P D D Alves

J A Mcculloch

Sergine Even

C Le Maréchal

A Thierry

N Grosset

V Azevedo

C A Rosa

E Vautor

Yves Le Loir

Y Le

Molecular characterisation of strains isolated from

come   L'archive ouverte pluridisciplinaire

(2) Agrocampus Ouest, UMR1253 STLO, 85 rue de Saint Brieuc, 35042 Rennes, France;

(3) Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Brazil;

(4) PPR, AFSSA Sophia Antipolis, France § : equal contribution *: corresponding author yves.leloir@rennes.inra.fr A c c e p t e d M a n u s c r i p t 2 Abstract Staphylococcus aureus is an important pathogen in domestic ruminants. The main objective of this study was to determine the similarity of epidemiologically unrelated S. aureus isolates from bovine, ovine, and caprine hosts regardless the locus of isolation (nares and udder). By pulsed-field gel electrophoresis, 7 major pulsotypes were identified among 153 isolates recovered from 12 different regions of France as well as from Brazil, the USA and Belgium.

Typing of the accessory gene regulator (agr) and capsular (cap) serotype was carried out on all the isolates and revealed the predominance of agrI and III and of cap8 regardless the ruminant host species. Screening for MRSA was carried out by disk diffusion and revealed a prevalence of only 3.2% of MRSA among the strains tested. These results suggest the existence of a host rather than tissue specificity among S. aureus isolates colonising the ruminant species and suggest a limited transmission of those isolates between large (bovine) and small (ovine-caprine) ruminants. The agr class and cap types correlated with pulsotype clusters rather than with a specific host species. Antimicrobial resistance appears not to have contributed to the predominance of any given genotypes, and MRSA prevalence appears very low in ruminant isolates.

Keywords: Staphylococcus aureus, Ruminant, host specificity, mastitis, PFGE

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

Introduction

S. aureus is one of the most frequent etiologic agents of mastitis in bovines, ovines and caprines, rendering livestock unable to adequately produce milk, which results in heavy economic losses for the dairy industry [START_REF] Seegers | Production effects related to mastitis and mastitis economics in dairy cattle herds[END_REF]. Studies characterising ruminant isolates of S. aureus are scarce compared to the much more abundant data on human isolates.

Devriese et al. first described phenotypic differences between S. aureus strains isolated from humans and other animal hosts including bovines and ovines, and proposed a scheme for the distinction of several host biotypes [START_REF] Devriese | Characteristics of Staphylococcus aureus strains isolated from different animal species[END_REF]. Differences between host biotypes are also reflected at the genotypic level as determined by macro-restriction analysis of the chromosome [START_REF] Hennekinne | Discrimination of Staphylococcus aureus biotypes by pulsed-field gel electrophoresis of DNA macrorestriction fragments[END_REF]. Due to the specificity of host-pathogen interactions needed to produce mastitis, it has been postulated that the nature of the virulon and the regulation of its expression are determining factors when it comes to the ability of a strain to produce mastitis [START_REF] Vautor | Genetic differences among Staphylococcus aureus isolates from dairy ruminant species: A single-dye DNA microarray approach[END_REF]. The recent release of the complete genome sequence of S. aureus ET-3, a bovine isolate, provides new insight into the genomic basis of a putative host adaptation and the existence of host specific genetic traits in S. aureus isolated from bovine hosts [START_REF] Herron-Olson | Molecular Correlates of Host Specialization in Staphylococcus aureus[END_REF]. We recently showed that diversification of the S. aureus core genome correlated with host origin in ruminants [START_REF] Ben Zakour | Genome-wide analysis of ruminant Staphylococcus aureus reveals diversification of the core genome[END_REF].

However, in most studies, the panel of ruminant S. aureus strains comprised mostly strains isolated from cases of mastitis. The mammary gland tissues present characteristics such that some authors hypothesized that the specific traits found to be common in bovine strains were related to a tissue-rather than to host-specificity [START_REF] Van Leeuwen | Host-and tissue-specific pathogenic traits of Staphylococcus aureus[END_REF]. In the present study, we characterised several S. aureus isolates obtained from different sites of colonisation / infection, from ruminants in different geographic regions, by PFGE, and further characterised the strains as to their agr group and capsular polysaccharide genotype. The aims of this characterisation were to verify whether the strains groups reflected a host-or tissue-A c c e p t e d M a n u s c r i p t 4 adaptation, whether there is a predisposition of certain cap or agr types to colonise or infect certain ruminant hosts.

Material and Methods

Bacterial strains.

In order to minimise bias when evaluating clonality by PFGE, we chose to include in the study strains from different geographic and temporal backgrounds. A total of 153 S. aureus isolates were either chosen from existing collections (n=114) or were collected specifically for this study (n=39). Isolates from bovine (65 strains), ovine (57), and caprine (31) hosts were collected between 1964 and 2006 in France (12 different regions, n=117), and Brazil (2 different regions, n=30). The panel of strains included also isolates from Belgium (n=5) and the USA (n=1). Strains chosen from existing collections were confirmed to be S. aureus by a specific PCR test [START_REF] Baron | Development of a PCR test to differentiate between Staphylococcus aureus and Staphylococcus intermedius[END_REF]. The strains collected in this study were isolated from the udder (mastitic milk) and nares of bovine, ovine, and caprine hosts as described previously [START_REF] Vautor | Genotyping of Staphylococcus aureus isolated from various sites on farms with dairy sheep using pulsed-field gel electrophoresis[END_REF]. Samples were first grown on selective Baird-Parker medium.

Species identification was carried out on coagulase positive staphylococci using previously described PCR tests [START_REF] Baron | Development of a PCR test to differentiate between Staphylococcus aureus and Staphylococcus intermedius[END_REF][START_REF] Morot-Bizot | Development of a multiplex PCR for the identification of Staphylococcus genus and four staphylococcal species isolated from food[END_REF]. Details of the sampling used in this study (locus of isolation and host) are given in Table 1.

PFGE.

Pulsed-Field Gel Electrophoresis for the characterisation of the strain lineage was carried out according to previously described protocols (Prevost et al., 1992b). The band profiles obtained for each strain were analysed using the Bionumerics software, version 2.0 (Applied-Maths, Belgium).

Determination of the agr group.

Classification of strains into agr interference groups was carried out by PCR according to Gilot et al. [START_REF] Gilot | Comparative analysis of agr locus diversification and overall genetic variability among bovine and human Staphylococcus aureus isolates[END_REF], which type 5 (primers Cap5k1 andCap5k2) or type 8 (primers Cap8k1 and Cap8k2) alleles, as described by Verdier et al. [START_REF] Verdier | Identification of the capsular polysaccharides in Staphylococcus aureus clinical isolates by PCR and agglutination tests[END_REF]. Each strain was submitted to a reaction using either the Cap5k1 and Cap5k2 or the Cap8k1 and Cap8k2 primer pair in independent reactions.

Determination of the susceptibility of strains to oxacillin. Susceptibilities of strains to

oxacillin were evaluated by means of the Kirby-Bauer disk diffusion method as described previously (CLSI, 2007).

Statistical analyses.

A χ 2 test was used to determine the significance of occurrence of genes in a host specific group by use of Statgraphics version 5.1. The nominal P value for statistical significance was 0.05.

Results and discussion

S. aureus strains grouped with regard to their host-but not tissue-origin.

Macrorestriction profile analysis using PFGE is one of the most discriminative methods for S. aureus when compared to other genotyping methods [START_REF] Kuhn | Double-locus sequence typing using clfB and spa, a fast and simple method for epidemiological typing of methicillin-resistant Staphylococcus aureus[END_REF]Prevost et al., 1992a;[START_REF] Van Belkum | Comparison of Four Genotyping Assays for Epidemiological Study of Methicillin-Resistant Staphylococcus aureus[END_REF]. All the strains studied here grouped up into 7 clusters (A to Gfigure 1) when analyzed by PFGE considering a similarity cut-off of 48 %. Each cluster was significantly correlated (P<0.05) to the subfamily of the host (bovine for large ruminants or A c c e p t e d M a n u s c r i p t 6 ovine-caprine for small ruminants) from which the strains were isolated. Three clusters were majorly composed of bovine isolates: 42 out of 45 strains (93 %) are bovine isolates in cluster B; 7/10 strains (70 %) in cluster C; and 7/13 strains (54 %) in cluster E. The four other clusters comprised a great majority of small ruminants isolates: 24 out of 24 strains (100 %) are ovine-caprine isolates in cluster A; 43/49 strains (88%) in cluster D; 7/9 strains (78 %) in cluster F; and 3/3 strains (100 %) in cluster G. Strains isolated from small ruminants could not be distinguished as to whether they were ovine or caprine based on their pulsotype, as they were evenly distributed along clusters A, D, F, and G. Strains isolated from ovine and caprine hosts presented a similarity of up to 100 %, whereas no bovine strain presented a 100% similarity to a small ruminant strain. Previous studies based on CGH analysis of S. aureus isolates including a few bovine strains suggested the existence of tissue-rather than hostspecific genetic traits [START_REF] Van Leeuwen | Host-and tissue-specific pathogenic traits of Staphylococcus aureus[END_REF]. However, in these studies, only a few bovine mastitis isolates were analyzed amongst a large panel of human strains. Our results are in contrast to another work showing that there is little or no host preference among S. aureus presenting different genotypes [START_REF] Mork | Comparison of Staphylococcus aureus genotypes recovered from cases of bovine, ovine, and caprine mastitis[END_REF], in which isolates were obtained from milk collected from cases of clinical and subclinical mastitis in ewes kept for meat production. In the present study, we included strains isolated from the nares and udder of various ruminant hosts and results showed that they were evenly distributed amongst each of the clusters (Figure 1). Thus S. aureus strains clearly grouped up into clusters which correlated to the type of host they were isolated from. In contrast, there was no significant correlation between clusters and the locus of isolation, geographic origin or date of isolation, as strains from the same geographic origin and similar dates of isolation were found to be spread among different clusters. Our results are in accordance with previous studies which reported that host biotypes, as determined according to Devriese's scheme, are reflected by PFGE profiles [START_REF] Hennekinne | Discrimination of Staphylococcus aureus biotypes by pulsed-field gel electrophoresis of DNA macrorestriction fragments[END_REF]. A recent study by [START_REF] Sung | Staphylococcus aureus host specificity: comparative genomics of human versus animal isolates by multi-strain microarray[END_REF] which evaluated the relatedness of strains of human and non-human (ruminants and equines) origin also found A c c e p t e d M a n u s c r i p t 7 that bovine strains tend to cluster into a few specific clonal clusters, which are different to predominantly human clusters. In this study, small ruminant strains were grouped into a bovine and equine cluster (ST771), however, the limited number of small ruminant strains analysed (n=4) make it impossible to conclude on the specificity of small ruminant strains.

