Leen Timbermont
email: leen.timbermont@ugent.be

Anouk Lanckriet

Frank Pasmans

Freddy Haesebrouck

Richard Ducatelle

Filip Van Immerseel

Intra-species growth-inhibition by Clostridium perfringens is a possible virulence trait in necrotic enteritis in broilers

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A c c e p t e d M a n u s c r i p t 2 Abstract Necrotic enteritis in broiler chickens is associated with Clostridium perfringens type A , carrying the NetB toxin. C. perfringens type A is also a member of the normal intestinal microbiota of broilers. Clinically healthy chickens carry several different Clostridium perfringens clones in their intestine. In flocks suffering from necrotic enteritis, however, mostly only one single clone is isolated from the gut of all the diseased animals. Selective proliferation of these clinical outbreak strains in the gut and spread within the flock seems likely, but an explanation has not yet been given. The hypothesis that necrotic enteritis associated Clostridium perfringens strains might suppress the growth of normal microbiota Clostridium perfringens strains, was therefore tested. Twenty-six Clostridium perfringens strains isolated from healthy broilers and 24 clinical outbreak isolates were evaluated for their ability to induce intra-species growth-inhibition in an in vitro setup. A significantly higher proportion of the Clostridium perfringens clinical outbreak strains inhibited the growth of other Clostridium perfringens strains compared to Clostridium perfringens strains isolated from the gut of healthy chickens. It is proposed that, in addition to toxin production, intra-species inhibition may be a virulence trait that contributes to the ability of certain Clostridium perfringens strains to cause necrotic enteritis in broilers.

Keywords: Clostridium perfringens / broiler / necrotic enteritis / growth-inhibition A c c e p t e d M a n u s c r i p t 3

Introduction

Since the ban on growth-promoting antibiotics in animal feed in the European Union, Clostridium (C.) perfringens associated necrotic enteritis is reemerging in broilers [START_REF] Grave | What has happened in Norway after the ban of avoparcin? Consumption of antimicrobials by poultry[END_REF][START_REF] Van Immerseel | Clostridium perfringens in poultry: an emerging threat for animal and public health[END_REF][START_REF] Williams | Intercurrent coccidiosis and necrotic enteritis of chickens: rational, integrated disease management by maintenance of gut integrity[END_REF]. Necrotic enteritis in poultry is associated with C. perfringens type A, carrying the NetB toxin [START_REF] Keyburn | NetB, a new toxin that is associated with avian necrotic enteritis caused by Clostridium perfringens[END_REF][START_REF] Van Immerseel | Rethinking our understanding of the pathogenesis of necrotic enteritis in chickens[END_REF]. C. perfringens type A is also a member of the normal intestinal microbiota of broilers.

Strains isolated from healthy broilers, however, do not induce necrotic enteritis in an experimental model using predisposing factors, in contrast to strains isolated from outbreaks of necrotic enteritis [START_REF] Timbermont | Origin of Clostridium perfringens isolates determines the ability to induce necrotic enteritis in broilers[END_REF].

In C. perfringens isolates from healthy birds, a high degree of genetic diversity is found, even between isolates from the same animal. In contrast, different isolates from a flock suffering from a clinical outbreak are generally of the same Pulsed Field Gel Electrophoresis (PFGE) type, regardless of the animal or the part of the intestine from which the strain was isolated [START_REF] Nauerby | Analysis by pulsed-field gel electrophoresis of the genetic diversity among Clostridium perfringens isolates from chickens[END_REF][START_REF] Gholamiandehkordi | Molecular and phenotypical characterization of Clostridium perfringens isolates from poultry flocks with different disease status[END_REF]. The reason for the presence of a single clone in necrotic enteritis outbreaks is not known. It is speculated that during an outbreak, certain C. perfringens strains have a competitive advantage over other C. perfringens strains in the broiler gut.

In the present study, strains isolated from healthy broilers and strains isolated from broilers suffering from necrotic enteritis were compared with respect to their capacity of intraspecies growth-inhibition in an in vitro inhibition assay.

Materials and methods

Bacteria

A c c e p t e d M a n u s c r i p t 4 Fifty C. perfringens type A strains belonging to different genotypes, as analyzed by PFGE, were included. Thirty-five strains were isolated from broiler chickens in Belgium: 26 strains from clinically healthy broiler chickens and 9 strains from broilers suffering from necrotic enteritis [START_REF] Gholamiandehkordi | Molecular and phenotypical characterization of Clostridium perfringens isolates from poultry flocks with different disease status[END_REF]. Fifteen Danish C. perfringens isolates from necrotic enteritis cases were kindly provided by Dr. L. Bjerrum [START_REF] Nauerby | Analysis by pulsed-field gel electrophoresis of the genetic diversity among Clostridium perfringens isolates from chickens[END_REF].

Strains of C. perfringens were grown on Colombia agar (Oxoid, Basingstoke, UK) containing 5% defibrinated sheep blood. Plates were incubated at 37°C in an anaerobic working cabinet (invivo 2 500, Ruskinn Life Sciences, Bridgend, UK), in an atmosphere of 8%H 2 :8%CO 2 :84%N 2 . Strains were stored at -80°C in lyophilisation medium (LYM) containing 7% glucose, 23% Brain Heart Infusion broth (BHI, Oxoid) and 70% defibrinated horse serum (Invitrogen, Merelbeke, Belgium).

In vitro growth-inhibition assay

The 50 C. perfringens strains were used in a checkerboard test for intra-species growthinhibition. Each strain was cultured anaerobically in BHI broth (Oxoid) for 24 h at 37°C. The overnight cultures were diluted in Phosphate Buffered Saline (PBS) to a density of McFarland No. 0.5, and 200 µl of these suspensions were spread with a sterile swab on the whole surface of BHI agar plates to obtain a bacterial lawn (12 cm x 12 cm). A single colony of each C. perfringens isolate was transferred with a sterile toothpick to the agar plates seeded with the different C. perfringens strains. Absence of growth of the bacterial lawn around a colony results in an inhibition zone (Fig. 1). After overnight incubation under anaerobic conditions, diameters of inhibition zones were measured in mm. The tests were performed in triplicate. The data were analysed with SPSS 16 software using the chi-square test to compare the number of strains of the normal microbiota group with number of strains of the necrotic enteritis group that were able to inhibit other strains. The student t-test was used to compare the mean inhibition zones of the healthy animal strains and the necrotic enteritis strains. Significance was determined at P < 0.05.

Statistical analysis

Results

Sixty percent of all tested strains inhibited growth of at least one other strain of C. perfringens (i.e., there was a zone of clearing around the stabbed colony). While some strains inhibited growth of many other strains, others had a very limited inhibitory spectrum. Fifteen (58%) of the 26 healthy animal strains and 4 (17%) of the 24 clinical outbreak strains were not able to inhibit the growth of any other strain. In contrast, 46% (11/24) of the clinical outbreak strains were able to inhibit more than 90% (more than 45/50) of the other strains while this was only the case for 15% (4/26) of the healthy animal strains (Fig. 2). The number of strains that were able to inhibit any number of other strains was significantly higher for the clinical outbreak strains than for the healthy animal strains (P < 0.05). If strains were able to inhibit the growth of other C. perfringens strains, they were able to inhibit both healthy animal strains and necrotic enteritis outbreak strains.

In the strains that were able to inhibit, the zones of inhibition varied for the strains from healthy broilers between 3 mm and 7 mm and for the necrotic enteritis strains between 3.5 mm and 5.5 mm. There was no significant difference in the average sizes of the inhibition zones: 4.9 mm and 4.3 mm for healthy animal strains and clinical outbreak strains, respectively.

