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

The purpose of this study was to compare in SPF pigs, the pathogenicity of an Actinobacillus 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 Mycoplasma 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.

D-alanylation of lipoteichoic acid contributes to the virulence of Streptococcus suis.

We generated by allelic replacement a DeltadltA mutant of a virulent Streptococcus suis serotype 2 field strain and evaluated the contribution of lipoteichoic acid (LTA) d-alanylation to the virulence traits of this swine pathogen and zoonotic agent. The absence of LTA D-alanylation resulted in increased susceptibility to the action of cationic antimicrobial peptides. In addition, and in contrast to the wild-type strain, the DeltadltA mutant was efficiently killed by porcine neutrophils and showed diminished adherence to and invasion of porcine brain microvascular endothelial cells. Finally, the DeltadltA mutant was attenuated in both the CD1 mouse and porcine models of infection, probably reflecting a decreased ability to escape immune clearance mechanisms and an impaired capacity to move across host barriers. The results of this study suggest that LTA D-alanylation is an important factor in S. suis virulence.

Transmission of pathogenic respiratory bacteria to specific pathogen free pigs at slaughter.

The purpose of this study was to evaluate the transmission of pathogenic respiratory bacteria to thirteen 5-month-old specific pathogen free (SPF) pigs, during the slaughtering process in a commercial slaughterhouse. Before transportation, the SPF pigs and the lorry were checked to confirm the absence of pathogenic respiratory bacteria. Nine SPF pigs (group 1) were in contact in a conventional slaughterhouse with finishing pigs, during 4h before slaughtering. Four SPF pigs (group 2) were slaughtered immediately at arrival in the slaughterhouse. Five bacterial pathogens (Mycoplasma hyopneumoniae, Actinobacillus pleuropneumoniae, Pasteurella multocida, Haemophilus parasuis and Streptococcus suis) were detected by PCR, after slaughtering, from nasal cavities, tonsils and trachea in the two groups of pigs. Lung samples were PCR negative. Three and four bacterial species were isolated from the pigs of group 2 and group 1, respectively. Cultures were negative from the lungs. All the bacterial species present in the SPF pigs were detected by PCR. P. multocida was isolated, from three samples of scalding water before the onset of slaughtering. Our results suggest that the SPF pigs became contaminated mainly by the slaughterhouse environment and the scalding water. Histological examinations revealed that during scalding, contaminated water could reach the trachea and the lungs of pigs. Checks conducted at slaughter for respiratory disorders have to be carried on, but nasal cavities and tonsils are not appropriate for bacteriological investigations. Moreover, bacteriological results obtained from the lungs of slaughtered pigs have to be used with carefulness.

CTX-M-1- and CTX-M-15-type beta-lactamases in clinical Escherichia coli isolates recovered from food-producing animals in France.

Clinical Escherichia coli strains with resistance or variable susceptibility to third-generation cephalosporins were detected in cattle, swine and poultry in France. These strains were shown to produce extended-spectrum beta-lactamases (ESBLs), with CTX-M-1- and CTX-M-15-type beta-lactamases being responsible for this phenotype. The bla(CTX-M-1) gene was encountered most commonly and was characterised in seven E. coli strains isolated from cattle, swine and poultry, whereas bla(CTX-M-15) was identified in one E. coli isolated from cattle. These genes were located on a conjugative plasmid and were linked to the insertion sequence ISEcp1, which could have contributed to dissemination of the resistance gene. No epidemiological link between the strains was determined by pulsed-field gel electrophoresis, although two plasmids were identical in two strains isolated from swine and in two strains isolated from cattle and poultry. Thus, this study describes the emergence of ESBLs in animals in France, with a probable similar prevalence rate to that observed in humans. This is a major concern because of the possibility of transfer of these genes between animal species as well as to humans, leading to treatment failures in veterinary and human medicine.

Molecular characterization of Streptococcus suis strains by 16S-23S intergenic spacer polymerase chain reaction and restriction fragment length polymorphism analysis.

