Corinne Marois
email: c.marois@ploufragan.afssa.fr

Marcelo Gottschalk

Hervé Morvan

Christelle Fablet

François Madec

Marylène Kobisch

Experimental infection of SPF pigs with Actinobacillus pleuropneumoniae serotype 9 alone or in association with Mycoplasma hyopneumoniae

Keywords: Actinobacillus pleuropneumoniae serotype 9, Mycoplasma hyopneumoniae, dual infection, SPF pigs, experimental infection

The purpose of this study was to compare in SPF pigs, the pathogenicity of an A. pleuropneumoniae serotype 9 strain 21 (isolated from the palatine tonsils of a healthy gilt on a French nucleus pig farm, with no clinical signs or lung lesions but a highly positive reaction to A. pleuropneumoniae serotype 9 antibodies) with a pathogenic A. pleuropneumoniae strain 4915 serotype 9 (isolated in France from an outbreak of porcine pleuropneumonia). The pathogenicity of one M. hyopneumoniae strain alone or associated with A. pleuropneumoniae strain 21 was also compared. Eight groups of 7 pigs were infected (at 6 or 10 weeks of age) and a control group was kept non-infected. Results showed that sensitivity to A. pleuropneumoniae was related to the age of the pig (6 weeks vs 10 weeks) whatever the strain. Surviving pigs infected at 6 weeks of age developed severe clinical signs, lung lesions typical of A. pleuropneumoniae and they seroconverted. In contrast, symptoms and lung lesions were almost non-existent in pigs infected with strain 21 at 10 weeks of age, but a seroconversion was observed with very high ELISA titres. These results were in accordance with those observed in the nucleus pig farm. Infection with M. hyopneumoniae alone induced typical mycoplasmal symptoms, pneumonia and seroconversion. Symptoms and lung lesions were the most noticeable in pigs infected with M. hyopneumoniae at 6 weeks of age and with A. pleuropneumoniae 4 weeks later. Our results

show that the presence of A. pleuropneumoniae serotype 9 in a pig herd may be clinically unnoticed and that M. hyopneumoniae may potentiate A. pleuropneumoniae infection.

Introduction

Porcine respiratory disease complex (PRDC) is a major concern in the pig production throughout the world and is due to a combination of multiple bacterial and viral agents. Two pathogenic bacteria, Mycoplasma hyopneumoniae (the primary agent of swine enzootic pneumonia) [START_REF] Ross | Mycoplasmal Diseases[END_REF][START_REF] Thacker | Mycoplasmal Diseases[END_REF] and Actinobacillus pleuropneumoniae (the etiologic agent of swine pleuropneumonia), alone or associated, can induce severe respiratory disorders in pigs [START_REF] Kobisch | Pathologie pulmonaire du porc : un modèle expérimental associant Mycoplasma hyopneumoniae et Actinobacillus pleuropneumoniae[END_REF][START_REF] Gottschalk | Actinobacillus pleuropneumoniae[END_REF]). An acute A. pleuropneumoniae infection can induce severe clinical signs and lung lesions. The infection may become chronic or subclinical without previous signs of the disease, outbreaks may suddenly appear or subclinical infections may remain silent. A. pleuropneumoniae is able to persist in pig tissues, particularly in tonsillar crypts and in sequestered necrotic lungs. Thus, the early identification of subclinically infected pig herds is necessary to control carrier pigs and prevent A. pleuropneumoniae transmission between herds, especially from nucleus pig farms to multipliers [START_REF] Gottschalk | Actinobacillus pleuropneumoniae[END_REF]. The virulence of A. pleuropneumoniae is known to be variable: biotype I has been divided into 13 serotypes and biotype II into 2 serotypes, for a total of 15 serotypes [START_REF] Gottschalk | Actinobacillus pleuropneumoniae[END_REF]. Serotype 2 and serotype 9, two virulent serotypes, are prevalent in outbreaks in European countries and particularly in France [START_REF] Gottschalk | Actualités sur la pleuropneumonie porcine[END_REF]. A. pleuropneumoniae produces four RTX toxins (ApxI-ApxIV) associated with virulence. All serotypes of A. pleuropneumoniae encode for at least two RTX toxins [START_REF] Frey | Actinobacillus pleuropneumoniae RTX toxins: uniform designation of haemolysins, cytolysins, pleurotoxin and their genes[END_REF][START_REF] Frey | Virulence in Actinobacillus pleuropneumoniae and RTX toxins[END_REF][START_REF] Schaller | Characterization of apxIVA, a new RTX determinant of Actinobacillus pleuropneumoniae[END_REF][START_REF] Kuhnert | Phylogenetic relationship of equine Actinobacillus species and distribution of RTX toxin genes among clusters[END_REF]. It is relatively easy to isolate A. pleuropneumoniae from pneumonic lesions in freshly dead animals but bacteriological detection is more difficult in chronic infections or in healthy carrier pigs. The presence of A. pleuropneumoniae in nasal cavities and tonsils can be revealed by specific PCR tests [START_REF] Savoye | A PCR assay used to study aerosol transmission of Actinobacillus pleuropneumoniae from samples of live pigs under experimental conditions[END_REF][START_REF] Fittipaldi | Evaluation and field validation of PCR tests for detection of Actinobacillus pleuropneumoniae in subclinically infected pigs[END_REF]. Serological monitoring has been used widely to control A. pleuropneumoniae infection in pig farms. The most commonly used are ELISA tests with LC-LPS as antigens [START_REF] Gottschalk | Evaluation of a saline boiled extract, capsular polysaccharides and long-chain lipopolysaccharides of Actinobacillus pleuropneumoniae serotype 1 as antigens for the serodiagnosis of swine pleuropneumonia[END_REF][START_REF] Dubreuil | Actinobacillus pleuropneumoniae surface polysaccharides: their role in diagnosis and immunogenicity[END_REF].