Discussion

A c c e p t e d M a n u s c r i p t C. perfringens in a broiler flock suffering from necrotic enteritis. Our results show that inhibition of other C. perfringens strains is a trait that is significantly more developed in clinical outbreak strains, compared with healthy animal strains. A minority of healthy animal strains were able to inhibit other C. perfringens strains isolated from healthy birds and necrotic enteritis strains. In a recent study of [START_REF] Barbara | Necrotic enteritis-producing strains of Clostridium perfringens displace non-necrotic enteritis strains from the gut of chicks[END_REF], none of the normal flora strains (n=17) could inhibit the growth of other C. perfringens strains. Also a minority of necrotic enteritis outbreak strains did not have the intra-species growth-inhibitory phenotype. Possibly in these strains other virulence traits may compensate for the lack of inhibitory capacity. Another possible explanation for the lack of in vitro inhibitory phenotype is that expression of the gene encoding for the growthinhibiting phenomenon can be down-regulated by certain signal transduction systems or regulators (Dupuy and Mamouros, 2006). The gene for the growth-inhibiting factor might also be located on a plasmid and the absence of growth-inhibition may be caused by the loss of this plasmid after in vitro cultivation [START_REF] Rood | Molecular genetics and pathogenesis of Clostridium perfringens[END_REF].

The results of the radial diffusion assay suggest that the intra-species inter-strain growthinhibition is caused by a substance secreted by the bacterium into its micro-environment. The nature of this substance is hitherto unclear. It is known that C. perfringens is able to produce bacteriocins capable of lysing other C. perfringens strains. [START_REF] Watson | The potential of bacteriocin typing in the study of Clostridium perfringens food poisoning[END_REF] observed that 79% of strains implicated in food poisoning outbreaks produced bacteriocins; however, only 18% of 322 isolates from the feces of healthy persons, human and animal infections, various foods, and the environment produced bacteriocins suggesting a relationship between bacteriocin production and the ability to cause food poisoning.

In this study, secretion of intra-species growth-inhibitory substances by C. perfringens is proposed to be an additional virulence trait that can possibly contribute to the pathogenesis of [START_REF] Collier | Coccidia-inuced mucogenesis promotes the onset of necrotic enteritis by supporting Clostridium perfringens growth[END_REF]. Intra-species interstrain growth inhibition may constitute an important advantage for certain strains in the competition for nutrients in the intestinal tract. It is well documented that C. perfringens requires at least 11 different essential amino acids for its multiplication [START_REF] Petit | Clostridium perfringens: toxinotypes and genotype[END_REF]. Suppressing its competitors may allow unlimited access to the nutrients and thus the possibility of explosive multiplication. The massive production of specific toxins and enzymes, such as the NetB toxin and proteolytic enzymes [START_REF] Olkowski | Responses of broiler chickens orally challenged with Clostridium perfringens isolated from field cases of necrotic enteritis[END_REF][START_REF] Olkowski | Subclinical necrotic enteritis in broiler chickens: Novel etiological consideration based on ultra-structural and molecular changes in the intestinal tissue[END_REF][START_REF] Keyburn | NetB, a new toxin that is associated with avian necrotic enteritis caused by Clostridium perfringens[END_REF] at sites of multiplication of these bacteria in the gut would then lead to necrotic lesions, and thus the release of more nutrients, completing the cycle.

In conclusion, C. perfringens outbreak isolates are significantly more capable of intraspecies growth inhibition compared to strains isolated from healthy birds. It is proposed that intra-species growth-inhibition by C. perfringens is a possible virulence trait in necrotic enteritis in broilers.

  selective proliferation of one single clone in the broiler gut. Indeed, when certain environmental predisposing factors, such as a coccidial infection, are present, nutrients utilizable by C. perfringens are leaking in the lumen

Figure 2

 2 Figure 1

  Figure 1A

  Figure 1B

Acknowledgements

We would like to thank Veerle Flama for her skilful technical assistance. Dr. L. Bjerrum is acknowledged for providing strains. This work was supported by the Institute for Science and Technology, Flanders (IWT). F. Van Immerseel is supported by a Postdoctoral Research Grant of the Research Foundation -Flanders (FWO) and by the Research Fund of Ghent University (BOF).

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


Mathias Corteel

João J Dantas-Lima

Mathieu Wille²

Victoria Alday-Sanz

Maurice B Pensaert

Patrick Sorgeloos²

Hans J Nauwynck
email: hans.nauwynck@ugent.be

Molt stage and cuticle damage influence White Spot Syndrome Virus immersion infection in penaeid shrimp

Keywords: White Spot Syndrome Virus, waterborne transmission, Penaeus vannamei, Penaeus monodon, molt stage

Transmission of white spot syndrome virus (WSSV) in shrimp has been reported to occur by feeding and immersion. In the present study, the impact of the molt process and artificial lesions in the cuticle on shrimp susceptibility to WSSV was examined using intramuscular and immersion routes.

For the intramuscular route, Penaeus (Litopenaeus) vannamei shrimp (n=450) were injected with 10 -2.3 up to 10 2.7 shrimp infectious dose 50% end point (SID 50 ) of WSSV in early and late post-molt, inter-molt, early and late pre-molt; resp. A-, B-, C-, D1-and D2-stage. The resulting infection titers demonstrated that no difference (p>0.05) in susceptibility existed between different molt stages when virus was injected.

For the waterborne route, shrimp in different molt stages were immersed in sea water containing 10 4 SID 50 ml -1 of WSSV. In a first study, P. vannamei (n=125) incubated in cell culture flasks, became infected with WSSV mostly in post-molt stages. In a second study, 2 groups of P. vannamei (n=100) and P. monodon (n=100) were transferred into plastic bags to prevent damage to the cuticle; and in 1 group a pleopod was cut off prior to incubation.

Induction of damage increased infection significantly (p<0.05) in A-stage from 0-40% to 60-100%, in B-stage from 0-20% to 40-60%, in C-stage from 0-20 to 20-60%, while infection was 0% in D-stages with both immersion methods. This study proved that shrimp are more susceptible to WSSV infection via immersion after molting than in the period before molting and wounding facilitates infection.

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

Introduction

White spot syndrome virus (WSSV) is one of the most wide-spread viruses in penaeid shrimp aquaculture and is considered to be responsible for a large portion of crop failures (for reviews on WSSV, see: [START_REF] Sanchez-Martinez | White spot syndrome virus in cultured shrimp: A review[END_REF][START_REF] Escobedo-Bonilla | A review on the morphology, molecular characterization, morphogenesis and pathogenesis of white spot syndrome virus[END_REF]. Since the first reports on the virus, it has become generally accepted that transmission between shrimp and other Decapod Crustacea can occur via 3 routes: (1) oral uptake of tissues from infected hosts; (2) waterborne, when virus is transmitted via the water by immersion or cohabitation and (3) per ovum (vertical) and possibly intra-ovum from broodstock to offspring.

When reviewing literature on WSSV, one finds a high number of experimental studies demonstrated that feeding of WSSV-infected shrimp tissues is an effective way to infect shrimp and other decapods. Especially the early reports on WSSV helped to build the image that the virus is highly contagious, even though many researchers had to administer WSSV-infected tissues more than one feeding, sometimes as long as 7 days. For the waterborne route, many studies reported that immersion and even cohabitation exposure readily allowed WSSV to cause infection, although older shrimp were reported to be less susceptible.

It is important to note, however, that most of the studies published so far were performed with non-specific pathogen-free (SPF) animals, without knowing the administered doses of WSSV and without screening the inoculum for the presence of other pathogens. Often, possible secondary transmissions after inoculation were not ruled out, temperature of the rearing water was not under control and most importantly, WSSV infections were rarely confirmed.

These facts make it difficult to reproduce those studies or make reliable conclusions.