We developed a new molecular method of typing Streptococcus suis based on polymerase chain reaction (PCR) amplification of a large fragment of rRNA genes, including a part of the 16S and 23S genes and the 16S-23S intergenic spacer region (ISR), followed by restriction fragment length polymorphism (RFLP) analysis with RsaI or MboII endonuclease. The 16S-23S ISRs of 5 S. suis isolates were sequenced and compared. Size and sequence polymorphisms were observed between the S735 reference strain and the 4 wild-type strains. The genetic relationships between 138 independent S. suis strains belonging to various serotypes, isolated from swine or human cases, were determined. The discriminatory power of the method was > 0.95, the threshold value for interpreting typing results with confidence (0.954 with RsaI and 0.984 with RsaI plus MboII). The in vitro reproducibility was 100%. The strains isolated from humans were less genetically diverse than the strains isolated from pigs. For the first time, 2 molecular patterns (R6, M9) were significantly associated with S. suis serotype 2 strains. This genetic tool could be valuable in distinguishing individual isolates of S. suis during epidemiologic investigations.

Persistence of Mycoplasma gallisepticum in chickens after treatment with enrofloxacin without development of resistance.

The ability of the avian pathogen Mycoplasma gallisepticum to persist despite fluoroquinolone treatment was investigated in chickens. Groups of specific pathogen free chickens were experimentally infected with M. gallisepticum and treated with enrofloxacin at increasing concentrations up to the therapeutic dose. When M. gallisepticum could no longer be re-isolated from chickens, birds were stressed by inoculation of infectious bronchitis virus or avian pneumovirus. Although M. gallisepticum could not be cultured from tracheal swabs collected on several consecutive sampling days after the end of the enrofloxacin treatments, the infection was not eradicated. Viral infections reactivated the mycoplasma infection. Mycoplasmas were isolated from tracheal rings cultured for several days, suggesting that M. gallisepticum persisted in the trachea despite the enrofloxacin treatment. The minimal inhibitory concentration (MIC) of enrofloxacin for most of the re-isolated mycoplasmas was the same as that of the strain with which the birds were inoculated. Furthermore, no mutation could be detected in the fluoroquinolone target genes. These results suggest that M. gallisepticum can persist in chickens without development of resistance despite several treatments with enrofloxacin.

Dilemma of virulence of Streptococcus suis: Canadian isolate 89-1591 characterized as a virulent strain using a standardized experimental model in pigs.

Virulence of Streptococccus suis capsular type 2 strain 89-1591 has been controversial in literature. A standardized experimental model with specific-pathogen free piglets was used for a new evaluation of this strain. Twenty-nine piglets were allotted in 4 separated groups. Group 1 consisted of negative control animals which received broth medium. Groups 2, 3, and 4 were intravenously challenged with 2 mL of S. suis, strains 1330, 89-1591, and 166', respectively. The strain 1330 is a recognized avirulent Canadian strain. The strain 166' is a reference French virulent isolate. Pigs inoculated with strain 1330 did not present clinical signs of a S. suis infection. Contamination in organs and bacterial blood circulation were rare and lesions were almost non-existent. Infection of pigs with S. suis strain 89-1591 (group 3) and 166' (group 4) caused severe clinical problems, animals infected with S. suis 166' were the most affected. Pigs presented with clinical signs such as high body temperature, lameness, nervous symptoms, and even mortality. Lesions associated with S. suis were numerous for both strains, but more evident in animals of group 4. It can be concluded that S. suis strain 89-1591 is virulent, although its virulence seems to be lower than that of the French strain. Results of an experimental infection with strain 89-1591 may depend on different factors such as the route of inoculation and the immunological status of the animals used. Using conventional animals, with an unknown status regarding previous S. suis infections, equivocal results may be obtained, and this may explain differences reported by some authors with the same strain.

Identification of genes involved in biosynthesis of Actinobacillus pleuropneumoniae serotype 1 O-antigen and biological properties of rough mutants.