Previously, we described an experimental infection of 10-week-old SPF piglets, with a pathogenic A. pleuropneumoniae strain (4915, belonging to serotype 9), isolated in France from an outbreak of porcine pleuropneumonia. The clinical signs were acute: hyperthermia, respiratory distress and death in some cases. The severe lung lesions included fibrinous pleurisy and lung haemorrhages in the acute stage, pleural adhesions and focal pulmonary necrosis in the chronic stage. The surviving pigs became seropositive in less than 6 days [START_REF] Jobert | Experimental aerosol transmission of Actinobacillus pleuropneumoniae to pigs[END_REF].

The objectives of the present study were: (i) to compare the pathogenicity in SPF pigs, of A.pleuropneumonia strain 4915, with that of A. pleuropneumoniae strain 21 (serotype 9), isolated in France from the palatine tonsils of a healthy gilt from a nucleus pig farm. This farm presented no clinical signs or lung lesions related to A. pleuropneumoniae but a relatively highly positive prevalence for A. pleuropneumoniae serotype 9 antibodies at the end of the finishing period and (ii) to investigate the interactions of this A. pleuropneumoniae strain with M. hyopneumoniae, under experimental conditions.

Materials and methods

Bacterial strains and culture conditions

A. pleuropneumoniae strains (4915 and 21) were grown on PPLO medium supplemented with 10 g/mL nicotinamide adenine dinucleotide, 1 mg/mL glucose, 5% decomplemented horse serum (PPLOsup) for 6 h at 37°C [START_REF] Jobert | Experimental aerosol transmission of Actinobacillus pleuropneumoniae to pigs[END_REF]. The titre of the A. pleuropneumoniae cultures was expressed in colony forming units (CFU/mL). The two strains were identified by biochemical tests and PCR [START_REF] Savoye | A PCR assay used to study aerosol transmission of Actinobacillus pleuropneumoniae from samples of live pigs under experimental conditions[END_REF]. Serotyping by coagglutination test with type-specific hyperimmune serum showed that they belonged to serotype 9 [START_REF] Gottschalk | Actinobacillus pleuropneumoniae[END_REF]. These A. pleuropneumoniae strains were also positive for their capacity to produce two RTX toxins (ApxI and ApxII) as detected by PCR [START_REF] Frey | Virulence in Actinobacillus pleuropneumoniae and RTX toxins[END_REF]. M. hyopneumoniae (strain 116) was isolated from an outbreak of enzootic pneumonia in France and was cultivated in Friis broth medium (FBM) at 37 °C, as previously described [START_REF] Marois | Isolation of Mycoplasma hyopneumoniae from different sampling sites in experimentally infected and contact SPF piglets[END_REF]. The titre of the M. hyopneumoniae cultures was expressed as colour changing units per millilitre (CCU/mL).

