Characterization of Pasteurella Multocida and Related Isolates from Rabbits

Zurich Open Repository and Archive University of Zurich Main Library Strickhofstrasse 39 CH-8057 Zurich www.zora.uzh.ch

Year: 2007

Characterization of Pasteurella multocida and related isolates from rabbits

Stahel, Anina B J

Posted at the Zurich Open Repository and Archive, University of Zurich ZORA URL: https://doi.org/10.5167/uzh-204344 Dissertation Published Version

Originally published at: Stahel, Anina B J. Characterization of Pasteurella multocida and related isolates from rabbits. 2007, University of Zurich, Vetsuisse Faculty. Aus dem Institut für Veterinärbakteriologie der Vetsuisse-Fakultät Universität Zürich

(Direktor: Prof. Dr. M. M. Wittenbrink)

Arbeit unter der Leitung von Prof. Dr. R. K. Hoop

Characterization of Pasteurella multocida and related isolates from rabbits

Inaugural-Dissertation

zur Erlangung der Doktorwürde der Vetsuisse-Fakultät Universität Zürich

vorgelegt von

Anina Barbara Jennifer Stahel

Tierärztin von Zürich / Hinwil ZH

genehmigt auf Antrag von

Prof. Dr. R. K. Hoop, Referent Prof. Dr. R. Stephan, Korreferent

Zürich 2007

To everyone who seeks knowledge

These days people seek knowledge, not wisdom. Knowledge is of the past, wisdom is of the future.

Vernon Cooper of the Lumbee Tribe

Science knows no country, because knowledge belongs to humanity, and is the torch which illuminates the world.

Louis Pasteur

CONTENTS

1. Summary 8

2. Introduction 9

3. Literature survey 12 3.1. Taxonomy of Pasteurella multocida 12 3.2. Bacterial and cultural characteristics 12 3.3. Virulence factors 13 3.4. Identification and typing 14 3.4.1. Biotyping 14 3.4.2. Serotyping 14 3.4.2.1. Capsular serotyping 14 3.4.2.2. Somatic serotyping 15 3.4.3. Molecular identification 15 3.4.4. Molecular typing 16 3.4.4.1. Restriction Endonuclease Analysis (REA) 16 3.4.4.2. Ribotyping 16 3.4.4.3. Pulsed Field Gel Electrophoresis (PFGE) 17 3.4.4.4. PCR fingerprinting 17 3.4.4.5. Amplified Fragment Length Polymorphism (AFLP) 18 3.4.4.6. Sequence analysis 19 3.5. Epidemiology 20 3.5.1. Occurrence and prevalence 20 3.5.2. Transmission 20 3.5.3. Pathogenesis 21 3.5.4. Predisposing factors 21

3.6. Clinical manifestations 22 3.6.1. Rhinitis, sinusitis 22 3.6.2. Pneumonia, pleuritis, pericarditis 22 3.6.3. Otitis 22 3.6.4. Conjunctivitis 23 3.6.5. Abscesses 23 3.6.6. Genital tract infections 23 3.6.7. Septicemia 24 3.6.8. Other infections 24 3.7. Diagnosis 24 3.7.1. Isolation of bacteria 24 3.7.2. Serodiagnosis 24 3.8. Treatment 25 3.9. Prevention 25 3.9.1. Barrier housing 25 3.9.2. Vaccination 26

4. Material and methods 27 4.1. Sources of isolates 27 4.2. Sequence analysis of the 16S rRNA and the rpoB gene 27 4.3. Phenotyping 28 4.4. Molecular characterization by REP-PCR 28

5. Results 30 5.1. Sequence analysis of the 16S rRNA and the rpoB gene 30 5.2. Phenotyping 31 5.3. Molecular characterization by REP-PCR 31

6. Discussion 33 6.1. Identification 33 6.2. Genotyping 37

7. Further investigations 38

8. Tables 40 Table I. 40 Table II. 41 Table III. 42 Table IV. 43 Table V. 44

9. Figures 45 Figure 1a. 45 Figure 1b. 46

10. Pictures 48 Picture 1 48 Picture 2 48 Picture 3 48

11. References 49

12. Acknowledgements 61

1. Summary

Several bacterial species belonging to the family Pasteurellaceae act as pathogens in rabbits. In particular, Pasteurella multocida is considered to be important and outbreaks caused by this species result in considerable economic losses. Proper identification of P. multocida at species and subspecies level is problematic and often not very precise. Moreover, Pasteurellaceae species isolated from rabbits are generally poorly characterized.

41 isolates from two different rabbit populations – six isolates from a breeding and fattening organization with group management and 35 isolates from post-mortem cases with pasteurellosis – were identified by DNA sequence comparison of the 16S rRNA and the rpoB genes and a selection of biochemical reactions. Furthermore, all 41 samples were genotyped by repetitive extragenic palindromic (REP)-PCR.

The phylogenetic analyses were distinct molecular methods for characterization and differentiation of inter- and intra-species variations within strains originating from rabbits. Phenotyping diverged from DNA-sequence-based identification. In particular fermentation of sorbitol, trehalose and dulcitol were inaccurate indicators for Pasteurella multocida classification. Genotyping by REP-PCR resulted in identical or similar characteristic banding patterns for strains of a specific Pasteurella species or subspecies.

Apart from strains identified as P. multocida ssp. multocida and P. multocida ssp. septica, P. canis and an unknown P. multocida ssp. were isolated. Strains showing no profound analogy with any currently described species within the family Pasteurellaceae were detected from clinically affected rabbits.

Keywords: Pasteurella, rabbit, 16S rRNA, rpoB, phenotype, REP-PCR

2. Introduction

Respiratory diseases, often induced by bacterial infection, are predominant causes of death in rabbits. Pasteurella multocida infection is the most significant and severe bacterial disease throughout the world (DeLong and Manning, 1994). In pet rabbits P. multocida is considered opportunist or secondary pathogen, in colonies however, this organism can emerge as a primary pathogen and result in considerable economic losses (Schimmel et al., 1996; Virag et al., 2005). Non-infected and resistant animals, chronic healthy carriers, animals with local infections (e.g. rhinitis, otitis media), pneumonia and septicemia can be distinguished (DeLong and Manning, 1994).

The current identification of P. multocida is mainly based on complex phenotypic characterization which is often time consuming and difficult to interpret (Christensen et al., 2007). Phenotypic variations in single key characters of P. multocida have been reported (Christensen et al., 2004a; Fegan et al., 1995). They complicate the proper assignment on species and subspecies level and make routine diagnostics uncertain.

An accurate assignation of P. multocida on the subspecies level, namely P. multocida ssp. multocida, P. multocida ssp. septica and P. multocida ssp. gallicida is possible using DNA-DNA hybridization (Mutters et al., 1985). However, DNA-DNA hybridization is known to be time-consuming and laborious and has often been criticized due to high experimental errors and the failure at generating cumulative databases (Rosselló-Mora, 2006). New methods to replace DNA-DNA hybridization for rapid identification and determination of novel taxa have been developed (Christensen et al., 2007; Stackebrandt et al., 2002).

A more powerful and promising tool for classification is the DNA-sequence-based identification using the 16S rRNA and rpoB genes (Christensen et al., 2007; Stackebrandt et al., 2002). The comparative sequence analyses of these genes help to clarify the phylogenetic relationship within the family Pasteurellaceae. So far 16S rRNA gene sequence comparison has played the major role in the definition of bacterial species. Changes in sequence of the highly conserved 16S rRNA gene are known to

mark evolutionary distance and relatedness of organisms (Christensen et al., 2004b; Clarridge, 2004; Kuhnert et al., 2000). However, DNA sequence comparison of the rpoB and further housekeeping genes provide additional information for genetic identification (Christensen et al., 2004b; Korczak et al., 2004).

Pulsed field gel electrophoresis (PFGE), restriction endonuclease analysis (REA) and REP-PCR are more recent molecular techniques contributing to the epidemiological characterization of various strains (Blackall et Miflin, 2000; Christensen et al., 2007; Hunt et al., 2000; Stackebrandt et al., 2002). REP-PCR is known for its ease of application and interpretation, high discrimination power, good intra- and moderate interlaboratory reproducibility and the possibility to perform the PCR without extensive DNA extraction. It appears to be an ideal method for the investigation of genetic relatedness between Pasteurellaceae strains (Amosin et al., 2002; Biswas et al., 2004; Gunawardana et al., 2000; Healy et al., 2005; Olive et Bean, 1999; Shivachandra et al., 2005; Townsend et al., 1997; Versalovic et al., 1991; Virag et al., 2004; Woods et al., 1993).

In the present study we investigated the phenotypic and genetic relationship of 41 isolates belonging to the Pasteurellaceae family, obtained from rabbits by selected biochemical tests, comparative 16S rRNA and rpoB genes sequence analysis and REP- PCR.