Probably the best-controlled experimental studies on WSSV transmission so far, were published by Soto and Lotz [START_REF] Soto | Transmission of white spot syndrome virus (WSSV) to Litopenaeus vannamei from infected cephalothorax, abdomen, or whole shrimp cadaver[END_REF][START_REF] Lotz | Model of white spot syndrome virus (WSSV) epidemics in Litopenaeus vannamei[END_REF][START_REF] Soto | Transmission, virulence, and recovery coefficients of white spot syndrome virus (WSSV) and Taura syndrome virus (TSV) infections in Kona stock Litopenaeus vannamei[END_REF] and A c c e p t e d M a n u s c r i p t far more effective treatment than immersion in infected water. Remarkably however, even when P. vannamei were isolated to ensure they had equal chance to consume the infected tissues offered to them, not all shrimp became infected (50-60%). [START_REF] Prior | Controlled bioassay systems for determination of lethal infective doses of tissue homogenates containing Taura syndrome or white spot syndrome virus[END_REF] succeeded in determining the lethal intramuscular dose of a WSSV stock and also tried to develop a controlled bio-assay by immersion of P. vannamei. Although very large amounts of infectious virus were added to the water (as shown by the injection study), mortality rates stayed below 40%. Recently, another study clearly illustrated the difficulty to infect animals by WSSV immersion challenge [START_REF] Gitterle | Optimization of experimental infection protocols for the estimation of genetic parameters of resistance to White Spot Syndrome Virus (WSSV) in Penaeus (Litopenaeus) vannamei[END_REF], while a study on an ornamental shrimp's susceptibility to WSSV resulted in a discussion of the problems encountered with experimental feeding challenges [START_REF] Laramore | Susceptibility of the peppermint shrimp Lysmata wurdemanni to the white spot syndrome virus[END_REF]. [START_REF] Gitterle | Optimization of experimental infection protocols for the estimation of genetic parameters of resistance to White Spot Syndrome Virus (WSSV) in Penaeus (Litopenaeus) vannamei[END_REF] showed that merely adding virus inoculum to the water was not sufficient to result in P. vannamei infection but needed to place the shrimp in tanks in which orally infected shrimp had previously died to finally obtain successful transmission. Finally, in the PhD thesis by Dr. Bonny [START_REF] Bayot | Epidemiology of infectious diseases in cultured white shrimp Penaeus vannamei, with emphasis on white spot disease[END_REF], less than 17% of P. vannamei shrimp became infected upon individual challenge with WSSV via oral route and none or merely 3% by immersion.

The overall conclusion from these publications is that there are restrictions on the ability of WSSV to gain entry into its host. With feeding of virus-infected tissues to shrimp, this is to be expected as the lack of control on the dose of virus actually reaching the site of entry, inherently creates irreproducible results. The fact that any portion of the animals might not be feeding (due to molting, stress, …) for instance, can easily prevent an equal chance to become infected. Another factor which cannot be ignored is that all tissues known to be susceptible to WSSV replication are protected from the outside world by cuticle [START_REF] Escobedo-Bonilla | Pathogenesis of a Thai strain of white spot syndrome virus (WSSV) in juvenile, specific pathogen-free Litopenaeus vannamei[END_REF]. This is also true for the gills and the epithelium of stomach and hindgut [START_REF] Bell | A handbook of normal penaeid shrimp histology[END_REF].

A c c e p t e d M a n u s c r i p t 5 Although little details are known about the structure and function of the cuticle of penaeid shrimp, it is well-known that it changes dramatically in time [START_REF] Chan | Characterization of the molt stages in Penaeus vannamei: setogenesis and hemolymph levels of total protein, ecdysteroids and glucose[END_REF]Compère et al., 2004;[START_REF] Promwikorn | Changes in tiger shrimp, Penaeus monodon[END_REF]. During the course of its life, a shrimp passes through consecutive molt cycles. Therefore, in a study examining transmission of pathogens in shrimp, it could be important to take the molt stage into account (Le [START_REF] Moullac | Haematological and phenoloxidase activity changes in the shrimp Penaeus stylirostris in relation with the moult cycle: protection against vibriosis[END_REF][START_REF] Mugnier | Combined effect of exposure to ammonia and hypoxia on the blue shrimp Litopenaeus stylirostris survival and physiological response in relation to molt stage[END_REF].

Considering the inability to reproducibly cause infection in shrimp exposed to WSSV by immersion, the present study was set-up to investigate the factors determining WSSV infection by waterborne route. In a first hypothesis we tested whether the susceptibility of shrimp to WSSV infection changes during the course of their molt cycle. The virus was delivered intramuscularly, thus passing the cuticle in order to compare the internal susceptibility between the different molt stages. In a second approach, the barrier function of the cuticle against natural infection by waterborne virus was tested in a series of immersion inoculation experiments of shrimp in different molt stages. Groups of artificially damaged shrimp were compared with control shrimp to test the hypothesis that the cuticle presents a barrier against WSSV and that wounding can promote infection.

Materials and Methods

Experimental animals and conditions

The shrimp used in this study were Penaeus (Litopenaeus) vannamei from Molokai Sea Farms Int., Hawaii, USA and P. monodon, from Moana Technologies Nucleus Breeding Centre, Hawaii, USA. The batches of shrimp from Moana Technologies were certified to be SPF by Jim Brock, DVM. Those from Molokai Sea Farms Ghent University, Belgium. They were fed with Artemia nauplii twice daily for a period of 3 weeks and were then weaned onto a commercial pelleted feed (A2 monodon high performance shrimp feed, INVE Aquaculture SA, Belgium), fed twice daily at a total rate of 5 % of their mean body weight (MBW). Water temperature was kept at 27 ± 1°C and salinity at 35 ± 1 gl -1 .

Regular water changes kept total ammonia-N below 0.5 mg l -1 and nitrite-N below 0.15 mg l -1 .

The room was illuminated 12 hours per day by dimmed TL-light. For the viral challenge experiments, shrimp were transported to the facilities of the Laboratory of Virology, Faculty of Veterinary Medicine, Ghent University, where the experiments were performed under biosafety conditions.

Molt stage determination

Molt stages were determined based on the descriptions by [START_REF] Robertson | Practical molt staging of Penaeus setiferus and Penaeus stylirostris[END_REF][START_REF] Chan | Characterization of the molt stages in Penaeus vannamei: setogenesis and hemolymph levels of total protein, ecdysteroids and glucose[END_REF]. Briefly, shrimp were restrained for a few seconds and their uropods were examined by inverted microscope. At a magnification of 100 to 200X, the exopodites of uropods were analysed on the appearance of the cuticle, epidermis and molt processes such as apolysis and the formation of new cuticle. Shrimp were separated into 5 major molt stages: early and late post-molt (A and B), inter-molt (C) and early and late pre-molt (D1 and D2).

Post-molt stages are characterized by an epidermis in close contact with all of the still thin cuticle. The epidermis is present in the setae in A-stage and retracts in B-stage, while it constantly secretes additional layers to the cuticle. In the inter-molt stage the epidermis lies in a straight line at the bottom of the setae while the construction of the cuticle is finalized. The pre-

Virus

The WSSV Thai-1 isolate was used in the present study. This isolate has been studied before [START_REF] Jiravanichpaisal | Experimental infection of white spot syndrome virus in freshwater crayfish Pacifastacus leniusculus[END_REF][START_REF] Escobedo-Bonilla | In vivo titration of white spot syndrome virus (WSSV) in specific pathogenfree Litopenaeus vannamei by intramuscular and oral routes[END_REF][START_REF] Escobedo-Bonilla | Standardized white spot syndrome virus (WSSV) inoculation procedures for intramuscular or oral routes[END_REF][START_REF] Escobedo-Bonilla | Pathogenesis of a Thai strain of white spot syndrome virus (WSSV) in juvenile, specific pathogen-free Litopenaeus vannamei[END_REF][START_REF] Rahman | Virulence of white spot syndrome virus (WSSV) isolates may be correlated with the degree of replication in gills of Penaeus vannamei juveniles[END_REF]. It was collected from naturally infected Penaeus monodon in Thailand in 1996 and passaged in crayfish Pacifastacus leniusculus [START_REF] Jiravanichpaisal | Experimental infection of white spot syndrome virus in freshwater crayfish Pacifastacus leniusculus[END_REF]. Crayfish gill suspension containing WSSV Thai-1 was kindly provided by K. Söderhäll (Uppsala University, Sweden) and amplified in SPF P. vannamei juveniles to produce virus stocks. The median infectious titer of the stock used for all experiments in this study was determined to be 10 6.0 shrimp infectious dose 50% end point (SID 50 ) per ml, following the in vivo intramuscular titration procedure in SPF P. vannamei described by [START_REF] Escobedo-Bonilla | In vivo titration of white spot syndrome virus (WSSV) in specific pathogenfree Litopenaeus vannamei by intramuscular and oral routes[END_REF].