Lineages might be adapted to colonise a certain type of host independent of the site of colonisation/infection. The fact that there was significant correlation between the sub-family of the animal host and lineage, in spite of the strains having been isolated from very different geographical locations (all major regions of France, plus isolates from Brazil, Belgium, and the USA) and in different years (from 1964 to 2006), strongly indicates that a certain strain has a penchant for the type of host it is successful in living on. Our study is as of yet the most comprehensive study involving strains isolated from small ruminants. It is important to realise that bovine strains are different to ovine and caprine strains, for this must bear an impact on the adoption of strategies for the prevention of mastitis in small or large ruminants.

Determination of agr group.

The ability of S. aureus, as a species, to endure in different niches, playing different roles in the relationship with an animal host is testament to either the regulation of the expression of a panoply of accessory genes, or a variability of strains within the species, with lineages presenting a collection of genes specializing in a particular modus vivendi [START_REF] Ben Zakour | Genome-wide analysis of ruminant Staphylococcus aureus reveals diversification of the core genome[END_REF][START_REF] Fitzgerald | Evolutionary genomics of pathogenic bacteria[END_REF]. The expression of the accessory genes in S. aureus is under the control of a series of systems that interact with each other to form a network. Of all these systems, the accessory gene regulator (agr), a twocomponent quorum-sensing system has arguably been the most studied in S. aureus. Four interference classes related to genetic polymorphisms in the agr locus have thus far been described, namely, agr groups I, II, III and IV (Jarraud et al., 2002a;[START_REF] Ji | Cell Density Control of Staphylococcal Virulence Mediated by an Octapeptide Pheromone 10[END_REF]. So far, agr variability has been only rarely studied in isolates obtained from ruminants [START_REF] Gilot | Comparative analysis of agr locus diversification and overall genetic variability among bovine and human Staphylococcus aureus isolates[END_REF][START_REF] Reinoso | Genotyping of Staphylococcus aureus isolated from humans, bovine subclinical mastitis and food samples in Argentina[END_REF][START_REF] Vautor | Genetic differences among Staphylococcus aureus isolates from dairy ruminant species: A single-dye DNA microarray approach[END_REF]. In this study, altogether, the most prevalent agr group found was agr I (n=91, 59.5%), followed by agr III (n=40, 26.1%) and agr II (n=21, 13.7%). Only a single strain bearing an agr IV polymorphism was found (0.6%) and corresponded to a bovine isolate. If stratified by host type (small or large ruminant), within bovines, the prevalence of agr groups I, II, III and IV were 54.7%, 17.2%, 26.6% and 1.6%, respectively, whereas within small ruminants, their prevalence was 62.9%, 11.2%, 25.8% and 0%. The ratios of the prevalences of each agr group are approximately the same when considering only small or only large ruminants. This suggests that agr type does not play a role in host specificity. When considering discrete genotype clusters, it appears that, for a given host, some genotypes have a high prevalence of agr I whereas for some others agr III is clearly prevalent. It has been previously shown that S. aureus strains belonging to agr group I have a greater ability of invading epithelial cells and persist in the mammary gland, respectively, suggesting that they are better at causing clinical or subclinical mastitis than strains of other agr groups [START_REF] Buzzola | Differential abilities of capsulated and noncapsulated Staphylococcus aureus isolates from diverse agr groups to invade mammary epithelial cells[END_REF]. Indeed, this same study found a disproportionately high prevalence (88%) of agr type I strains causing mastitis in bovines. Our study shows that agr group distribution among ruminant isolates correlated with genotype cluster rather than with a given host. Of note, the distribution of agr groups was tissue-independent since isolates from mastitis milk, udder skin or nares were found in each cluster. This observation is in agreement with Jarraud et al. (Jarraud et al., 2002b), who demonstrated a relationship between the genetic background of the strains and the agr allele group. We previously reported the prevalence of agr III (46% vs 44% for agr I) in small ruminant isolates and a correlation between geographical region and predominance of a given agr group was hypothesized to explain predominance in some lineages [START_REF] Vautor | Genetic differences among Staphylococcus aureus isolates from dairy ruminant species: A single-dye DNA microarray approach[END_REF]. Here, strains originating from various geographical regions grouped within the same clusters and belonged to the same agr group. This suggests that the link between agr allele groups and a given region is likely due to the predominance of a given lineage within the region considered.

Determination of the capsular polysaccharide type.

The cap operon responsible for the biosynthesis of capsular polysaccharides (CP) expressed on the surface of S. aureus belongs to the virulon (O'Riordan and [START_REF] O'riordan | Staphylococcus aureus capsular polysaccharides[END_REF]. There are 11 serotypes of CP, however, the majority of strains isolated from humans express either CP type 5 or type 8, for which PCR a test has been developed (O' Riordan and Lee, 2004;[START_REF] Verdier | Identification of the capsular polysaccharides in Staphylococcus aureus clinical isolates by PCR and agglutination tests[END_REF]. In contrast, a variable prevalence of different CP types has been observed in strains isolated from ruminants from geographically different regions of the world [START_REF] Guidry | Prevalence of capsular serotypes among Staphylococcus aureus isolates from cows with mastitis in the United States[END_REF][START_REF] Poutrel | Prevalence of capsular polysaccharide types 5 and 8 among Staphylococcus aureus isolates from cow, goat, and ewe milk[END_REF][START_REF] Sordelli | Capsule expression by bovine isolates of Staphylococcus aureus from Argentina: genetic and epidemiologic analyses[END_REF] and the actual role of CP in mastitis is questioned [START_REF] Tuchscherr | Capsule-negative Staphylococcus aureus induces chronic experimental mastitis in mice[END_REF]. Here, strains were CP typed using a PCR test enabling differentiation between CP type 5 and 8. The prevalence of the types of CP (5, 8 or non-typeable) for each lineage can be seen in Figure 1. Altogether, the capsular type 8 was predominant in this study and accounted for 65.4 % of the panel. CP type 5 and the non-typeable CP types accounted for 30.7 % and 3.9 % respectively. When considering the host species, CP type 8 was clearly predominant in small ruminants, with an overall prevalence of 83.1 % in ovine-caprine isolates. In contrast, CP type 5 was slightly predominant in bovine isolates (56.3 %) and the prevalence of one type over the other seemed dependent of the genotype cluster. The bovine clusters B and C were indeed majorly CP type 5. These results are in accordance with previous studies on CP typing of S. aureus mastitis isolates showing that most bovine isolates were CP type 5 whereas ovine and caprine isolates were CP type 8 [START_REF] Guidry | Prevalence of capsular serotypes among Staphylococcus aureus isolates from cows with mastitis in the United States[END_REF][START_REF] Poutrel | Prevalence of capsular polysaccharide types 5 and 8 among Staphylococcus aureus isolates from cow, goat, and ewe milk[END_REF].

The low prevalence of non-typeable strains in this study contrasted with previous work where up to 76.5% of bovine mastitis strains [START_REF] Sompolinsky | Encapsulation and capsular types in isolates of Staphylococcus aureus from different sources and relationship to phage types[END_REF] were found to be nontypeable. However, in this latter study only 17 strains were typed and the geographical region of isolation is not mentioned although this criterion, as shown above, is of great importance and may, in this case, bias the results. MRSA strains present a are much more clonal distribution than MSSA [START_REF] Enright | The evolutionary history of methicillin-resistant Staphylococcus aureus (MRSA)[END_REF]. Thus, our initial worry was that if we had a high prevalence of MRSA amongst our strains, this might pose a bias to our results, for MRSA strains would tend to cluster up. Nevertheless, only 5 strains (3.2%; 3 bovine strains and 2 caprine strains; dispatched in 5 different PFGE clusters) presented resistance to oxacillin, an antibiotic used for the detection of Methicilin Resistant S. aureus (MRSA). Our findings contrast with recent studies in which MRSA was found in the nares of 39% and 10.9% of pigs and horses tested, respectively [START_REF] De Neeling | High prevalence of methicillin resistant Staphylococcus aureus in pigs[END_REF][START_REF] Van Den | High occurrence of methicillinresistant Staphylococcus aureus ST398 in equine nasal samples[END_REF]. Together with other studies [START_REF] Juhasz-Kaszanyitzky | MRSA transmission between cows and humans[END_REF], our results confirmed that MRSA prevalence is still low in ruminants.

Conclusions

S. aureus strains are phenotypically and genomically variable. Pulsotyping of ruminant isolates showed that S. aureus strains clustered with regard to their host origin (bovine and ovine-caprine) and regardless the locus of colonization or infection. Accessory gene regulator polymorphism and the type (or presence) of capsular polysaccharide are facets of strain-tostrain difference. The prevalence of each agr group and cap type within each lineage varied with the lineage, however, when analysed within the context of each sub-family of host (small or large ruminant), the prevalences of each agr group or cap type are about the same either in bovines, or ovines/caprines. This hints at the hypothesis that adaptation to a host is not a result of the regulation of a set of accessory genes, but rather the presence/absence or allelic variation of certain genes. The fact that stains found in small ruminants are different from those found in bovines is important enough to warrant the research and development of different immunoprophylactic products against mastitis in small and large ruminants. The low prevalence of resistance to antibiotics found in ruminants suggests that domesticated livestock is not a reservoir of genes which provide resistance to antimicrobial drugs.

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

In conclusion, this study shows that the nature of S. aureus strains differs between large and small ruminants and suggests the existence of a host rather than tissue specificity. Further studies are being pursued for the determination of the molecular nature of this host specificity and strategies for mastitis prevention in small or large ruminants should take account of these differences.

Acknowledgements.