The isolates came from the nares or sinus of slaughtered group management rabbits from different breeding and fattening farms of one Swiss rabbit meat organization and were collected during the years 2004, 2005 and 2006. The commercial rabbitries house ZIKA hybrids, a crossbreed between four high-capacity breeds. A ZIKA doe, which is replaced all 2 years, has approximately eight litters per year with 6 to 12 kits. The fattening rabbits are slaughtered between 70 and 90 days and weigh about 2.7-3.2 kg. They are held indoors, in groups of 8 does with one male and their offspring per breeding group and max. 28 animals per fattening group. The animals move around freely and find structures and contacts to others, this being unique for breeding rabbits in Europe. The floor space is organized in different areas for feeding and drinking, nesting and resting with a supplement of straw (Pictures 1-3; Benz, 2005). One main

breeder of ZIKA rabbits imports the breeding pairs from Southern Germany, and distributes the kits to other breeding farms; their offspring is sold to fattening farms all over Switzerland. The all-in all-out procedure along with strict separation of breeding and fattening stables is a pre-condition for a successful prophylaxis of diseases. However, over the last few years an increase of infectious diseases including pasteurellosis has been registered in these animal friendly housing systems (Benz, 2005; Probst, 2005; Meier, 2002; Lanz, 2000).

Simultaneously isolates from altered organs of post-mortem cases with pasteurellosis sent to our department by various rabbit owners were incorporated in the survey. The rabbits were of all age groups and showed pathologic findings such as rhinitis, otitis media, pneumonia, pericarditis, pleuritis, conjunctivitis, phlegmone, abcesses, meningoencephalitis, mastitis, endometritis, salpingitis and septicemia. The isolates were collected during 1991 and 1992 and during 2004 and 2005.

3. Literature survey

3.1. Taxonomy of Pasteurella multocida

The type species P. multocida („many killing“), together with the species P. canis, P. stomatis, P. dagmatis, and the unnamed taxon Pasteurella species B, form the core group of Pasteurella sensu stricto of the genus Pasteurella in the family Pasteurellaceae. Further species of the genus Pasteurella are P. gallinarum, P. avium, P. volantium, P. langaa, P. anatis and another unnamed taxon Pasteurella species A (Christensen et Bisgaard, 2003; Mutters et al., 1989).

The species of Pasteurella multocida can be divided into three subspecies, namely P. multocida ssp. multocida, P. multocida ssp. septica and P. multocida ssp. gallicida (Rimler et Rhoades, 1989). The current classification is mainly based on DNA-DNA hybridization and phenotypic characterization. DNA-DNA-binding values of 84-100%, 89-100% and 91-100% were found within the three subspecies (Mutters et al., 1985). A more powerful tool for classification is the DNA-sequence-based identification using the 16S rRNA and rpoB genes (Christensen et al., 2007; Korczak et al., 2004; Korczak et al., 2006; Kuhnert et al., 1996; Kuhnert et al., 2002; Stackebrandt et al., 2002). The comparative sequence analysis of these genes helps to clarify the phylogenetic relationship within the family Pasteurellaceae.

3.2. Bacterial and cultural characteristics

P. multocida is a gram-negative, nonmotile asporogenous coccobacillus of 0.2-0.4 x 0.6-2.5 µm that shows bipolar staining. The bacteria grow well on blood or dextrose starch agar but not on MacConkey’s agar. After aerobic incubation for 24 hours at 37°C the colonies are circular, convex and smooth with a diameter of 0.5-2.0 mm. Most isolates have large capsules forming mucoid colonies that appear to flow together. Less

often isolates have small or absent capsules and vary in coloration from blue to green iridescent. P. multocida emits a distinctive odor, which is liken to that of indole (Christensen et Bisgaard, 2003; Kpodékon et al, 1999; Manning et al., 1989; Rimler et Rhoades, 1989).

3.3. Virulence factors

Described virulence factors of P. multocida include adhesion factors, iron regulation, phagocyte resistance and an exotoxin, known as the P. multocida toxin (Ewers et al., 2004; Ewers et al. 2006).

Apart from the capsule, lipopolysaccarides responsible for endotoxic activity and genes coding for outer membrane and porin proteins (oma87, psl, omph), other non-fimbriae- associated and fimbriae-associated adhesins are known. The filamentous hemagglutinin gene (pfhA) and type 4 fimbriae (ptfA) are key elements for the attachment and colonization of epithelial host cells. Further virulence-associated genes detected so far are iron acquisition related factors (exbBD-tonB, tbpA, hgbA, hgbB), responsible for the provision of energy and binding of hemoglobin, transferrin or hemin as iron source and superoxid dismutases (sodA, sodC) for disruption of the mucocillary clearance. Some capsular type D and seldom type A strains produce an exotoxin, which can induce pneumonia, pleuritis, lymphoid atrophy and osteoclastic bone resorption in rabbits (Chrisp and Foged, 1991; DiGiacomo et al., 1993; Digiacomo et al., 1989; Ewers et al., 2004; Nielsen et al., 1986; Rimler and Brogden, 1986). To detect P. multocida toxin (PMT) producing strains an ELISA can be used based on monoclonal antibodies against the toxin (Foged et al., 1988). Several PCR assays targeting the toxA gene coding for the toxin have been described (Hotzel et al., 1997; Kamp et al., 1996; Lichtensteiger et al., 1996; Nagai et al., 1994). The PCR test described by Kamp et al. (1996) seems to be the most sensitive and effective method for large-scale analysis of nasal and tonsillar swabs; the method of Lichtensteiger et al. (1996) is simpler for smaller studies despite false positive results.

3.4. Identification and typing

3.4.1. Biotyping

Until now differences in biochemical reactions are used for the classification and nomenclature of Pasteurellaceae (Christensen et Bisgaard, 2003; Christensen et al., 2007; Mutters et al., 1985; Mutters et al., 1989; Rimler et Rhoades, 1989; Stackebrandt et al., 2002). Major phenotypical characteristics of various Pasteurella species are shown in table I.

P. multocida differs from other species of the genus Pasteurella by several biochemical reactions: positive reactions for ornithine decarboxylase, indole and mannitol and lack of fermentation of maltose and dextrin. According to Mutters et al. (1985), the main characteristics of the P. multocida subspecies are variations in sorbitol and dulcitol fermentation. P. multocida ssp. multocida ferments sorbitol, but reacts negative for dulcitol, P. multocida ssp. septica can neither ferment sorbitol nor dulcitol and P. multocida ssp. gallicida shows positive reactions for both sorbitol and dulcitol fermentation.

It must however be noted that high phenotypic variability has been reported for P. multocida strains, making classification difficult and imprecise (Biberstein et al., 1991; Bisgaard et al., 1991; Christensen et al., 2004a; Fegan et al., 1995; Korbel et al, 1992; Kuhnert et al., 2000; Mohan et al., 1994; Muhairwa et al., 2001; Petersen et al., 1998; Sachse et al., 1990; Truong et Schimmel, 1994).

3.4.2. Serotyping

3.4.2.1. Capsular serotyping Capsular serotyping is based on the passive haemagglutination of erythrocytes sensitized by capsule antigen (Carter, 1955). 5 capsule types can be distinguished: A, B, D, E and F (Rimler and Rhoades, 1989), nontypable isolates have been reported in rabbits (Chengappa et al., 1982; Lu et al., 1983; Mushin et Schoenbaum, 1980).

Most P. multocida isolates from rabbits represent the capsular type A, some isolates are of type D and appear to be especially pathogenic for mice (Manning et al., 1989). Serogroup F isolates have been reported (Jaglic et al., 2004; Jaglic et al., 2005).

3.4.2.2. Somatic serotyping Somatic serotyping is based on gel diffusion precipitin tests (Heddleston et al., 1972); another not widely used method is the tube agglutination (Namioka et al., 1978). The Heddleston system identifies 16 serotypes, namely the types 1-16.

In Europe the P. multocida serotype A:3 is the most common serotype in commercial rabbitries (Kpodékon et al., 1999), in the United States the serotypes A:12 and A:3 are the most prevalent types in rabbits (Chengappa et al., 1982; Manning et al., 1989).

Serotyping however is not sufficient for the characterization of P. multocida isolates based on pathogenic or epidemiological factors. Strains of the same serotype have revealed no difference in pathogenicity (Kpodékon et al., 1999).

3.4.3. Molecular identification

Species-specific PCR assays have been developed for the detection of P. multocida. The PCR tests target different genes such as the transcriptional regulator encoding genes Pm0762 and Pm1231 (Liu et al., 2004), the 23S rRNA gene (Miflin et Blackall, 2001) or a DNA sequence unique to P. multocida (KMT1) of a gene with unknown function (Townsend et al., 1998). The PCR of Liu et al. (2004) was not validated on other Pasteurella species, the PCR tests of Miflin et Blackall (2001) and Townsend et al. (1998) both give false positive reactions with P. canis biovar 2, the 23S rRNA-based PCR assay also demonstrates a positive reaction with P. avium biovar 2 strains. This misclassification has been opposed by a newer study of genotypic classification identifying P. canis biovar 2 and P. avium biovar 2 isolates to belong to P. multocida (Christensen et al., 2004a). Another approach is a PCR targeting the psl gene coding for the P6-like outer membrane protein (Kasten et al., 1997). However, this PCR has not been checked for specificity with various other Pasteurella species and false positive

reactions are found with Haemophilus influenzae strains. To increase sensitivity an additional hybridization with psl is required.