In vivo titration by intramuscular inoculation using shrimp in different molt stages

P. vannamei juveniles (MBW = 5.6 ± 2.7 g; n = 450) were taken from stock cultures maintained at ARC and screened for their molt stage as described in 2.2. Thirty shrimp were selected in each of the 5 major molt stages (A, B, C, D1 and D2) and inoculated intramuscularly with 50 μl of a 10-fold serial dilution of the WSSV stock (10 -2 to 10 -7 ), with 5 shrimp per dilution. After the inoculation, shrimp were housed individually in covered 10 l aquaria, filled with artificial seawater at a salinity of 35 g l -1 , provided with constant aeration A c c e p t e d M a n u s c r i p t 8 and maintained at 27°C by air heaters. Approximately 2.5% of BW of a commercial shrimp diet was provided to each shrimp in 2 rations per day. Moribund and dead shrimp were recorded, removed from the aquaria and processed for detection of WSSV infection. The experiment was terminated at 120 hpi, when surviving shrimp were sacrificed and analyzed for WSSV infection. The experiment was performed in triplicate.

Study of WSSV infection by immersion route

Immersion inoculation inside cell culture flasks

The aim of this experiment was to develop a model for WSSV infection by immersion.

A total of 125 SPF P. vannamei were used. As the batch of shrimp grew up, 5 groups of shrimp with a MBW of 1, 4, 6, 11 and 20 g were taken from the stock culture at ARC and screened for their molt stage as described in 2.2. For each size group, 5 shrimp per molt stage were immersed. The WSSV inoculum used to immerse the shrimp was a 1% dilution of the WSSV stock. It was prepared in a volume of 25 ml artificial seawater (35 g l -1 ) per g bodyweight, resulting in a dose of 10 4 SID 50 ml -1 . Shrimp of 1 g were put inside '25 cm 2 ' cell culture flasks (Nunc A/S, Denmark) containing 25 ml of the inoculum. Animals of 4, 6 and 11 g were put inside '75 cm 2 ' cell culture flasks containing respectively 100, 150 and 275 ml of the inoculum. Shrimp of 20 g were put inside '175 cm 2 ' cell culture flasks containing 500 ml of the inoculum.

Flasks were placed on a lateral side in order to allow the shrimp to stay in a physiological position. The duration of the immersion was 3 hours and water was aerated with an airstone.

After the inoculation, the procedures were identical to those in 2.4, but no food was given the first 12 h after the immersion to avoid additional oral up-take of virus via the food.

Shrimp were monitored for clinical signs every 12 h and dead shrimp were removed and processed for detection of virus replication. The experiment was terminated 5 days post A c c e p t e d M a n u s c r i p t immersion. At this time, all surviving shrimp were euthanized and processed for virus detection. Mortality and infection rates were compared between the molt stages and between the sizes. 2.5.2. Immersion inoculation inside plastic bags of shrimp with and without damaged cuticle.

In this experiment, damage was induced to 1 group of shrimp by cutting off a pleopod while shrimp of the control group were left undamaged. Both groups were put inside plastic bags to limit physical damage as much as possible. The aim was to evaluate whether mechanical damage would allow a higher incidence of WSSV infections in shrimp.

A total of 100 P. vannamei and 100 P. monodon were used in this experiment. For each species, 2 size groups of 50 shrimp were tested with a MBW of 2 and 15 g. Shrimp were taken from the stock culture at ARC and screened for their molt stage as described in 2.2. An attempt was made to minimize damage to the cuticle by carefully catching and handling the animals. Of each species and size, 10 shrimp of each molt stage were selected and placed individually in 4 l transparent polyethylene bags (220x330nm, 50my, Binpac) filled with sea water. These were placed inside buckets lined with shock-absorbing plastic for transport to the facilities of the Laboratory of Virology. At the start of the immersion, the water in the plastic bags containing the individual shrimp was replaced by 50 ml of the inoculum for 2 g shrimp and 375 ml for 15 g shrimp. The inoculum was prepared as described for 2.5.1. Per molt stage, 5 shrimp were then briefly recaptured and 1 pleopod of the first abdominal segment was cut off by bistouri blade at the level of the coxa. During the immersion, bags were hung in mid-air in order to allow the animals to stay in a physiological position in the layer of inoculum on the bottom and a tube with an aeration stone was inserted to allow aeration of the water. After 3 hours of incubation, the inoculum was drained from the bag and shrimp were placed straight into aquaria. The setup of the remainder of the experiment was identical to that described for experiment 2.5.1.

A c c e p t e d M a n u s c r i p t 10 Mortality and infection rates were compared between the molt stages, artificially damaged and intact shrimp and their respective sizes.

Detection of WSSV infection by indirect immunofluorescence(IIF)

The procedure to detect WSSV infection by IIF was described before [START_REF] Escobedo-Bonilla | In vivo titration of white spot syndrome virus (WSSV) in specific pathogenfree Litopenaeus vannamei by intramuscular and oral routes[END_REF]. In brief, the cephalothoraxes of dead shrimp were dissected longitudinally, embedded in 2% methylcellulose and quickly frozen at -20 °C. Cryosections (5 µm) were made and immediately fixed in 100% methanol at -20 ºC for 20 min. Sections were washed three times for 5 min each in phosphate buffered saline (PBS) and incubated with 2 µg ml -1 of the monoclonal antibody 8B7 (Diagxotics Inc. USA) directed against viral protein VP28 [START_REF] Poulos | Development and application of monoclonal antibodies for the detection of white spot syndrome virus of penaeid shrimp[END_REF] for 1 h at 37 ºC. Then, sections were washed three times for 5 min each in PBS and incubated with fluorescein isothiocyanate (FITC)-labelled goat anti-mouse IgG (F-2761, Molecular Probes, The Netherlands) for 1 h at 37 ºC. Sections were finally washed in PBS, rinsed in deionised water, dried and mounted with a solution of glycerine and 1, 4-diaza-bicyclo2,2,2-octane (DABCO) (ACROS organics, USA). Slides were analyzed by fluorescence microscopy (Leica DM RBE).

Statistical analysis

The virus titers of the intramuscular titration (2.2) were compared between molt stages using the Wilcoxon rank-sum non-parametric test [START_REF] Zar | Biostatistical analysis[END_REF].

Differences between molt stages within groups of 5 to 10 shrimp per group (2.3 and 2.4) were tested for significance using Fisher's exact test [START_REF] Kirkwood | Essential medical statistics[END_REF].

A c c e p t e d M a n u s c r i p t infection rates was tested between the molt stages and between the control and the pleopod cut groups by Pearson's Chi Square tests with Yates' correction.

All calculations were performed using S-plus version 6.1 (Lucent Technologies).

Results

In vivo titration by intramuscular inoculation (Table 1)

IIF analysis of dead and surviving shrimp revealed the following virus infection titers: 10 6 , 10 6.5 and 10 6.8 for A-stage (10 6.50.4 ); 10 6.6 , 10 6.8 and 10 7.5 for B-stage (10 7.10.4 ); 10 6.5 , 10 6.7 and 10 6.8 for C-stage (10 6.70.2 ); 10 6.8 , 10 6.8 and 10 7.1 for D1-stage (10 6.90.2 ) and 10 6.3 , 10 6.7 and 10 7 for D2-stage (10 6.70.3 ) (Table 1). No significant differences in infection titers were observed between the stages (p>0.05).