Authors

1

  Molecular characterisation ofStaphylcoccus aureus strains isolated from small and large ruminants reveals a host rather than tissue specificity P. D. D. Alves 1,2,3 § , J. A. McCulloch 1,2 § , S. Even 1,2 , C. Le Maréchal 1,2 , A. Thierry 1,2 , N. Grosset 1,2 , V. Azevedo 3 , C. A. Rosa 3 , E. Vautor 4 and Y. Le Loir 1,2 * (1) INRA, UMR1253 STLO, 85 rue de Saint Brieuc, 35042 Rennes, France;

  primer common to all agr groups and four primers, each one specific to each agr group. 2.4. Determination of the capsular polysaccharide type. The capsular polysaccharide type was determined by means of PCR with primers directed to sequences of the staphylococcal capsular polysaccharide gene (cap) specific to either the

  are very grateful to Pascal Pachot (Agrocampus Ouest, France) for advice on statistical analyses and to Germana K. Chassa (UFMG, Brazil) for support and constructive discussions all along this work. Priscila D. Alves and John A. McCulloch are the recipients of a CAPES grant from the Brazilian government (CAPES-COFECUB project 539/06). Caroline Le Maréchal is the recipient of a PhD grant from INRA and AFSSA (IMISa project).

Figure Legend Figure 1 .

 Legend1 Figure Legend

Figure 1

 1 Figure 1

Table 1 -

 1 S. aureus isolates used in this study.

	Sample site		No. of isolates
		Bovine	Caprine	Ovine
	nares	14	5	39
	udder	51	26	18
	Total	65	31	57


Helena Eriksson
email: helena.eriksson@sva.se

Désirée S Jansson

Karl-Erik Johansson

Viveca Båverud

Jan Chirico

Anna Aspán

Characterization of Erysipelothrix rhusiopathiae isolates from poultry, pigs, emus, the poultry red mite and other animals

Keywords: Erysipelothrix sp., poultry, pigs, PFGE, serotyping, antimicrobial susceptibility

Erysipelothrix rhusiopathiae is the causative agent of erysipelas in mammals and birds, especially pigs and poultry. In order to investigate the suitability of different subtyping methods for genetic and phenotypic similarities among Swedish isolates of the organism, 45 isolates from poultry (n=23), pigs (n=17), emus (n=2) and the poultry red mite Dermanyssus gallinae (n=3) were investigated by serotyping, pulsed-field gel electrophoresis (PFGE) and antimicrobial susceptibility testing. Sequence analysis of the 16S rRNA gene was performed on eleven isolates from nine animal species. The results indicated a random scattering of serotypes throughout the dendrogram based on PFGE banding patterns following SmaI digestion. In three cases, isolates with an identical PFGE pattern were of differing serotypes.

No differentiation into subgroups by antimicrobial susceptibility testing by broth microdilution was possible as results were similar for all isolates. The Minimum Inhibitory Concentrations for most antimicrobials, including penicillin and oxytetracycline, were low.

The 16S rRNA gene sequences (1443 nts) from eight of eleven selected isolates of Erysipelothrix spp. were identical to that of the type strain E. rhusiopathiae ATCC 19414 T .

The other three isolates differed from the type strain by two or three nucleotides. While this method may be useful for identification of Erysipelothrix spp., it is unsuitable for epidemiological investigations. Similarities in PFGE banding patterns between isolates from chickens and mites supported the hypothesis that D. gallinae may act as a reservoir and vector for E. rhusiopathiae. Further PFGE studies on E. rhusiopathiae isolates are appropriate to investigate the epidemiology of poultry erysipelas.

A c c e p t e d M a n u s c r i p t Introduction Erysipelothrix (E.) rhusiopathiae is a gram-positive rod-shaped bacterium belonging to the family Erysipelotrichaceae [START_REF] Verbarg | Erysipelothrix inopinata sp. nov., isolated in the course of sterile filtration of vegetable peptone broth, and description of Erysipelotrichaceae fam[END_REF]. Different serotypes of the bacterium occur worldwide and infection has been reported in many species [START_REF] Brooke | Erysipelothrix rhusiopathiae: bacteriology, epidemiology and clinical manifestations of an occupational pathogen[END_REF].

Healthy pigs can carry E. rhusiopathiae in lymphoid tissues, and may therefore constitute a reservoir of the bacterium (Wood, 1999). In turkey and chicken flocks E. rhusiopathiae may cause severe disease outbreaks (erysipelas) [START_REF] Eriksson | Erysipelas in layers -a growing problem in aviary and organic housing systems[END_REF]. A closely related species, E. tonsillarum is considered to be non-pathogenic for pigs and chickens [START_REF] Takahashi | Comparison of the pathogenicity for chickens of Erysipelothrix rhusiopathiae and Erysipelothrix tonsillarum[END_REF] but may cause clinical disease in dogs [START_REF] Takahasi | Taxonomic evidence that serovar 7 of Erysipelothrix strains isolated from dogs with endocarditis are Erysipelothrix tonsillarum[END_REF].

Serotyping is the traditional tool for subtyping of E. rhusiopathiae. Pulsed-field gel electrophoresis (PFGE) has been used for examining the genetic diversity among strains of E. rhusiopathiae [START_REF] Opriessnig | Erysipelothrix rhusiopathiae: genetic characterization of midwest US isolates and live commercial vaccines using pulsed-field gel electrophoresis[END_REF] and for studies of clonality during outbreaks of erysipelas in poultry [START_REF] Købke | Investigations on clonality and stability of clones of Erysipelothrix rhusiopathiae in turkeys and free range layers flocks affected by erysipelas[END_REF]. Antimicrobial susceptibility testing has been useful for subtyping in other bacterial species as subtypes have been correlated to susceptibility groups [START_REF] Aspevall | Antimicrobial susceptibility pattern of Clostridium difficile and its relation to PCR ribotypes in a Swedish university hospital[END_REF]. In previous studies antimicrobial susceptibility testing of E. rhusiopathiae has been performed almost exclusively on isolates from pigs (Yamamoto et al., 2001). 16S rRNA gene sequencing is often used for phylogenetic studies and characterization of bacterial isolates. For some groups of bacteria nucleotide differences between sequences make the method suitable for epidemiological investigations [START_REF] Heldtander | Genetic diversity and evolution of Mycoplasma capricolum subsp. capripneumoniae strains from eastern Africa assessed by 16S rDNA sequence analysis[END_REF].

The purpose of this study was to investigate the suitability of different subtyping methods for genetic and phenotypic similarities between Swedish isolates of E. rhusiopathiae from poultry, pigs, emus and the poultry red mite Dermanyssus gallinae. In addition, sequence analysis of the 16S rRNA gene was performed on eleven isolates from nine animal species.

Materials and Methods

Isolates investigated

Altogether 45 isolates of E. rhusiopathiae were characterized by serotyping, PFGE and antimicrobial susceptibility testing. Twenty-five isolates from chickens (n=20), turkeys (n=3) and emus (n=2) were collected from outbreaks of erysipelas during 1998 to 2004. The isolates from outbreaks in chickens were from flocks housed in indoor litter based systems ( 14isolates from 11 farms) or on organic farms were the birds had access to outside pens and/or pasture (6 isolates from 3 farms). Two of the isolates from turkeys were from backyard flocks whereas the third isolate represented an outbreak among commercial breeder turkeys housed indoors. All avian isolates were from birds diagnosed with septicaemia at routine necropsy and represented one to four randomly selected isolates from all farms from which E. rhusiopathiae were isolated at the National Veterinary Institute (SVA, Uppsala, Sweden) during the study period. An additional three isolates from the hematophagous poultry red mite (D. gallinae) were collected during outbreaks in flocks of laying hens as described by [START_REF] Nordenfors | Evaluation of a sampling trap for Dermanyssus gallinae (Acari: Dermanyssidae)[END_REF]. Seventeen porcine isolates of E. rhusiopathiae, collected between 1996 and 2003 from pigs in seventeen different herds geographically scattered over Sweden, were also investigated. The isolates from pigs were selected from the strain collection at the National Veterinary Institute (SVA, Uppsala, Sweden) based on a history of characteristic macroscopic lesions of erysipelas noted at necropsy or slaughter. The isolates were compared with the type strain of E. rhusiopathiae (ATCC 19414 T ) which was obtained from the Culture Collection at the University of Göteborg (CCUG) as CCUG 221 T .

Sequence analysis of the 16S rRNA gene and serotyping were performed on eleven isolates from the strain collection at the SVA from nine different animal species previously diagnosed A c c e p t e d M a n u s c r i p t 5 as infected with E. rhusiopathiae (Table 1). One of these (Bd 6531/96, from a dog) was previously typed and deposited at CCUG (CCUG 36858). These isolates were derived from Swedish cases diagnosed between 1981 and 2003, and chosen for analysis with the aim to give as wide variation as possible as regards year of isolation and host. Three isolates from laying hens were included as erysipelas is considered an emerging disease in this population in Sweden. All isolates were stored in serum broth with 15% glycerol at -70ºC, until further testing.

Culture and biochemical identification method

Samples were cultured on horse blood agar plates (blood agar base no. 2 [LabM], 39.5 g/L; citrated horse blood [SVA], 50 ml/L) and on bromcresol purple lactose agar plates (balanced peptone, 10 g/L [LabM]; NaCl, 5 g/L [Merck]; sodium ammoniumphosphate, 1 g/L [Merck]; Lab Lemco Powder, 4 g/L [Oxoid]; Agar no. 2, 10 g/L [LabM]; lactose solution 20%, 50 ml/L [SVA]; bromcresol purple solution 1.6%, 1 ml/L [SVA]). Bacterial growth was examined after 24 and 48 h incubation at 37ºC in 5% CO 2 . Colonies were identified by colony morphology, Gram staining and biochemical tests [START_REF] Quinn | Erysipelothrix rhusiopathiae[END_REF]. Isolates identified as E. rhusiopathiae were catalase-negative and oxidase-negative. The isolates usually fermented lactose, glucose and maltose, were H 2 S positive and arginine was hydrolysed. The isolates did not ferment rhamnose or sucrose and did not produce indole or urease. The isolates were stored as described above. Each strain was streaked on 5% horse blood agar plates and incubated at 37ºC overnight, before further analyses were made.

Serotyping

All isolates were serotyped by the tube agglutination method at the National Veterinary Institute, Copenhagen, Denmark, as previously described by Wellmann et al. (1983).