A recent study describes a 5’ Taq nuclease assay targeting the 16S rRNA gene of P. multocida, suitable for detection in field samples without prior cultivation. Cross- reactions with other Pasteurella species or other Pasteurellaceae were not found (Corney et al., 2007).

3.4.4. Molecular typing

3.4.4.1. Restriction Endonuclease Analysis (REA) A specific genetic locus is amplified by PCR, digested with various restriction enzymes, separated on an agarose gel and visualized following ethidium bromide staining. Distinctive digestion patterns allow the differentiation of isolates independent of biotyping, serotyping and other conventional typing methods.

Several restriction enzymes have been used for fingerprinting of P. multocida isolates. Therefrom HhaI and HpaII seem to be the most suitable and widely spread enzymes, HpaII showing a higher discriminatory power, resulting in finer typing with well distinguishable profiles.

REA is useful for fast and easy applicable screening with direct visual comparison of a small number of very closely related strains (Blackall et Miflin, 2000; Hunt et al., 2000; Olive et Bean, 1999).

3.4.4.2. Ribotyping Ribotyping, like REA, is based on restriction enzyme digestion of genomic DNA. Electrophoresis is followed by Southern blotting and hybridization with a labeled DNA probe specific for rRNA gene sequences, resulting in only selected DNA fragments being visible.

For ribotyping of P. multocida the restriction enzyme HpaII and the 16S + 23S rRNA from E. coli seem most suitable.

Compared to REA, the reduction of complex banding patterns and the simplicity of interpretation however, limits the ability to distinguish between closely related strains (Blackall et Miflin, 2000; Hunt et al., 2000; Olive et Bean, 1999).

3.4.4.3. Pulsed Field Gel Electrophoresis (PFGE) For pulsed field gel electrophoresis intact bacterial cells are embedded in agarose plugs, subjected to in situ detergent-enzyme lysis and digested with a rare cutting restriction enzyme. The resulting large DNA fragments are separated in a modified electrophoresis apparatus with periodical change in electric field.

PFGE with the restriction enzyme ApaI has been used on avian, ovine, bovine, rabbit and human P. multocida isolates.

PFGE is considered the „gold standard“ fingerprinting method for molecular epidemiology, demonstrating a high discriminatory power and good reproducibility. Limitations are set by the time for the whole procedure, the equipment needs and the costs (Blackall et Miflin, 2000; Hunt et al., 2000; Olive et Bean, 1999).

3.4.4.4. PCR fingerprinting The random amplified polymorphic DNA (RAPD) assay uses short random sequenced primers in order to amplify a bacterial genome region between the two annealed primers. The amplification products are separated by gel electrophoresis, a specific pattern of bands results.

This method however shows a lack of reproducibility and standardization (Olive et Bean, 1999).

Repetitive extragenic palindromic (REP)-PCR is a further PCR based typing method. REP-PCR is known for its ease of application and interpretation, high discrimination power, good intra- and moderate interlaboratory reproducibility and the possibility to

perform the PCR without extensive DNA extraction. It appears to be an ideal method for molecular typing.

REP elements are palindromic genome elements of 33-40 base pair repeats, which take up about 1% of the whole genome and include 500-1000 copies (Stern et al., 1984). They are found in a wide range of enteric bacteria, distributed unequally across the genome. These repeat sequences are used as templates and serve as an efficient primer- binding site for PCR analysis, amplifying the genome regions between the REP elements. The amplification products are uneven in size depending on the location of the REP elements and result in a multiplicity of bands apparent after electrophoresis and visualization.

REP-PCR was applied successfully for epidemiological typing of P. multocida and separation of isolates from various outbreaks (Amosin et al., 2002; Biswas et al., 2004; Gunawardana et al., 2000; Healy et al., 2005; Shivachandra et al., 2005; Townsend et al., 1997; Versalovic et al., 1991; Virag et al., 2004; Virag et al. 2005; Woods et al., 1993). Even, a classification of P. multocida isolates on subspecies level has been demonstrated (Chen et al., 2002).

REP-PCR and REA are considered the two most suitable methods for epidemiological investigation of P. multocida, requiring minimal equipment and allowing direct visual comparison of the typing patterns (Blackall et Miflin, 2000; Olive et Bean, 1999).

Enterobacterial repetitive intergenic consensus (ERIC) sequences are other described repeat sequences used for PCR fingerprinting (Blackall et Miflin, 2000; Olive et Bean, 1999).

3.4.4.5. Amplified Fragment Length Polymorphism (AFLP) Amplified fragment length polymorphism demonstrates a selective amplification of DNA fragments generated by restriction enzyme digestion with subsequent visualization using fluorescent labeling techniques and automated DNA sequencing equipment.

AFLP is a technique with high reproducibility and good discriminatory power, however it is more labor intensive and acquires a well-equipped laboratory (Blackall et Miflin, 2000; Olive et Bean, 1999).

3.4.4.6. Sequence analysis For sequencing and phylogenetic analysis of P. multocida isolates two genes seem to be ideal marker genes, the 16S rRNA and rpoB gene.

Only few other genes are as highly conserved as the 16S rRNA gene; changes in sequence are known to mark evolutionary distance and relatedness of organisms. The 16S rRNA gene sequence is composed of conserved regions at the beginning and end of the gene, to which universal primers will anneal and a more variable region in between, used for comparative sequence analysis (Clarridge, 2004; Kuhnert et al., 1996; Kuhnert et al., 2002).

The rpoB gene codes for the universal bacterial RNA polymerase ß-subunit. Comparison of the rpoB sequences has been used for phylogenetic analysis among some bacteria and species identification. Primers are designed complementary to conserved regions flanking the variable sequence-encoding region. The rpoB sequencing shows a higher discriminatory power and a higher level of divergence between sequences of different strains than phylogenetic trees obtained by 16S rRNA genes (Korczak et al., 2004; Korczak et al., 2006; Mollet et al., 1997).

DNA-sequence-based identification and comparative sequence analysis using the 16S rRNA and rpoB gene is a powerful tool for classification of strains on genus, species and subspecies level.

3.5. Epidemiology

3.5.1. Occurrence and prevalence

Pasteurella multocida exists since the species rabbit is known and on all continents of the world. Many rabbit colonies are endemically infected. According to several surveys the prevalence of P. multocida in the nares of evidently healthy mature New Zealand White rabbits ranges from 31% to 94% (DiGiacomo et al., 1983; Holmes et al., 1983; Lu et al., 1978; Mraz et al., 1980; Nakagawa et al., 1986). The prevalence increases with age at least through young adulthood or until about 5 months of age. In preweanlings (<1 month old) 4% of nasal cultures are positive, 0-63% in weanlings (1-3 months old) and 47-85% in older rabbits. The infection rate in kits is high when maternally acquired antibodies decrease and active immunity is not fully developed. Pharyngeal cultures are more likely to be positive than nasal cultures. This reflects the suggestion that P. multocida proliferates initially in the oropharynx with further spreading to the nasal passages. Therefore nasal positive rabbits are mostly nasopharyngeal positive, but not all nasopharyngeal positive animals carry Pasteurella in the nares (Patton et al., 1984).

3.5.2. Transmission

Transmission of P. multocida mainly occurs by direct contact and airborne spread. The fastest way of spread, in as few as 8 days, is the direct contact between uninfected and infected rabbits. Aerosol transmission between animals housed in the same room but in different cages is slower and takes up to 12 weeks; rabbits housed in separate rooms may be infected within 3-6 months (DiGiacomo et al., 1983). Contact with contaminated fomites such as watering valves or other objects is the likely cause of transmission over longer distances. The duration of survival of the organism on inanimate objects is not known. Genital tract infections in does or bucks have been reported as a further source of infection during mating and parturition. However, due to

successful elimination of the organism by caesarean re-derivation there is no evidence of vertical transmission (Manning et al., 1989).

3.5.3. Pathogenesis

P. multocida primarily enters the host through the nares and colonizes the mucous film covering the mucous membrane. Mostly a balance is achieved between bacterial proliferation and mucocillary clearance; the organism emerges as commensal and the rabbits are subclinical carriers of infection. If this balance is disrupted the infection spreads by direct extension to contiguous tissues (via paranasal sinuses, nasolacrimal duct to conjunctiva, eustachian tube to middle ear and via trachea, bronchi to the lungs) or by hematogenous spread to middle ear, lungs and other internal organs (Deeb et al., 2004; Manning et al., 1989).

3.5.4. Predisposing factors

Under conditions of immunodeficiency and stress P. multocida can emerge as pathogenic organism. Predisposing factors such as changes in nutrition, environment, management or social structure, as well as concomitant infections or physical and chemical injuries of the mucosa due to suboptimal temperature, ventilation and air humidity with increase of ammonia, dilute acetic acid and dust exposure, can lead to an outbreak of disease. Especially in times of pregnancy, parturition and lactation the does are exposed to higher levels of stress; weaning, the purchase of new animals and overcrowding are further straining factors (Deeb et al., 2004; Kpodékon et al., 1999; Manning et al., 1989).

