Arshnee Moodley

Luca Guardabassi

Clonal spread of methicillin-resistant coagulase-negative staphylococci among horses, personnel and environmental sites at equine facilities

Keywords: Methicillin-resistant coagulase-negative staphylococci at equine facilities antibiotic resistance, commensal bacteria, veterinary hospitals, contamination

A cross-sectional study was carried out to investigate species distribution and clonality of methicillin-resistant coagulase-negative staphylococci (MRCoNS ) isolated from the nasal cavity of 39 horses and 32 veterinary or caretaker staff, and from 76 environmental surfaces at three equine facilities in Denmark. MRCoNS obtained by selective isolation were characterized by 16S rDNA sequencing, PCR detection of mecA, and pulsed-field gel electrophoresis (PFGE). MRCoNS were found in 32 (82%) horses, 20 (63%) humans and 50 (66%) environmental samples. The most common species was S. vitulinus (n=56), followed by S. sciuri (n=25), S. haemolyticus (n=14). Undistinguishable PFGE patterns were observed in MRCoNS of equine, human and environmental origin. A S. vitulinus clone and a S. haemolyticus clone were isolated from multiple horses, staff members and environmental sites within a farm and a veterinary hospital, respectively. The results indicated that methicillinresistant strains of these two species can be shared by and possibly exchanged between horses and personnel, either directly or through contaminated environments.

A c c e p t e d M a n u s c r i p t Introduction Coagulase-negative staphylococci (CoNS) comprise a diverse group of commensals found on the skin and mucosae in both animals and humans but some species are recognised as important opportunistic pathogens in humans [START_REF] Martins | Methicillin resistance in Staphylococcus aureus and coagulase-negative staphylococci: epidemiological and molecular aspects[END_REF]. S. epidermidis and S. haemolyticus are the two most important human pathogenic species, mainly associated with post surgical infections, indwelling and implanted foreign bodies [START_REF] Kloos | Update on clinical significance of coagulasenegative staphylococci[END_REF]. The role of CoNS as veterinary pathogens is less known but some species have been implicated in mastitis, wound infections and skin abscesses in various animal species [START_REF] Van Duijkeren | Methicillinresistant staphylococci isolated from animals[END_REF][START_REF] Taponen | Clinical characteristics and persistence of bovine mastitis caused by different species of coagulase-negative staphylococci identified with API or AFLP[END_REF]. In a Danish study (Winther et al., 2008), CoNS were cultured from 14% (16/114) clinical specimens from horses, including wound, respiratory, ocular and uterine infections.

Methicillin-resistant staphylococci, in particular methicillin-resistant S. aureus (MRSA), are resistant bacteria of great concern to public health. Methicillin resistance is more common in CoNS than in S. aureus and approximately 50-90% of human clinical isolates are reported as being resistant [START_REF] Von Eiff | Nationwide German multicenter study on prevalence of antibiotic resistance in staphylococcal bloodstream isolates and comparative in vitro activities of quinupristin-dalfopristin[END_REF][START_REF] Martins | Methicillin resistance in Staphylococcus aureus and coagulase-negative staphylococci: epidemiological and molecular aspects[END_REF]. Methicillin-resistant CoNS (MRCoNS) are common inhabitants on the nasal mucosa of healthy animals, especially horses (Yasuda et al., 2000;[START_REF] Vengust | Methicillin-resistant staphylococcal colonization in clinically normal dogs and horses in the community[END_REF]Bacgicil et al., 2007). In this cross-sectional study, the objectives were i) to determine whether MRCoNS clones can be shared by horses and veterinary or caretaker staff; ii) to identify the species involved; and iii) to assess MRCoNS contamination in the environment of equine facilities.

Materials and Methods

Sampling

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

The sampling sites were two equine referral hospitals on two Danish islands (Hospital A on Zealand and Hospital B on Funen) and one equine farm owned by the Danish Army. Human participation in the study was on a strict voluntary basis and personal information was kept anonymous. The 32 humans tested were healthy and not on antibiotic therapy at the time of sampling. Animal samples were randomly collected from all horses at the farm (n=20) and from accessible patients admitted at Hospitals A (n=9) and B (n=10). Horses at the farm were clinically healthy and not on antibiotic therapy at the time of sampling. Most patients at the two hospitals were subjected to antibiotic therapy. All samples were collected on a single day at each facility. Cotton swabs were inserted into each nostril, rolled on the mucosal membranes for 5 sec, and stored in Stuarts' medium (Statens Serum Institut, Copenhagen, Denmark) prior to laboratory analysis. Environmental swabs were collected from 50 objects handled by the staff, such as sinks and taps, stable doors, equine handling equipment, door and cupboard handles, surgical equipment, computer keyboards and telephones, treatment tables, kettles and radios.

MRCoNS isolation and identification

Methicillin-resistant staphylococci were detected after overnight enrichment in MRSA selective broth (Statens Serum Institute, Copenhagen, Denmark) at 37C, followed by plating on Oxacillin Resistance Screening Agar Base (ORSAB, Oxoid, Hampshire, United Kingdom).

After 24-48 hr incubation at 35C, a single presumptive methicillin-resistant staphylococcal colony (blue or white on ORSAB) was isolated from each sample and confirmed as methicillin-resistant by PCR detection of mecA (Zhang et al., 2004). Diversity of mecApositive isolates was assessed by Random Amplification of Polymorphic DNA (RAPD) analysis [START_REF] Bagcigil | Occurrence, species distribution, antimicrobial resistance and clonality of methicillin-and erythromycinresistant staphylococci in the nasal cavity of domestic animals[END_REF] and isolates exhibiting distinct RAPD profiles were identified by partial 16S rDNA sequencing using primers 1AF: 5'-AGAGTTTGATYMTGGCT-3' and A c c e p t e d M a n u s c r i p t 519R: 5'-GTRTTACCGCGGCTGCTG-3'.. Nucleotide sequences were compared with those on Genbank and sequences with the highest similarity score to a type strain was deemed the species identity.

PFGE analysis

PFGE was performed according to Murchan et al. (2003) with minor modifications [START_REF] Bagcigil | Occurrence, species distribution, antimicrobial resistance and clonality of methicillin-and erythromycinresistant staphylococci in the nasal cavity of domestic animals[END_REF]. A low-range PFGE Marker (New England Biolabs, Ipswich, England) was used as a molecular weight marker and for normalisation between gels. Gels were analysed using GelCompar II software (Applied Maths, Kortrijk, Belgium), and cluster analysis was performed by UPGMA based on the Dice similarity coefficient, with optimization and position tolerance set at 0.5% and 1.5%, respectively. A similarity cut-off value of 90% was used to define PFGE types. Within each PFGE type, isolates exhibiting minor band differences were assigned to distinct subtypes.

Results

MRCoNS were isolated from 32 (82%) horses, 20 (63%) humans and 50 (66%) environmental samples (Table 1). We identified 12 RAPD types and 16S rDNA sequencing of isolates representative of each type revealed the occurrence of six species: S. vitulinus (n=56), S. sciuri (n=25), S. haemolyticus (n=14), S. equorum (n=4), S. epidermidis (n=2) and S. saprophyticus (n=1). S. vitulinus was the most common MRCoNS species in horses (46%), humans (28%) and the environment (38%). The species distribution differed greatly between the three equine facilities. S. vitulinus accounted for 80% and 70% of MRCoNS isolates from the equine farm and Hospital B, respectively, but was less frequent among isolates from Hospital A (22%). S. haemolyticus and S. sciuri was isolated more frequently at Hospital A (30% and 49%, respectively) than at the other two sampling sites (< 10%). S. epidermidis and A c c e p t e d M a n u s c r i p t S. saprophyticus were only isolated from humans at the farm. S. equorum was only isolated at Hospital B, from a horse, a veterinarian and samples from the stall and radio. MRCoNS were isolated from all environmental sites and the most contaminated sites were wash and treatment areas (Table 1).