Experimental design

Experimental infections

Sixty-three SPF pigs (hysterectomy derived piglets) were obtained from the experimental pig herd of the French Food Safety Agency of Ploufragan. Animal experiments were performed in accordance with current legislation and ethical and welfare recommendations (Agreement B-22-745-1). Very strict biosecurity measures were implemented in order to avoid undesirable contaminations of the pigs: air filtration system and airlocks for each unit, unit-specific clothes and compulsory showering before and after visiting the pigs [START_REF] Cariolet | Rappel des différentes méthodes d obtention de porcelets assainis : conditions de maintien du statut sanitaire et valorisation de ces animaux[END_REF]. All animals were confirmed to be serologically negative to A. pleuropneumoniae and M. hyponeumoniae.

Groups of seven pigs were randomly allocated to nine separate rooms (Table 1). At six weeks of age, pigs in group 2 were inoculated intranasally (1 mL per nostril), each pig received 1.4 x 10 8 CFU of A. pleuropneumoniae strain 4915 . Pigs in groups 3, 7 and 9 were experimentally infected, in the same conditions, with 1.3 x 10 8 CFU of A. pleuropneumoniae strain 21 for each pig. Pigs in groups 5 and 8 were infected with the latter strain, at 10 weeks of age. In addition, pigs in groups 4, 8 and 9 were experimentally infected with M. hyopneumoniae at six weeks of age and those in groups 6 and 7 four weeks later (Table 1). They were inoculated intratracheally (5 mL per day), as previously described [START_REF] Kobisch | Experimental infections of swine[END_REF]. Each pig was infected, on 2 consecutive days, with 5 x 10 9 CCU by tracheal intubation. The pigs in group 1 were controls (uninfected pigs).

Clinical monitoring

Daily clinical examinations consisted of taking rectal temperature (normally 39.5°C in an SPF pig but noted as hyperthermia when 40.5°C) and looking for symptoms such as dyspnea, coughing (from daily counts of the number of coughs for 15 min), cyanosis, nasal discharge or foaming. Body weight was recorded once a week.

Samples and analysis

Blood samples were taken once a week from live pigs for serological analysis (D0 to D63). Sera were stored at 20°C and tested with a blocking ELISA (DAKO ELISA, Kitvia, Labarthe-Inard, France) to detect M. hyopneumoniae antibodies. The percentage of inhibition for each serum was calculated with the following formula: Percent inhibition = 100-[100 (sample mean OD buffer control mean OD)]. A sample was classified as positive if the percent of inhibition was > 50%. Sera were also analysed to detect A. pleuropneumoniae antibodies, by an ELISA technique using long chain purified polysaccharides (LC-LPS) specific for serogroup 1-9-11 (Swinecheck App, Biovet, AES Laboratoire, Combourg). The positive threshold was fixed at an optical density (OD) of 0.55.

If mortality occurred or after euthanasia, maximum D30-D65 after M. hyopneumoniae or A. pleuropneumoniae infection, the pigs were necropsied and their thoracic organs were thoroughly examined (Table 2). Pneumonia and pleurisy were scored as previously described by [START_REF] Madec | Fréquence, intensité et localisation des lésions pulmonaires chez le porc charcutier[END_REF]. The maximum total scores possible for each lung were 28 for pneumonia and 4 for pleurisy.

Swabs were collected from trachea, palatine tonsils and lungs, from all the pigs. The swabs were placed in 2 mL of Buffered Pepton Water Broth (samples). A. pleuropneumoniae was cultured from 50 L of each sample placed on PPLOsup agar and incubated overnight at 37°C in 5% CO 2 . A. pleuropneumoniae like colonies were identified by PCR [START_REF] Savoye | A PCR assay used to study aerosol transmission of Actinobacillus pleuropneumoniae from samples of live pigs under experimental conditions[END_REF]. M. hyopneumoniae was cultured from samples by diluting 100 L of each sample in 900 L of FBM supplemented with bacitracin (150 g/mL), amphotericin B (2.5 g/mL), ampicillin (100 g/mL) and colistin (7.5 g/mL) to avoid contamination by non-specific bacteria and optimise M. hyopneumoniae recovery. Each sample was grown in FBM, 10-fold diluted up to 10 -3 and incubated at 37°C until the culture developed an acid colour change or up to 30 days. When a colour change of the FBM was observed, M. hyopneumoniae cultures were confirmed by a specific PCR (test described by [START_REF] Verdin | A nested PCR assay for the detection of Mycoplasma hyopneumoniae in tracheobronchiolar washings from pigs[END_REF] and modified by [START_REF] Marois | Isolation of Mycoplasma hyopneumoniae from different sampling sites in experimentally infected and contact SPF piglets[END_REF].