3.6. Clinical manifestations (according to Deeb et al., 2004; Kpodékon et al., 1999; Manning et al., 1989)

3.6.1. Rhinitis, sinusitis

Rhinitis and sinusitis are the most common manifestations of pasteurellosis in rabbits. Primary sign is serous, mucoid or mucopurulent nasal discharge, followed by exudate adhering to the fur around the nares and the medial aspects of the forepaws due to grooming. Affected rabbits may breathe audibly and sneeze, but feature no other signs of respiratory distress. In conventionally managed colonies rhinitis is mostly a chronic condition; up to 60% of the rabbits may be affected, with peaks of incidence in spring and fall. Approximately 50% of the rabbits harboring P. multocida in the nares show no clinical signs of infection (DiGiacomo et al., 1983).

3.6.2. Pneumonia, pleuritis, pericarditis

Infections of the lower respiratory tract are usually chronic and asymptomatic. Specific clinical signs of pleuropneumonia, pericarditis or thoracic abscesses are seldom found, the diagnosis is mostly made at necropsy. About 20% of apparently healthy 8-10 week old rabbits were shown to have macroscopic lesions of pneumonia (Flatt et Dungworth, 1971). Nonspecific signs such as anorexia and depression should arouse suspicion of infection.

3.6.3. Otitis

The infection probably reaches the middle ear by the eustachian tubes. Therefore, most rabbits with otitis media simultaneously suffer from rhinitis. Otitis media is usually asymptomatic and represents a major reservoir of P. multocida, often unreachable for antibiotics. Once the infection has spread to the inner ear acute clinical signs of

torticollis, nystagmus and ataxia can occur. In conventional rabbitries the prevalence of otitis media at necropsy was shown to be about 33%; however, fewer than 5% of the rabbits demonstrated torticollis (Snyder et al., 1973).

3.6.4. Conjunctivitis

Conjunctivitis has been mentioned as a common manifestation of pasteurellosis, second only to rhinitis. The most probable route of infection is via the nasolacrimal duct. Clinical signs include ocular discharge, chemosis and hyperemia.

3.6.5. Abscesses

Abscesses are most commonly found in subcutaneous tissues of the head, neck or shoulders; further sites of infection are the mandibula and hock joints, the retrobulbar tissues and other internal organs such as lungs, heart, brain and reproductive tract. Spreading of the bacteria occurs by direct extension, hematogenous or lymphatic, and wound contamination.

3.6.6. Genital tract infections

Infections of the genital tract often proceed asymptomatic and chronic, however in breeding does a low conception rate can be an indicator of disease. In does pyometra is common, bucks may suffer from orchitis and epididymitis. Venereal transmission is the most important way of transmission.

3.6.7. Septicemia

Septicemia is the commonest cause of sudden death. It has been reported that septicemia followed a placement of rabbits in an old poultry farm in a time shorter than 24 hours.

3.6.8. Other infections

Cases of osteomyelitis and meningoencephalitis have been described, but occur seldom in commercial rabbitries.

3.7. Diagnosis

3.7.1. Isolation of bacteria

An inexpensive and easy applicable method for the detection of P. multocida in rabbits is the bacterial culture of nasal swabs. The swabs are inserted 1-4 cm into the nares along the nasal septum, inoculated on blood agar plates and incubated aerobic at 37°C for 24 hours (Deeb et al., 2004; Manning et al., 1989). As approximately 30% of infected animals are not detected by a single culture, one suggests that three sequential swabs should be taken within one week (Holmes et al., 1986; Manning et al., 1987). Only a positive result is proof for an infection with P. multocida.

3.7.2. Serodiagnosis

Because cultures of the nares may underdiagnose low numbers of organisms in subclinical carriers or in difficult to access sites such as the pharynx, several serological tests have been developed, including agar gel diffusion tests (Heddleston et al., 1972) enzyme-linked immunosorbent assays (Holmes et al., 1986; Klaassen et al., 1985;

Lukas et al., 1987) and a dot-immunobinding assay (Manning et al., 1987). To avoid false negative results due to lack of seroconversion and to ensure a Pasteurella-free status, the test should be repeated after 2-3 weeks. False positive results are based on an infection with related bacteria or on maternally acquired antibodies, which should have disappeared within 8 weeks (Deeb et al., 2004; Manning et al., 1989).

3.8. Treatment

Various antibiotics are available for the treatment of pasteurellosis in rabbits. The therapy of choice seems to be penicillin; most P. multocida isolates reacting sensitive to this antibiotic. Other described antibiotics are tetracyclines, trimethoprim-sulfonamide or enrofloxacin, which may even be more effective than penicillin (Broome et Brooks, 1991; Suckow et al., 1996).

However, the treatment of pasteurellosis in rabbits is difficult. Many rabbits remain asymptomatic carriers, despite decrease in clinical signs; recurrences of disease are common (Deeb et al., 2004; Manning et al., 1989).

3.9. Prevention

3.9.1. Barrier housing

In order to establish Pasteurella-free rabbitries several factors have to be considered. Optimal husbandry in barrier facilities with prevention of disease transmission by equipment and personnel, proper feeding practices, avoidance of stress especially during weaning and ideal ambient temperature, air humidity and ventilation, are some of the important principles of controlling pasteurellosis.

Further methods for total sanitation of rabbit colonies are culling of infected animals after repeated bacteriological and serological screening, cesarean derivation, hand raising and early weaning with or without the use of antimicrobials (Kpodékon et al., 1999; Manning et al., 1989).

3.9.2. Vaccination

An inactivated vaccine containing Pasteurella multocida A, Bordetella bronchiseptica and the Pasteurella toxoid is available in Germany since 1997 (Cunivak Past, Impfstoffwerk Dessau-Tornau GmbH).

A primary vaccination of the colony before the start of the breeding season is recommended on a yearly basis; the breeding does and bucks should be revaccinated in a semi-year turnover and before higher risk of exposure (e.g. exhibition). Juveniles should be vaccinated after weaning. In rabbitries with high infection rates a revaccination of the does during the first trimester of pregnancy and of the bucks in shorter time intervals is of interest (Holubek, 2000a; Holubek, 2000b; Holubek, 2004; Schmidt et Holubek, 2001).

Under good hygienic conditions the vaccination contributes to a long-term reduction of clinical signs and a dilution of the pathogen in chronic infected rabbit colonies. The rabbits however remain carriers.

4. Material and methods

4.1. Sources of isolates

Six isolates were gathered from the nares or sinus of slaughtered group management rabbits during the years 2004 to 2006. The animals came from different breeding and fattening farms of one Swiss rabbit meat organization. There was no information about the clinical status of the animals available.

35 isolates were from altered organs of post-mortem cases with pasteurellosis, sent to our department by various rabbit owners. The rabbits were of all age groups and showed pathologic findings such as rhinitis, otitis media, pneumonia, pericarditis, pleuritis, conjunctivitis, phlegmone, abcesses, meningoencephalitis, mastitis, endometritis, salpingitis and septicemia. 24 of these isolates were collected during 1991 and 1992 and eleven during 2004 and 2005.

4.2. Sequence analysis of the 16S rRNA and the rpoB genes

Sequences were determined as described previously (Korczak et al., 2004; Kuhnert et al., 2002). Sequencing products were run on an ABI Prism 3130xl Genetic Analyzer (Applied Biosystems, CA, USA) followed by editing using the SEQUENCHER software (GeneCodes, MI, USA). The sequences obtained were cross-compared against GenBank (Benson et al., 2000) and SmartGene (Simmon et al., 2006) databases. Phylogenetic analysis of the proofread sequences and selected GenBank entries representing each described genus within the Pasteurellaceae family was performed using neighbor-joining tree building with the BioNumerics program version 4.61 (Applied Maths, Belgium). The determined sequences were submitted to GenBank (National Center for Biotechnology Information, Bethesda, MD).

4.3. Phenotyping

The isolates were inoculated on 5% sheep blood agar (Oxoid, Hampshire, UK) and incubated under aerobic conditions at 37°C for 24 hours.

The biochemical characterization was carried out as described previously (Mutters et al., 1985). Briefly the genus Pasteurella was identified using the following reactions: Nicotinamide adenine dinucleotide (NAD)-dependence, hemolysis, indole production, oxidase, catalase, and synthesis of lysine. For further assignation on the species level, dulcitol, sorbitol, mannitol, trehalose, maltose, urease and ornithine decarboxylase (ODC) tests were performed.

Reactions were observed after inoculation of one loop of 1-2 colonies in 2 ml of growth medium with appropriate indicator and incubation at 37°C for 48 hours; sorbitol reactions were recorded up to 14 days.

4.4. Molecular characterization by REP-PCR

REP-PCR was performed using the two opposing primers REP1R-IDt: REP1R-IDt: 5’– NNNNGCNGCNGTAGNCCG–3’ and REP2-IDt: 5’-NCGNCTTATCNGGCCTAC-3’ targeting noncoding repetitive extragenic palindromic sequences of bacteria (Versalovic et al., 1991).