High variability of PFGE patterns was observed within the three major species. The degree of inter-species diversity in band patterns was notably greater for S. vitulinus (up to 60% and 58%, respectively) than for S. haemolyticus (up to 25%). Twenty-four distinct PFGE types were identified among the 56 S. vitulinus isolates (See Figure S1, supplementary file). A dominant S. vitulinus clone (Type A) was isolated from four horses, one human and various environmental sites within the farm, including wash areas, door handles, horse restraints, and stall bars (Figure 1a). Other less frequent indistinguishable PFGE patterns were shared between equine or human and environmental S. vitulinus isolates from the same equine facility. One indistinguishable S. vitulinus clone (Type E) was isolated from a veterinarian at Hospital B and a horse and stall bar at the Farm. At Hospital A, clonal spread of S. haemolyticus and S. sciuri was observed. Detection of the S. sciuri clone was limited to equine and environmental samples, whereas the S. haemolyticus clone occurred in all sample types (Figures 1b-c). The four S. equorum isolates from Hospital B and the two S. epidermidis isolates from the farm displayed genetically unrelated PFGE patterns (data not shown).

Discussion

MRCoNS were widely disseminated among horses, humans and environmental sites at the three equine facilities. The PFGE results demonstrated that specific methicillin-resistant S. vitulinus and S. hamolyticus clones were shared by various horses and humans with the same farm or hospital environment, indicating possible interspecies transmission, either directly or A c c e p t e d M a n u s c r i p t through the contaminated environment. Although methicillin-resistance in staphylococci is a public health concern, not all staphylococcal species are equally important as human or veterinary pathogens. S. haemolyticus is one of the most important pathogenic species among CoNS isolated from human infections [START_REF] Kloos | Update on clinical significance of coagulasenegative staphylococci[END_REF]. This species is also sporadically isolated from equine infections [START_REF] Van Duijkeren | Methicillinresistant staphylococci isolated from animals[END_REF]. On the contrary, S. vitulinus has not been associated with human nor animal infections.

Although clone sharing suggests that MRCoNS transmission between horses and humans may have occurred at two of the three equine facilities, the present study cannot be used to infer the direction of transmission. However, some speculations can be made based on the natural habitats of the species involved. Methicillin-resistant S. vitulinus is known to be a frequent commensal in the nasal cavity of horses [START_REF] Bagcigil | Occurrence, species distribution, antimicrobial resistance and clonality of methicillin-and erythromycinresistant staphylococci in the nasal cavity of domestic animals[END_REF] and is not associated with carriage in healthy humans [START_REF] Nagase | Isolation and species distribution of staphylococci from animal and human skin[END_REF][START_REF] Busscher | The prevalence of methicillin-resistant staphylococci in healthy horses in the Netherlands[END_REF]. Therefore, the S. vitulinus clone isolated from humans is likely to have originated from the horses or from the contaminated environment. In contrast, the S. haemolyticus clone at Hospital A could have originated from either horses or humans since this species is part of the normal nasal flora of both hosts.

Two previous studies [START_REF] Nagase | Isolation and species distribution of staphylococci from animal and human skin[END_REF][START_REF] Busscher | The prevalence of methicillin-resistant staphylococci in healthy horses in the Netherlands[END_REF] have reported S. sciuri as the most prevalent CoNS in horses. This observation is in contrast with our findings since S. vitulinus was the frequently isolated from horses at all three equine facilities. This discrepancy could be due to methodological factors. It should be noted that S. vitulinus and S. sciuri are members of the S. sciuri group and discrimination between the two species is difficult by conventional phenotypic methods [START_REF] Stepanovic | A comparative evaluation of phenotypic and molecular methods in the identification of members of the Staphylococcus sciuri group[END_REF]) such as those used in the two earlier studies [START_REF] Nagase | Isolation and species distribution of staphylococci from animal and human skin[END_REF][START_REF] Busscher | The prevalence of methicillin-resistant staphylococci in healthy horses in the Netherlands[END_REF]. The high frequency of S. vitulinus A c c e p t e d M a n u s c r i p t detected in this study by 16S rDNA sequencing suggests that isolates belonging to this species could have been misidentified as S. sciuri in the previous studies.

Historically, the presence of CoNS in clinical specimens is thought to be a result of contamination in both human and veterinary medicine. During the last decade, some species, mainly S. epidermidis and S. haemolyticus, have gained increasing attention as nosocomial pathogens in humans. Although no information is available on the frequency of nosocomial pathogens in veterinary hospitals, postoperative infections are a common complication in equine surgery and can result in intensive postoperative care, prolonged hospitalization, increased costs and mortality [START_REF] Santschi | Prevention of postoperative ifections in horses[END_REF]. [START_REF] Schnellmann | Presence of new mecA and mph(C) variants conferring antibiotic resistance in Staphylococcus spp. isolated from the skin of horses before and after clinic admission[END_REF] showed that horses entering a hospital become readily colonised by CoNS after hospitalisation. Persistence and clonal spread of methicillin-resistant S. haemolyticus have been demonstrated within a veterinary teaching hospital in Norway [START_REF] Sidhu | Persistence of multidrug-resistant Staphylococcus haemolyticus in an animal veterinary teaching hospital clinic[END_REF]. CoNS are able survive on many hospital surfaces such as floors, tables, fabric, gloves and medical devices [START_REF] Neely | Survival of enterococci and staphylococci on hospital fabrics and plastic[END_REF].

The property of CoNS to adapt to veterinary hospital environments was confirmed by our study. Both veterinary hospitals were found to be widely contaminated with CoNS, including surgical and medical equipment (Table 1).

Our study illustrates that MRCoNS clones, including clinically-relevant species such as S. haemolyticus, can be shared by and possibly exchanged between horses and humans in close contact, and that the environment of veterinary hospitals can be widely contaminated with these bacteria. Although human and animal health risks associated with these findings remain unknown, CoNS human infections are usually caused by the patient's own colonizing strain (Frebourg et al. 1999 andCosta et al. 2004). The study also provides interesting information Table 1: Occurrence and species distribution of methicillin-resistant coagulase-negative staphylococci (MRCoNS) among horses, humans and environmental sites at the three equine facilities under study.

  s c r i p t on ecology and epidemiology of MRCoNS within veterinary hospitals, and underlines the importance of infection prevention and control best practices in equine medicine. Winther L., Aalbaek B., Baptiste K.E., Guardabassi L. 2008. Antimicrobial susceptibility of Danish equine pathogens (2006-2008). Poster presentation, abstract book pp. 112. 4th International Conference on Antimicrobial Agents in Veterinary Medicine, Prague, 24-28 August 2008.Yasuda, R., Kawano, J., Onda, H., Takagi, M., Shimizu, A., Anzai, T. 2000. Methicillinresistant coagulase-negative staphylococci isolated from healthy horses in Japan. Am J Vet Res. 61, 1451-1455.Zhang, K., Sparling, J., Chow, B.L., Elsayed, S., Hussain, Z., Church, D.L., Gregson, D.B., Louie, T., Conly, J.M. 2004. New quadriplex PCR assay for detection of methicillin and mupirocin resistance and simultaneous discrimination of Staphylococcus aureus from coagulase-negative staphylococci. J.Clin. Microbiol. 42,[4947][4948][4949][4950][4951][4952][4953][4954][4955]

Figure legend Figure 1 :

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Acknowledgments

The study was supported by the EU Marie Curie Early Stage Training program TRAINAU (contract MEST-CT-2004-007819). We would like to thank Louise Krohn for help with sampling and isolation.


Nicole Schütze

Rüdiger Raue

Mathias Büttner

Dr Gottfried Alber
email: alber@rz.uni-leipzig.de

Inactivated parapoxvirus ovis activates canine blood phagocytes and T lymphocytes

A c c e p t e d M a n u s c r i p t 2 Abstract Inactivated parapoxvirus ovis (iPPVO) shows strong immunomodulatory activities in several species and is used in veterinary medicine as an immunostimulatory biological for the prevention and/or treatment of infectious diseases. In this study the immunostimulatory capacity of iPPVO on the innate immune system was investigated in vitro by the evaluation of induction of the oxidative burst and modulation of phagocytosis by canine blood leukocytes (polymorphonuclear cells and monocytes) of dogs. In addition, the activation of canine T lymphocytes was also studied. After stimulation with iPPVO the phagocytosis of FITClabeled Listeria monocytogenes was increased in canine blood monocytes and neutrophils.