Immersion inoculation inside cell culture flasks (Table 2)

Of the 1 g shrimp, only 1 shrimp in the A-stage group started to become anorectic and lethargic at 36 hpi. This was the only shrimp to die due to WSSV before the end of the experiment. All other shrimp were euthanized at 120 hpi and were negative for WSSV on IIF.

When the immersion was performed with 4 g shrimp, all survived the experiment uninfected.

At a size of 6 g, 3 out of 5 A-stage shrimp started to show clinical signs at 36 hpi and died at 60-84 hpi. When the experiment was performed with 11 g, all A-and one B-stage shrimp showed clinical signs and died due to WSSV infection between 48 and 120 hpi. Two D2-stage animals molted during the immersion and one died before the end of the 3 hours procedure.

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

This was the only mortality during the course of the experiment which was not caused by WSSV infection. In the experiment performed on 20 g shrimp, all A-, 2 out of 5 B-and 1 Cstage shrimp showed clinical signs after 36 hpi. These shrimp died between 48 and 72 hpi and were confirmed to be infected with WSSV, while all other shrimp survived and were uninfected. The difference in infection rate was significantly higher in A-stage than in the other stages in 11 g shrimp, and between A-and C-, D1-and D2-stage in 20 g animals (p<0.05).

During this experiment, it was noticed that the 11 and 20 g post-molt shrimp had suffered injuries to appendages during the immersion procedure. Because of this observation, an alternative immersion procedure using plastic bags was designed in an attempt to limit selfgenerated damage to the shrimp.

Immersion inoculation inside plastic bags (Table 3)

One pleopod of the first abdominal segment could be removed at the level of the coxa by bistouri blade without causing any clinical signs or mortality. Damaged sites showed melanization within 12-24 hours after injuries had occurred. Melanizations which were present on the animals after natural damage and prior to immersion were recorded. Thus, the physical damage occurring during the immersion procedure could be estimated.

Throughout the experiment, anorexia was recorded in D2-stage shrimp 24 to 48 h before molting and in A-stage shrimp. Uninfected animals started eating normally again by the end of A-stage. Infected animals displaying anorexia on the other hand also became lethargic between 48 to 72 hours post immersion (hpi), generally 24 hours before dying.

In 2 g juvenile P. vannamei immersed in plastic bags without cutting of pleopods, 2 shrimp in A-stage and 1 in C-stage died. Of the shrimp with cut off pleopods, 3 in A-and B- In all cases, dead shrimp were WSSV positive on IIF, and surviving shrimp were WSSV negative.

Only in 15 g P. vannamei with cut pleopods, significant differences were calculated between A-stage on one hand and C-, D1-and D2-stage on the other (Fisher's exact test; p<0.05). When the infection rates of species and sizes were pooled (Table 3B), the Chi Square test on the results showed the following: 1) a significantly higher infection rate in A-stage than in D1-or D2-stage of the control groups (p<0.05); 2) a highly significant difference between A-and D1or D2-stage in the pleopod cut groups (p<0.001); 3) no significant difference between A-, B-or C-stage in the pleopod cut groups (p>0.05); 4) significantly more infected shrimp in B-and Cstage than in D1-and D2-stage of the pleopod cut groups (p<0.05); 5) significantly more infected shrimp in A-, B and C-stages with cut pleopods than in the control group (p<0.05).

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

Discussion

In preliminary WSSV immersion experiments leading up to this study, an influence of the molt cycle on the susceptibility to the virus had been observed. In the present study, an in vivo titration of the virus stock in shrimp in different molt stages was first performed by intramuscular route. This experiment showed that no significant intrinsic difference in susceptibility to WSSV existed between shrimp in the different molt stages. Hence, the underlying mechanism responsible for the difference in susceptibility to WSSV between molt stages had to be examined using trials mimicking natural transmission.

A new immersion inoculation procedure was set up to study the infection of WSSV by waterborne route. Studies on the waterborne route of WSSV transmission in literature all employed simply aquaria for inoculations of shrimp, except for [START_REF] Prior | Controlled bioassay systems for determination of lethal infective doses of tissue homogenates containing Taura syndrome or white spot syndrome virus[END_REF] who used cell culture flasks. At first sight, cell culture flasks seemed to be adequate tools to perform an immersion procedure as these containers are sterile, do not inactivate virus and allow observation of the animals. However, prevention of uncontrollable physical damage to the animals during transport in buckets and the immersion procedure in cell culture flasks proved to be difficult. All shrimp instinctively struggled by contracting their tail during catching and handling in an attempt to escape and jumped violently against the walls of the containers. Only post-molt (A-and B-stage) shrimp suffered visible damage. Most affected were appendages such as rostrum, telson, uropods, antennae, pleo-and pereiopods. The damage was mainly comprised of fractures of the cuticle, noticed by deformities and hemolymph bleeding from the fractures. Sometimes this resulted in loss of appendages. Especially the larger 11 and 20 g shrimp were suffering injuries due to the relatively small access of the flasks.

As an alternative immersion recipient, polyethylene bags were tested in this study.

When shrimp were carefully placed inside plastic bags before transport and the water replaced Overall, the incidence of infection and mortality was clearly higher in shrimp immersed in WSSV inoculum during the post-molt stages than in pre-molt stages. It was postulated that immersion inoculation of shrimp in hard-walled containers could result in infection in larger shrimp in post-molt stages, because of damage to the cuticle which is softer and thinner in these stages. An inoculation procedure using plastic bags resulted in much less infection in post-molt stages as the animals were handled more carefully. A clear correlation between damage of the cuticle and infection was demonstrated by cutting a pleopod at the start of immersion. The incidence of infection was increased 2 to 8-fold between undamaged and artificially damaged groups. Similar results could also be obtained by cutting the rostrum in A-stage shrimp (data not shown). However, even with the infliction of a wound, no infection was ever recorded in shrimp which had been pre-molt at the time of exposure to waterborne virus. While differences were seen in infection rates between ages in shrimp immersed in cell culture flasks, no such differences were recorded between 2 or 15 g shrimp inoculated inside plastic bags.

The actual portal of entry of WSSV from the water into a host has never been described, but some assume that the gills are the best candidates [START_REF] Chang | Identification of white spot syndrome associated baculovirus (WSBV) target organs in the shrimp Penaeus monodon by in situ hybridization[END_REF][START_REF] Witteveldt | Protection of Penaeus monodon against white spot syndrome virus by oral vaccination[END_REF][START_REF] Arts | Haemocyte reactions in WSSV immersion infected Penaeus monodon[END_REF]. The experimental findings of the present study demonstrate that an artificially induced wound in the cuticle increases the rate of WSSV infection upon immersion.