The PCR was performed with bacterial cell lysates. 3 to 4 colonies from blood agar plates were transferred in 50 μl Nuclease-free water (Promega, WI, USA) and boiled in a heating block at 100°C for 10 min. After centrifugation with 10000 x g for 5 min, the supernatant was collected and used as a template for the PCR. The PCR mixture contained: 1 x PCR buffer with 4 mM MgCl2 and 1.25 U Taq DNA polymerase (TaqBead™ Hot Start Polymerase, Promeaga, WI, USA) as well as 200 μM of each of the four dNTPs (Promega, WI, USA) and 50 pmol of each opposing primer in 50 μl final volume. 2.5 μl of bacterial cell lysate was added as template. REP-PCR was run on a DNA 2720 Thermal Cycler (Applied Biosystems, CA, USA) with an initial denaturation step at 95°C for 7 min, followed by 35 cycles of denaturation at 94°C for 1

min, annealing at 40°C for 1 min and extension at 65°C for 8 min. The PCR was terminated with a final extension at 65°C for 16 min (Rademaker et De Bruijn, 1997).

The amplified genome elements were electrophorised at 70 V for 240 min on a 1.5% agarose gel (Agarose Standard, Eurobio, Courtaboeuf, France) with ethidium bromide in 1 x TAE. The molecular size marker ranged from 100 to 3000 bp bands (peQLab, peqGold 100 bp DNA-Leiter plus, Erlangen, Germany). Bands were visualized by UV transillumination and the size of the fragments was analzed with a computer-aided bioimage system (Alpha Innotech, CA, USA).

REP-PCR was repeated twice to check reproducibility. The assigning of the profile produced by each strain was referred to as distinct when it differed by ≥ 1 band.

5. Results

5.1. Sequence analysis of the 16S rRNA and rpoB genes

After truncation of the primer sequences, the 16S rRNA gene sequencing mostly resulted in a fragment of 1364 bp; ten samples (Clin. 829 to Clin. 7) gave a fragment of 1356 bp and were placed together in the same distinct cluster. All rpoB sequences were of the same length of 520 bp.

The phylogenetic relationship of the 16S rRNA and the rpoB gene sequences of our 41 isolates are illustrated in Figs. 1a. and 1b., respectively. The investigated strains clustered in a very similar way in both trees.

In the 16S rRNA-based tree the strains were grouped in five clusters. Most of the samples were located in a very homogeneous cluster represented by the type strain of P. multocida ssp. multocida and P. multocida ssp. gallicida and showed a very high identity to both type strains of 99.93-100 % and 99.79-99.85 %, respectively.

The rpoB gene provided a higher resolution than the 16S rRNA and split the strains located in the P. multocida ssp. multocida / P. multocida ssp. gallicida cluster of the 16S rRNA-based tree in two clusters. Nine strains showed 99.23-99.81 % sequence identity to the type strain of P. multocida ssp. multocida and only 96.46-97.26 % sequence identity to the type strain of P. multocida ssp. gallicida. Interestingly, eleven further strains formed an own cluster that was clearly separated from P. multocida ssp. multocida (sequence identity 95.45-96.66 %), P. multocida ssp. gallicida (sequence identity 95.85-98.25 %), and P. multocida ssp. septica (sequence identity 92.74- 93.79 %).

In both trees, the same six isolates clustered together with the type strain of P. multocida ssp. septica showing high sequence identities of 99.71-100 % for the 16S rRNA and 99.03-100 % for the rpoB genes.

Furthermore, four isolates sharing identical 16S rRNA and rpoB sequences were placed in a cluster together with the Pasteurella canis type strain and differed from it in 1 and 8 base pairs, respectively.

The isolates with shorter identical 16S rRNA gene sequences of 1356 bp and one unique isolate differed strongly from the other Pasteurella strains and formed two clusters distinct from currently known species within the family Pasteurellaceae.

All sequences were submitted to GenBank (National Center for Biotechnology Information, Bethesda, MD) under the accession numbers ranging from EF579813 to EF579894.

5.2. Phenotyping

All 41 isolates were NAD-independent, non-hemolytic, oxidase and catalase positive and negative for lysine and urease. Differences were seen in the production of indole, the fermentation of dulcitol, sorbitol, mannitol, trehalose and maltose and in the presence of ODC.

A total of 12 distinct biochemical types (BT) were identified (Table II.).

5.3. Molecular characterization by REP-PCR

Analysis by REP-PCR revealed nine different patterns (Table III.).

The REP-PCR type I possessed bands sized 180 bp, 220 bp, 310 bp, 440 bp, 490 bp, 590 bp, 710 bp, 830 bp, 1370 bp, and 1400 bp. The second REP-PCR type featured bands at 180 bp, 220 bp, 310 bp, 440 bp, 460 bp, 490 bp, 590 bp, 710 bp, 830 bp and 1400 bp. The REP-PCR type III was found to be very similar to the type II with exception of additional bands at 370 bp, 410 bp and 630 bp and the band at 590 bp missing.

The REP-PCR types IV to VI demonstrated similar patterns with bands at 180 bp, 220 bp, 250 bp, 440 bp, 590 bp, 830 bp, 1130 bp, 1740 bp and 2160 bp. In REP-PCR type IV three additional bands at 490 bp, 980 bp and 2410 bp were observed and REP-PCR type V demonstrated one additional band at 490 bp.

The REP-PCR types VII to IX differed strongly from the other types. The determined pattern of REP-PCR type VII consisted of three weak bands at 240 bp, 340 bp and 460 bp, four consecutive bands at 590 bp, 630 bp, 710 bp and 780 bp and an intensive one at 1580 bp. The REP-PCR type VIII showed three weak bands at 220 bp, 240 bp and 890 bp and strong bands at 450 bp, 550 bp, 1370 bp, 1400 bp, 1720 bp and 1740 bp. In the last REP-PCR type IX, fragments appeared at 170 bp, 240 bp, 260 bp, 290 bp, 690 bp, 850 bp, 900 bp, 1100 bp, 1600 bp and 1930 bp.

Correlation between the high-resolution rpoB sequencing, biochemical differentiation and REP-PCR genotyping of the strains is shown in table IV.

6. Discussion

6.1. Identification

Studies identifying Pasteurella isolates from rabbits have been published previously. However, most surveys were performed on basis of biochemical identification before reclassification of the genus sensu stricto in 1985 (Mutters et al., 1985) and demonstrate only brief tables of phenotypic results. Therefore, an accurate classification and retrospective comparison with present Pasteurella species and subspecies is difficult.

The conventional phenotyping of our 41 Pasteurella isolates demonstrated a high heterogeneity, resulting in the recognition of 12 different biochemical types. The BT 1, BT 2, BT 9 and BT 10 of P. multocida, as well as the BT 11 identified as P. canis biotype 1, have been described by Mutters et al. (1985). BT 3 to BT 8 reflect previously described biochemical variations of P. multocida ssp. multocida strains. Phenotypical variability within the species Pasteurella multocida has been reported for other animals such as cattle (Biberstein et al., 1991; Bisgaard et al., 1991; Christensen et al., 2004a; Sachse et al., 1990; Truong et Schimmel, 1994), pig (Biberstein et al., 1991; Blackall et al., 1997; Jamuludin et al, 2005; Truong et Schimmel, 1994), poultry (Fegan et al., 1995; Muhairwa et al., 2001), turkey (Fegan et al., 1995), dog (Biberstein et al., 1991; Mohan et al., 1994; Muhairwa et al., 2001), cat (Biberstein et al., 1991; Korbel et al., 1992, Kuhnert et al, 2000; Muhairwa et al., 2001; Petersen et al., 1998) and bird (Biberstein et al., 1991; Korbel et al., 1992; Petersen et al., 1998) from various countries and continents. In rabbits, strains comparable with our BT 3, BT 4 and BT 7 were identified (Biberstein et al., 1991). Finally, our isolates grouped as BT 12 represent an unknown biochemical pattern, not described in previous studies.

Comparative sequence analysis of the 16S rRNA and rpoB genes is a distinct molecular method for the classification of Pasteurella strains. In this study the isolates clustered into six main phylogenetic groups, demonstrating the ability to differentiate inter- and intra-subspecies variations. According to our study the sequencing of the rpoB resulted

in a higher resolution and appears to be a good phylogenetic marker for genetic identification with a higher discriminatory power than the 16S rRNA gene sequencing. The same observation of the rpoB gene being a powerful additional tool for DNA- sequence-based identification and a promising approach for the improvement of the taxonomy of Pasteurellaceae has been described previously (Christensen et al., 2004b; Korczak et al., 2004).

Interestingly diverse biochemical types of phenotypically identified P. multocida ssp. multocida isolates of our study were homogeneous by means of 16S rRNA and rpoB gene sequencing. However, the biochemical classification was not always in correlation with molecular typing. Fermentation patterns of sorbitol, trehalose and dulcitol, which have been established as indicators for subspecies differentiation of P. multocida (Mutters et al., 1985), diverged from the phylogenetic subspecies clustering, making classification based on phenotypic reactions difficult.