Actinobacillus pleuropneumoniae is an important pathogen of swine. Lipopolysaccharide (LPS) has been identified as the major adhesin of A. pleuropneumoniae and it is involved in adherence to porcine respiratory tract cells. We previously generated seven rough LPS mutants of A. pleuropneumoniae serotype 1 by using a mini-Tn10 transposon mutagenesis system [Rioux S, Galarneau C, Harel J et al. Isolation and characterization of mini-Tn10 lipopolysaccharide mutants of Actinobacillus pleuropneumoniae serotype 1. Can J Microbiol 1999; 45: 1017-1026]. The purpose of the present study was to characterize these mutants in order to learn more about LPS O-antigen biosynthesis genes and their organization in A. pleuropneumoniae, and to determine the surface properties and virulence in pigs of these isogenic mutants. By mini-Tn10 insertions in rough mutants, four putative genes (ORF12, ORF16, ORF17, and ORF18) involved in O-antigen biosynthesis in A. pleuropneumoniae serotype 1 were found within a region of 18 ORFs. This region is homologous to the gene cluster of serotype-specific O-polysaccharide biosynthesis from A. actinomycetemcomitans strain Y4 (serotype b). Two mutants showed homology to a protein with identity to glycosyltransferases (ORF12); two others had the mini-Tn10 insertion localized in genes encoding for two distinct proteins with identity to rhamnosyltransferases (ORF16 and ORF17) and three showed homology to a protein which is known to initiate polysaccharide synthesis (ORF18). These four ORFs were also present in A. pleuropneumoniae serotypes 9 and 11 that express an O-antigen that serologically cross-reacts with serotype 1. Evaluation of some biological properties of rough mutants seems to indicate that the absence of O-chains does not appear to have an influence on the virulence of the bacteria in pigs and on the overall surface hydrophobicity, charge and hemoglobin-binding activity, or on LAL activation. An acapsular mutant was included in the present study in order to compare the influence of O-chains and capsule polysaccharides on different cell surface properties. Our data suggest that capsular polysaccharides and not O-chains polysaccharides have a major influence on surface properties of A. pleuropneumoniae serotype 1 and its virulence in pigs.

[Streptococcus suis bacteremia]. / Bactériémie à Streptococcus suis.

INTRODUCTION: Streptococcus suis infection is recognised despite it rareness as a zoonotic occupational disease in humans, and is often associated with meningitis, more rarely with bacteremia. OBSERVATION: A Streptococcus suis bacteremia occurred in a hunter and was complicated by septic shock with disseminated intravascular coagulation, rhabdomyolysis and purpura fulminans. Contamination had occurred through inoculation of a cut on the thumb when the hunter was slaughtering a wild boar. The blood cultures isolated Streptococcus suis. The patient died 36 hours after admission, despite intensive care and adapted antibiotic treatment with penicillin A and macrolide. CONCLUSION: Streptococcus suis bacteremia are uncommon but serious in humans. Despite adapted treatment, evolution may be fatal, so their conditions of occurrence must be well known by hunters and practitioners.

Development of two real-time polymerase chain reaction assays to detect Actinobacillus pleuropneumoniae serovars 1-9-11 and serovar 2.

Two real-time, or quantitative, polymerase chain reaction (qPCR) assays were developed to detect Actinobacillus pleuropneumoniae serovars 1-9-11 (highly related serovars with similar virulence potential) and serovar 2, respectively. The specificity of these assays was verified on a collection of 294 strains, which included all 16 reference A. pleuropneumoniae strains (including serovars 5a and 5b), 263 A. pleuropneumoniae field strains isolated between 1992 and 2009 in different countries, and 15 bacterial strains other than A. pleuropneumoniae. The detection levels of both qPCR tests were evaluated using 10-fold dilutions of chromosomal DNA from reference strains of A. pleuropneumoniae serovars 1 and 2, and the detection limit for both assays was 50 fg per assay. The analytical sensitivities of the qPCR tests were also estimated by using pure cultures and tonsils experimentally spiked with A. pleuropneumoniae. The detection threshold was 2.5 × 10(4) colony forming units (CFU)/ml and 2.9 × 10(5) CFU/0.1 g of tonsil, respectively, for both assays. These specific and sensitive tests can be used for the serotyping of A. pleuropneumoniae in diagnostic laboratories to control porcine pleuropneumonia.

Plasmid-borne florfenicol and ceftiofur resistance encoded by the floR and blaCMY-2 genes in Escherichia coli isolates from diseased cattle in France.