Enhanced burst rates by canine monocytes stimulated with iPPVO were observed and the MHC-II expression on canine CD14+ monocytes was elevated following stimulation with iPPVO compared to the stabiliser control. Canine CD4+ T cells were activated for oligoclonal proliferation in response to iPPVO. This study shows that iPPVO is able to stimulate both phagocytotic and T-cell-dependent immune mechanisms in canine blood leukocytes. keywords: cell activation, phagocytosis, oxidative burst, monocyte/macrophages, virus A c c e p t e d M a n u s c r i p t

Introduction

The family of Poxviridae contains the largest known DNA viruses. Due to their complex genetic structure poxviruses possess various mechanisms for immune evasion by utilizing a variety of genes encoding virulence proteins that counteract the host immune response.

Otherwise attenuated poxviruses have immunogenic properties and induce strong innate host immune response [START_REF] Burgers | Construction, characterization, and immunogenicity of a multigene modified vaccinia Ankara (MVA) vaccine based on HIV type 1 subtype C[END_REF][START_REF] Gherardi | Recombinant poxviruses as mucosal vaccine vectors[END_REF], probably due to facilitated cellular uptake by macropinocytosis [START_REF] Mercer | Vaccinia virus uses macropinocytosis and apoptotic mimicry to enter host cells[END_REF]. Parapoxvirus ovis (PPVO) shows a restricted host range for sheep and goats with a tropism to the skin and mucous membranes and lack of systemic virus spread [START_REF] Büttner | Parapoxviruses: from the lesion to the viral genome[END_REF][START_REF] Mckeever | Studies of the pathogenesis of orf virus infection in sheep[END_REF]. The virus provides proteins that are viral variants of soluble host immune factors such as an inhibitor of ovine GM-CSF and IL-2 (GIF), viral IL-10, that is very similar to ovine IL-10 and others such as viral vascular endothelial growth factor (VEGF) [START_REF] Deane | Orf virus encodes a novel secreted protein inhibitor of granulocyte-macrophage colony-stimulating factor and interleukin-2[END_REF][START_REF] Imlach | Orf virusencoded interleukin-10 stimulates the proliferation of murine mast cells and inhibits cytokine synthesis in murine peritoneal macrophages[END_REF][START_REF] Lateef | Orf virus-encoded interleukin-10 inhibits maturation, antigen presentation and migration of murine dendritic cells[END_REF][START_REF] Scagliarini | In vitro activity of VEGF-E produced by orf virus strains isolated from classical and severe persistent contagious ecthyma[END_REF]. The stringent host specificity of parapoxvirus ovis results from capture of host-derived protective immunomodulatory factors [START_REF] Haig | Immunomodulation by virulence proteins of the parapoxvirus orf virus[END_REF][START_REF] Haig | Immunity and counter-immunity during infection with the parapoxvirus orf virus[END_REF]. For an effective immune response against PPVO, CD4+ T-cells and a humoral immune response seem to be relevant. However, the duration of acquired PPVO-specific immunity is transient commonly resulting in re-infection of the host [START_REF] Haig | Ovine diseases[END_REF][START_REF] Lloyd | In vivo T-cell subset depletion suggests that CD4+ T-cells and a humoral immune response are important for the elimination of orf virus from the skin of sheep[END_REF].

The known immunostimulating properties on the innate immune system combined with a weak and transient immune response to the virus makes PPVO an optimal candidate for an immunoactivator. These characteristics are even preserved after inactivation. The immunostimulant Zylexis ® is based on the chemically inactivated PPVO (iPPVO) strain D1701 originally isolated from a diseased lamb [START_REF] Mayr | Control of contagious ecthyma in sheep by means of a new paranteral cell culture derived live vaccine[END_REF]. Before its use as an immunostimulant the strain D1701 had been attenuated by serial cell culture passages which led to substantial genomic changes [START_REF] Cottone | Analysis of genomic rearrangement and subsequent gene deletion of the attenuated Orf virus strain D1701[END_REF]. The attenuated and genetically A c c e p t e d M a n u s c r i p t 4 stable strain D1701 was successfully utilized for the aid in the prevention and treatment of infectious diseases in non-permissive species, especially in several herpes virus infection models [START_REF] Castrucci | The use of immunomodulators in the control of infectious bovine rhinotracheitis[END_REF][START_REF] Weber | Inactivated parapoxvirus ovis (Orf virus) has antiviral activity against hepatitis B virus and herpes simplex virus[END_REF][START_REF] Ziebell | The use of Baypamun N in crowding associated infectious respiratory disease: efficacy of Baypamun N (freeze dried product) in 4-10 month old horses[END_REF]. A major anti-viral effect may result from the strong interferon (IFN)-inducing capacity of PPVO [START_REF] Büttner | Interferon induction in peripheral blood mononuclear leukocytes of man and farm animals by poxvirus vector candidates and some poxvirus constructs[END_REF].

In this study the immunomodulatory effects of iPPVO were investigated on selected mechanisms of the innate immune response by canine leukocytes. The internalisation and degradation of pathogens in phagocytotic cells form a first barrier of the immune system against pathogens [START_REF] Serbina | Monocyte-mediated defense against microbial pathogens[END_REF][START_REF] Egan | Understanding the multiple functions of Gr-1(+) cell subpopulations during microbial infection[END_REF]. The engulfment of pathogens by phagocytosis as well as the production of bactericidal active reactive oxygen intermediates (ROI) is primarily achieved by monocytes/macrophages and neutrophils. The latter process is described as oxidative burst because the responsible enzyme complex NADPH oxidase assembles in activated phagocytes and increases consumption of oxygen [START_REF] Roberts | Oxidation of NADPH by Polymorphnuclear Leucocytes during phagocytosis[END_REF]. The impact of iPPVO during early immune responses such as phagocytosis and oxidative burst has not completely been characterized particularly in dogs.

Following uptake and destruction of pathogens by antigen-presenting cells (APC) the pathogens are processed to antigenic peptides and bound to major histocompatibility complex (MHC) molecules for presentation to T cells. In the process the MHC molecules are upregulated. For MHC-II molecules the class II transactivator (CIITA) is the master regulator of MHC-II transcription that modulates the expression of MHC-II genes [START_REF] Drozina | Expression of MHC II genes[END_REF].

In this context it was reported that in PPVO-infected sheep an accumulation of MHC-II+ dendritic cells in skin lesions was found [START_REF] Haig | The immune and inflammatory response to orf virus[END_REF].

Consequently, the activation of T-cells by iPPVO was a further topic of this investigation. In general, naive T cells that recognize pathogen-derived antigenic peptides/MCH complexes with their T cell receptor become activated and start to proliferate. Following infection with PPVO in sheep, lymphoproliferation in draining lymph nodes and also in efferent lymph was A c c e p t e d M a n u s c r i p t 5 found some days later [START_REF] Yirrell | Response of efferent lymph and popliteal lymph node to epidermal infection of sheep with orf virus[END_REF]. In a non-permissive host system such as swine in vitro T cell proliferation following stimulation with iPPVO was also observed. In this system the predominant proliferation of T helper cells was induced by iPPVO functioning in a superantigen-like manner independent of antigen processing by APC, demonstrated by a general inhibition of proliferation by an inhibitory anti-MHC II antibody (Fachinger et al., 2000a).

In this report we demonstrate by several in vitro assays for the first time that iPPVO enhances phagocytosis and oxidative burst in canine neutrophils and/or monocytes as well as induces the up-regulation of MHC-II and the proliferation of T helper cells.

Materials/ Methods

Reagents

Lipopolysaccharide (LPS) from Salmonella Abortus-equi (Alexis ® , Gruenberg, Germany); phorbol 12-myristate 13-acetate (PMA, Alexis ® , Gruenberg, Germany); dihydrorhodamine 123 (Invitrogen, Karlsruhe, Germany), fluorescein isothiocyanate (FITC) and cytochalasin D (SIGMA-Aldrich, Taufkirchen, Germany) were purchased. Bacteria Listeria monocytogenes strain EGD were heat-inactivated (hi) at 60 °C for 1 h and 5 x 10 8 colony forming units (cfu), incubated with 0.5 mg/ml FITC for 1 h at 37 °C for labelling and afterwards washed 3 times with PBS.

