lead to contaminated material brought into the broiler house. In our study, cattle reared on two farms (7 farms sampled, N=16) were Campylobacter positive, but harbored C. hyointestinalis. In contrast, identical strains have The significance of rodents, mice in particular, as vectors and reservoirs of Campylobacter has been shown by [START_REF] Meerburg | Presence of Salmonella and Campylobacter spp. in wild small mammals on organic farms[END_REF]. In our study, Campylobacter infected mice or rats were not found (N=3). Anyhow, since most farms apply rodent control programs, rodents are no longer considered as a significant risk factor for introducing Campylobacter in broiler houses [START_REF] Berndtson | A 1-year epidemiological study of campylobacters in 18 Swedish chicken farms[END_REF]. More difficult to control are insects. All insects sampled in 12 flocks (N=33) in our study did not carry Campylobacter, while other studies reported on this and considered this a considerable risk for infection of broilers with C. jejuni and C. coli [START_REF] Hald | Flies and Campylobacter infection of broiler flocks[END_REF][START_REF] Hald | Influxed insects as vectors for Campylobacter jejuni and Campylobacter coli in Danish broiler houses[END_REF]). In the study by [START_REF] Hald | Influxed insects as vectors for Campylobacter jejuni and Campylobacter coli in Danish broiler houses[END_REF], the prevalence of Campylobacter spp.-positive flies in summer was considerably higher on a farm with pig production compared to these without other livestock, for which the prevalence was below 1.0%. In our study, pig production was apparent in only one farm, in which the flock status was Campylobacter negative and sampling occurred at the end of September. At this period, the prevalence in flies dropped to 2.0% for a farm with pig production in the study by [START_REF] Hald | Influxed insects as vectors for Campylobacter jejuni and Campylobacter coli in Danish broiler houses[END_REF]. Also, the caterpillars (2 flocks sampled, N=2), snails (1 flock sampled, N=2) and worms (5 flocks sampled, N=8) sampled in our study did not harbour Campylobacter.

In conclusion, molecular typing is an important tool to provide knowledge on the transmission of Campylobacter at farm level. Our results show that multiple genotypes of C. jejuni may be present in a commercial broiler flock during rearing. New types probably appeared either by subsequent introductions of Campylobacter or by frequent mutations of the dominant clones. The outdoor environment constitutes a risk for infecting the broilers: the ditch water was found contaminated prior to while the water puddles at the same moment as

  study, 18 Belgian broiler flocks and their environment inside and outside the poultry house were sampled and cultured for the presence of Campylobacter. Isolates were typed by fluorescent amplified fragment length polymorphism (FAFLP), pulsed field gel electrophoresis (PFGE), and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) of the flagellin (flaA and flaB) gene and their antimicrobial resistance was determined to determine contamination patterns.

  isolates with their source of isolation, genotypes and antibiotic resistance profile. Six flocks were colonized exclusively with C. jejuni and one flock (flock 2) was initially colonized with C. jejuni (day 14) but replaced by C. coli at depletion. The nipple water inside the broiler house of all colonized flocks was initially found positive either at the same sampling time as the flocks (flock 12) or at a next sampling time (flocks 2, 3, 7, 9, 11). In flock 16, the nipple water was not found contaminated during the study because the flock colonization was not found earlier than at 42 days with only 3 of 12 pooled cecal samples positive for Campylobacter.

  , 38.9% (N=18) of the broiler flocks became colonized with thermotolerant Campylobacter spp. during rearing. Other studies report prevalence at the farm level of 27%

  cattle next to the broiler house and subsequently in the broiler house[START_REF] Newell | Sources of Campylobacter colonization in broiler chickens[END_REF]. Samples of dog (10 flocks sampled, N=16), horse (3 flocks sampled, N=6), sheep (3 flocks sampled, N=5), deer (1 flock sampled, N=2) or pig faeces (1 flock sampled, N=1) were negative for Campylobacter in our study.

  . This study also clearly demonstrated that once Campylobacter is introduced in the flock, the nipple water is subsequently contaminated leading to a further spread of the organisms throughout the whole flock.

  Figure legend. Dendrogram derived from FAFLP patterns of 88 C. jejuni and 4 C. coli

Figure

  Figure

f number of positive samples per number of samples tested.g Genotype determined by a combination of FAFLP, PFGE-SmaI, FlaA-DdeI and FlaA-HinfI in that order. The number of samples from which each genotype was isolated, is given between brackets. If more than one

Acknowledgements

This study was financed by the Belgian State, Services of the Prime Minister -Federal Services for Scientific, Technical and Cultural Affairs in the framework of the programme "Scientific support of a prenormative investigation of the food sector in the frame of a durable development" and by the Federal Public Service Health, Food Chain Safety and Environment, project R-04/002-CAMPY. Petra Vanmol, Elly Engels, Veronique Ottoy, Jessy Claeys and Sandra Vangeenberghe are acknowledged for excellent practical assistance.

se

se se

-,18,1,7 (1) J,19,9,9 (1) ---14 d 0/5 0/1 0/1 0/1 OF , 0/1 H , 0/1 PU , 0/1 W 28 d 0/4 0/1 0/1 0/1 C , 0/1 D , 0/1 OF , 0/1 DH , 0/1 H , 0/2 PU 42 d 0/5 0/1 0/1 0/1 D , 0/2 DH , 0/1 OF n.a., not applicable -, not determined se, sensitive to all tested antibiotics a Flock number and number of poultry houses on the rearing farm (between brackets); age of the birds at sampling in days