Finally, microscopic lung examinations were conducted on each pig. Lung samples (a piece of the right middle lobe) were fixed in 10% buffered formalin: paraffin-embedded sections were cut at 5 m, stained by a trichrome coloration (hematoxylin, eosin and safran) and examined by light microscopy.

Statistical analysis

The data obtained from the experimental study (average daily weight gains, macroscopic and microscopic lesions and serological results) for each group were compared simultaneously by Kruskall-Wallis test and in 2 by 2 tables by Kolmogorov-Smirnov test. Differences in the number of pigs (with clinical signs, macroscopic or microscopic lesions and with positive bacteriological or serological analyses) between groups were assessed by Fisher exact test (n 5) or chi-square test (n > 5) of independence in 2 by 2 tables. These tests were carried out with Systat 9.0 program for Windows (Systat Software GmbH, Erkrath, Germany). Differences were considered significant when P 0.05.

Results

Clinical signs and macroscopic lesions

As shown in Table 1, clinical signs and pulmonary lesions varied with the group of pigs. The necropsy plan is presented in Table 2.

No clinical signs or lesions were observed in non-infected animals (group 1). The average daily weight gain (ADG) of these pigs was 932 g.

3.1.1. Positive controls of the experiment: our standardized experimental models Pigs infected at 6 weeks of age with A. pleuropneumoniae strain 4915 (group 2), developed hyperthermia (41-41.4°C), dyspnea and anorexia. All pigs with respiratory distress died two days after infection, so the ADG could not be evaluated. Macroscopic lung lesions, typical of A. pleuropneumonia infection (pneumonia, pleurisy, fibrinous pleuropneumonia and haemorrhage mostly located in the cranial, middle and caudal lobes) and hypertrophy of the tracheo-bronchial lymph nodes were observed in 7 pigs.

All pigs infected at either 6 or 10 weeks of age with M. hyopneumoniae strain 116 (groups 4 and 6) developed coughing with a significantly higher frequency in group 4 (P 0.05). Typical mycoplasmal pneumonia, mostly located in the apical and cardiac lobes, was noted and no significant differences were observed between the two groups. The ADG of both groups, compared with that of group 1, was significantly affected, particularly that of group 4 (P 0.05).

Experimental infections with A. pleuropneumoniae strain 21

Pigs infected with A. pleuropneumoniae alone, at 6 weeks or at 10 weeks of age (groups 3 and 5)

The results differed considerably between these two groups. The symptoms and lung lesions observed in pigs of group 3 were typical of A. pleuropneumoniae infection and three pigs died (24-48h after infection). Lung lesions were located in the diaphragmatic lobes and in some cases, in the apical and cardiac lobes. With the exception of pleurisy scores and mortality rates, no significant differences were apparent between groups 2 and 3 from the overall results.

No clinical signs or lung lesions were present in group 5, with the exception of tracheo-bronchial lymph nodes hypertrophy in 4 pigs and hyperthermia for a single day (40.7°C), in only one pig, 6 days after infection. The ADG in groups 3 and 5 were similar. But, a significant difference (P 0.05) was noted between the ADG of the pigs of these two groups and those of the negative control group (group 1).

Pigs infected with A. pleuropneumoniae in association with M. hyopneumoniae (groups 7, 8 and 9) Group 7: pigs infected with A. pleuropneumoniae at 6 weeks of age and with M. hyopneumoniae at 10 weeks of age. One pig died two days after A. pleuropneumoniae infection. Hyperthermia and lung lesions were similar to those of group 3 but the coughing score was higher in group 7. Lung lesions were located in the apical and cardiac lobes (M. hyopneumoniae infection) as well as in the diaphragmatic lobes (A. pleuropneumoniae infection). Pulmonary necrosis was observed in four pigs.

The ADG of pigs in group 7 were significantly different from those of group 1 (P 0.05) but the ADG values for groups 3, 6 and 7 were similar.