Virus

PPVO was chemical inactivated (iPPVO). Pharmaceutical formulation of commercial available iPPVO (Zylexis ® ) for dogs and cats with at least 230 IFN units of iPPVO strain D1701 per dose, the stabalizer control (polygeline) as well as purified iPPVO (pu iPPVO;

A c c e p t e d M a n u s c r i p t 6 without stabilzer) were supplied by Pfizer Animal Health, UK. One dose of pharmaceutical formulation (in the following declarated as iPPVO) contains 1 x 10 7 TCID 50 of PPVO (preinactivation) and was resuspended in 1 ml of PBS or medium. For most experiments a 2-fold dilution series of one dosis was used starting from 1:4 up to 1:32 (2.5 x 10 6 , 1.25 x 10 6 , 0.63 x 10 6 and 0.31 x 10 6 TCID 50 /ml).

Animals/blood

Blood was obtained from healthy dogs (breeds: beagles, n = 8; hybride, n = 1) by venepuncture of the anterior cephalic vein into heparinised vacutainer tubes (4 µg/ml lithiumheparin; Kalbe-Labortechnik, Nümbrecht-Elsenroth). Dogs belonged to the College of Veterinary Medicine, University of Leipzig. All procedures were performed in accordance with local legislations and animal welfare legislations.

Phagocytosis assay

Heparinized blood (150 µl/sample) was stimulated with iPPVO or stabiliser control, LPS (5 µg/ml), or PBS for 20 min at 37 °C in a waterbath. For specific inhibition of phagocytosis Cytochalasin D (5 µg/ml) was co-incubated. 100 µl of FITC-labelled L. monocytogenes was added at a multiplicity of infection (MOI) of 10 relative to leukocytes (microscopically counted in Tuerk's reagent in a Neubauer counting chamber) and incubated for a further 15 min at 37 °C. Phagocytosis was stopped by chilling samples on ice for 5 min. For lysis of erythrocytes, blood samples were incubated with lysis buffer (150 mM NH 4 Cl, 8 mM

Oxidative burst

Blood samples were stimulated with diluted iPPVO or stabiliser, LPS (5 µg/ml) or PMA (185 ng/ml) for 15 min at 37 °C in a water bath. The oxidative burst activity of the leucocytes was assessed by adding the respiratory burst-sensitive dye dihydrorhodamine 123 (DHR, 8.9 µg/ml) and incubating for 10 min at 37 °C [START_REF] Rothe | Dihydrorhodamine 123: a new flow cytometric indicator for respiratory burst activity in neutrophil granulocytes[END_REF]. Subsequently lysis of erythrocytes was carried out and cells were washed with PBS twice at 4 °C. The samples were measured by flow cytometry within 30 min. PMN and monocytes were gated by FSC/SSC and analysed separately. The burst rate equates to the percentage of oxidative burst active cells within one cell population (PMN or monocytes).

MHC-II expression

Peripheral blood leucocytes (PBL) were purified from heparinised blood by density centrifugation (1.077 g/ml; Histopaque, SIGMA-Aldrich). Cells (7 x 10 5 per well) were seeded out in 24 well, flat bottom plates (TTP, Midsci; St. Louis, MO, USA) and stimulated for 48 h with iPPVO (1 x 10 6 TCID 50 /ml) or pu iPPVO in RPMI medium supplemented with 10 % FCS, 2 µM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin. In an analogous manner, cells were stimulated with the same dilutions of stabiliser control or cultivated in medium. After 48 h cells were harvested with a cell scraper (TPP, Midsci; St. Louis, MO, USA). Before staining for FACS analysis cells were treated with a mixture of rat/mouse/dog normal serum (5 % of each in PBS) to block Fc receptors. To define the monocyte population, cells were stained with anti-human-CD14 Alexa Fluor ® 647 antibody (found to be cross-

CFSE proliferation assay

PBL were purified from heparinized blood by densitiy centrifugation using Histopaque® (Sigma Aldrich). Cells (1 x 10e7/ml 0.1 % BSA in PBS) were stained with 5 µM carboxy fluorescein diacetate succinimidyl ester (CFSE) at 37 °C in darkness for 10 min. Staining was stopped by adding the same volume of FCS for 3 min. The cells were washed three times with RPMI. Stained cells (1 x 10 5 per well) were seeded into a 96-well round-bottom plate (TTP, Midsci; St. Louis, MO, USA) and stimulated with diluted iPPVO or stabiliser control. As further controls medium and concanavalin A (ConA, 5 µg/ml) were used. Post cultivation for 7 d at 37 °C and 5 % CO 2 , cells were harvested and stained with anti-canine-CD4 PE and -CD8 Fluor ® 647 antibodies or rat-IgG2a PE and rat-IgG1 Fluor ® 647 antibodies as isotype controls, respectively. Cells were measured by FACS (Calibur ® , BD, Heidelberg, Germany).

All FACS analyses were performed with the CellquestPro software.

Statistical analysis

Statistical analysis of parametric data (Gaussian distribution) was performed by ANOVA.

Bonferroni´s multiple comparison test was used for post test to compare groups of interest. To analyse separate dilutions of iPPVO in oxidative burst assay paired t-tests was used for post test.

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

Results

IPPVO enhances the phagocytotic activity of canine monocytes and PMN

We investigated modulation of phagocytotic activity in canine leucocytes by iPPVO. Canine peripheral blood leucocytes, both monocytes and PMN showed a noticeable increase of phagocytotic activity after stimulation with iPPVO (Fig. 1A). As demonstrated by a dilution series of iPPVO, there is a dose-dependent increase of the phagocytosis rate. In comparison to LPS, iPPVO raised phagocytosis rate significantly (*** P<0.001; ** P<0.01).

Cytochalasin D, a potent inhibitor of actin polymerisation, which results in inhibition of phagocytosis, was used as an additional control. IPPVO-stimulated phagocytotic activity of canine leukocytes was found to be strongly reduced in the presence of cytochalasin D, to levels equivalent to those of the PBS control (Fig. 1B).

iPPVO induces oxidative burst in canine monocytes

To study the ability of iPPVO to induce oxidative burst in canine leukocytes, we stimulated blood samples from several dogs with iPPVO in comparison to stabiliser and PBS control. For detection of ROIs we utilized the oxidative burst sensitive substrate dihydrorhodamine123.

Canine monocytes showed dose-dependent moderate, but statistically significant (***P<0.001, **P<0.01, *P<0.05) increase of burst rates following stimulation with iPPVO in comparison to accordant stabiliser control (Fig. 2). In contrast, PMN were not activated for oxidative burst by iPPVO, whereas positive controls, LPS and PMA, clearly induced the oxidative burst in both cell populations, monocytes and PMN.

IPPVO induces MHC-II up-regulation on canine monocytes

MHC-II is up-regulated during activation of antigen presenting cells. In order to investigate the change of MHC-II molecules expression level in response to iPPVO canine monocytes

A c c e p t e d M a n u s c r i p t 10 were stimulated with pu iPPVO or the pharmaceutical formulation of iPPVO and compared to medium or stabiliser control. The MHC-II expression increased on iPPVO-stimulated cells in comparison to stabiliser control, as demonstrated in the histogram (Fig. 3A). The same result was found for pu iPPVO (without the stabiliser polygeline) stimulated cells compared to medium cultivated cells. Analyses of geometric means of fluorescence intensitiy (MFI) for MHC-II expression demonstrates a statistically significant difference between stimuli pu iPPVO vs. medium and iPPVO vs. stabiliser control (*** P<0.001, Fig. 3B).