Cutting off a pleopod creates an open wound which can allow either (1) infection of cells at the site of the wound or (2) entry of WSSV into the hemolymph followed by direct systemic spread or on the other hand (3) reduce the competence of shrimp to resist WSSV infection. In the first two scenarios, entry of the virus would occur through the opening in the cuticle itself. If one A c c e p t e d M a n u s c r i p t 16 considers the (ultra)structure of the cuticle of crustacea such as shrimp, it is not difficult to imagine that the cuticle constitutes an impregnable barrier against viruses from food or the environment (Compère, pers.comm.). Although damage to the cuticle appears to be the key to WSSV infection from the water, the situation is more complex. Even when an open wound is present in shrimp, this does not always lead to infection, especially in molt stages when the exoskeleton is well-developed (i.e. inter-and pre-molt). Factors which determine whether WSSV can ultimately invade a shrimp could be: (1) morphological and physiological (cuticle and epidermal cells) or (2) (a)specific defence-related (coagulation time, phagocytosis, phenoloxidase and reactive oxygen species activity etc.). All these factors are likely or are already known to vary between different stages of the molt cycle [START_REF] Charmantier | Effect of molt stage and hypoxia on osmoregulatory capacity in the penaeid shrimp Penaeus vannamei[END_REF][START_REF] Moullac | Haematological and phenoloxidase activity changes in the shrimp Penaeus stylirostris in relation with the moult cycle: protection against vibriosis[END_REF][START_REF] Liu | The immune response of the white shrimp Litopenaeus vannamei and its susceptibility to Vibrio infection in relation with the moult cycle[END_REF][START_REF] Chiou | Expression and characterisation of tiger shrimp Penaeus monodon penaeidin (mo-penaeidin) in various tissues, during early embryonic development and moulting stages[END_REF][START_REF] Promwikorn | Changes in tiger shrimp, Penaeus monodon[END_REF][START_REF] Mugnier | Combined effect of exposure to ammonia and hypoxia on the blue shrimp Litopenaeus stylirostris survival and physiological response in relation to molt stage[END_REF]. The third alternative explanation for the increased chance for WSSV infection in damaged shrimp, would be that wounding has a direct or indirect effect on the capacity of shrimp to resist to WSSV infection. Indeed, removal of a pleopod will induce stress, which could have an effect on the subsequent immune response of the shrimp. The inflicted damage and subsequent clotting, hemocyte migration and exocytosis at the site of the wound, and immune responses to other microorganisms which may enter, can all alter possible defence against WSSV infection.

Overall, the findings in the present paper give the impression that there are important restrictions on the ability of WSSV to gain entry to its host and question whether the water in which shrimp live is a natural medium for the spread of the virus, as long as the cuticle of shrimp is a firm barrier. This clearly differs from some reports on WSSV infections from water in literature [START_REF] Kanchanaphum | Experimental monodon[END_REF][START_REF] Witteveldt | Protection of Penaeus monodon against white spot syndrome virus by oral vaccination[END_REF][START_REF] Arts | Haemocyte reactions in WSSV immersion infected Penaeus monodon[END_REF], while it is supported by other [START_REF] Prior | Controlled bioassay systems for determination of lethal infective doses of tissue homogenates containing Taura syndrome or white spot syndrome virus[END_REF][START_REF] Bayot | Epidemiology of infectious diseases in cultured white shrimp Penaeus vannamei, with emphasis on white spot disease[END_REF][START_REF] Gitterle | Optimization of experimental infection protocols for the estimation of genetic parameters of resistance to White Spot Syndrome Virus (WSSV) in Penaeus (Litopenaeus) vannamei[END_REF]. Differences in

Conclusion

This study revealed that the molt stage of penaeid shrimp does not influence their susceptibility to WSSV infection when the virus is injected, but that on the other hand shrimp in post-molt stages of the molt cycle become more easily infected with WSSV from water than in pre-molt stages. The procedure by which shrimp were immersed in WSSV inoculum strongly affected the chances for infection. The rate of infection was significantly higher in animals with damages to the exoskeleton due to immersion in hard-walled containers or with a pleopod removed. From these findings we postulate that the cuticle is a barrier against WSSV infection and wounding can increase the susceptibility of shrimp.

Acknowledgements

The first author was supported by a scholarship from the Institute for the Promotion of

  according to inspection services by the Aquaculture Development Program, State of Hawaii. Batches of 10,000 PL-10 shrimp were shipped to Belgium and reared in a recirculation system at the Laboratory of Aquaculture & Artemia Reference Center (ARC),

  as the epidermis retracts from the cuticle in stage D1 and begins formation of a new cuticle. In the final stage before the molt, D2, the newly forming cuticle and setae become visible.

  C-stage died between 48 and 72 hpi. All other shrimp of the various molt stages with pleopods left intact or cut survived until the end of the experiment at 120 hpi.Of 15 g P. vannamei with no pleopod cut, only 1 out of five A-and B-stage animals died at 72 hpi. Cutting a pleopod increased the mortality to 5 in A-stage (48 to 120 hpi), 2 out of 5 in B-stage (96 hpi) and 1 in C-stage (120 hpi). All other shrimp survived until 120 hpi.In 2 g juvenile P. monodon immersed with pleopods intact, only 1 shrimp in A-stage died. Of those with cut off pleopods, 3 in A-and B-and 2 in C-stage died between 48 and 120 hpi. All other shrimp survived until the end of the experiment.Of 15 g P. monodon with pleopods left intact, 2 out of five A-and 1 B-stage shrimp died (48 or 72 hpi). Cutting a pleopod induced mortality in 3 shrimp in A-stage (48 to 72 hpi), 2 in B-stage (48 to 72 hpi) and 3 in C-stage (72 to 84 hpi). All other shrimp survived until the end of the experiment.

  the amount of resistance and jumping of the shrimp was reduced and much less obvious injuries could be observed while the shrimp hung suspended in mid-air. Even though the bags proved to be useful, it remained impossible to completely prevent the occurrence of damages in the soft post-molt shrimp.

  ability of WSSV isolates, administered dose and methodology are the likely explanations for these variable results.

  Innovation through Science and Technology in Flanders (IWT-Vlaanderen, Belgium), the second author by the Foundation for Science and Technology (FCT, Portugal).

Table 1 .

 1 Infection titers of White Spot Syndrome Virus stock by intramuscular inoculation in P. vannamei in different molt stages (3 repetitions of 5 shrimp per dilution). Average titers were not significantly different between molt stages (p>0.05).

	Molt Stage	Dilution of WSSV	Mortality	Confirmed infected by IIF	Infection titer
	A	10 -2 10 -3 10 -4 10 -5 10 -7 10 -6	15/15 15/15 0/15 0/15 15/15 10/15	15/15 15/15 0/15 0/15 15/15 10/15	10 6.50.4 SID 50 ml -1
	B	10 -2 10 -3 10 -4 10 -5 10 -7 10 -6	15/15 15/15 0/15 6/15 15/15 11/15	15/15 15/15 0/15 6/15 15/15 11/15	10 7.10.4 SID 50 ml -1
	C	10 -2 10 -3 10 -4 10 -5 10 -7 10 -6	15/15 15/15 0/15 2/15 15/15 10/15	15/15 15/15 0/15 2/15 15/15 10/15	10 6.70.2 SID 50 ml -1
	D1	10 -2 10 -3 10 -4 10 -5 10 -7 10 -6	15/15 15/15 0/15 4/15 15/15 13/15	15/15 15/15 0/15 4/15 15/15 13/15	10 6.90.2 SID 50 ml -1
	D2	10 -2 10 -3 10 -4 10 -5 10 -7 10 -6	15/15 15/15 0/15 4/15 15/15 10/15	15/15 15/15 0/15 4/15 15/15 10/15	10 6.70.3 SID 50 ml -1

Table 1

 1


Halina E Tegetmeyer

AND Kerstin Fricke

Nina Baltes
email: nina.baltes@tiho-hannover.de

An isogenic Actinobacillus pleuropneumoniae AasP mutant exhibits altered biofilm formation but retains virulence

Keywords: Actinobacillus pleuropneumoniae, AasP, adherence, autotransporter

AasP, an autotransporter serine protease of A. pleuropneumoniae, has been shown to be expressed in necrotic porcine lung tissue. Based on the hypothesis that AasP might play an important role in A. pleuropneumoniae adhesion and virulence by processing other surfaceassociated proteins, the predicted catalytic site of AasP was deleted and the isogenic mutant, AP76ΔaasP, was compared to the wild type strain in a biofilm assay as well as an aerosol infection model. AP76ΔaasP showed increased adherence compared to the wild type strain under standard culturing conditions as well as under NAD restriction. No significant differences between AP76 wild type and AP76ΔaasP were observed upon experimental infection of pigs, indicating that AasP does not play a crucial role in A. pleuropneumoniae virulence.