DNA preparation and pulsed-field gel electrophoresis

One horse blood agar plate of bacteria with an overnight culture was harvested for each isolate. The bacteria were suspended in 250 µl of lysis buffer (10 mM Tris-HCl, pH 8.0, 1 M NaCl, 200 mM EDTA, 0.5% Sarcosyl and 0.2% sodium deoxycholate) and immediately mixed with an equal volume of 1.5% low melt point agarose, and the mixture was left to solidify in agarose plug moulds at 4ºC for 10 minutes. Gel plugs were incubated overnight in 2.5 ml of lysis buffer, supplemented with lysozyme (Roche; Basle, Switzerland) to a final concentration of 1.0 mg/ml, in a shaking incubator at 37ºC. New lysis buffer was added, this time supplemented with proteinase K (Roche) to a final concentration of 1.0 mg/ml, and incubation was performed for 20 h in a shaking incubator at 54ºC. After three washes with pre-warmed double-distilled water and four washes with pre-warmed TE-buffer (10 mM Tris, 1 mM EDTA, pH 7.4) at 54ºC for 15 min each, genomic DNA was digested with the macrorestriction enzyme SmaI (GE Healthcare; Amersham, England), according to the manufacturer's instructions. Plug slices were loaded onto a 1.2% agarose gel, and genomic DNA was separated by electrophoresis for 20 h in a CHEF DRII apparatus (Biorad; Hercules, Cal., USA), while the pulse switch time was increased from 0.5 s to 40 s. PFGE patterns were analysed with the GelCompar II software (Applied Maths, Sint-Martens-Latem, Belgium). A dendrogram was prepared by the Unweighted Pair Group Method with Arithmetic mean (UPGMA), dice coefficient and 0.5% optimization with 1.4% band position tolerance.

Antimicrobial susceptibility testing

A 96-well broth microdilution panel, VetMIC TM Large animal panel (National Veterinary Institute, SVA, Uppsala) was used for antimicrobial susceptibility testing with concentration A c c e p t e d M a n u s c r i p t 7 ranges as per Table 2. The antimicrobial agents were dried in two-fold serial dilutions in the panel according to Table 2.

All E. rhusiopathiae isolates were recovered from frozen (─70°C) stocks and subcultured at least twice on horse blood agar plates before susceptibility testing. One well-filled 1-µl loop of colony material from an overnight culture was suspended in 4 ml of NaCl. From the suspension, two 20-µl loopfuls were transferred to 10 ml of brain heart infusion broth [Difco] supplemented with 10% fetal calf serum, giving a final inoculum density of 1 x 10 6 CFU/ml. Each well in the VetMIC TM panel was inoculated with 50 µl. The wells were sealed with transparent adhesive tape and then incubated at 37°C for 18 h. The Minimum Inhibitory Concentration (MIC) was read as the lowest concentration where no visible growth, or the most significant reduction of growth, was observed. The assays were performed in duplicate for each isolate.

For purity control, each bacterial suspension was streaked on a horse blood agar plate and incubated at 37°C for 18 h. Two wells were used as growth controls in each panel without antimicrobial agents, but containing dried dilution buffers. The inoculation density was determined by viable counts on horse blood agar plates after incubation at 37°C for 24 h. The Hinton broth, precultured for 4 h at 37°C and then diluted to a final inoculum density of 1 x 10 5 CFU/ml and incubated at 37°C for 1618 h.

Sequence analysis of the 16S rRNA gene

The 16S rRNA gene of the eleven selected isolates was amplified with primers suitable for members of the phylum Firmicutes [START_REF] Johansson | Genetic diversity of Clostridium perfringens type A isolates from animals, food poisoning outbreaks and sludge[END_REF]. The amplicons were used for cycle sequencing with labelled terminators (Big Dye; Applied Biosystems, Foster City, Cal., USA) and with the sequencing primers described by [START_REF] Johansson | Genetic diversity of Clostridium perfringens type A isolates from animals, food poisoning outbreaks and sludge[END_REF]. The sequencing products were analysed with an ABI Prism 3100 genetic analyser (Applied Biosystems) and contigs (clones with contiguous nucleotide sequences) were generated by using the Contig Express program included in the Vector NTI Suite (InforMax, Bethesda, Md, USA). The contigs were edited manually by using the Genetic Data Environment software [START_REF] Smith | GDE: Genetic data environment, version 2.2[END_REF] before further analysis and deposition in GenBank. The sequences of the 16S rRNA gene of the isolates were used for similarity searches in GenBank by applying the BLASTn program.

Results

Serotyping

The most prevalent E. rhusiopathiae serotype from pigs, poultry, emus and D. gallinae (17 of 45 isolates) was serotype 1a while the second most prevalent (serotype 2b; n=12) was only found among pig isolates. In addition, six isolates were reported as serotype 1ab (isolates that reacted with sera for both serotype 1a and serotype 1b). Serotypes 1b, 4, 5 and 6 were represented by two isolates each. Two of the pig isolates as well as five of the isolates selected for 16S rRNA sequencing were not typeable. Detailed results are shown in Fig. 1 isolates (100% PFGE homology) were found. Three of these groups contained more than one isolate from the same poultry or emu farm, together, in all three cases, with isolates from other farms. Two out of three isolates from the poultry red mite D. gallinae were found to be identical to the isolate from the corresponding outbreak in laying hens. The third mite isolate clustered closely (90% homology) to the corresponding poultry isolate. The other two of these five PFGE groups contained isolates from pigs that had no known epidemiological relationship to each other.

Antimicrobial susceptibility testing

The MIC distributions of all antimicrobial agents are presented in Table 2. The results were similar for all strains and differentiation into subgroups was not possible. For most of the antimicrobial agents tested, including penicillin and oxytetracycline, the MICs were low. The aminoglycosides gentamicin, neomycin, and streptomycin had uniformly high MICs, greater than or equal to the highest concentration tested. The MICs obtained for all the antimicrobial agents tested in duplicate did not differ for any isolate.

For the reference strain E. rhusiopathiae ATCC 19414 T , the MICs of all antimicrobial agents from four tests did not differ ( The results of the sequencing of the 16S rRNA gene of the eleven isolates are presented in Table 1. The length of the sequences was 1443 nucleotides and they have been deposited in GenBank under the accession numbers listed in Table 1. All eleven isolates have been deposited in CCUG (Table 1). Eight of the sequences were identical to the 16S rRNA sequence of E. rhusiopathiae, strain ATCC 19414 T . One of the sequences (from strain B 470/87) differed by two nucleotides from the sequence of the type strain. Two of the sequences (from strains B 2344/87 and Bd 6531/96) were identical to the 16S rRNA sequence of E. tonsillarum, strain ATCC 43339 T .

Discussion

In this study, isolates of E. rhusiopathiae were investigated by serotyping, PFGE, antimicrobial susceptibility testing and 16S rRNA sequencing. PFGE was found to be a suitable method for genotypic studies of E. rhusiopathiae and provided further support for the previously reported hypothesis that D. gallinae may act as a vector of or reservoir for the bacteria, especially among laying hens.

In our study, serotyping clearly distinguished strains from certain hosts as pigs were the only host to carry serotype 2b and emus the only host with serotype 5. Therefore most of the pig isolates were of a serotype not found in poultry while most of the poultry isolates were of a serotype (1a or 1ab), less common in pigs. Interestingly, no correlation between serotype and PFGE pattern could be observed as serotypes were randomly scattered throughout the dendrogram. Furthermore, isolates with the same PFGE pattern were of different serotypes in three cases. The significance of this finding is uncertain. However, based on the limited number of serotypes found in our material we conclude that serotyping is less suitable for A c c e p t e d M a n u s c r i p t 11 studies on epidemiological relationships between flocks. PFGE however appears to be a useful method for this purpose. In five cases, PFGE linked isolates from different farms with no known epidemiological connection. This could be due to lack of historical epidemiological information on possible contacts between these farms or to a limitation of the PFGE method in separating the strains. This latter problem could be overcome by using one or several additional restriction enzymes.

A novel finding in this study was that isolates with different PFGE patterns came from the same laying hen farms (farms B and F in Fig. 1). The five isolates from farm B represent outbreaks in four different organic laying hen flocks in the years 1998, 1999, 2001 and 2004. For farm F, the five isolates represent almost simultaneous outbreaks on the farm in separate flocks, housed in a litter-based aviary system, in 2001 to 2002. The differing PFGE patterns might be attributable to a genetic instability of E. rhusiopathiae in a flock over time. This has been discussed previously by [START_REF] Cross | Serological classification of Australian strains of Erysipelothrix rhusiopathiae isolated from pigs, sheep, turkeys and man[END_REF] regarding changes of serotype.

Another explanation could be that the outbreaks were caused by different strains with different PFGE types. Extended field studies or experimental challenge studies need to be undertaken to ascertain the genetic stability of E. rhusiopathiae.

In two outbreaks on different farms (farms B and F), isolates with identical PFGE-banding patterns were found in laying hens and D. gallinae. The relationship between isolates from mites and affected laying hens found by PFGE (Fig. 1) supports our previously reported hypothesis that mites may act as a vector of, or reservoir host for E. rhusiopathiae (Chirico et al., 2003). D. gallinae is very difficult to control and has so far proved to be extremely difficult to eradicate from commercial laying hen operations (J. Chirico, personal communication).

A c c e p t e d M a n u s c r i p t 12 Pig and poultry isolates were randomly scattered throughout the PFGE dendrogram, and were not separated into two distinct populations, which could suggest that pigs may act as a source of the infection for poultry. However, no identical pairs of isolates were found between the two species, thus we cannot draw the conclusion that the same isolate of E. rhusiopathiae can infect both pigs and poultry.

Antimicrobial susceptibility testing was not a useful method for subtyping of E. rhusiopathiae, as most isolates from both poultry and pigs in this study gave similar results.

To our knowledge this is the first report of antimicrobial susceptibility of chicken isolates of E. rhusiopathiae. In previous studies, the agar dilution method has been used on isolates from pigs [START_REF] Takahashi | Antibiotic resistance of Erysipelothrix rhusiopathiae isolated from pigs with chronic swine erysipelas[END_REF][START_REF] Takahashi | Serotype, antimicrobial susceptibility, and pathogenicity of Erysipelothrix rhusiopathiae isolates from tonsils of apparently healthy slaughter pigs[END_REF]Yamamoto et al., 1999Yamamoto et al., , 2001) ) and a variety of sources including humans [START_REF] Fidalgo | Susceptibility of Erysipelothrix rhusiopathiae to antimicrobial agents and home disinfectants[END_REF]. However, for most laboratories this method is a timeconsuming and tedious procedure for the often small number of isolates involved. In this study, broth microdilution was a reliable and reproducible method for susceptibility testing of E. rhusiopathiae. The MIC endpoints were easy to read and the method was readily performed. In the present investigation, all isolates had low MICs for penicillin, the drug of choice for treating E. rhusiopathiae infections [START_REF] Prescott | Beta-lactam Antibiotics: Penam Penicillins[END_REF]. All isolates also had low MICs of oxytetracycline, the antimicrobial most widely used in poultry [START_REF] Hofacre | Antimicrobial drug use in poultry[END_REF]. Field experience from Sweden supports this finding, as some flocks with acute outbreaks of erysipelas have been successfully treated with oxytetracycline, which is the only approved antimicrobial for layers in Sweden today [START_REF] Eriksson | Erysipelas in layers -a growing problem in aviary and organic housing systems[END_REF]. In previous studies on isolates from pigs the MICs of oxytetracyline varied [START_REF] Takahashi | Antibiotic resistance of Erysipelothrix rhusiopathiae isolated from pigs with chronic swine erysipelas[END_REF][START_REF] Takahashi | Serotype, antimicrobial susceptibility, and pathogenicity of Erysipelothrix rhusiopathiae isolates from tonsils of apparently healthy slaughter pigs[END_REF]Yamamoto et al., 1999Yamamoto et al., , 2001)).