Group 8: pigs infected with M. hyopneumoniae at 6 weeks of age and with A. pleuropneumoniae at 10 weeks of age. Four pigs died 2 or 3 days after A. pleuropneumonia infection and a fibrinous and haemorrhagic pleuropneumoniae was apparent. Clinical signs and lung lesions were typical of both M. hyopneumoniae and A. pleuropneumoniae infections. Coughing score and pneumonia mean score were significantly higher in group 8 than in group 7(P 0.05). The comparison with group 5 gave similar results. The ADG of the survivors was significantly affected in group 8, in comparison with the negative control group and with pigs in groups 5 and 7.

Group 9: pigs simultaneously infected with A. pleuropneumoniae and M. hyopneumoniae at 6 weeks of age. One pig died one day after the double infection. Clinical signs and lung lesions were similar to those observed in groups 3 and 7, but significantly different from those observed in group 8 (P 0.05).

The ADG was affected, in comparison with the negative control group and with group 7 (P 0.05), but no differences were noted with pigs of groups 3 and 8.

Microscopic lung lesions

Results are presented in Table 3 and Figure 1. Pigs infected with M. hyopneumoniae alone or in association with A. pleuropneumoniae: infiltrating lymphocytes were observed in the peribronchiolar, peribronchial and perivascular areas at the beginning of M. hyopneumoniae infection. This was followed by interstitial pneumonia, lymphoid nodules associated with the airways and collapse of the alveoli. These two phases were noted in pigs infected with M. hyopneumoniae, independently of the day of necropsy. With the exception of two pigs that died, either firstly or simultaneously infected with A. pleuropneumoniae (groups 7 and 9), all other pigs developed typical mycoplasmal lesions.

Microscopic examination did not reveal any differences between the lungs of pigs infected with M. hyopneumoniae alone (groups 4 and 6) or with both bacterial species (groups 7, 8 and 9).

Pigs infected with A. pleuropneumoniae, alone or in association with M. hyopneumoniae: the histopathologic changes of the lungs, in the early phase of A. pleuropneumoniae infection, were characterised by haemorrhage, vascular thrombosis, oedema, necrosis and the presence of fibrinous exudate. Acute lung lesions were apparent in similar numbers of pigs in groups 2, 3 and 8 but significant differences (P 0.05) were observed between group 2 and groups 5, 7 and 9.

In the chronic phase, marked fibrosis around areas of necrosis and fibrinous pleuritis were observed.

In group 5, two pigs without any macroscopic lung lesions, showed very mild and superficial interlobular fibrosis. One pig in group 9, with macroscopic pneumonia, did not show any microscopic lesions in the section observed. Similar numbers of pigs were affected in groups 3, 5, 7, 8 and 9.

Finally, no lesions were observed in the negative control group.

M. hyopneumoniae and A. pleuropneumoniae detection

M. hyopneumoniae and A. pleuropneumoniae were not recovered from any non-infected pigs (Table 4).

M. hyopneumoniae was re-isolated and identified by PCR, in all infected pigs (groups 4, 6, 7, 8 and 9) and no significant differences were observed between these groups. In group 7, one pig (that died two days after A. pleuropneumoniae infection) could not be experimentally infected with M. hyopneumoniae. The most appropriate sites for the detection of M. hyopneumoniae appeared to be the trachea and lungs rather than the tonsils.

A. pleuropneumoniae was recovered from all infected pigs (groups 2, 3, 5, 7, 8 and 9). Sampling the lungs and trachea was most effective in the early phase of infection, whereas swabbing the tonsils was more appropriate in the chronic phase. A. pleuropneumoniae was not isolated from the lungs of pigs in group 5.

Serological results

Serological results are shown in Figure 2. All non-infected pigs were seronegative throughout the experiment. In all pigs infected with M. hyopneumoniae, seroconversion occurred 2-3 weeks after infection (Figure 2A). At the end of the experiment, the ELISA titres in the different groups of pigs were similar (mean results:100 percent inhibition). All surviving pigs infected with A. pleuropneumoniae were seropositive three or four weeks after infection (groups 3, 7 and 9) or two weeks after infection (groups 5 and 8). After 42 days, the ELISA titres (mean OD almost 1.0) for the different groups of pigs were similar (Figure 2B).