iPPVO enhances proliferation of canine CD4+ T cells

Since T cell proliferation was descibed in permissive and non-permissive hosts [START_REF] Yirrell | Response of efferent lymph and popliteal lymph node to epidermal infection of sheep with orf virus[END_REF]Fachinger et aL., 2000a) we were interested in the ability of iPPVO to stimulate innate lymphocyte response and addressed this question for canine T cells by a proliferation assay with CFSE-labeled PBL. Profound T cell proliferative activity following stimulation with iPPVO was found in the CD4+ T cell fraction. This activity was substantially increased compared to stabiliser or medium control and showed dose dependency (ANOVA P<0.001 and Bonferroni´s multiple comparison test, *** P<0.001, * P<0.05; Fig. 4A). Surprisingly CD4/CD8 double positive (CD4+/CD8+) T cells were detectable in this proliferation assay. This cell population was found in medium, stabiliser control, and following stimulation with ConA or iPPVO. The iPPVO-induced proliferation rate of CD4+/CD8+ T cells was higher than in CD4+ T cells (Figure 4A). In contrast, most cytotoxic CD8+ T cells (CD8 single positive) did not proliferate in response to iPPVO. Only PBL from two dogs, that were found also to be highly responsive in the CD4+ and CD4+/CD8+ populations, proliferated by iPPVO stimulation, but the differences to the stabiliser control were not statistically significant. The analysis with CFSE demonstrated that only a portion of the total T cells divided. Most of these proliferating cells formed a cluster, i.e. most cells went through the same number of cell divisions (Fig 4B). Moreover, we also tested iPPVO for its ability to

Discussion

In this study it was shown that after iPPVO stimulation the phagocytotic activity was enhanced in canine monocytes and PMN. However, the induction of oxidative burst showed only a significant increase in the monocyte population. In human neutrophils enhancement of both phagocytosis and oxidative burst were demonstrated, whereas monocytes were not investigated [START_REF] Förster | Highly attenuated poxviruses induce functional priming of neutrophils in vitro[END_REF]. However, Fachinger et al. (2000b) could not find any modifying capacity by iPPVO stimulation on phagocytotic activity and oxidative burst in porcine PMN and monocytes. It has to be noted that these investigations were performed using two commercial test kits for human whole blood which were not validated for porcine blood.

The enhanced phagocytic activity of monocytes and PMN does not mean an obligatory recognition of iPPVO by these cell types, since cytokines (TNF-, GM-CSF) or mediators (like leucotrien B4) released by these cell types or other cell populations can enhance phagocytosis [START_REF] Cross | CR3-dependent phagocytosis by murine macrophages: different cytokines regulate ingestion of a defined CR3 ligand and complement-opsonized Cryptococcus neoformans[END_REF][START_REF] Peres | Specific leukotriene receptors couple to distinct G proteins to effect stimulation of alveolar macrophage host defense functions[END_REF] From depletion experiments in infected lambs it is known that CD4+ T-helper cells are important for the immune response against PPVO in sheep, whereas the role of CD8+ cytotoxic T cells is not completely understood [START_REF] Lloyd | In vivo T-cell subset depletion suggests that CD4+ T-cells and a humoral immune response are important for the elimination of orf virus from the skin of sheep[END_REF]. We tested the proliferation of PBL isolated from dogs never treated with iPPVO before to exclude a memory response resulting from previous priming. A dose-dependent proliferation of CD4+ T cells but mostly not of CD8+ T cells was observed. This is in agreement with results from a proliferation study with porcine PBL. In this study it was shown that T-helper cells (CD4high), expressing activation marker CD25 and MHC-II are predominant after cultivation A c c e p t e d M a n u s c r i p t 13 in the presence of iPPVO (Fachinger et aL., 2000b). In a subsequent study this group demonstrated that iPPVO functions as a superantigen, i.e. iPPVO was not processed by APC but activated T cells by binding directly to MHC molecules (Fachinger et aL., 2000a).

Surprisingly we could identify a further T-cell subpopulation in 7d-cultivated PBL, i.e. CD4+/CD8+ double positive T cells. This CD4+/CD8+ T cell subpoulation, that could not only be stimulated by iPPVO, but also by ConA, showed an increased proliferation activity in comparison to single CD4+ or CD8+ cells. Canine CD4+/CD8+ double positive T cells were found previously in a similar PBL cultivation system consisting of ConA together with recombinant human IL-2 [START_REF] Kato | A novel culture method of canine peripheral blood lymphocytes with concanavalin a and recombinant human interleukin-2 for adoptive immunotherapy[END_REF]. Human peripheral CD4+/CD8+ T cells were characterized as anti-viral effector memory cells [START_REF] Nascimbeni | Peripheral CD4(+)CD8(+) T cells are differentiated effector memory cells with antiviral functions[END_REF]. From this we suggest that CD4+/CD8+ T cells are activated T cells. Use of CFSE to assess proliferation of CD4+ T cells allows tracing of cell divisions [START_REF] Lyons | Determination of lymphocyte division by flow cytometry[END_REF]) contrary to previous work, which utilised 3 H-thymidine incoorporation (Fachinger et aL., 2000b). By our method an oligoclonal proliferation was revealed, that is characterised by synchronised cell divisions, since proliferating CD4+ T cells cluster at the same CFSE intensity (see Fig. 4B). Since oligoclonal activation is one of the key features of superantigens [START_REF] Irwin | Enterotoxin residues determining T-cell receptor V beta binding specificity[END_REF], it is tempting to speculate that iPPVO activates canine T cells in a superantigen-like manner.

Further, another group demonstrated by stimulation with the superantigen staphylococcus enterotoxin B that reactive T cells (V8+) undergo a discrete number of cell divisions before cells go into apoptosis [START_REF] Renno | Cutting edge: apoptosis of superantigen-activated T cells occurs preferentially after a discrete number of cell divisions in vivo[END_REF]. The exact mechanism of iPPVO-induced proliferation of canine CD4+ T cells as well as the proliferating cell population need further characterisation.

In summary, the induction of several early innate immune mechanisms as well as proliferation of CD4+ and CD4+CD8+ T cells by iPPVO was shown in canine cells for the first time. This broad activation profile promotes iPPVO as a potentent immunoactivator.

Figure 2: Induction of oxidative burst by iPPVO in canine monocytes

Oxidative burst in canine leucocytes post stimulation with iPPVO, stabiliser, PMA (185 ng/ml), or LPS (5 µg/ml) was detected with ROI sensitive DHR (8.9 µg/ml) by FACS.

Burst rate represents percentage of rhodamine-positive cells in the indicated cell population.

Data are derived from 6 independent experiments using three dogs. Statistical analysis was performed with ANOVA (P<0.001) and paired t-tests for the separate dilutions (*** P<0.001, ** P<0.01, * P<0.05).

Figure 3: MHC-II up-regulation on canine monocytes in the presence of iPPVO

PBL purified from peripheral blood were cultivated for 48 h and stimulated with purified (pu) iPPVO (1 x 10 6 TCID 50 /ml) or the pharmaceutical formulation of iPPVO (diluted 1:4).

As controls cells were cultivated in the presence of LPS (1 µg/ml), in medium or stabiliser (1:4). All CD14+ cells were analysed for expression level of MHC-II on the cell membrane.

KHCO 3 ,

 3 1 mM EDTA; pH 7.0) for 10 min. The lysis was stopped by adding PBS (4 °C), and cells were washed twice (4 °C). Cells were fixed with 1 % v/v formaldehyde in PBS and measured within 60 min by flow cytometry (FACS Calibur, Beckton-Dickinson, Heidelberg, Germany). Extracellular FITC-labeled bacteria were quenched by the addition of 1 mg/ml trypan blue before measurement. Polymorphonuclear cells (PMN) and monocytes were gated

  A c c e p t e d M a n u s c r i p t 7 by forward/sideward scatter (FSC/SSC) and analysed separately. The phagocytosis rate is the percentage of phagocytic active cells within one cell population (PMN or monocytes). Since phagocytosis rates of PBS controls differed between experiments, net phagocytosis rates were used to normalize all experiments (PR = PR treatment -PR PBS ).

A c c e p t e d M a n u s c r i p t 8 reactive

 8 with dog CD14 by the manufactures; Serotec, Düsseldorf, Germany). The cells were counterstained with anti-canine-MHC-II FITC, clone YKIX-334-2[START_REF] Cobbold | Monoclonal antibodies that define canine homologues of human CD antigens: summary of the First International Canine Leukocyte Antigen Workshop (CLAW)[END_REF] or rat-IgG2a FITC isotype control antibody (Serotec, Düsseldorf, Germany).

  canine B cells. CD19+ B cells did not show any proliferation after 7 d cultivation in the presence of iPPVO (data not shown). From this results we conclude that in contrast to ConA, which induces a polyclonal expansion, iPPVO stimulates an oligoclonal proliferation of canine CD4+ single and CD4+/CD8+ double positive T cells, which were synchronized in their cell divisions.