A c c e p t e d M a n u s c r i p t 13 The 16S rRNA genes of eleven isolates from nine different Swedish animal species were sequenced in this study. The fact that these sequences contained only minor nucleotide differences indicates that the method is unsuitable for epidemiological investigations of erysipelas outbreaks. Moreover, there are only three nucleotide differences between the 16S rRNA sequences of the type strains of E. rhusiopathiae and E. tonsillarum. The results should therefore be interpreted with caution, as rather few 16S rRNA sequences of E. rhusiopathiae and E. tonsillarum have, so far, been deposited in GenBank. Furthermore, three of the sequences deposited in GenBank as E. rhusiopathiae (AB055907, AB055909 and AB055910) contained more nucleotide differences to the type strain than to E. tonsillarum. Notably, two isolates in this study, from a rat [START_REF] Feinstein | Naturally occurring erysipelas in rats[END_REF]) and a dog, were identified biochemically as E. rhusiopathiae, based on their inability to ferment sucrose, but results obtained by 16S rRNA sequencing indicated that these isolates were actually E. tonsillarum.

Our finding that sucrose-negative strains of E. tonsillarum might be misdiagnosed as E. rhusiopathiae are consistent with those of [START_REF] Okatani | Randomly amplified polymorphic DNA analysis of Erysipelothrix spp[END_REF] who questioned both the fermentation of sucrose and serotyping as appropriate methods for distinguishing the two species. A simple method suitable for differentiating E. tonsillarum from E. rhusiopathiae in routine diagnostic laboratories is needed.

Conclusions

PFGE was in our study found to be a suitable method for epidemiological studies of outbreaks of erysipelas. Our results further support the previously proposed hypothesis that D. gallinae may act as a reservoir for and possibly as a vector of E. rhusiopathiae. Collecting isolates from affected poultry flocks for further epidemiological studies by PFGE is recommended. To establish the stability in PFGE-patterns extended field studies or experimental challenge Yamamoto, K., Kijima, M., Takahashi, T., Yoshimura, H., Tani, O., Kojyou, T., Yamawaki, Y., Tanimoto, T., 1999. Serovar, pathogenicity and antimicrobial susceptibility of Erysipelothrix rhusiopathiae isolates from farmed wild boars (Sus scrofa) affected with septicemic erysipelas in Japan. Res. Vet. Sci. 67, 301-303. Yamamoto, K., Kijima, M., Yoshimura, H., Takahashi, T., 2001. Antimicrobial susceptibilities of Erysipelothrix rhusiopathiae isolated from pigs with swine erysipelas in Japan, 1988-1998. J. Vet. Med. B 48, 115-126. a MIC 50 and MIC 90 are the concentrations at which 50% and 90% of the isolates respectively were inhibited.

MIC (mg/ml)

Table 2

  type strain E. rhusiopathiae ATCC 19414 T , was included in each test run. The reference strains Staphylococcus aureus ATCC 29213 and Enterococcus faecalis ATCC 29212 were tested in duplicate. The three reference strains were tested as described for the clinical isolates of E. rhusiopathiae. As a further quality check of the panels, tests were performed with the reference strains E. faecalis ATCC 29212, Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC A c c e p t e d M a n u s c r i p t 8 27853 and S. aureus ATCC 29213. These four strains were tested in cation-adjusted Mueller

  gel electrophoresis A dendrogram based on PFGE-banding patterns is presented in Fig. 1. The isolates were distributed among several different PFGE patterns. Five PFGE groups with multiple identical

  to be undertaken. Sequence analysis of the 16S rRNA gene might be useful for differentiation between E. rhusiopathiae and E. tonsillarum but this requires further study. ., Kucsera, G., Nørrung, V., 1983. Comparative studies on different methods in typing strains of Erysipelothrix rhusiopathiae. I. Methods and influence of some factors on their results. Zentralbl. Bakteriol. Hyg. Abt. 1 Orig. A 254:42-54. Wood, R.L., 1999. Erysipelas. In: Straw, B.E., D'Allaire, S., Mengeling, W.L., Taylor, D.J. (Eds.), Diseases of Swine. 8th ed. Iowa State University Press, Ames, pp. 419-430.

Figure 1 .

 1 Figure captions

Table 1 .

 1 and

Table 2 )

 2 . The MICs of the reference strains E. faecalis ATCC 29212, E. coli ATCC 25922, P. aeruginosa ATCC 27853 and S. aureus ATCC 29213 were within the recommended ranges (National Committee for Clinical Laboratory Standards, 2002; Clinical and Laboratory Standards Institute, 2005).

Table 2 .

 2 Results of MIC determination of eleven antimicrobial agents for 45 Erysipelothrix rhusiopathiae isolates from poultry, pigs, emus and mites by broth microdilution using the VetMIC TM Large animal panel (SVA, Uppsala) MICs for the reference strain E. rhusiopathiae ATCC 19414 T are also included

Antimicrobial agent Range tested Range results MIC 50 ª MIC 90 ª

	Ampicillin	1-8	 1	 1	 1
	Ceftiofur	0.25-2	0.25	0.25	0.25
	Enrofloxacin	0.12-1	0.12 -0.25	0.12	0.25
	Florfenicol	2-16	4-8	4	8
	Gentamicin	2-16	> 16	> 16	> 16
	Neomycin	4-32	> 32	> 32	> 32
	Oxacillin	0.5-1	0.5	0.5	0.5
	Oxytetracycline	1-8	1	1	1
	Penicillin	0.12-1	 0.12	 0.12	 0.12
	Spiramycin	4-32	4	4	4
	Streptomycin	4-32	32 ->32	>32	>32

Acknowledgements

The authors wish to thank Lisa Lindberg, Helena Ljung, Marianne Persson and Anna-Lena Sahlin for excellent technical assistance. This work was financially supported by the National Veterinary Institute, Sweden.


J M Minke

R Bey

PhD J P Tronel

S Latour

G Colombet

J Yvorel

PhD C Cariou

A L Guiot

PhD V Cozette

P M Biostatistician

Guigal

PhD Dvm

S Dvm

Latour

G Dvm

Colombet

Onset and duration of protective immunity against clinical disease and renal carriage in dogs provided by a bi-valent inactivated leptospirosis vaccine

Keywords: Leptospira interrogans serovars canicola and icterohaemorrhagiae, bacterin, vaccine, clinical leptospirosis, renal carriage, onset and duration of immunity

Onset and duration of protective immunity against clinical disease and renal carriage in dogs provided by a bi-valent inactivated leptospirosis vaccine

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

Introduction

Leptospirosis is an important zoonosis of worlwide distribution caused by infection with spirochaetes belonging to the pathogenic species of Leptospira. Infection typically results from direct or indirect contact with urine of infected animals. The clinical signs associated with Leptospira infection range from subclinical to acute disease characterized by anorexia, vomiting, lethargy, muscle pain, dehydration, jaundice, abdominal pain, diarrhoea, bloody urine, and death. Renal failure is the predominant finding in symptomatic dogs, with a small percentage also showing evidence of liver disease (Greene, 1998, review;[START_REF] Boutilier | Leptospirosis in dogs: a serologic survey and case series 1996 to 2001[END_REF]. Clinically recovered dogs frequently become asymptomatic renal carriers, and as such can be an important source of human leptospirosis (Center for Disease Control 1972[START_REF] Trevejo | Epidemic leptospirosis associated with pulmonary hemorrhage-Nicaragua[END_REF]. Leptospira (L) interrogans serovars icterohaemorrhagiae and canicola are the two serovars traditionally associated with disease in dogs (Hartman, 1984[START_REF] Trevejo | Epidemic leptospirosis associated with pulmonary hemorrhage-Nicaragua[END_REF], but new serovars play an increasingly important role [START_REF] Scanziani | Serological survey of leptospiral infection in kennelled dogs in Italy[END_REF][START_REF] Ward | Serovar-specific prevalence and risk factors for leptospirosis among dogs: 90 cases (1997-2002)[END_REF][START_REF] Moore | Canine leptospirosis, United States, 2002-2004[END_REF][START_REF] Geisen | Canine leptospirosis infections -clinical signs and outcome with different suspected Leptospira serogroups (42 cases)[END_REF][START_REF] Stokes | Prevalence of serum antibodies against six Leptospira serovars in healthy dogs[END_REF]. Hence, leptospirosis is now recognised as an important re-emerging disease in dogs [START_REF] Bolin | Diagnosis of leptospirosis: a reemerging disease of companion animals[END_REF][START_REF] Ward | Prevalence of and risk factors for leptospirosis among dogs in the United States and Canada: 677 cases (1970-1998)[END_REF]. While short-term clinical protection has been demonstrated experimentally in dogs after vaccination with several vaccines [START_REF] Huhn | Immunity to leptospirosis: bacterins in dogs and hamsters[END_REF]Andre-Fontaine et al, 2003;[START_REF] Klaasen | Duration of immunity in dogs vaccinated against leptospirosis with a bivalent inactivated vaccine[END_REF]Schreiber et al, 2005a[START_REF] Schreiber | Prevention of renal infection and urinary shedding in dogs by a Leptospira vaccination[END_REF], there is much debate on whether leptospirosis vaccines protect against the renal carrier state (Andre Fontaine et al, 2003) or provide long term immunity (Cohne et al., 2001). As far as we know, we were the first to demonstrate 10 months duration of immunity against L. interrogans serovar canicola provided by a classical bacterin [START_REF] Tronel | Efficacy of Leptodog vaccine in dogs demonstrated by experimental challenge: Evaluation at short term and duration of immunity[END_REF]. Since then only one paper has been published demonstrating duration of immunity of 13 months for a commercial bacterin [START_REF] Klaasen | Duration of immunity in dogs vaccinated against leptospirosis with a bivalent inactivated vaccine[END_REF]). In the current study, we confirm and extend our previous observations and demonstrate that two doses of EURICAN  L provide both rapid onset and duration of protective immunity of at least 14 months against both serovars icterohaemorrhagiae and canicola. The vaccine was evaluated for protection against clinical disease and prevention of the renal carrier state.