Discussion

In our experimental conditions, SPF pigs intranasally inoculated, at six weeks of age, with 1.4x10 8 CFU of A. pleuropneumoniae strain 4915 (serotype 9, isolated from an outbreak of porcine pleuropneumoniae), developed severe clinical signs as well as lung lesions typical of the acute phase of infection. All pigs died 24 to 48 h after experimental infection and A. pleuropneumoniae was recovered from their respiratory tract. These results are in accordance with those of our previous study showing that SPF pigs experimentally infected, at 10 weeks of age, with the same A. pleuropneumoniae strain, developed acute clinical signs and severe lung lesions (Jobert al., 2000).

Nevertheless, in this first experiment with A. pleuropneumoniae strain 4915, the pigs received ampicillin to avoid death. Thus, surviving pigs showed lung lesions corresponding to the chronic phase of infection. According to our results in the present study, the sensitivity of pigs to A. pleuropneumoniae infection is probably age-related: six weeks vs ten weeks. Thus, the results obtained by [START_REF] Jobert | Experimental aerosol transmission of Actinobacillus pleuropneumoniae to pigs[END_REF] are in agreement with those obtained in the present study with A. pleuropneumoniae strain 21 (also a serotype 9 producing ApxI and ApxII toxins): SPF pigs infected at 6 weeks of age with 1.3x10 8 CFU of A. pleuropneumoniae showed very severe clinical signs and lung lesions, analogous to those induced with A. pleuropneumoniae strain 4915. On the other hand, lung lesions and symptoms were almost non-existent in the group of pigs infected at 10 weeks of age.

Nevertheless, the ADG of the pigs was affected. In group 3, surviving pigs were seropositive three to four weeks after infection and all the pigs of group 5 seroconverted after two weeks. In both cases, the ELISA titres were similar and very high (mean OD almost 1) at the end of the experiment. These results are in accordance with those observed in the nucleus pig farm from which A. pleuropneumoniae strain 21 had been isolated. The young sows of this pig farm became contaminated, were A. pleuropneumoniae positive in the palatine tonsils and clearly seroconverted without any manifestation of A. pleuropneumoniae infection. This might be explained by the excellent housing conditions and hygienic environment of this particular breeding herd.

Checks for respiratory disorders in breeding herds are made during routine farm visits by veterinarians in the context of clinical surveillance [START_REF] Gottschalk | Actinobacillus pleuropneumoniae[END_REF]. Serological testing of a representative number of animals from the herd and detection of A. pleuropneumoniae by bacteriological isolation or PCR, are very useful for epidemiological investigations. Positive serological results must be taken seriously even if clinical signs are absent from the herd. Asymptomatic carrier pigs are a major source for introduction of A. pleuropneumoniae into an uninfected herd (Chiers et al., 2002a;Chiers et al., 2002b). Some authors indicated that asymptomatic pigs carrying bacteria in the tonsils do not generally develop measurable antibody titres and the lower respiratory tract appears to be involved to obtain a humoral response (Chiers et al., 2002a;Chiers et al., 2002b;[START_REF] Maas | Use of an Actinobacillus pleuropneumoniae multiple mutant as a vaccine that allows differentiation of vaccinated and infected animals[END_REF]. However, it has been clearly demonstrated that clinically healthy animals colonized at tonsils can develop a strong serological reaction [START_REF] Gottschalk | Actinobacillus pleuropneumoniae[END_REF]. [START_REF] Krejci | Systemic and local antibody responses after experimental infection with Actinobacillus pleuropneumoniae in piglets with passive or active immunity[END_REF] also showed that the presence of A. pleuropneumoniae in the tonsils could induce immunity of the airway mucosa and, in some cases, prevent bacterial colonisation of the lower part of the respiratory tract.

According to [START_REF] Haesebrouck | Actinobacillus pleuropneumoniae infections in pigs: the role of virulence factors in pathogenesis and protection[END_REF], the presence of antibodies in the serum of pigs does not provide complete protection against A. pleuropneumoniae infection. However, these antibodies may prevent severe forms of the disease [START_REF] Cruijsen | Phagocytosis and killing of Actinobacillus pleuropneumoniae by alveolar macrophages and polymorphonuclear leukocytes isolated from pigs[END_REF][START_REF] Krejci | Systemic and local antibody responses after experimental infection with Actinobacillus pleuropneumoniae in piglets with passive or active immunity[END_REF].