In the phylogenetic tree based on 16S rRNA sequencing twenty representative strains clustered with the type strain of P. multocida ssp. multocida and P. multocida ssp. gallicida. The close relation between the two subspecies, as noted in previous studies (Korczak et al., 2004; Kuhnert et al., 2000; Petersen et al., 2001), was therefore confirmed. The isolates represented seven biochemical profiles, P. multocida ssp. multocida BT 2, BT 3, BT 4 and BT 6, P. multocida ssp. gallicida BT 10, as well as the two not clearly assignable biochemical types BT 7 and BT 8. Isolates of BT 7, as well as BT 8, can be interpreted as sorbitol negative variants of P. multocida ssp. multocida or as trehalose negative variants of P. multocida ssp. septica (Fegan et al., 1995). Sorbitol negative biovars have previously been reported in Australian poultry (Fegan et al., 1995), in a pig from New Zealand (Jamaludin et al., 2005), as well as in cattle, a dog, a cat, an eagle and a rabbit in California (Biberstein et al., 1991). In this study the sequencing of the 16S rRNA gene confirmed the relatedness to P. multocida ssp. multocida. The clear biochemical separation of P. multocida ssp. multocida and P. multocida ssp. septica as suggested by Mutters et al. (1985) is impossible in some cases, as has been demonstrated previously by several authors (Christensen et al., 2004a; Davies, 2004;

Hunt Gerardo et al., 2001; Kuhnert et al., 2000; Petersen et al., 2001). DNA-sequence- based identification, rather than sorbitol fermentation, seems more suitable for accurate classification of P. multocida ssp. septica and differentiation from ssp. multocida and ssp. gallicida.

The rpoB gene provided a higher resolution than the 16S rRNA sequencing. Nine of the twenty strains showed a closer sequence identity to the type strain of P. multocida ssp. multocida than to the ssp. gallicida. The other eleven isolates formed a separate group, which phylogenetically could not be assigned to a known subspecies of P. multocida and probably represents a new subspecies within the P. multocida species. By means of phenotyping a single strain was confirmed as ssp. gallicida. All other isolates were biochemical variants of P. multocida ssp. multocida.

16S rRNA and rpoB sequencing placed six of the 41 tested isolates in a cluster with the P. multocida ssp. septica reference strain. The clear separation of P. multocida ssp. septica from the other two subspecies has been confirmed in previous studies (Chen et al., 2002; Christensen et al., 2004b; Korczak et al., 2004; Kuhnert et al., 2000; Petersen et al., 2001). Sequencing of the 16S rRNA gene, as well as of the rpoB gene, was therefore shown to be an accurate method to distinguish between P. multocida ssp. multocida and P. multocida ssp. septica isolates. Phenotyping, however, diverged from molecular characterization; two of the phylogenetic confirmed strains of P. multocida ssp. septica were identified as a trehalose positive biochemical type of P. multocida ssp. multocida. Similar results have been published previously, suggesting the metabolism for trehalose to be distinctly different between P. multocida ssp. multocida and P. multocida ssp. septica (Chen et al., 2002; Muhairwa et al., 2001). A study investigating P. multocida strains from humans by using 16S rRNA sequencing and REP-PCR found the metabolism for trehalose to be distinctly different between P. multocida ssp. multocida and ssp. septica (Chen et al., 2002); ribotyping of isolates from poultry, dogs and cats in Tanzania demonstrated that trehalose positive strains of P. multocida ssp. multocida are genomically closer related to P. multocida ssp. septica than to trehalose negative strains of P. multocida ssp. multocida, irrespective of sorbitol fermentation (Muhairwa et al., 2001).

By means of 16S rRNA, rpoB gene sequencing and biochemical differentiation, four of the 41 strains were assigned to P. canis biotype 1. The isolation of this species from clinical and pathological rabbit specimens has been noted previously (Biberstein et al., 1991). P. canis biotype 1 is normally isolated from cats and dogs or bite wounds from these animals (Christensen et Bisgaard, 2003). The role of these hosts in the transmission of P. canis strains to rabbits should be further investigated. However, a previous study in Tanzania could not show direct evidence that clones of P. multocida are widely exchanged between dogs, cats and free ranging poultry (Muhairwa et al., 2001).

Ten isolates represented a DNA-sequence-based homogeneous group of an unknown species belonging to the family Pasteurellaceae. Most strains were phenotypically unassignable and referred to as BT 12; two strains however were classified as BT 2 and BT 6 variants of P. multocida ssp. multocida.

By means of 16S rRNA and rpoB sequencing another single unidentified strain was observed. The strain was phenotypically assigned to the BT 5, a maltose positive variation of P. multocida ssp. multocida. Maltose positive ssp. multocida and ssp. septica isolates have been identified in cats as well as in common blackbirds bitten by cats in neighboring Southern Germany (Korbel et al., 1992). Furthermore, such isolates were found in blackbirds, a turkey, a cat, a man (Petersen et al., 1998), cattles and pigs (Truong et Schimmel, 1994), in Australian poultry (Fegan et al., 1995) and cats from Tanzania (Muhairwa et al., 2001).

In conclusion, the 16S rRNA and rpoB marker genes were used successfully to investigate the phylogenetic relationships within the analyzed strains, the resolution of the rpoB gene being clearly higher. They confirmed that phenotypical characterization of such a heterogeneous bacteria species remains difficult. The identification of different subspecies by means of sorbitol, trehalose and dulcitol fermentation is an inaccurate indicator of genetic relatedness and emphasizes the importance of considering a reclassification of the family Pasteurellaceae.

6.2. Genotyping

For further genotyping of the identified Pasteurella strains, all 41 isolates were analyzed by REP-PCR. Three of the five main phylogenetic clusters, obtained by sequencing of the 16S rRNA gene and four of the six different phylogenetic clusters, obtained by sequencing of the rpoB gene, respectively, resulted each in an unique REP- PCR group. REP-PCR typing could therefore not further divide these clusters.

Multiple genotypes were found in the six strains of P. multocida ssp. septica and in the eleven isolates of an unknown P. multocida ssp. identified through rpoB sequencing. Genotyping of P. multocida ssp. septica resulted in three REP-PCR patterns, which demonstrated a similar banding. The genotyping of one isolate of the unknown P. multocida ssp. differed from the REP-PCR pattern of the other ten isolates.

The REP-PCR types I to VI obtained from P. multocida isolates differed strongly from the REP-PCR types VII of P. canis and VIII and IX of unknown Pasteurellaceae. Also, REP-PCR types assigned to the same P. multocida subspecies showed very similar banding patterns, differing in two to three bands only. Therefore, certain bands seem to be distinct for a specific Pasteurella species or subspecies, making REP-PCR a precise, reproducible and easy applicable method with an excellent discriminatory power for the inter- and intra-species genotyping of Pasteurella strains (Chen et al., 2002; Christensen et al., 2007; Rademaker et al., 1997; Stackebrandt et al., 2002; Townsend et al., 1997; Versalovic et al., 1991).

7. Further investigations

Meanwhile, 187 additional isolates, 117 from group management rabbits and 70 from post-mortem cases with pasteurellosis, were analyzed by biochemical differentiation and REP-PCR typing (Table V.).

The majority (82%) of all 228 isolates were characterized as REP-PCR type I, which relying on sequencing results of our previous work, appears to be typical for P. multocida ssp. multocida. This subspecies seems the most prevalent in pasteurellosis of rabbits.

Seven isolates (3%) represented the REP-PCR types IV, V and VI. Comparative sequence analysis of the 16S rRNA and rpoB genes placed these isolates in the cluster of the P. multocida ssp. septica reference strain.

Eleven isolates of REP-PCR type II and the single isolate of REP-PCR type III (5%) were assigned to an unknown P. multocida subspecies.

Eleven isolates of REP-PCR type VII (5%), according to sequencing belonged to P. canis.

Ten remaining isolates of REP-PCR type VIII and a single one of type IX (5%) seemed to form two clusters of an unknown species belonging to the family Pasteurellaceae.

An indication of the epidemiological potential of REP-PCR was observed. REP-PCR demonstrates the presence of two genotypes in an integral organization of breeding and fattening rabbits. With 112 of 123 isolates (91%), a particular genotype (REP-PCR type I) of P. multocida ssp. multocida is widely spread throughout the population of group management rabbits. The top down practice with one main breeder delivering animals to all other farmers, led to the distribution of this particular strain to different rabbitries. The identification of such a single strain, as has previously been demonstrated in a closed rabbit production unit in Germany (Schimmel et al., 1996), clarifies presumptive route of infection and eases vaccine fabrication.

On the other hand the involvement of nine different genotypes from clinical material originating from rabbits collected within two distinct time periods and from many independent rabbit holders documents the heterogeneity of Pasteurella isolates within the small rabbit population in Switzerland (300'000 animals from 30'000 owners). Only 75 of all investigated 105 isolates (72%) were grouped as REP-PCR type I of P. multocida ssp. multocida. Interestingly, strains representing P. multocida ssp. septica and unassignable P. multocida species and subspecies strains were found and appear to play a role as etiologic agent of clinical disease.

In conclusion, our study is the first extended study of a large collection of Pasteurella isolates from rabbits. We have confirmed that P. multocida ssp. multocida is the most common subspecies present in Swiss rabbits, represented by 187 of the 228 isolates we examined.

In addition, we have shown REP-PCR typing to be in high correlation with the sequencing of conserved regions of the genom and therefore represents a quick and easy alternative for taxonomic classification of Pasteurella and related bacteria. According to our study, REP-PCR also has potential as an epidemiological tool.