Materials and methods

Experimental design

Four separate vaccination-challenge experiments, including 74 puppies, were performed to study onset and duration of immunity provided by EURICAN L (referred to as studies 1-4, Table 1). Study 1 contained nine vaccinates and eight controls, study 2 nine vaccinates and ten controls, study 3 seven vaccinates and eight controls and study 4 nine vaccinates and ten controls (Table 1). Institutional Animal Care and Use Committee approvals were obtained before conducting the studies. In all studies, puppies were vaccinated twice subcutaneously, 4 weeks apart. Puppies were 8-9 weeks of age at the time of first vaccination. Dogs from studies 1 and 3 were challenged with L. interrogans serovar canicola at 2 weeks and 14 months, respectively, after the primary vaccination program of two doses. Dogs from studies 2 and 4 were challenged with L. interrogans serovar icterohaemorrhagiae at 2 weeks and 14 months, respectively, after the second vaccination of the primary vaccination course. Because it is difficult to induce clinical leptospirosis in adult dogs, two 2-4 months-old pups were added to studies 3 and 4 at the time of challenge to assess the severity of the infection. Following challenge, dogs were examined for the presence of clinical signs characteristic of leptospirosis. For leptospires isolation, blood and urine samples were collected at regular intervals, and a kidney and liver (study 4) sample were aseptically taken at necropsy. Blood was also sampled for serological, haematological, and biochemical analysis (study 4 only). At the end of the observation period or at death, dogs were necropsied, and organs were removed for histological examination

Vaccines

Routine production batches of EURICAN L (Merial, Lyon, France), a whole cell, nonadjuvanted vaccine prepared from inactivated cultures of L. interrogans serovars icterohaemorrhagiae and canicola, were used. All batches complied with the potency requirements of monograph 0447 of the European Pharmacopoiea (2002). In studies 1 and 2, the vaccine was administered simultaneously, but at a separate site, with a vaccine containing a recombinant canine distemper virus and modified live canine adenovirus type 2, canine parvovirus, canine coronavirus, and canine parainfluenza type 2 virus. In studies 3 and 4, EURICAN L was used as diluent to reconstitute a freeze-dried pellet containing a modified

Animals

Seventy-four specific pathogen free (SPF) 8-to 16-week-old male and female beagle pups were purchased from Harlan Sprague Dawley (Indianapolis, USA or Zeist, The Netherlands) or from Ferme des Gouttes (Charles Rivers Laboratories, Inc., France). Dogs were barrier maintained and fed a high-quality commercial dry ration with unlimited access to water. Dogs were identified by a microchip implanted subcutaneously and/or by ear tattoo.

Challenge strains

L. interrogans serovar canicola, strain Moulton (National Veterinary Services Laboratory (NVSL), Ames, Iowa, USA) was used as challenge inoculum in studies 1 and 3. L. interrogans serovar icterohaemorrhagiae, strain CFI (NVSL) and strain 193 (Pasteur Institute, Paris, France) were used as challenge inocula in studies 2 and 4, respectively. The identity of all serovars was confirmed by the Pasteur Institute, Paris, France, using restriction fragment analysis.

Challenge protocol

After an initial culture in Ellinghausen-McCullough-Johnson-Harris (EMJH) medium, the strains were back-passaged twice (studies 3 and 4) or four times (studies 1 and 2) in hamsters to prevent loss of virulence through adaptation to culture conditions. Moribund hamsters were humanely euthanised and their livers and kidneys or spleens (study 4) were aseptically removed and homogenated in sterile saline. After sedimentation by centrifugation, the supernatant was diluted 1:10 in sterile saline and inoculated in the dogs of studies 1 and 2.

Each dog received 8 mL of challenge suspension containing approximately 5x10 8 and 1x10 9 organisms of L. interrogans serovars icterohaemorrhagiae and canicola, respectively by the intraperitoneal route. In studies 3 and 4, after harvest, the challenge strains were passaged once in vitro (EMJH medium) to allow a more precise quantification of the bacterial suspension. Each dog received 11 mL (study 3) or 12 mL of challenge suspension with 0.5 mL instilled in the ventral conjunctival sac of each eye and the remainder administered intra-A c c e p t e d M a n u s c r i p t 6 peritoneally. The total challenge dose per dog was 2.1x10 9 and 5.6x10 9 organisms for L. interrogans serovars icterohaemorrhagiae and canicola, respectively.

Clinical examination

All animals were observed daily for 14 (studies 1 and 2) or 35 days (studies 3 and 4) after challenge for signs consistent with leptospirosis, including, depression, anorexia, conjunctivitis, iritis, vomiting, diarrhoea, jaundice, petechiae, and signs of urinary disease (haematuria). Signs were scored by use of a standardized protocol (Table 2). Rectal temperatures were taken and recorded daily for 14 days after challenge, and temperatures of 39.5°C or more were considered as hyperthermia. Dogs from studies 3 and 4 were weighed once a week until the end of the study or death. A weight loss of more than 5% was considered significant. During the post-challenge clinical examination, any animals displaying serious and irreversible clinical signs that lead to suffering were humanely euthanized.

Serology

Whole blood was collected at regular intervals before and after vaccination and challenge.

Selected sera were tested for the presence of microscopic agglutination titres (MAT) by the College of Veterinary Medicine, Diagnostic Laboratory, University of Minnesota, St Paul, USA (studies 1 and 2) or AFFSA, Laboratoire de Recherche Vétérinaire, Alfort, Paris, France (study 4). Sera were tested against L. interrogans serovars icterohaemorrhagiae and canicola using standardized procedures. Since serology has limited value for evaluating the efficacy of vaccines against leptospirosis, sera from study 3 were not tested. Antibody titers were expressed as the reciprocal of the highest serum dilution that induced at least 50% (studies 1 and 2) or 75% (study 4) agglutination. For the calculation of geometric mean titer (GMT), values under the lower limit of quantification (LLOQ) were replaced by LLOQ/2.

Haematology

EDTA blood samples were collected from dogs of studies 3 and 4 on at least 2 days before challenge and then daily for 7 days after challenge. Counts of platelets were performed using a MS-9 cell counter analyser (Melet Schloesing, France). Platelet counts were compared to reported standard values for dogs (2-9x10 11 -platelets/L (Merck Veterinary Manual, 2005)).

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

Blood biochemistry

The following tests were only performed on dogs from study 4. Whole blood samples were collected before challenge and on day 4 and 6 after challenge. Sera were analyzed for urea nitrogen, creatinine, total bilirubin, serum glutamic oxalacetic transaminase (SGOT), and serum glutamine pyruvic transaminase (SGPT) by the Laboratoire Marcel Mérieux, Lyon, France. Urea nitrogen, creatinine, and total bilirubin were compared to normal values provided by the same laboratory. Because many pre-challenge values for SGOT and SGPT were outside the reported "normal" values, only large modifications of the baseline values were taken into account.

Detection of leptospiraemia

Blood samples were collected on heparin tubes before challenge (day -2/day 0) and on day 1, 2, 3, 4, 5, 6, 7, and 10 after challenge. In study 4, an additional blood sample was taken on day 35. Blood samples were immediately inoculated in semisolid medium (1-3 drops of blood in 8 mL of medium (studies 1 and 2)) or in liquid EMJH medium (1 mL of blood in 9 mL medium (studies 3 and 4)) and transferred to the lab. Serial 10-fold dilutions (up to 10 -3 ) were made in the same media and incubated at 30°C. All the cultures were incubated for 6-9 weeks and observed weekly for the presence of leptospires using dark field microscopy.

Detection of leptospires in urine and organs

Urine samples were collected before challenge (day -2/day 0) and at 2, 3 and 5 weeks after challenge (studies 3 and 4) or by direct bladder tap at the time of euthanasia (studies 1 and 2).

In studies 3 and 4, urine samples were collected either spontaneously after subcutaneous injection of the diuretic furosemide (DIMAZON ® , Intervet, France) (0.5 to 1 mL/kg bodyweight) (females) or after probing with a urethra catheter (males). 5 Fluorouracil was added at a concentration of 100 µg/mL to the urine samples of study 4. Samples from kidneys (all studies) and livers (study 4) were collected aseptically.

Approximately 5-8 grams of organ tissue were macerated into 10 mL of culture medium and vortexed. Tissue debris was allowed to settle, and serial 10-fold dilutions were made through 1:1,000. Urine and organ cultures were made as described for the blood cultures.

Post-mortem examination

Immediately after euthanasia or death, the animals were necropsied and subjected to a macroscopic examination. Samples of kidneys and livers were fixed with 10% buffered formalin or frozen and processed for microscopic examination following standard procedures.

Only organ samples from study 4 were submitted for microscopic examination. Histological sections were stained with haematoxylin-eosin (HE) and with Warthin-Starry silver stain for the detection of leptospires.

Analysis of the results

Statistical analyses were carried out using STATGRAPHICS ® software and SAS ® release 12 software. The level of significance was set at P 0.05.

Clinical scores

The severity of clinical signs (sickness score) was compared among the vaccinated and control groups within one study by assigning the dogs to one of two disease categories: no or mild clinical disease and moderate-to-severe clinical disease. The sickness score was calculated by using the daily scores for each clinical sign on the basis of an algorithm, which gave a triple weighting to the scores for jaundice and haematuria. Thus, sickness score = 1x (daily score for conjunctivitis/iritis) + 1x (daily score for anorexia) + 1x (daily score for diarrhoea/vomiting) + 1x (daily score for general appearance) + 3x (daily score for jaundice) + 3x (daily score for haematuria). Each dog was classified according to the most severe daily score recorded during the after challenge observation period with a score of 0 for no disease, 1 to 2 for mild disease, 3 to 4 for moderate disease, and >4 for severe disease. Differences in the incidence of moderate-to-severe disease (scores 3) among groups were analyzed by use of a Fischer's exact test. A daily score between 0 and 3 was attributed to each animal from studies 3 and 4, according to the result of the blood culture (0=negative, 1=positive at dilution 10 -1 , 2=positive at dilution 10 -2 , 3=positive at dilution 10 -3 ). The duration of leptospiraemia and cumulative scores for the first 7 days post challenge (Area under the time-titer curve) were compared between vaccinated and control dogs using a one-sided student t-test (study 3) or Wilcoxon test (study 4).