  [START_REF] Friebe | Immunomodulatory effects of inactivated parapoxvirus ovis (ORF virus) on human peripheral immune cells: induction of cytokine secretion in monocytes and Th1-like cells[END_REF] showed that stimulation of human peripheral mononuclear cells (PBMC) and whole blood with iPPVO and ConA induced secretion of the proinflammatory cytokines TNF-α and IL-6. However, further investigations are necessary to clarify the mechanisms of the iPPVO actions particularly in several species.

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

 12 and adaptive immunity are connected via antigen processing and presentation, which results in the presentation of antigenic peptides to T cells on the MHC molecules. MHC class II (MHC-II) determinants present antigens to CD4+ T helper cells, which are the main regulators of the adaptive immune response. In PPOV infected sheep a dense accumulation of MHC-II+ dendritic cells in the skin lesions was observed (Haig et aL., 1997). Here the upregulation of the MHC-II expression on canine monocytes was shown by iPPVO in vitro. IFN- induces MHC-II transcription via CIITA (Drozina et aL., 2005). An iPPVO-induced release of IFN- was shown by several groups (Fachinger et al., 2000b; Friebe et al., 2004; Voigt et al., 2007; Weber et aL., 2003). It seems possible that the observed MHC-II upregulation on canine monocytes is mediated by IFN- production induced by iPPVO. Nitric oxide (NO) is a downstream mediator from IFN- (Blanchette et al., 2003). However, NO release from cultivated PBL following stimulation with iPPVO was not observed (data not shown). In this context it needs to be noted that using CD14 as a marker to define the monocyte population does not definitely exclude dendritic cells (DC) from this cell population. It is unclear, whether canine blood derived DC express CD14 in vivo as demonstrated for in vitro DC differentiation. The expression of CD14 on monocyte-derived DC appears to depend on differentiation factors such as Flt-3L (fms-like tyrosine kinase 3 ligand) (Ibisch et al., 2005; Wang et al., 2007; Mielcarek et al., 2007).

Figure captions Figure 1 :

 captions1 Figure captions

  Cells were counterstained with anti-canine-MHC-II mAb or isotype control before. The fluorescence intensity was measured by flow cytometry. A) Histogram for electronically

  shows the fluorescence intensity of bound anti-MHC-II-FITC and isotype control (rat IgG2a-FITC). B) Mean + SD of the geometric means of fluorescence intensity of the MHC II fluorescence channel from all tested dogs (n = 8). Statistical analysis was performed with ANOVA (P<0.001) and Bonferroni´s multiple comparison test (*** P<0.001).

Figure 4 :

 4 Figure 4: Proliferation of canine CD4+ T-cells in the presence of iPPVO

Figure 1 Page 21 of 23 A

 123 Figure 1

Figure 2 A

 2 Figure 2

Figure 3 A

 3 Figure 3

Figure 4

 4 Figure 4

Acknowledgement

We thank J. Richter for technical assistance and I. Hochheim from the Institute of Pharmacology for professional blood sampling. These studies were funded by Pfizer Animal Health.

Conclusion:

In this report we show nonspecific immunostimulatory activity of iPPVO for canine leukocytes, that contributes to evidence of effectiveness of iPPVO in the nonpermissive host dog.


Alieda Van Essen-Zandbergen

Hilde Smith

Kees Veldman

Dik Mevius

In vivo transfer of an incFIB plasmid harbouring a class 1 integron with gene cassettes dfrA1-aadA1

Transfer of resistance genes from bacteria from food producing animals to human pathogens is a potential risk to human health. The aim of this study was to determine in vivo transfer of a plasmid harbouring a class 1 integron containing gene cassettes dfrA1-aadA1 from Salmonella to E. coli and the influence of the use of antimicrobials on this transfer. Thirty four-days-old SPF chickens colonized with E. coli K12 were divided into 3 groups of 10 animals each, and placed in separate isolators.

Eleven days after inoculation with E. coli K12 the chickens were inoculated orally with 10 4 CFU of S.

IncFIB plasmid harbouring a class 1 integron containing gene cassettes dfrA1-aadA1 from Salmonella to E. coli, with or without selective pressure of antibiotics in chickens.

enterica spp. enterica serovar Typhimurium containing a plasmid harbouring a class 1 integron with gene cassettes dfrA1-aadA1. Two days after the administration of S. Typhimurium 1 group was treated orally with doxycycline, 1 group orally with trimethoprim/sulphamethoxazole and 1 group remained untreated (control group). E. coli K12, S. Typhimurium and the transconjugants were isolated from cloacal samples on selective MacConkey agar plates. Transfer of the plasmid was confirmed by plasmid characterization, PCR, PFGE and hybridization. Plasmid mediated transfer of a class 1 integron was observed almost immediately after inoculation with S. Typhimurium. Treatment of the chickens with antibiotics had neither a positive nor a negative effect on the transfer rates. In addition to the resistance genes located on the integron, resistance genes encoding for tetracycline and amoxicillin resistance transferred from the donor strain as well. The resistance genes and the integron were located on a 130 kb sized IncFIB plasmid. Our data demonstrate in vivo transfer of an

Manuscript

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

Introduction

Antimicrobial resistance and, in particular, multidrug resistance (MDR) is an increasing problem worldwide. Exposure to MDR bacteria via the food chain is considered a potential risk to human health either by food borne infections with resistant pathogens or by horizontal transfer of resistance genes from bacteria from food producing animals to human pathogens [START_REF] Box | Integrons in Escherichia coli from food-producing animals in The Netherlands[END_REF][START_REF] Leverstein-Van Hall | Multidrug resistance among Enterobacteriaceae is strongly associated with the presence of integrons and is independent of species or isolate origin[END_REF][START_REF] Poppe | Acquisition of resistance to extended-spectrum cephalosporins by Salmonella enterica subsp. enterica serovar Newport and Escherichia coli in the turkey poult intestinal tract[END_REF]. Integrons are genetic elements able to link resistance genes as gene cassettes. If integrons are located on plasmids or transposons they transfer linked resistance genes within or between bacterial species [START_REF] Fluit | Resistance integrons and super-integrons[END_REF]. Linkage of resistance genes is considered to be involved in the transfer of MDR through co-selection by using individual antimicrobials [START_REF] Fluit | Class 1 integrons, gene cassettes, mobility, and epidemiology[END_REF].

In both MDR Salmonella and MDR E. coli from broiler chickens plasmids containing class 1 integron with gene cassettes dfrA1-aadA1 is frequently found in the Netherlands (van [START_REF] Van Essen-Zandbergen | Occurrence and characteristics of class 1, 2 and 3 integrons in Escherichia coli, Salmonella and Campylobacter spp. in the Netherlands[END_REF][START_REF] Vo | A novel Salmonella genomic island 1 and rare integron types in Salmonella Typhimurium isolates from horses in The Netherlands[END_REF], as well as in many other countries [START_REF] Poppe | Characterization of antimicrobial resistance of Salmonella Newport isolated from animals, the environment, and animal food products in Canada[END_REF][START_REF] Riano | Detection and characterization of extended-spectrum beta-lactamases in Salmonella enterica strains of healthy food animals in Spain[END_REF]. In vitro transfer of plasmids harbouring this integron from E. coli of avian origin and Salmonella to E. coli K12 has been described (van [START_REF] Van Essen-Zandbergen | Occurrence and characteristics of class 1, 2 and 3 integrons in Escherichia coli, Salmonella and Campylobacter spp. in the Netherlands[END_REF]. Also in vivo transfer of plasmids in Enterobacteriaceae has been described in mouse intestine [START_REF] Garcia-Quintanilla | Conjugal transfer of the Salmonella enterica virulence plasmid in the mouse intestine[END_REF][START_REF] Schjørring | Transfer of antimicrobial resistance plasmids from Klebsiella pneumoniae to Escherichia coli in the mouse intestine[END_REF] and in turkey intestine [START_REF] Poppe | Acquisition of resistance to extended-spectrum cephalosporins by Salmonella enterica subsp. enterica serovar Newport and Escherichia coli in the turkey poult intestinal tract[END_REF]. However, information on in vivo transfer in young broiler chickens is lacking including the influence of the use of antimicrobials on this transfer. To study in vivo transfer of plasmid mediated class 1 integron containing gene cassettes dfrA1-aadA1 and the influence of antibiotics an animal trial in young chickens was performed. Since doxycycline and trimethoprim/sulphamethoxazole are commonly used in broiler chickens, these antibiotics were used in the animal experiment.