Table I. Biochemical characteristics of Pasteurella multocida and the Pasteurella group

Species/Subspecies NAD Hemolysis Indole Oxidase Catalase Lysine Dulcitol Sorbitol Mannitol Trehalose Maltose Urease ODC Table I. Tables Pasteurella multocida

ssp. multocida --+++--++v--+

ssp. septica --+++---++--+

ssp. gallicida --+++-+++---+

Pasteurella dagmatis --+++----+++-

Pasteurella canis --+ / -++----v--+ Biot. 1 / Biot. 2

Pasteurella stomatis --+++----+---

Actinobacillus pneumotropica --+++----v+++ 40 Pasteurella aerogenes ---++-----++-

Mannheimia haemolytica -+-++--++-+--

Actinobacillus ureae ---++---++++-

Pasteurella gallinarum ---++-- --++--

Pasteurella anatis ---++---++---

Pasteurella langaa ----+---+----

Pasteurella avium v--++-- --+---

Pasteurella volantium +--+---v+++-v

Pasteurella species A +--++---v+v--

Pasteurella species B --+++-+--++-+

NAD = nicotinamide adenine dinucleotide - dependence ODC = ornithine decarboxylase + = >90% of strains positive; - = >90% of strains negative; v = variable reactions

Table II.

Table II. Biochemical differentiation of Pasteurella isolates from Swiss rabbits

Unknown Pasteurella multocida P. canis Pasteurellaceae

ssp. multocida ssp. septica ssp. gallicida biotype 1

Biochemical 1 2 3 4 5 6 7 8 9 10 11 12 types

Indole + + + + + - + - + + + - Dulcitol ------+ - + Sorbitol + + + + + + - - - + - - Mannitol + + + + + + + + + + - - Trehalose + - + - + - - - + - + + Maltose - - - - + ------+ ODC + + - - + + + + + + + -

Table III.

Table III. Basic fragment patterns of REP-PCR types I to IX

Molecular weight REP-PCR types (+/- 10) bp I II III IV V VI VII VIII IX

2410 - - - + - - - - - 2160 - - - + + + - - - 1930 ------+ 1740 - - - + + + - + - 1720 ------+ - 1600 ------+ 1580 ------+ - - 1400 + + + - - - - + - 1370 + ------+ - 1130 - - - + + + - - - 1100 ------+ 980 - - - + - - - - - 900 ------+ 890 ------+ - 850 ------+ 830 + + + + + + - - - 780 ------+ - - 710 + + + - - - + - - 690 ------+ 630 - - + - - - + - - 590 + + - + + + + - - 550 ------+ - 490 + + + + + - - - - 460 - + + - - - + - - 450 ------+ - 440 + + + + + + - - - 410 - - + ------370 - - + ------340 ------+ - - 310 + + + ------290 ------+ 260 ------+ 250 - - - + + + - - - 240 ------+ + + 220 + + + + + + - + - 180 + + + + + + - - - 170 ------+

+: band; -: no band

Table IV. Table IV. Correlation between rpoB sequencing, biochemical differentiation and REP-PCR genotyping of the 41 strains

Pasteurella multocida P. canis Unknown Pasteurellaceae

ssp. multocida ssp. septica ssp. unknown biotype 1

43 234627 8 12914 011 6 125

I V VI IVV II III VII VIII IX

Table V. Table V. Correlation between rpoB sequencing, biochemical differentiation and REP-PCR genotyping of the 228 isolates

Pasteurella multocida P. canis Unknown Pasteurellaceae

ssp. multocida ssp. septica ssp. unknown biotype 1

2346 7 8 1 912 4 0112 6 12 5 44

I V VI IVV II III VII VIII IX

Group management n=123* 76(1)† 36(1) 1(1) 10(3)

Clinical cases n=105 34(1) 1(1) 3(1) 3(2) 32(1) 2(1) 2(1) 1(1) 3(3) 1(1) 5(5) 5(4) 1(1) 1(1) 1(1) 1(1) 8(8) 1(1)

n* number of isolates, all isolates were tested biochemically and by REP-PCR ()† number of selected isolates for rpoB sequencing

9. Figures

Figure 1a.

Phocoenobacter uteri, NCTC12872T, X89379 Mannheimia haemolytica , NCTC9380T, M75080 Actinobacillus lignieresii , NCTC4189T, AY362892 Histophilus somni , 8025T, AF549387 Volucribacter psittacicida , CCUG47536T, AY216868 Gallibacterium anatis, CCUG15563T, AF228001 Avibacterium gallinarum , NCTC11188T, AY362921 Nicoletella semolina, CCUG43639T, AY508816 Lonepinella koalarum , ATCC700131T, AY170218 Bibersteinia trehalosi , NCTC10370T, AY362927 *Aggregatibacter actinomycetemcomitans , HK1651 Haemophilus influenzae , Rd KW20, NC_000907 Clin. 29, EF579843 [IX; BT5; Trachea; Rh, Ot, Co, Ph, Pn, Pe; 1991] Clin. 829, EF579853 [VIII; BT12; Heartblood; Se; 2004] Clin. 175, EF579850 [VIII; BT12; Trachea; Pn; 1992] Clin. 217, EF579852 [VIII; BT12; Lung; Rh, Ot, Pn, Pl; 1992] Clin. 109, EF579849 [VIII; BT12; Trachea; Rh; 1991] Clin. 103, EF579847 [VIII; BT12; Nares ; Rh; 1991] Clin. 107, EF579848 [VIII; BT12; unknown ; unknown ; 1991] Clin. 8, EF579845 [VIII; BT12; unknown ; unknown ; 1991] Clin. 216, EF579851 [VIII; BT6; Trachea; Rh; 1992] Clin. 51, EF579846 [VIII; BT12; Nares; Rh, Pn; 1991] Clin. 7, EF579844 [VIII; BT2; Lung ; Rh, Ot, Pn, Pl; 1991] Pasteurella canis , CCUG12400T, AY362919 Group. 479/ 1, EF579841 [VII; BT11; Sinus; unknown ; 2005] Group. 868/ 2, EF579842 [VII; BT11; Sinus; unknown ; 2004] Group. 241/ 11, EF579840 [VII; BT11; Nares; unknown ; 2005] Clin. 59, EF579839 [VII; BT11; Nares ; Rh, Pn; 1991] Clin. 809, EF579838 [V; BT9; Heartblood; Ab, Se; 2004] Clin. 105, EF579833 [V; BT1; Lung; Pn, Se; 2005] Clin. 561, EF579837 [VI; BT1; Liver; Se; 2005] Clin. 126, EF579834 [IV; BT9; Lung; Pn, Pe, Se; 2005] Pasteurella multocida ssp. septica , CCUG17977T, AF294411 Clin. 334/1, EF579835 [IV; BT9; Lung; Rh; 2005] Clin. 334/2, EF579836 [IV; BT9; Lung; Rh, Ph; 2005] Pasteurella multocida ssp. multocida, CCUG17976T, AF294410 Clin. 610, EF579819 [I; BT6; Lung; Pn, Pl; 2004] Pasteurella multocida ssp. gallicida, CCUG17978T, AF294412 Clin. 48, EF579815 [I; BT2; Lung; Rh, Ot, Pn, Pe, Pl; 1991] Clin. 120, EF579827 [II; BT2; Trachea; Rh, Pn; 1991] Clin. 35, EF579813 [I; BT3; Lung; Rh, Pn, Pl; 1991] Clin. 36, EF579814 [I; BT4; Trachea; Rh, Se; 1991] Clin. 37, EF579823 [II; BT4; Trachea; Rh, Ot, Ma, Pn, Pe, Pl; 1991] Clin. 197, EF579818 [I; BT6; Bulla; Ot, Se, 2005] Clin. 115, EF579825 [III; BT10; Heartblood; Se; 2005] Clin. 57, EF579816 [I; BT7; Nares ; Rh, Pn; 1991] Group. 37/ 9, EF579820 [I; BT7; Sinus; unknown ; 2006] Clin. 95, EF579817 [I; BT8; Nares ; Rh, Ot; 1991] Group. 257/ 1, EF579821 [I; BT2; Sinus; unknown ; 2005] Clin. 118, EF579826 [II; BT2; Heartblood; Rh, En, Sa, Se; 1991] Clin. 147, EF579828 [II; BT2; Trachea; Rh, Ot, Pn; 1991] Clin. 151, EF579829 [II; BT2; Lung; Rh, Pn, Pe, Pl; 1991] Clin. 203, EF579830 [II; BT2; Nares; Rh, Ot; 1992] Group. 142/ 16, EF579832 [II; BT4; Sinus; unknown ; 2005] Clin. 32, EF579822 [II; BT4; Trachea; Rh, Ot, Pn; 2005] Clin. 53, EF579824 [II; BT4; Trachea; Rh, Ot, Pn, Pe, Pl; 1991] Clin. 220, EF579831 [II; BT4; Abscess; Ab; 1992] Escherichia coli, MG1655, NC_000913 2%

Figure 1b.