Renal carrier state

Any dog with at least one positive urine or kidney culture was defined as a renal carrier.

Differences in the incidence of renal carriers among groups were analyzed by use of a Fischer's exact test.

Platelet counts

Platelet counts of vaccinated and adult control groups were compared using a Wilcoxon test (study 4) or a mixed model with repeated measurements (study 3). SGOT and SGPT values of vaccinated and control groups were compared using a Wilcoxon test and Chi-square test, respectively.

Results

Humoral responses to vaccination and challenge

Prior to vaccination, none of the dogs had detectable antibody titres against L interrogans serovars icterohaemorrhagiae or canicola. All vaccinated dogs from studies 1 and 2 had detectable antibody titres on the day of challenge against L. interrogans serovar canicola (GMT=549, range: 80-1280) and L. interrogans serovar icterohaemorrhagiae (GMT=47, range 20-80). A booster effect was observed in one out of nine and eight out of nine dogs after Leptospira interrogans serovars icterohaemorrhagiae and canicola challenge, respectively.

A c c e p t e d M a n u s c r i p t icterohaemorrhagiae (GMT=1280) and L. interrogans serovar canicola challenge (GMT=3044, range 1280-10240). After both challenges, antibody titres were higher on average in surviving controls than in vaccinates.

Four out of nine dogs from study 4 had detectable antibody titres against L. interrogans serovar icterohaemorrhagiae 4 weeks after the second vaccination (range: 100-200).

Antibodies persisted until challenge in only one dog. A booster response was observed in all vaccinates after L. interrogans serovar icterohaemorrhagiae challenge. In the same study, seven out of nine dogs had detectable antibody titres against L. interrogans serovar canicola 4 weeks after the second vaccination (range: 200-400), and two out of nine animals still had low MAT antibody titres 5 days before challenge.

Clinical signs

The incidences of moderate to severe disease in vaccinated and control dogs from studies 1-4 are shown in Table 3.

All eight control pups from study 1 became ill after L. interrogans serovar canicola challenge; seven pups developed severe and one pup moderate disease. The affected pups were depressed and were frequently observed curled up in their food bowls. Some of these animals were vomiting, slightly dehydrated, and had haematuria. They also had foul smelling bloody diarrhoea. One pup developed jaundice on day 4 post-challenge. Four pups with severe clinical disease were humanely euthanised between day post challenge (DPC) 5 and 6. In In study 4, one of the two control puppies added at the time of challenge died on DPC 6 and the other developed severe disease (sickness score of 6) but recovered. Unexpectedly, the challenge appeared to be very severe for the adult controls. Three out of 10 control dogs had to be humanely euthanised because of depression, diarrhoea, dehydration, and jaundice (one dog) on DPC 7 (two dogs) and 23, respectively. Five controls had mild disease consisting of conjunctivitis, depression, and anorexia. Two controls had no disease. Clinical signs in the vaccinated dogs were mild (conjunctivitis in one dog) or absent. The incidence of moderate to severe disease was not significantly different between the vaccinated and control dogs (P=0.124, Fisher's exact test). Due to the small number of dogs, the a posteriori power of the test was too low (0.15) to detect a significant difference. Twenty-one dogs per group would be needed to detect the same difference (30%) with a probability equal to 80%.

Haematology

No thrombocytopenia was recorded after challenge in the vaccinated dogs from studies 3 and 4, except for one vaccinated dog on day 1 post L. interrogans serovar icterohaemorrhagiae challenge. In contrast, 40% (study 4) to 75% (study 3) of the controls became thrombocytopenic after challenge. Over the 1 to 7 days post-challenge period, the platelet count was significantly lower in the controls than in the vaccinates after L. interrogans serovar canicola challenge (P=0.0001, Mixed model), and the difference was close to significance (P=0.08, Wilcoxon's test) after L. interrogans serovar icterohaemorrhagiae challenge.

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

Biochemistry

Sharp increases in urea, creatinine, bilirubin, SGOT, or SGPT values were found after challenge in three adult controls from study 4 and in the two control puppies that were added to study 4 at the time of challenge. All these dogs developed severe clinical disease and all three adult dogs and one of the two puppies succumbed to the challenge. In contrast, none of the vaccinates had increased urea, creatinine, or bilirubin values. Large modifications of baseline values of SGPT were recorded in two vaccinated dogs.

Leptospiraemia

An overview of the results is provided in Figures 1A (study 1) and 1B (study 2) and Tables 4A ( Study3) and 4B (study 4). All blood samples from studies 1-4 were negative for leptospires before challenge. Leptospires could be isolated from the blood of all controls from studies 1 and 2 for at least 3 days following challenge. Leptospiraemia persisted for up to 6 and 10 days for Leptospira interrogans serovars icterohaemorrhagiae and canicola, respectively. None of the vaccinated dogs from studies 1 and 2 developed leptospiraemia indicating that infection was not established in any of the vaccinated dogs.

All control puppies from studies 3 and 4 added at the time of challenge developed leptospiraemia. Leptospires could be isolated from all vaccinated and control dogs of study 3.

The total amount of leptospira isolated from the blood over the first 7 days after challenge was significantly lower in the vaccinated dogs than in the control dogs (P<0.0001, one-sided student t-test). In addition, the duration of leptospiraemia was significantly shorter in the vaccinated dogs compared to the control dogs (P=0.0002, student t-test).

All control dogs and seven out of nine vaccinated dogs from study 4 developed leptospiraemia. Both amount and duration of leptospiremia were significantly reduced in the vaccinated dogs compared to the control dogs (P=0.0001 for both, Wilcoxon's test).

Isolation of leptospires from urine, kidney and livers

An overview of the results is given in Figures 1A (study 1) and 1B (study 2) and Tables 4A (study 3) and 4B (study 4). All urine samples from studies 1-4 were negative for leptospires before challenge. Leptospires could be isolated from the kidneys of all control dogs and from the urine of 37.5% and 30% of the control dogs in studies 1 and 2, respectively. In contrast,

A c c e p t e d M a n u s c r i p t none of the vaccinated dogs from studies 1 and 2 had any positive urine or kidney cultures at the time of euthanasia. The proportion of dogs with renal infection, characterized by the presence of leptospires in urine and/or kidneys, was significantly lower in the vaccinated dogs compared to the controls dogs in both studies (Table 5)

Seven out of 8 adult control dogs from study 3 shed L. interrogans serovar canicola in the urine, and the kidneys of three control dogs were cultured positive. Leptospires could be isolated from the urine of two vaccinated dogs and from the kidney of one vaccinated dog.

The incidence of renal carriers was significantly lower in the vaccinated dogs than in the control dogs (P=0.035, Fisher's exact test, Table 5).

Urine could be collected from only nine adult control dogs of study 4. L. interrogans serovar icterohaemorrhagiae could be recovered from the urine of eight dogs and from the kidneys of five dogs, but not from the livers. None of the vaccinated dogs shed leptospires in the urine, and leptospires could not be isolated from any of the kidneys or livers at post-mortem examination. The incidence of renal carriage was significantly lower in the vaccinated dogs compared to the control dogs (P=0.0006, Fisher's exact test, Table 5).

Necropsy and histopathology

Necropsy

The macroscopic lesions detected during necropsy were similar for all dogs from studies 1-4 that died or were humanely euthanised due to terminal illness. Gross findings included haemorrhages on the surface of the lungs and the abdominal cavity and the presence of red stained fluid in the pleural and abdominal cavity. The kidneys were enlarged and friable.

When urine was present from these animals, it was tinged with blood up to severely haematuric. Typically, faecal material was liquid and tinged with blood having a fetid odour.

In some dogs, the sclera, gingival, and subcutaneous tissues were jaundiced. Apart from some reactive mesenteric lymph nodes, control dogs that survived the experimental infection appeared normal on gross visual examination, as well as did all vaccinated dogs.

Microscopic examination

Prominent lesions in the kidneys of terminally ill control dogs included subacute to severe interstitial glomerulo-nephritis and tubular degeneration. Moderate to severe diffuse hepatic

Discussion

A number of factors must be considered in the design and evaluation of efficacy trials for canine leptospirosis vaccines. These factors include the age of the dogs, recommended vaccination schedule, selection of challenge strain, and challenge method. The ultimate goal of vaccination against leptospirosis is to protect dogs against clinical disease, as well as against the establishment of a renal carrier state. The latter protection is especially important because carrier dogs can be a public health hazard when in close contact to humans (Center for Disease Control, 1972[START_REF] Trevejo | Epidemic leptospirosis associated with pulmonary hemorrhage-Nicaragua[END_REF]. Therefore, leptospirosis vaccines should be tested in models that reliably produce the series of clinical signs and renal colonization pattern that the vaccine is designed to prevent or reduce. Canine leptospirosis has been a difficult disease to reproduce under experimental conditions and usually requires the use of young puppies and a high challenge dose [START_REF] Keenan | Pathogenesis of experimental leptospira interrogans serovar bataviae, infection in the dog: microbiological, clinical, hematologic and biochemical studies[END_REF]. Furthermore, clinical signs may vary depending on the isolate [START_REF] Greenlee | Clinical and pathologic comparison of acute leptospirosis in dogs caused by two strains of Leptospira kirschneri serovar grippotyphosa[END_REF], altered expression of bacterial proteins resulting from culture passage [START_REF] Greenlee | Clinical and pathologic comparison of acute leptospirosis in dogs caused by two strains of Leptospira kirschneri serovar grippotyphosa[END_REF], and the timing of harvest after hamster passage (Minke, personal observation). Even when taking these factors into account, reported infection in control dogs often results in no evident [START_REF] Klaasen | Duration of immunity in dogs vaccinated against leptospirosis with a bivalent inactivated vaccine[END_REF] or only subclinical disease [START_REF] Broughton | Prevention of renal carriage of leptospirosis in dogs by vaccination[END_REF]Andre-Fontaine et al, 2003;[START_REF] Schreiber | Prevention of renal infection and urinary shedding in dogs by a Leptospira vaccination[END_REF].