) pleuropneumoniae is the causative agent of porcine pleuropneumonia, an economically important disease of fattening pigs. The ability to persistently colonize pigs after infection and even after vaccination with bacterins is a major obstacle for eradication of the organism [START_REF] Fenwick | Porcine pleuropneumonia[END_REF][START_REF] Haesebrouck | Actinobacillus pleuropneumoniae infections in pigs: the role of virulence factors in pathogenesis and protection[END_REF], Chiers et al., 1998). We recently identified an autotransporter serine protease, AasP (GenBank accession no. DQ490067), which appears to remain associated with the membrane and is present in membrane fractions in large amounts [START_REF] Baltes | Selective capture of transcribed sequences (SCOTS) of Actinobacillus pleuropneumoniae in the chronic stage of disease reveals an HlyX-regulated autotransporter protein[END_REF]. Its function is largely unknown to date, but one possible role might be the processing of other autotransporter proteins, namely adhesins, similar to SphB1 in Bordetella pertussis [START_REF] Coutte | Subtilisinlike autotransporter serves as maturation protease in a bacterial secretion pathway[END_REF]. AasP has recently been found to be involved in the processing of OmlA [START_REF] Ali | Functional characterization of AasP, a maturation protease autotransporter protein of Actinobacillus pleuropneumoniae[END_REF]. While autotransporter adhesins have not been characterized in A. pleuropneumoniae to date, two have been identified, one of which is interrupted by the insertion of an IS element (Baltes andGerlach, 2004, Tegetmeyer et al., 2008). A. pleuropneumoniae has been shown to form biofilms, and genes responsible for matrix polysaccharide production have been identified [START_REF] Kaplan | Genes involved in the synthesis and degradation of matrix polysaccharide in Actinobacillus actinomycetemcomitans and Actinobacillus pleuropneumoniae biofilms[END_REF][START_REF] Kaplan | Biofilm formation is prevalent among field isolates of Actinobacillus pleuropneumoniae[END_REF]. However, the role of biofilms in A. pleuropneumoniae infection has not been extensively studied in the pig. A regulatory mutant lacking regulator protein arcA was shown to be impaired in biofilm formation and virulence [START_REF] Buettner | Effects of endobronchial challenge with Actinobacillus pleuropneumoniae serotype 9 of pigs vaccinated with inactivated vaccines containing the Apx toxins[END_REF]. In the present study, we investigate the influence of AasP on A. pleuropneumoniae adherence in vitro, and on virulence.

MATERIALS AND METHODS

Bacterial strains, plasmids, and primers.

The strains, plasmids, and primers used in this work are listed in Table 1.

Media and growth conditions.

A c c e p t e d M a n u s c r i p t 4 Escherichia coli strains were cultured in LB-medium supplemented with ampicillin (100 g/ml). A. pleuropneumoniae strains were routinely cultured in PPLO medium (Difco GmbH, Augsburg, Germany) supplemented with nicotinamide dinucleotide (NAD; 10 µg/ml; Merck, Darmstadt, Germany), L-glutamine (100 µg/ml; Serva, Heidelberg, Germany), L-cysteinehydrochloride (260 µg/ml; Sigma-Aldrich), L-cystine-dihydrochloride (10 µg/ml; Sigma-Aldrich), Dextrose (1 mg/ml), and Tween ® 80 (0.1%).

Manipulation of DNA.

DNA-modifying enzymes were purchased from New England Biolabs (Bad Schwalbach, Germany) and used according to the manufacturer's instructions. Taq polymerase was purchased from Gibco-BR Life Technologies (Karlsruhe, Germany). Chromosomal DNA for PCR and Southern Blotting as well as plasmid DNA was prepared by standard protocols [START_REF] Sambrook | Molecular cloning: a laboratory manual[END_REF]. PCR, Southern Blotting, transformation and gel electrophoresis were done by standard procedures [START_REF] Sambrook | Molecular cloning: a laboratory manual[END_REF].

Sequence analyses.

Nucleotide sequencing was performed by SeqLab GmbH, Göttingen, Germany. Promoter prediction was performed using the Virtual Footprint promoter analysis tool [START_REF] Munch | Virtual Footprint and PRODORIC: an integrative framework for regulon prediction in prokaryotes[END_REF] available at http://prodoric.tu-bs.de/vfp/).

Construction of transconjugation plasmid pSER705

Primer pairs oSER101-Psp/oSER106-Bsm and oSER102-Not/oSER105-Bsm were used to generate PCR products which were ligated together after digestion with BsmBI. The ligation product was amplified using primer pair oSER101-Psp/ oSER102-Not, which was then digested with PspOMI and NotI and ligated into pEMOC2 to generate transconjugation plasmid pSER705.

Adherence assay.

Adhesion of A. pleuropneumoniae to abiotic surfaces under different conditions was tested in an adherence assay that has been described for Listeria monocytogenes (Djordjevic et al., A c c e p t e d M a n u s c r i p t 5 2002). Overnight cultures of AP76 and AP76ΔaasP were diluted 1:100 with fresh PPLO media containing additives for the respective conditions (standard: 10 µg/ml NAD; reduced NAD: 0.1 µg/ml; iron restriction: 0.1 mM dipyridyl and 10 µg/ml NAD). Sterile 96 well flat bottom tissue culture plates (Sarstedt, Germany) were seeded with 4 x 200 µl of bacterial suspension or the respective supplemented media per growth condition, and incubated at 37°C, 5% CO 2 for a total of six or 24 hours. Optical density (OD) was measured in a microtiter plate reader (GENios Pro, Tecan GmbH, Crailsheim, Germany) at 630 nm. After incubation, the medium was removed and nonadherent bacteria were removed by rinsing the wells with distilled water three times. After drying, biofilms were stained with 80 µl of 1 % crystal violet solution per well for 30 minutes, followed by four washing steps with distilled water. After at least 30 minutes of air drying, crystal violet was dissolved by adding 150 µl destaining solution (50% ethanol, 30 % glacial acetic acid, 20% acetone) and OD was measured at 550 nm after 15 minutes of incubation. Average OD values from control wells were subtracted from average OD values from respective inoculated wells both for bacterial culture (630 nm) and crystal violet (550 nm) ODs. For quantitative comparison of adherence, OD values of crystal violet readings were divided by culture OD values to correct for different growth OD values between the two A. pleuropneumoniae strains. Four wells were set up for each strain and each growth condition, and the experiment was repeated three times.

Animal experiments.

Animal experiments were carried out using an aerosol infection model previously described [START_REF] Baltes | Actinobacillus pleuropneumoniae iron transport and urease activity: effects on bacterial virulence and host immune response[END_REF]. 16 A. pleuropneumoniae seronegative male landrace pigs of 7-8 weeks of age were randomly divided into two groups. The challenge dose was prepared from cultures at an OD 600 of 0.49 (AP76wt) and 0.48 (AP76ΔaasP). 13 ml of an 1:30000 dilution of these cultures were aerosolized per four pigs. Body temperatures were monitored daily. Pigs were cared for in accordance with the principles outlined in the European Convention for the and the experiments were approved by an ethics committee. Lung lesion scores were evaluated according to Hannan et al. [START_REF] Hannan | Tylosin tartrate and tiamutilin effects on experimental piglet pneumonia induced with pneumonic pig lung homogenate containing mycoplasmas, bacteria and viruses[END_REF].

Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes

RESULTS AND DISCUSSION

In order to determine whether the function of AasP was crucial for A. pleuropneumoniae adherence and virulence, we constructed an isogenic mutant lacking the predicted catalytic site (Gly 349 to Gly 359 ). Using E. coli strain ß2155 [START_REF] Dehio | Maintenance of broad-host-range incompatibility group P and group Q plasmids and transposition of Tn5 in Bartonella henselae following conjugal plasmid transfer from Escherichia coli[END_REF], plasmid pSER705 was used to introduce the deletion in the aasP gene using the single-step transconjugation system described earlier [START_REF] Oswald | A single-step transconjugation system for the introduction of unmarked deletions into Actinobacillus pleuropneumoniae serotype 7 using a sucrose sensitivity marker[END_REF]. The mutant, AP76ΔaasP, lacks 123 bp spanning the putative catalytic site of AasP as confirmed by PCR, sequence analysis and Southern blot (data not shown). AP76ΔaasP does not differ from the wild type strain macroscopically or microscopically, and growth of AP76ΔaasP in liquid PPLO medium supplemented with 10 µg/ml NAD, incubated with shaking, was unaffected compared to the parent strain.

In the in vitro adherence assay, both AP76wt and AP76ΔaasP showed increased adhesion during iron and NAD restricted growth compared to standard culturing conditions (Fig. 1A).

While increased adherence of A. pleuropneumoniae has previously been described for NAD restricted growth conditions (van Overbeke et al., 2002), this has not been demonstrated for iron restricted conditions. AP76ΔaasP showed a higher degree of adherence than the wild type both under standard and NAD restricted conditions, these results were statistically significant in the Wilcoxon signed-rank test (p < 0.05). Under iron restricted conditions, AP76ΔaasP showed less adherence than the wild type strain. This effect was statistically significant only after 24 hours, not after 6 hours. Interestingly, growth of AP76ΔaasP in this stationary culture was significantly impaired in comparison to the wild type under standard and NAD restricted conditions (Fig. 1B). Under iron restricted conditions, the wild type strain A c c e p t e d M a n u s c r i p t 7 showed better growth than AP76ΔaasP after 6 hours, but reached a lower OD than AP76ΔaasP after 24 hours. However, this effect was not statistically significant. In summary, AP76ΔaasP appears to grow slower, but adhere more efficiently than AP76wt under standard and NAD restricted conditions. Under iron restricted conditions, the impact of the deletion on growth is not statistically significant, but the mutant is slightly impaired in adhesion after 24 hours. Our results imply that AasP function does not seem to play a fundamental role in A. pleuropneumoniae adhesion to abiotic surfaces. The only factor crucial for A. pleuropneumoniae adhesion that has been identified so far is LPS [START_REF] Rioux | Isolation and characterization of LPS mutants of Actinobacillus pleuropneumoniae serotype 1[END_REF][START_REF] Paradis | Inhibition of adherence of Actinobacillus pleuropneumoniae to porcine respiratory tract cells by monoclonal antibodies directed against LPS and partial characterization of the LPS receptors[END_REF]. Our findings also show that AasP is unlikely to have adhesin properties itself, because the mutant still shows increased adhesion under conditions that have previously shown to increase adhesion, and more importantly, the mutant adheres better than the wild type strain under standard and NAD restricted conditions. Rather than AasP being an adhesin, it is more likely that AasP processes other proteins, possibly adhesins, like it has been shown for autotransporter serine protease SphB1 of Bordetella pertussis [START_REF] Coutte | Role of adhesin release for mucosal colonization by a bacterial pathogen[END_REF].

However, OmlA, the only substrate of AasP identified so far [START_REF] Ali | Functional characterization of AasP, a maturation protease autotransporter protein of Actinobacillus pleuropneumoniae[END_REF], has no known role in biofilm formation. A role for AasP in PGA polysaccharide (biofilm matrix) biosynthesis or degradation can also not be excluded at this point.

Assuming a role of AasP in adhesin processing, the observed increased adherence upon elimination of AasP function might be due to adhesive structures not being released from the bacterial surface, preventing detachment of bacteria. Fine tuning of mucosal colonization by adhesin release has been shown to be an important function of SphB1 of Bordetella pertussis [START_REF] Coutte | Role of adhesin release for mucosal colonization by a bacterial pathogen[END_REF].

Transcription of aasP has previously been found to be upregulated under iron restriction conditions [START_REF] Deslandes | Transcriptional profiling of Actinobacillus pleuropneumoniae under iron-restricted conditions[END_REF].Upstream of the aasP start codon, two putative binding sites for global iron regulator fur can be found using the Virtual Footprint software; one is centered at 239 bp upstream (GATTTTGAATGTCATTTCT), the other is closer to the start Under iron restricted conditions, AP76ΔaasP adheres less effectively than AP76wt after 24 hours. These contrasting effects of the AasP deletion under standard conditions and NAD restriction versus iron restriction might indicate that AasP mediated protein cleavage is dependent on growth conditions, or that AasP has more than one substrate, and the substrate(s), rather than AasP itself, are dependent on growth conditions.

We assessed the pathogenic potential of A. pleuropneumoniae AP76ΔaasP in an aerosol infection experiment. One animal in the AP76ΔaasP group had to be excluded from the trial prior to infection due to severe lameness. Following infection, one animal in the AP76ΔaasP group died on day 2. This animal was excluded from statistical analyses because lung lesion scores in such cases are extraordinarily high and therefore difficult to compare to those of animals who die after day 4. A second animal from the AP76ΔaasP was euthanized on day 5 post infection. No significant difference was found between both groups concerning lung lesion scores (Fig. 2) or reisolation rates (Table 2), indicating that AP76ΔaasP is fully virulent. The effects of the aasP deletion that we saw in adhesion assays are either masked or compensated in vivo by the action of of other virulence associated factors, most importantly the ApxIII toxin (the ApxIV toxin ORF is interrupted in AP76 by insertion element ISApl1 in AP76, [START_REF] Tegetmeyer | ISApl1, a novel insertion element of Actinobacillus pleuropneumoniae, prevents ApxIV-based serological detection of serotype 7 strain AP76[END_REF].

In summary, loss of AasP function appears to have an effect on A. pleuropneumoniae adhesion to abiotic surfaces, but not on virulence. Our findings imply that AasP does not play a crucial role in A. pleuropneumoniae pathogenicity, but may still be involved in "fine tuning" of adhesion. A c c e p t e d M a n u s c r i p t

  Series, no. 123: http://conventions.coe.int/treaty/EN/Menuprincipal.htm),

  at 122 bp upstream (CATAAATTCTTTCACTCTC). These fur boxes share 15/19 and 11/19 bp, respectively, with the fur consensus sequence of E. coli(GATAATGATTTTCATTAAC, de Lorenzo et al., 1987)).

ACKNOWLEDGMENTS

  FIGURE LEGENDS

  I., Chiers, K., Charlier, G., Vandenberghe, I., Van Beeumen, J., Ducatelle, R., and Haesebrouck, F., 2002. Characterization of the in vitro adhesion of Actinobacillus pleuropneumoniae to swine alveolar epithelial cells. Vet. Microbiol. 88, 59-74. A c c e p t e d M a n u s c r i p

Figure1A

  Figure1

  Figure 2

  PspOMI and NotI restriction sites, respectively, surrounding the putative catalytic site of aasP, resulting in a 1750 bp amplification product in the wild type,

Table 1

 1

Table 2 :

 2 Challenge experiment of A. pleuropneumoniae AP76 wild type (wt) and deletion mutant A. pleuropneumoniae AP76 aasP in an aerosol infection model animal died on day 2 post infection, another one was euthanized on day 5 b) one animal was excluded from the trial before infection because of severe lameness

	A. pleuropneumoniae strains	number of animals with lung lesions	arithmetic mean of lung lesion scores	number of animals and localization of reisolation of A. pleuropneumoniae post mortem (day 7 post infection) tonsil lymph node heart lung
	AP76 wt	3/8	3.21	5/8	3/8	0/8	4/8
	AP76 aasP	3 a) /7 b)	7.56	5/7	3/7	0/7	3/7
	a) one

Table 2

 2

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