This study was designed to determine the genetic basis of florfenicol and ceftiofur resistance in Escherichia coli isolates recovered from French cattle. In these isolates, ceftiofur resistance was conferred by bla(CMY-2) located on three distinct conjugative plasmids on a specific DNA fragment, ISEcp1-bla(CMY-2)-blc- sugE. Two of the plasmids also carried the floR gene conferring resistance to florfenicol. The floR gene was shown to be associated with the insertion sequence ISCR2. Mobile elements appear to contribute to the mobilization of floR and bla(CMY-2) genes in E. coli. The presence of bla(CMY-2) and floR on the same plasmid highlights the potential risk for a co-selection of the bla(CMY-2) gene through the use of florfenicol in food animal production.

Potential use of an unencapsulated and aromatic amino acid-auxotrophic Streptococcus suis mutant as a live attenuated vaccine in swine.

Streptococcus suis is responsible for severe economic losses to the swine industry. Prevention of the diseases caused by this pathogen is hampered by the inability of available vaccines to generate a protective response in pigs. A non-virulent, aromatic amino acid-auxotrophic and unencapsulated mutant strain of S. suis was generated in this study and a preliminary evaluation of its protective capacities was conducted in swine. Deletion of the cognate promoter of the aro operon in S. suis virulent strain S735 resulted in the abolishment of the expression of the four members of the operon, aroA, aroK, pheA and orf10. The resulting mutant strain BD101 was auxotrophic for aromatic amino acids as demonstrated by its failure to grow in a chemically defined medium unless it was supplemented with these compounds. In addition, as a result of the deletion of the cognate promoter of the aro operon, mutant BD101 lost its encapsulated phenotype. A protection assay was performed by immunisation of pigs with live strain BD101. Vaccination resulted in minor clinical signs but did not substantially impair the growth of vaccinated animals. Immunisation of animals with live mutant BD101 induced a considerable antibody response against S. suis. Vaccinated pigs presented only minor clinical signs and a survival rate of 100%, while 57% of non-vaccinated animals died, after a challenge with the virulent parent strain S735. In order to prevent S. suis infections in swine, it may be useful to further evaluate strain BD101 as a vaccine candidate.

Fhua and HgbA, outer membrane proteins of Actinobacillus pleuropneumoniae: their role as virulence determinants.

For the recently described serotype 15 of biotype I and serotypes 13 and 14 of biotype II of Actinobacillus pleuropneumoniae, fhuA and hgbA were detected by polymerase chain reaction and DNA sequencing. To determine the substrate specificity of the iron receptors FhuA and HgbA and to study their role in the virulence of A. pleuropneumoniae, we used two isogenic A. pleuropneumoniae serotype 1 deletion mutants of fhuA and hgbA. Different sources of iron and siderophores were tested in growth promotion assays. FhuA and HgbA are specific for their ligands ferrichrome and hemoglobin, respectively. The virulence of the two deletion mutant strains was evaluated in experimental infections using specific pathogen-free piglets. While the fhuA mutant (DG02) was as highly virulent as the parental strain S4074, the virulence of the hgbA mutant (DeltahgbA) was reduced. Our data indicate that both FhuA and HgbA are conserved among all serotypes and biotypes of A. pleuropneumoniae and that HgbA, the receptor for porcine hemoglobin, may play a role in virulence.

Experimental evidence of indirect transmission of Mycoplasma synoviae.

The aim of the study was to analyse experimental transmission of Mycoplasma synoviae, an avian pathogen. Three experiments using specific pathogen-free day-old chicks placed in isolators were conducted. In the first experiment, the birds were introduced in an isolator previously contaminated with a M. synoviae broth culture. After 34 days, these birds were eliminated and, for the second trial, the chicks were introduced in the same isolator without disinfecting. In the third assay, the chicks were placed in an isolator containing a mixture of food, feathers and dust collected less than an hour earlier from a M. synoviae infected laying hen flock. In the second and third experiments in order to exacerbate the M. synoviae infection, the birds were inoculated with infectious bronchitis (IB) virus. The presence of M. synoviae in the environment and in tracheal swabs was monitored by culture, a multiplex PCR (mPCR) detecting M. synoviae and Mycoplasma 16S rDNA and a multiplex RT-PCR (mRT-PCR) detecting the M. synoviae mRNA coding for a membrane protein and Mycoplasma 16S rRNA. In in vitro experimental conditions, M. synoviae mRNA and 16S rRNA were detected up to 20 min and 23 h respectively after mycoplasma death. In the first assay, the first infected bird was detected on the 13th day. In the second trial, culturable M. synoviae or viable M. synoviae were detected in the isolator for 3 or 4 to 5 days respectively after depopulation of the birds of the first assay whereas the first culture positive tracheal swabs were detected on the 33rd day, after IB inoculation. In the third experiment, the first infected birds were detected on the 54th day. Thus, the different assays showed that M. synoviae contaminated material (dust, feathers and food) can infect chicks, sometimes after remarkably long silent periods.