In only a few studies has severe lethal disease been reported following experimental infection of dogs with L. interrogans serovar canicola (Schreiber et al, 2005a;[START_REF] Kerr | Protection against the renal carrier state by a canine leptospirosis vaccine[END_REF] or L. interrogans serovar icterohaemorrhagiae [START_REF] Kerr | Protection against the renal carrier state by a canine leptospirosis vaccine[END_REF]. In our studies, puppies experimentally infected with Leptospira interrogans serovars icterohaemorrhagiae and canicola developed a spectrum of disease that ranged from mild to lethal in severity.

Renal, hepatic and haematological signs dominated the clinical presentation and supported the polysystemic nature of leptospira infection. The overall mortality rate in control puppies was 60% and 58% for Leptospira interrogans serovars icterohaemorrhagiae and canicola, respectively. Under these extreme challenge conditions, clinical signs in the vaccinated pups A c c e p t e d M a n u s c r i p t were rare, and when observed, mild and transient in nature. Clinical disease in adult dogs was less severe, but unexpectedly, we were able to induce morbidity and mortality in adult dogs as well, further demonstrating the severity of our challenge models. These results are in sharp contrast with those published by [START_REF] Klaasen | Duration of immunity in dogs vaccinated against leptospirosis with a bivalent inactivated vaccine[END_REF], where no evident clinical symptoms associated with canine leptospirosis were observed in the adult control dogs. The reason for this difference is not clear but may be attributable to the choice of challenge strain and/or challenge dose. Hematological parameters and blood biochemistry were not intended to be a major criterion to assess the efficacy of the vaccine, but they supported the diagnosis of leptospirosis. Thrombocytopenia was the main hematological abnormality observed in control dogs after challenge, while vaccinated dogs were protected against thrombocytopenia. This hematological disorder is a common finding in canine leptospirosis (Greene, 1998) and has been reported after experimental challenge [START_REF] Tronel | Efficacy of Leptodog vaccine in dogs demonstrated by experimental challenge: Evaluation at short term and duration of immunity[END_REF][START_REF] André-Fontaine | Comparison of the efficacy of three commercial bacterins in preventing canine leptospirosis[END_REF][START_REF] Klaasen | Duration of immunity in dogs vaccinated against leptospirosis with a bivalent inactivated vaccine[END_REF]Schreiber et al, 2005a[START_REF] Schreiber | Prevention of renal infection and urinary shedding in dogs by a Leptospira vaccination[END_REF]. Blood biochemistry illustrated the alteration of hepatic and renal functions in control dogs. Significant changes in urea nitrogen, creatinine, bilirubin, SGOT, and SGPT were observed only in the control dogs with severe clinical signs. The increased levels of SGPT in two vaccinated dogs did not correlate with the clinical observations. It cannot be ruled out that the massive challenge induced transient liver damage in those dogs. At necropsy, macroscopic examination of the animals was consistent with clinical signs, and typical lesions of leptospirosis were observed in dogs succumbing to the challenge. In addition, microscopic analysis showed that even surviving controls had lesions of interstitial nephritis compatible with leptospirosis, whereas no specific lesions were observed in the vaccinated dogs.

The second objective of our studies was to determine whether EURICAN L would protect dogs against the development of a renal carrier state. As in many instances, isolation results on kidney and urine samples were not concordant in our studies, we defined a renal carrier as a dog with at least one positive urine or kidney culture. Discrepancies between urine and kidney isolation results have also been reported in the literature and were attributed to the presence of specific inhibiting enzymes from kidney cells [START_REF] Faine | Topley and Wilson's Microbiology and Microbial Infections[END_REF], high urine osmolarity and pH [START_REF] Nervig | Use of furosemide to obtain bovine urine samples for leptospiral isolation[END_REF], and the fact that leptospires are shed intermittently [START_REF] Nervig | Use of furosemide to obtain bovine urine samples for leptospiral isolation[END_REF]). Overall we found that 100% of the control pups and 83% of the adult controls became renal carriers. Despite the heavy challenges, none of the 18 vaccinated puppies and only two out of the 16 vaccinated adult dogs developed a renal carrier state. It should be stressed that the challenge doses that we used were probably much higher than those observed in a natural infection, suggesting that the protection against renal carriage



  EURICAN is a registered trademark of Merial in France and elsewhere A c c e p t e d M a n u s c r i p t 4

  virus, canine adenovirus type 2, canine parvovirus and canine parainfluenza type 2 virus. This second combination vaccine is commercialized under the name EURICAN DHPPi2L.

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

  DIMAZON is a registered trademark of Intervet Internationl B.V. in the United Kingdom and elsewhere. STATGRAPHICS is a registered trademark of Statistical Corporation in the United States of America; SAS is a registered trademark of SAS Institute Inc. in the United States of America and elsewhere A c c e p t e d M a n u s c r i p t Leptospiremia Because no leptospiremia was found in the vaccinated pups from studies 1 & 2, no statistical analysis was performed.

  contrast, vaccinated pups showed no or only mild transient signs. Two vaccinated pups had slight conjunctivitis and one pup developed mild digestive signs lasting for one day. The incidence of moderate to severe disease was significantly lower in the vaccinated pups than in the control pups (P=0.00004, Fisher's exact test) All 10 control pups from study 2 developed clinical signs following L. interrogans serovar icterohaemorrhagiae challenge. Six control pups were humanely euthanised because of severe disease between DPC 4 and 7. Clinical signs were similar between dogs challenged with L. interrogans serovars icterohaemorrhagiae and canicola and included depression, anorexia, haemorrhagic diarrhoea, vomiting, icterus, and haematuria. Three control pups showed only mild clinical signs consisting of depression and mild diarrhoea, and one control pup showed no clinical signs. Only one vaccinated pup was depressed on DPC 2 and 8. The incidence of A c c e p t e d M a n u s c r i p t moderate to severe disease was significantly lower in the vaccinated pups than in the control pups (P=0.0077, Fisher's exact test) The two control puppies added at the time of challenge in study 3 died from severe leptospirosis on DPC 4 and 5, validating the challenge. As expected, clinical signs in the adult controls were less severe than in the control pups. Nevertheless, four control dogs developed moderate and one dog severe leptospirosis. The latter dog was humanely euthanised on DPC 7 after having shown characteristic signs of frank leptospirosis. Clinical signs in the moderately ill dogs included conjunctivitis, mild diarrhoea, dehydration, and weight loss. Five out of seven vaccinated dogs did not show any clinical signs during the observation period. One vaccinated dog showed conjunctivitis on DPC 18, and two dogs had mild diarrhoea for 2 consecutive days starting on DPC 23. The incidence of moderate to severe disease was significantly different between the vaccinated and control dogs (P=0.0186, Fisher's exact test).

  in dogs with jaundice, mostly consisting of an acute degenerative hepatitis characterized by hepato-cellular dissociation and necrosis. Interestingly, in three surviving control dogs of study 4, there was evidence of sub-acute multi-focal interstitial nephritis compatible with leptospirosis infection. No specific lesions were found in the vaccinated dogs. Only the kidneys from dogs diagnosed with acute renal failure stained positive by Warthin-Starry silver indicating the presence of leptospires.

  Abbreviations: V= vaccinated; C= control. * In brackets the number of dogs that died or had to be euthanized after challenge. ** Fisher's exact test Study 1 = onset of immunity L. interrogans serovar canicola Study 2 = onset of immunity L. interrogans serovar icterohaemorrhagiae Study 3 = duration of immunity L. interrogans serovar canicola Study 4 = duration of immunity L. interrogans serovar icterohaemorrhagiae

Figure 1A :

 1A Figure 1A : Results of blood, urine and kidney cultures after challenge of puppies with L. interrogans serovar canicola. Puppies were challenged two weeks after a primary course of two doses of vaccine (study 1: onset of immunity)

  Figure 1A :

  Figure 1B :

Table 1 :

 1 Experimental design

	Study	Designation	Group	# dogs	Challenge
					Time after V2	Serovar
	1	Onset of immunity	V C	9 8	2 weeks	Lc
	2	Onset of immunity	V C	9 10	2 weeks	Li
	3	Duration of immunity	V C	7 8*	14 months	Lc
	4	Duration of immunity	V C	9 10*	14 months	Li
	*2 control pups were added at the time of challenge V=Vaccinated, C=Control, V2 = second

vaccination Lc = L. interrogans serovar canicola, Li = L. interrogans serovar icterohaemorrhagiae

Acknowledgements

The authors wish to thank Merial R&D Department for their help and Bob Nordgren for his critical reading of the manuscript.

A c c e p t e d M a n u s c r i p t 16 might be almost complete in the field. The literature has conflicting reports on the efficacy of leptospiral bacterins to protect against the renal carrier state. Much of the variability is likely the result of differences in the immunogenicity of the bacterins used, as was demonstrated by Andre Fontaine et al (2003). In that study, only one of the three commercial vaccines completely protected dogs against the establishment of a renal carrier state shortly after primo-vaccination in a challenge model that induced no mortality and severe clinical disease in only one out of the six control puppies. Culture appeared to more sensitive in our hands than silver staining to detect leptospires in the kidney, with the additional advantage that infectious material is detected, rather than fragments of the bacteria. We did not explore alternative detection methods like PCR or immuno-fluorescence.

Typical serological findings in the present studies were the relatively low and short-lived antibody responses against both serovars after vaccination. Several studies have reported low antibody responses after administration of leptospirosis inactivated vaccines (Andre-Fontaine et al, 2003;[START_REF] Schreiber | Prevention of renal infection and urinary shedding in dogs by a Leptospira vaccination[END_REF], Klaasen et al, 2003;[START_REF] Steger-Lieb | An old disease with a new face: canine leptospirosis does not loose its relevance[END_REF]. Furthermore, no correlation could be established between antibody titers after vaccination and protection against experimental infection. The absence of correlation has been classically described in other studies as well [START_REF] Broughton | Prevention of renal carriage of leptospirosis in dogs by vaccination[END_REF]Andre-Fontaine et al, 2003;Klaasen et al, 2003;Schreiber et al, 2005a,).

It is concluded that a primary course of two doses of EURICAN L provided quick onset and long-term protection against both clinical leptospirosis and the renal carrier stage. This vaccine should provide veterinarians with a powerful tool to prevent clinical disease in dogs and zoonotic transmission of leptospirosis to humans.