The aim of this study was to determine in vivo transfer of a plasmid harbouring a class 1 integron containing gene cassettes dfrA1-aadA1 from Salmonella to E. coli in chickens with or without the influence of doxycycline or trimethoprim/sulphamethoxazole.

Materials and methods

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

Experimental design of the in vivo transmission studies

In the animal trial S. enterica spp. enterica serovar Typhimurium S115.62 containing a plasmid harbouring a class 1 integron with a 1550 bp size variable region harbouring gene cassettes dfrA1-aadA1 was used as donor strain. This strain demonstrates resistance to amoxicillin, tetracycline, sulphamethoxazole and trimethoprim (van [START_REF] Van Essen-Zandbergen | Occurrence and characteristics of class 1, 2 and 3 integrons in Escherichia coli, Salmonella and Campylobacter spp. in the Netherlands[END_REF]. In the animal trial E. coli K12 was used as recipient because of its ability to acquire a class 1 integron harbouring plasmid that contained gene cassettes dfrA1-aadA1. Moreover E. coli K12 can be distinguished from commensal E. coli due to its rifampicin resistance and because it is indol negative. Bacterial cultures were grown overnight at 37ºC on Heart Infusion Agar (AcuMedia Manifactures, inc. Lancing, Michigan, USA) with 5% sheep blood prior to colonisation of the chickens.

Before the animal experiment, as a pilot study, 5 four-days-old Specified Pathogen Free (SPF) White Leghorn American (WLA) chickens were inoculated orally with 10

4 CFU E. coli K12 and 7 days later with 10 4 CFU S. Typhimurium S115.62. The purpose of this pilot study was to demonstrate colonisation of young chickens with the laboratory adapted E. coli K12 and to study the effect of administration of a potentially pathogenic S. Typhimurium. On day 5, 8, 9 and 10 after hatching E. coli K12 was isolated from cloacal samples using dry cotton swabs. On day 15, 16, 17 and 21 after hatching E. coli K12, S. Typhimurium and transconjugants were isolated by inoculating cloacal samples on selective MacConkey agar plates. The selective MacConkey agar plates contained either no contain antibiotics, 50 mg/L of rifampicin (batch no. 085K1929), 256 mg/L of trimethoprim (batch no.064K1256) or a combination of 50 mg/L of rifampicin and 256 mg/L of trimethoprim. The antibiotics used for the selective plates were obtained from Sigma-Aldrich (Poole, Dorset, UK).

Each swab was suspended in 1 ml of buffered peptone water, which was considered to be the 1:10 w/v suspension. The average weight of faecal samples on cloacal swabs taken from young chickens was 0.093 g ± 0.068 g. E. coli, S. Typhimurium and transconjugants were isolated from the undiluted, the 10 -1 , 10 -2 and 10 -3 diluted faecal suspension in a semi-quantitative manner. The viable count in the samples discriminated 5 different quantitative levels. Level 1 contained to 0-2 x 10 The donor strain, the recipient strain and a selection of transconjugants were quantitatively tested for susceptibility to sulphamethoxazole, trimethoprim, tetracycline, amoxicillin, chloramphenicol, neomycin, cefotaxime, gentamicin and ceftazidime with the broth microdilution method according to the CLSI guidelines (NCCLS, 2004), E. coli ATCC 25922 was included for quality control purposes.

The antimicrobial resistance genes present in the donor strain, the recipient strain and in a selection of transconjugants were determined by the miniaturized microarray Indentibac AMR04 (VLA Weybridge, Surrey, UK ) as described previously [START_REF] Batchelor | Development of a miniaturised microarray-based assay for the rapid identification of antimicrobial resistance genes in Gram-negative bacteria[END_REF].

The in vitro growth curves of the donor strain, the recipient strain and a selection of transconjugants were determined in Luria-Bertani medium (LB medium) by using the Bioscreen C (OY Growth Curves AB Ltd, Helsinki, Finland) following the instructions provided by the manufacturer. The donor strain, the recipient strain and the transconjugants were diluted 10 -1

-10 -8 in 200 µl LB-medium, the Optical Density of all dilutions was measured every 5 min for 24 h.

The donor strain and the transconjugants were tested for the presence of integron class 1 and for the presence of the 1550 bp variable region harbouring the gene cassettes dfrA1-aadA1 by PCR, as described previously (van [START_REF] Van Essen-Zandbergen | Occurrence and characteristics of class 1, 2 and 3 integrons in Escherichia coli, Salmonella and Campylobacter spp. in the Netherlands[END_REF].

Plasmid characterization

A c c e p t e d M a n u s c r i p t 5

The plasmid content of donor strain and the transconjugants were analysed using PCR Based

Replicon Typing (PBRT) with primer pairs targeting replicon regions, as described previously [START_REF] Carattoli | Identification of plasmids by PCR-based replicon typing[END_REF].

To determine the size of the plasmids, S1-PFGE [START_REF] Barton | A general method for detecting and sizing large plasmids[END_REF] was used. Total DNA of the donor strain and transconjugants was incorporated into SeaKem Gold agarose (Cambrex Bio Science Rockland Inc., Rockland, ME, USA), and digested for 45 min at 37ºC with 8 U S1-nuclease (Amersham Biosciences). PFGE was performed in a CHEF DRIII system on a 1% SeaKem Gold agarose gel using the following electrophoreses conditions: switch time 1-25 s, run time 17 h, voltage 6 V, angle 120º. Low range and middle range PFGE size standards (New England BioLabs) were included as molecular weight standards. DNA was stained with ethidium bromide.

Pulse Field Gel Electrophoreses and hybridization with a DIG labelled intI1 probe

To confirm the identity of the isolated strains, XbaI -PFGE was performed on the original S.

Typhimurium donor strain and the original E. coli K12 recipient, as well as on the isolated S.

Typhimurium, E. coli and transconjugants, as described by PulseNet (http://www.cdc.gov/pulsenet/protocols/ecoli_salmonella_shigella_protocols.pdf).

DNA from the gel was transferred to a positively charged nylon membrane Hybond N+ (Amersham Biosciences) using a fast blotting system (Bio-Rad Laboratories) following the instructions provided by the manufacturer and cross-linked by baking at 80ºC for 30 min. Hybridisation was performed overnight at 60ºC in Church hybridisation buffer [START_REF] Church | Genomic sequencing[END_REF] with a DIG-labelled intI1 PCR amplification product. Labelling the intI1 amplification product and detection was performed using the DIG-High Prime DNA Labelling and Detection Starter Kit I (Roche Applied Science) following the instructions provided by the manufacturer.

Results

In vivo transmission study

In the pilot study (data not shown) and in the in vivo transmission study (Figure 1a, b and c) E. coli K12 and S. Typhimurium were able to colonize the chickens' intestine immediately after inoculation and could be re-isolated in high rates of approximately 6x10 5 -6x10 6 CFU/gram faeces.

The chickens showed no clinical signs of disease other than a slight and transient diarrhea after In the present study transconjugants were observed almost immediately after inoculation with S. Typhimurium in the treated as well as in the untreated chickens (Figure 1a, b andc). This demonstrates that a high efficiency of in vivo conjugation existed. Treatment of the chickens with antibiotics had neither a positive nor a negative effect on the transfer rates.

Within 3 days after introduction of S. Typhimurium to the chickens' intestine (14 days after hatching) the viable count of the isolated rifampicin and trimethoprim resistant E. coli was equal to the total viable count of isolated E. coli. In the untreated group the viable count was equal on day 15 after hatching, demonstrating that al recipients became transconjugants and that no other commensal E. coli were isolated.

Pulse Field Gel Electrophoreses and hybridization with a DIG labelled intI1 probe

Figure 2 shows the XbaI-PFGE profiles from the original E. coli recipient, the original S.