Clin. 107, EF579889 [VIII; BT12; unknown ; unknown ; 1991] Clin. 109, EF579890 [VIII; BT12; Trachea; Rh; 1991] Clin. 217, EF579893 [VIII; BT12; Lung; Rh, Ot, Pn, Pl; 1992] Clin. 175, EF579891 [VIII; BT12; Trachea; Pn; 1992] Clin. 7, EF579885 [VIII; BT2; Lung; Rh, Ot, Pn, Pl; 1991] Clin. 8, EF579886 [VIII; BT12; unknown ; unknown ; 1991] Clin. 216, EF579892 [VIII; BT6; Trachea; Rh; 1992] Clin. 51, EF579887 [VIII; BT12; Nares ; Rh, Pn; 1991] Clin. 829, EF579894 [VIII; BT12; Heartblood; Se; 2004] Clin. 103, EF579888 [VIII; BT12; Nares ; Rh; 1991] *Aggregatibacter actinomycetemcomitans , HK1651 Lonepinella koalarum , ATCC700131T, AY170218 Bibersteinia trehalosi , NCTC10370T, AY314028 Mannheimia haemolytica , NCTC9380T, AY170217 Actinobacillus lignieresii , NCTC4189T, AY170215 Haemophilus influenzae , Rd KW20, NC_000907 Phocoenobacter uteri, NCTC12872T, AY170219 Nicoletella semolina, CCUG43639T, AY508861 Clin. 29, EF579884 [IX; BT5; Trachea; Rh, Ot, Co, Ph, Pn, Pe; 1991] Volucribacter psittacicida , CCUG47536T, EF077220 Gallibacterium anatis, CCUG15563T, AY314032 Avibacterium gallinarum , NCTC11188T, AY314041 Histophilus somni , 8025T, AY170207 Pasteurella canis , CCUG12400T, AY314038 Group. 479/ 1, EF579882 [VII; BT11; Sinus; unknown ; 2005] Group. 868/ 2, EF579883 [VII; BT11; Sinus; unknown ; 2004] Group. 241/ 11, EF579881 [VII; BT11; Nares; unknown ; 2005] Clin. 59, EF579880 [VII; BT11; Nares; Rh, Pn; 1991] Clin. 105, EF579874 [V; BT1; Lung; Pn, Se; 2005] Clin. 561, EF579878 [VI; BT1; Liver; Se; 2005] Clin. 126, EF579875 [IV; BT9; Lung; Pn, Pe, Se; 2005] Clin. 809, EF579879 [V; BT9; Heartblood; Ab, Se; 2004] Pasteurella multocida ssp. septica , CCUG17977T, AY362970 Clin. 334/1, EF579876 [IV; BT9; Lung; Rh; 2005] Clin. 334/2, EF579877 [IV; BT9; Lung; Rh, Pn; 2005] Pasteurella multocida ssp. gallicida, CCUG17978T, AY362969 Clin. 35, EF579854 [I; BT3; Lung; Rh, Pn, Pl; 1991] Clin. 36, EF579855 [I; BT4; Trachea; Rh, Se; 1991] Clin. 57, EF579857 [I; BT7; Nares; Rh, Pn; 1991] Clin. 48, EF579856 [I; BT2; Lung; Rh, Ot, Pn, Pe, Pl; 1991] Group. 37/ 9, EF579861 [I; BT7; Sinus; unknown ; 2006] Clin. 197, EF579859 [I; BT6; Bulla; Ot, Se; 2005] Clin. 95, EF579858 [I; BT8; Nares ; Rh, Ot; 1991] Group. 257/ 1, EF579862 [I; BT2; Sinus; unknown ; 2005] Pasteurella multocida ssp. multocida, CCUG17976T, AY170216 Clin. 610, EF579860 [I; BT6; Lung; Pn, Pl; 2004] Clin. 115, EF579866 [III; BT10; Heartblood; Se; 2005] Clin. 37, EF579864 [II; BT4; Trachea; Rh, Ot, Ma, Pn, Pe, Pl; 1991] Clin. 118, EF579867 [II; BT2; Heartblood; Rh, En, Sa, Se; 1991] Clin. 120, EF579868 [II; BT2; Trachea; Rh, Pn; 1991] Clin. 147, EF579869 [II; BT2; Trachea; Rh, Ot, Pn; 1991] Clin. 151, EF579870 [II; BT2; Lung; Rh, Pn, Pe, Pl; 1991] Clin. 203, EF579871 [II; BT2; Nares ; Rh, Ot; 1992] Group. 142/ 16, EF579873 [II; BT4; Sinus; unknown ; 2005] Clin. 32, EF579863 [II; BT4; Trachea; Rh, Ot, Pn; 2005] Clin. 53, EF579865 [II; BT4; Trachea; Rh, Ot, Pn, Pe, Pl; 1991] Clin. 220, EF579872 [II; BT4; Abscess; Ab; 1992] Escherichia coli, MG1655, NC_000913 4%

Fig. 1a. Phlyogenetic tree of 41 rabbit isolates and selected GenBank entries of the most related Pasteurellaceae based on 16S rRNA gene sequences. The species names, strain numbers and accession numbers are listed. E. coli was used as an outgroup. The scale bar represents percentage of sequence divergence. Strain acronyms: Clin. for clinical cases, Group. for group management [REP-PCR types I-IX; Biochemical types 1-12; Origin; Disease status: Rh=Rhinitis, Ot=Otitis media, Pn=Pneumonia, Pe=Pericarditis, Pl=Pleuritis, Co=Conjunctivitis, Ph=Phlegmone, Ab=Abscess, Me=Meningoencephalitis, Ma=Mastitis, En=Endometritis, Sa=Salpingitis, Se=Septicemia; Year of isolation]† *www.genome.ou.edu

Fig. 1b. Phlyogenetic tree of 41 rabbit isolates and selected GenBank entries of the most related Pasteurellaceae based on rpoB gene sequences. The species names, strain numbers and accession numbers are listed. E. coli was used as an outgroup. The scale bar represents percentage of sequence divergence. Strain acronyms: Clin. for clinical cases, Group. for group management [REP-PCR types I-IX; Biochemical types 1-12; Origin; Disease status: Rh=Rhinitis, Ot=Otitis media, Pn=Pneumonia, Pe=Pericarditis, Pl=Pleuritis, Co=Conjunctivitis, Ph=Phlegmone, Ab=Abscess, Me=Meningoencephalitis, Ma=Mastitis, En=Endometritis, Sa=Salpingitis, Se=Septicemia; Year of isolation]† *www.genome.ou.edu

10. Pictures

Pict. 1 Stable divided in different housing groups

Pict. 2 Breeding unit with nesting boxes

Pict. 3 Fattening unit

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12. Acknowledgements

I would like to express my gratitude to everyone, who contributed to this work. Special thanks goes to:

Prof. Dr. R. Hoop, Institute of Veterinary Bacteriology, NRGK, Vetsuisse Faculty University of Zurich, for giving me the opportunity to work on this interesting topic and for his supervision and assistance.

Prof. Dr. R. Stephan, Institute for Food Safety and Hygiene, Vetsuisse Faculty University of Zurich, for accepting to referee this dissertation.

Dr. B. Korczak Stuber, Institute of Veterinary Bacteriology, Vetsuisse Faculty University of Berne, for her assistance, scientific support and help in preparation of the manuscript.

Mrs. L. Bigler, Zentrum für tiergerechte Haltung, Zollikofen, Mr. F. Näf, Mr. U. Wullschleger and other owners of rabbit breeding and fattening farms, for their cooperation and assistance in providing part of the research material.

Mrs. R. Keller, Mrs. M. Kreienbühl, Mrs. D. Morger and Mrs. B. Zimmermann for their help with the dissections and the laboratory work.

Dr. M. Kaufmann, Dr. M. Rusch and Dr. C. Rutz, Institute of Veterinary Bacteriology, NRGK, Vetsuisse Faculty University of Zurich, for their professional and moral support.

Risch Bandli, Sigrid Boettcher, Glauco Camenisch, Silvia Dalessi, Maria Furger, Cornelia Neff and in particular Olivier Schenker for their encouragements and all the great times we spent together. Without them I could not have finished this work.

My family and friends for their constant patience and understanding.

Curriculum vitae

Personalien

Name Anina Barbara Jennifer Stahel Geburtsdatum 5. November 1979 Geburtsort Framingham, MA, USA Nationalität Schweiz, USA Heimatort Zürich / Hinwil ZH

Ausbildung

1983-1985 Walnut Park Montessori School, Newton MA, USA 1985-1987 Inter Community School Zumikon, ZH 1987-1992 Primarschule in Aesch, ZH 1992-1999 Gymnasium Kantonsschule Hohe Promenade in Zürich 1999 Matura Typus D

1999-2004 Studium der Veterinärmedizin an der Vetsuisse-Fakultät Universität Zürich, Schweiz 2004 Staatsexamen Veterinärmedizin an der Vetsuisse-Fakultät Universität Zürich, Schweiz

2005-2007 Doktorandin am Nationalem Referenzzentrum für Geflügel- und Kaninchenkrankheiten, Institut für Veterinärbakteriologie, Vetsuisse-Fakultät, Universität Zürich, Schweiz

Zürich, 27. August 2007