Truncation of the lipopolysaccharide outer core affects susceptibility to antimicrobial peptides and virulence of Actinobacillus pleuropneumoniae serotype 1.

We reported previously that the core oligosaccharide region of the lipopolysaccharide (LPS) is essential for optimal adhesion of Actinobacillus pleuropneumoniae, an important swine pathogen, to respiratory tract cells. Rough LPS and core LPS mutants of A. pleuropneumoniae serotype 1 were generated by using a mini-Tn10 transposon mutagenesis system. Here we performed a structural analysis of the oligosaccharide region of three core LPS mutants that still produce the same O-antigen by using methylation analyses and mass spectrometry. We also performed a kinetic study of proinflammatory cytokines production such as interleukin (IL)-6, tumor necrosis factor-alpha, IL1-beta, MCP-1, and IL8 by LPS-stimulated porcine alveolar macrophages, which showed that purified LPS of the parent strain, the rough LPS and core LPS mutants, had the same ability to stimulate the production of cytokines. Most interestingly, an in vitro susceptibility test of these LPS mutants to antimicrobial peptides showed that the three core LPS mutants were more susceptible to cationic peptides than both the rough LPS mutant and the wild type parent strain. Furthermore, experimental pig infections with these mutants revealed that the galactose (Gal I) and d,d-heptose (Hep IV) residues present in the outer core of A. pleuropneumoniae serotype 1 LPS are important for adhesion and overall virulence in the natural host, whereas deletion of the terminal GalNAc-Gal II disaccharide had no effect. Our data suggest that an intact core-lipid A region is required for optimal protection of A. pleuropneumoniae against cationic peptides and that deletion of specific residues in the outer LPS core results in the attenuation of the virulence of A. pleuropneumoniae serotype 1.

Genetic diversity of Streptococcus suis strains isolated from pigs and humans as revealed by pulsed-field gel electrophoresis.

The genetic diversity of 123 Streptococcus suis strains of capsular types 2, 1/2, 3, 7, and 9, isolated from pigs in France and from humans in different countries, was evaluated by pulsed-field gel electrophoresis (PFGE) of DNA restricted with SmaI. The method was highly discriminative (D = 0.98), results were reproducible, and the PFGE analysis was easy to interpret. Among all S. suis strains, 74 PFGE patterns were shown. At 60% homology, three groups (A, B, and C) were identified, and at 69% homology, eight subgroups (a to h) were observed. Strains isolated from diseased pigs or from humans were statistically clustered in group B, especially in subgroup d. By contrast, S. suis strains isolated from clinically healthy pigs were preferentially included in subgroup b of group A. Relationships could be established between capsular types 1/2, 3, and 9 and groups A, e, and B, respectively. S. suis strains isolated from humans were homogeneous, and a very high level of association between these strains and four DNA patterns was observed. The PFGE used in this study is a very useful tool for evaluating the genetic diversity of S. suis strains, and it would be used for epidemiological investigations.

Evaluation and field validation of PCR tests for detection of Actinobacillus pleuropneumoniae in subclinically infected pigs.