Typhimurium donor strain as well as the profiles from a selected number of Salmonella strains reisolated from the chickens and a number of isolated transconjugants. The profiles of the Salmonella strains re-isolated from chickens were similar to the profile of the original S. Typhimurium, demonstrating that the original S. Typhimurium was able to colonize in the chickens' intestine. The profiles of all isolated transconjugants were almost similar to the profile of E. coli K12, except for an extra band of approximately 50 kb in the profile of the transconjugants. The intI1-DIG labelled probe hybridized with this 50 kb band of the transconjugants as well as with a 50 kb band of the Salmonella strains (results not shown) confirming the presence of a class 1 integron on a transferable element.

Characterization of the isolates

PCR amplification showed that all re-isolated Salmonella strains and all transconjugants contained a class 1 integron with a 1550 bp variable region harbouring gene-cassettes dfrA1-aadA1.

Similar to the S. Typhimurium donor strain, all isolated transconjugants demonstrated resistance to amoxicillin, tetracycline, sulphamethoxazole and trimethoprim. The miniaturized microarray showed the presence of the resistance genes aadA1, dfrA1, intI1, sul1, sul2, tem1, tetA, A c c e p t e d M a n u s c r i p t 7 strA and strB in the donor strain and in the transconjugants as well. All isolated transconjugants were indol negative similar to the E. coli K12 recipient.

Transconjugants and E. coli K12 showed identical growth abilities in vitro. Difference was observed between the growth curves of the S. Typhimurium donor strain and the transconjugants, S.

Typhimurium reached the log-phase and the stationary-phase approximately 2 hours sooner than the transconjugants.

S1-PFGE and hybridization of the donor strain and transconjugants showed the presence of a 130 kb sized plasmid (results not shown), which hybridized with the intI1-DIG labelled probe. This plasmid was identified as IncFIB by PBRT.

Discussion

In vitro transfer of a plasmid harbouring a class 1 integron with gene cassettes dfrA1-aadA1 from E. coli and Salmonella to E. coli K12 has been described (van [START_REF] Van Essen-Zandbergen | Occurrence and characteristics of class 1, 2 and 3 integrons in Escherichia coli, Salmonella and Campylobacter spp. in the Netherlands[END_REF].

The present study demonstrates the in vivo plasmid mediated transfer of this integron from an amoxicillin, streptomycin, tetracycline, sulphamethoxazole and trimethoprim resistant Salmonella Typhimurium S115.62 to E. coli K12. In addition to the resistance genes aadA1 and dfrA1 encoding for streptomycin and trimethoprim resistance located on the variable region of the integron and the resistance gene sul1 encoding sulphamethoxazole resistance located the 3'conserved segment of the integron, resistance genes strA, strB, sul2, tem1 and tetA encoding for streptomycin, sulphanomide, amoxicillin and tetracycline resistance transferred from the donor strain as well.

Transconjugants were observed almost immediately after inoculation with S. Typhimurium in treated as well as in untreated chickens. This demonstrates a high efficiency of in vivo conjugation, similar data were described previously [START_REF] Garcia-Quintanilla | Conjugal transfer of the Salmonella enterica virulence plasmid in the mouse intestine[END_REF][START_REF] Poppe | Acquisition of resistance to extended-spectrum cephalosporins by Salmonella enterica subsp. enterica serovar Newport and Escherichia coli in the turkey poult intestinal tract[END_REF] Because genes encoding for resistance to tetracycline, trimethoprim and sulphamethoxazole were co-transferred on the 130 kb IncFIB plasmid, the doxycycline and trimethoprim/sulphamethoxazole used in the experiment had no inhibitory effect on the S.

Typhimurium donor strain nor on the transconjugants but may have affected the growth of the recipient E. coli K12 (figure 1). The recipient E. coli K12 could not be re-isolated from chickens after colonization of S. Typhimurium in the chickens' intestine, as described previously [START_REF] Schjørring | Transfer of antimicrobial resistance plasmids from Klebsiella pneumoniae to Escherichia coli in the mouse intestine[END_REF]. This may be due to a reduction in viable count of the recipient E. coli K12 below the detection coli strains were able to colonize the chickens intestine very efficiently, indicating that the transconjugant were better adapted to the chicken intestines compared to the original E. coli K12 isolate. Because transconjugants and E. coli K12 showed identical growth abilities in vitro these data suggest that in addition to genes encoding for antibiotic resistance also genes facilitating colonization of bacteria in chickens were transferred from S. Typhimurium to E. coli, which was previously described to occur in mice [START_REF] Rice | Transferable Capacity for Gastrointestinal Colonization in Enterococcus faecium in a Mouse Model[END_REF]. Maybe the transfer of these colonization genes contributed to the success of the transfer of plasmids. Since in this study, antibiotics had no effect on the transfer rates of the plasmids. The study of [START_REF] Schjørring | Transfer of antimicrobial resistance plasmids from Klebsiella pneumoniae to Escherichia coli in the mouse intestine[END_REF] showed that during treatment with antibiotics plasmids were transferred to indigenous E. coli and treatment with antibiotics had effect on the colonization rates of Klebsiella pneumoniae. But in this study animals with a normal bacterial flora were used, while the present study used SPF chickens instead. This may explain the different results observed according to the antibiotic use. A c c e p t e d M a n u s c r i p t

4

  To study the influence of antibiotics on in vivo transfer of the integron, 30 four-days-old SPF WLA chickens colonized with E. coli K12 were divided into 3 groups of 10 animals and placed in separate isolators on day 4 after hatching. On day 11 all chickens were inoculated orally with 10 4 CFU S. Typhimurium S115.62. From day 13 to day 17, 1 group of chickens was daily treated with 100 ppm doxycycline orally through the drinking water, 1 group was daily treated with 40 ppm trimethoprim and 200 ppm sulphamethoxazole orally through the drinking water, and 1 group remained untreated (control group). On day 5, 6, 7, 10 through 25 after hatching E. coli K12, S. Typhimurium and transconjugants were isolated in a semi-quantitative manner from cloacal samples on selective MacConkey agar plates as described above. Doxycycline (Soludox 50%) and trimethoprim/sulphamethoxazole (Methoxazol-T) were obtained from Eurovet Animal Health BV, Bladel, the Netherlands.

  S. Typhimurium. During the experiment only the original Salmonella donor strain, the original E .coli K12 recipient strain and E. coli K12 transconjugants were isolated from the chicken intestines.

  CFU/g faeces, by the inhibitory or killing effect of the antibiotics administered or by the very efficient and fast in vivo conjugation to all recipient E. coli K12 strains. This was observed in the chickens treated with antibiotics as well as in the untreated chickens. In contrast, transconjugant E.

  It can be concluded that in vivo transmission of a IncFIB plasmid harbouring a class 1 integron with gene cassettes dfrA1-aadA1 from S. Typhimurium to E. coli occurred in high efficiency in young SPF chickens with or without selection pressure by antibiotics.

Fig. 1 .

 1 Fig. 1. Dynamics of the mean viable count levels of E. coli K12 (○), S. Typhimurium S115.62 (■) and transconjugants (▲) in 10 broiler chickens treated ( ) orally with 100 ppm doxycycline (a); 40 ppm trimethoprim and 200 ppm sulphamethoxazole (b); untreated, control group (c). The broiler chickens were orally inoculated with E. coli K12 ( ) and with S. Typhimurium S115.62 ( ). Semi-quantitative viable count level 1 samples contained <2x10 2 CFU/gram faeces; level 2, samples contained 2x10 2 -6x10 3 CFU/gram faeces; level 3 samples contained 6x10 3 -6x10 4 CFU/gram faeces; level 4 samples contained 6x10 4 -6x10 5 CFU/gram faeces; and level 5 samples contained 6x10 5 -6x10 6 CFU/gram faeces.

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Acknowledgements

Positive controls for the PCR Based Replicon Typing of the plasmids were kindly provided by Alessandra Carattoli from the Instituto Superiore di Sanità, Rome, Italy.

Funding

This study was part of project no. 802.190.5000, Characterization and transmission of resistance genes, funded by the Ministry of Agriculture, Nature and Food Quality.

Conflict of interest statement

None to declare

Ethics approval

In accordance with the Act on Experimental Animals, use of experimental animals is granted to CVI by permit from the Dutch Government. Every experimental use is evaluated and scrutinised by an Ethical Review Committee (ERC). This Committee evaluates ethical issues of animal experiments,