Pigs infected with M. hyopneumoniae alone developed coughing, lung lesions and showed seroconversion and M. hyopneumoniae was re-isolated whatever the time of infection (6 weeks vs 10 weeks). The ADG of the pigs was affected and antibodies were present in all the pigs three weeks after infection. These results are similar to those described in previous studies [START_REF] Kobisch | Experimental infections of swine[END_REF][START_REF] Fano | Dynamics and persistence of Mycoplasma hyopneumoniae infection in pigs[END_REF][START_REF] Marois | Isolation of Mycoplasma hyopneumoniae from different sampling sites in experimentally infected and contact SPF piglets[END_REF].

Under the conditions of this study, when pigs were firstly infected with A. pleuropneumoniae or simultaneously with M. hyopneumoniae and A. pleuropneumoniae, at six weeks of age, clinical signs and lung lesions were severe and corresponded to the pathogenicity of the two bacterial strains combined. However, pigs experimentally infected with M. hyopneumoniae at six weeks of age and four weeks later with A. pleuropneumoniae (strain 21) were particularly affected. Very severe clinical signs (hyperthermia, death and coughing) and lung lesions, corresponding to the double infection, were observed in these animals. The ADG was the lowest of all the experimental groups (650g vs 932g in the negative control group). M. hyopneumoniae, associated with enzootic pneumonia, is also considered to play a primary role in PRDC [START_REF] Thacker | Mycoplasmal Diseases[END_REF]. M. hyopneumoniae is able to induce damage in the respiratory tract and to predispose pigs to other respiratory pathogens, especially bacteria and viruses [START_REF] Yagihashi | Effect of Mycoplasma hyopneumoniae infection on the development of Haemophilus pleuropneumoniae pneumonia in pigs[END_REF][START_REF] Ross | Mycoplasmal Diseases[END_REF][START_REF] Maes | Control of Mycoplasma hyopneumoniae infections in pigs[END_REF]. According to [START_REF] Thacker | Mycoplasmal Diseases[END_REF], M. hyopneumoniae and A. pleuropneumoniae co-infection is a common cause of PRDC. M. hyopneumoniae may potentiate the severity of A. pleuropneumoniae-induced lesions in co-infected pigs [START_REF] Ciprián | Mycoplasma hyopneumoniae: interaction with other agents in pigs, and evaluation of immunogens[END_REF]. These observations are in accordance with the results of the present study showing changes in pigs experimentally infected with M. hyopneumoniae and A. pleuropneumoniae.

The breeding herd, from which A. pleuropneumoniae strain 21 was isolated, was not infected with M. hyopneumoniae (routine checks conducted by serology). A. pleuropneumoniae was probably not associated with other respiratory pathogens in this farm with a high health status. A. pleuropneumoniae was only detected in the palatine tonsils of gilts. Routine checks by veterinarians are very useful in pig farms, particularly in breeding herds. Positive serological results, especially concerning A. pleuropneumoniae serotype 9, a major respiratory pathogen of pigs, should be interpreted with care. : 1 2 3 4 5 6 7 8 9 D1 andD2 0 7 3 0 0 0 1 0 1 D30 to D35 0 -0 3 0 0 0 4 2 D48 andD49 3 -0 4 0 0 0 3 4 D57 andD58 0 -1 -3 3 2 --D63 to D65 4 -3 -4 4 4 --

Figure 1 :

 1 Figure 1: Microscopic examinations in the lungs of four pigs (LN: Lymphoid Nodules, N:

Figure 2 :

 2 Figure 2 : Serological response from uninfected pigs and pigs experimentally infected with M.

  Figure 2A: antibodies to M. hyopneumoniae, group 1: uninfected pigs, groups 4, 6, 7, 8 and 9: infected with M. hyopneumoniae.

  Figure 2B: antibodies to A. pleuropneumoniae, group 1: uninfected pigs, groups 3, 5, 7, 8 and 9: infected with A. pleuropneumoniae (surviving pigs).