Eight PCR tests were evaluated for their abilities to detect Actinobacillus pleuropneumoniae in swine tonsils. At first they were compared regarding their specificities by using A. pleuropneumoniae and related bacterial species and their analytical sensitivities by using tonsils experimentally infected in vitro. PCRs were carried out both directly with tonsil homogenates (direct PCR) and after culture of the sample (after-culture PCR). Most tests demonstrated good specificities; however, some tests gave false-positive results with some non-A. pleuropneumoniae species. High degrees of variation in the analytical sensitivities among the tests were observed for the direct PCRs (10(9) to 10(2) CFU/g of tonsil), whereas those of most of the after-culture PCRs were similar (10(2) CFU/g of tonsil). In a second phase, the effects of sample storage time and storage conditions were evaluated by using tonsils from experimentally infected animals. Storage at -20 degrees C allowed the detection of the organism for at least 4 months. Finally, the omlA PCR test described by Savoye et al. (C. Savoye et al., Vet. Microbiol. 73:337-347, 2000) and the commercially available Adiavet App PCR test were further validated with field samples. Their effectiveness was compared to those of standard and immunomagnetic separation-based methods of bacterial isolation. In addition, a comparison of tonsil biopsy specimens (from living animals) and whole tonsils (collected at the slaughterhouse) was also conducted. A. pleuropneumoniae was neither isolated nor detected by PCR from a herd serologically negative for A. pleuropneumoniae. PCR was more sensitive than the standard isolation method with whole tonsils from three infected herds. After-culture PCR offered the highest degree of sensitivity (93 and 83% for the omlA and Adiavet App PCRs, respectively). The PCR detection rate was higher with whole tonsils than with tonsil biopsy specimens. Good agreement (kappa = 0.65) was found between the presence of A. pleuropneumoniae in tonsils and the individual serological status of the animals.

Streptococcus suis serotype 2 mutants deficient in capsular expression.

Streptococcus suis serotype 2 is responsible for a wide variety of porcine infections. In addition, it is considered a zoonotic agent. Knowledge about the virulence factors for this bacterium is limited but its polysaccharide capsule is thought to be one of the most important. Transposon mutagenesis with the self-conjugative transposon Tn916 was used to obtain acapsular mutants from the virulent S. suis type 2 reference strain S735. Clones were screened by colony-dot ELISA with a monoclonal antibody specific for a type 2 capsular epitope and clones that failed to react with the antibody were characterized. Two mutants, 2A and 79, having one and two Tn916 insertions respectively, were chosen for further characterization. Absence of capsule was confirmed by coagglutination, capillary precipitation and capsular reaction tests and by transmission electron microscopy. Absence of capsular polysaccharides correlated with increased hydrophobicity and phagocytosis by both murine macrophages and porcine monocytes compared to the wild-type strain. Furthermore, both mutants were shown to be avirulent in murine and pig models of infection. Finally, mutant 2A was readily eliminated from circulation in mice compared to the wild-type strain, which persisted more than 48 h in blood. Thus, isogenic mutants defective in capsule production demonstrate the importance of capsular polysaccharides as a virulence factor for S. suis type 2.

Pathophysiological changes occurring during Escherichia coli endotoxin and Pasteurella multocida challenge in piglets: relationship with cough and temperature and predicitive value for intensity of lesions.

The aims of this study were (1) to correlate cough and body temperature (BT) with the severity of bronchopneumonia in pigs, (2) to determine whether these clinical signs can be used to early diagnose bronchopneumonia and (3) to assess the predictive values of cough and BT regarding lung lesions. Bronchopneumonia was induced by administering E. coli endotoxin (LPS) combined with Pasteurella multocida type A (PmA) in the trachea of 13 piglets. Saline-instilled negative controls (n = 8), PmA inoculated (n = 6) and LPS instilled (n = 5) groups were also constituted. Cough and BT were recorded daily while the bronchopneumonia severity was assessed using bronchoalveolar lavage fluid (BALF) cytology, cytokines and measurement of lung lesion volume. Changes in expiratory breathing pattern were also measured (Penh). The combination of LPS and PmA induced a subacute bronchopneumonia characterised by macrophage, neutrophil, and lymphocyte infiltration, changes in Penh and an increase in the mRNA level of IFN-gamma while IL8, IL-18 and TNF-alpha mRNA levels remained unchanged. The daily body weight gain of infected animals was significantly reduced. Cough and BT changes were proportional to the intensity of the lung inflammatory process, functional respiratory changes and to the extent of macroscopic lesions. When comparing the individual values of cough and BT to thresholds defined for both parameters, an early diagnosis of pneumonia was possible. Considering the pooled data of each group, it was possible to define thresholds allowing an early segregation between the groups of diseased and healthy piglets. The daily values of cough and BT were predictive for the volume of lung lesions recorded at the end of the trial. In conclusion, cough and BT appear as potential indicators for the intensity and the evolution of the respiratory disease. They also seem to be good predictors for the magnitude of lung lesions and weight gain recorded at the study endpoint.