Figure 1 :

 1 Figure 1: Microscopic examinations in the lungs of four pigs (LN: Lymphoid Nodules, N: Necrosis, H: Haemorrhage, FE: Fibrinous Exudates, FN: Fibrosis around areas of Necrosis, FP: Fibrinous Pleuritis) (A) Lung of a control pig (group 1) (x50) (B) Lung of a pig infected with M. hyopneumoniae (group 4) (x25) (C) Lung of a pig infected with M. hyopneumoniae simultaneously with A. pleuropneumoniae (group 9). Early phase of infection (x50-Figure C1 and x100-Figure C2) (D) Lung of a pig infected with M. hyopneumoniae (6 weeks of age) and with A. pleuropneumoniae (10 weeks of age) (group 7). Chronic phase of infection (x50-Figure D1 and x25-Figure D2).

Figure 2 :

 2 Figure 2 : Serological response from uninfected pigs and pigs experimentally infected with M. hyopneumoniae or A. pleuropneumoniae or both.Figure 2A: antibodies to M. hyopneumoniae, group

  Figure 2A: antibodies to M. hyopneumoniae, group 1: uninfected pigs, groups 4, 6, 7, 8 and 9: infected with M. hyopneumoniae.

  Figure 2B: antibodies to A. pleuropneumoniae, group 1: uninfected pigs, groups 3, 5, 7, 8 and 9: infected with A. pleuropneumoniae (surviving pigs).

  Figure 2A

Table 1 :

 1 Experimental design, clinical and pathological observations

Table 2 :

 2 Planning of necropsies

	No. of pigs	Groups of pigs (n=7)
	necropsied at

Table 3 :

 3 Microscopic lung lesions Typical lung lesions corresponding to the early phase of A. pleuropneumoniae infection e Typical lung lesions corresponding to the chronic phase of A. pleuropneumoniae infection

									Groups of pigs (n=7)
					1	2		3	4	5	6	7	8	9
	Challenge at 6 weeks of age (Day 0)	UI	App 4915	App 21	Mhp 116	-	-	App 21	Mhp 116	App 21 Mhp 116
	Challenge at 10 weeks of age (Day 28)	UI	-		-	-	App 21	Mhp 116	Mhp 116	App 21	-
			D1 and D2	a		0/7	b	0/3				0/1	0/1
			D30 to D35					3/3			4/4	2/2
	Mycoplasmal lesions	c	D48 and D49 D57 and D58	0/3			0/1	4/4	0/3	3/3	2/2	3/3	4/4
			D63 to D65	0/4			0/3		0/4	4/4	4/4
			Total		0/7	0/7	0/7	7/7	0/7	7/7	6/7	7/7	6/7
			D1 and D2		7/7	3/3				1/1	1/1
			D30 to D35					0/3			4/4	0/2
	Early phase of App infection d		D48 and D49 D57 and D58	0/3			0/1	0/4	0/3	0/3	0/2	0/3	0/4
			D63 to D65	0/4			0/3		0/4	0/4	0/4
			Total		0/7	7/7	3/7	0/7	0/7	0/7	1/7	4/7	1/7
			D1 and D2		0/7	0/3				0/1	0/1
			D30 to D35					0/3			0/4	1/2
	Chronic phase of App infection e		D48 and D49 D57 and D58	0/3			1/1	0/4	1/3	0/3	2/2	3/3	4/4
			D63 to D65	0/4			3/3		1/4	0/4	3/4
			Total		0/7	0/7	4/7	0/7	2/7	0/7	5/7	3/7	5/7
	a Necropsy days b No. of pigs with microscopic lung lesions / No. of necropsied pigs

c Typical mycoplasmal lesions d

Table 4 :

 4 Bacteriological results (at time of necropsy)

	505
						Groups of pigs (n=7)
	Samples 1	2	3	4	5	6	7	8
	Challenge at 6 weeks of age (Day 0)	UI	App 4915	App 21	Mhp 116	-	-	App 21	Mhp 116	App 21 Mhp
	Challenge at 10 weeks of age (Day 28)	UI	-	-	-	App 21	Mhp 116	Mhp 116	App 21	-
	Number of positive Mhp cultures

Acknowledgements

The authors would like to thank the Regional Council of Brittany, the "Comité Régional Porcin and Boehringer Ingelheim Animal Health France, Fort Dodge Animal Health, Intervet S.A., Pfizer Animal Health and Schering-Plough Animal Health for their financial support. They also thank Roland Cariolet, Laëtitia Le Devendec, Véronique Tocqueville, Bernard Beaurepaire, Gérard Bénévent, Pierre Ecobichon and Jean-Claude Rault for their technical assistance.