Øystein Angen

John Thomsen

Lars Erik Larsen

Jesper Larsen

Branko Kokotovic

Peter M H Heegaard

Jörg M D Enemark

Respiratory disease in calves: Microbiological investigations on trans-tracheally aspirated bronchoalveolar fluid and acute phase protein response

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

shown to be specific for the two species M. haemolytica and M. glucosida. All 90 aspirations were also analyzed for bovine respiratory syncytial virus (BRSV), parainfluenza-3 virus, and bovine corona virus by antigen ELISA. Surprisingly, 63% of the apparently healthy calves harbored potentially pathogenic bacteria in the lower respiratory tract, 60% of these samples contained either pure cultures or many pathogenic bacteria in mixed culture. Among diseased calves, all samples showed growth of pathogenic bacteria in the lower respiratory tract. All of these were classified as pure culture or many pathogenic bacteria in mixed culture. A higher percentage of the samples were positive for all bacterial species in the group of diseased animals compared to the clinically healthy animals, however this difference was only significant for M. dispar and M. bovirhinis. M. bovis was not detected in any of the samples. BRSV was detected in diseased calves in two herds but not in the clinically healthy animals. Among the diseased calves in these two herds a significant increase in haptoglobin and serum amyloid A levels was observed compared to the healthy calves. The results indicate, that haptoglobin might be the best choice for detecting disease under field conditions. For H. somni and M. haemolytica, a higher percentage of the samples were found positive by PCR than by cultivation, whereas the opposite result was found for P. multocida.

Detection of P. multocida by PCR or cultivation was found to be significantly associated with the disease status of the calves. For H. somni a similar association with disease status was only observed for cultivation and not for PCR.

Introduction

Respiratory disease in calves causes great economic losses for the dairy and beef industry worldwide (Snowder et al., 2006). [START_REF] Blom | The influence of housing and climatisation on health and growth of young calves under farm conditions[END_REF] reported that the annual calf mortality between day 2 and 180 was 7% in Danish dairy herds. He reported that on average 4.5% of the calves died due to respiratory disease. A mortality rate in the range 1.5-4.2% has also been reported from other countries [START_REF] Ames | Dairy calf pneumonia. The disease and its impact[END_REF][START_REF] Andrews | Calf pneumonia costs! Cattle pract[END_REF].

In Denmark, bovine respiratory syncytial virus (BRSV) and coronavirus are the most common viral agents found in relation to calf pneumonia [START_REF] Larsen | Diagnosis of enzootic pneumonia in Danish cattle: Reverse transcription -polymerase chain reaction assay for detection of bovine respiratory syncytial virus in naturally and experimentally infected cattle[END_REF]. Histophilus somni, Pasteurella multocida, Mannheimia haemolytica, and Arcanobacterium pyogenes are the bacteria most commonly isolated from calf pneumonia [START_REF] Tegtmeier | Pathological and microbiological studies on pneumonic lungs from Danish calves[END_REF]. Several Mycoplasma species have been isolated in Denmark from bovine lungs. [START_REF] Friis | Isolation of mycoplasmas from Danish cattle[END_REF] found M. dispar, M. bovirhinis, and Ureaplasma spp. to be the most prevalent of these, whereas M. bovis and M. bovigenitalium were isolated infrequently.

The aim of the present investigation was to study the presence and interaction of bacteria and virus in the lower respiratory tract in calves with and without clinical symptoms of respiratory disease from six Danish herds. In addition, the serum concentrations of haptoglobin and serum amyloid A (SAA) were determined to describe the correlation between presence of infectious agents and acute phase protein response. In order to avoid contamination from bacteria resident in the upper respiratory tract, the sampling was performed by trans-tracheal aspiration. This method has been recommended as optimal for evaluation of the microbiological status of the lower respiratory tract in order to elucidate the etiology of pneumonia in an animal [START_REF] Espinasse | Use of transtracheal aspiration to identify pathogens in pneumonic calves[END_REF]Rebhun, 1995;Virtala et al., 1996;[START_REF] Pommier | Use of transtracheal aspiration to identify the pulmonary bacterial flora of pneumonic feedlot calves: results from 126 samples[END_REF][START_REF] Pommier | Transtracheal aspiration of bronchial secretions in cattle[END_REF]. Finally, an aim was to evaluate whether PCR tests are suitable for obtaining a reliable and quick diagnosis of pneumonia related to bacterial pathogens.

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

Materials and methods

Herds and samples

The investigation included six dairy herds, which in previous years had experienced problems with calf pneumonia during the winter period. In September 2002, 56 trans-tracheally aspirated samples were taken from clinically healthy animals in these herds (herds 1, 3, 4, and 6: 10 samples; herd 2: 9 samples; herd 5: 7 samples). In November, 34 samples from calves suffering from respiratory distress were taken from 4 of the herds (herd 1: 10 samples; herd 3: 13 samples; herd 5: 6 samples; herd 6: 5 samples). No animals were treated by antibiotics at the time of sampling. In two herds (herds 2 and 4), respiratory disease was not observed and, consequently, no samples were taken. In total, 90 trans-tracheal aspirations were obtained.

The first samples were taken during a warm and dry September from clinically healthy calves (rectal temperatures below 39.5C, no nasal discharge, no coughing, and an unprovoked respiration frequency lower than 40 per min). All calves sampled in November showed clinical symptoms of pneumonic disease. Clinical disease was defined as a rectal temperature above 39.5C in connection with nasal discharge, coughing, or an unprovoked respiration frequency higher than 40 per min. The age of the calves ranged from 14 days to 4 months. The average age of the clinically healthy calves sampled in September was 2.1 months (SD=1.1) and for the pneumonic calves sampled in November 2.8 months (SD=0.9). Only four of the calves were sampled on both occasions (one calf in each of herd 3 and 5, and two calves in herd 6).

Blood samples were taken from all clinically healthy calves in connection with the first visit in September. In addition, paired blood samples (3 weeks interval) were taken in November-December after onset of clinical disease in the 4 herds where pneumonia was observed.

An area of 3x3 cm located 7 to 10 cm distal to the larynx was shaved and decontaminated with 70% alcohol and iodophors. The calves were sedated by intramuscular A c c e p t e d M a n u s c r i p t 5 injection of 0.1-0.2 mg/kg xylasine. After injection of local analgesic (0.5 ml 2% lidocain), a longitudinal incision of 1 cm was made in the midline directly above the trachea. Perforation of trachea was done with an Intraflon ® 12G between 2 cartilage rings. A male dog urinary catheter was inserted into the Intraflon ® and pushed down into the airway until a slight resistance was felt.

Between 20 and 40 ml sterile 0.9% NaCl were injected through the catheter and followed by immediate aspiration. This resulted normally in 5 -7 ml aspirated fluid.

One ml aspirated fluid was centrifuged for 3 min at 16.000 g. The supernatant was removed and a loopful (approx. 10l) of the sedimented material was used for bacterial cultivation.

The growth of the different bacterial species was recorded on a semi-quantitative scale (pure culture, many bacteria in mixed culture, mixed culture, few bacteria in mixed culture, no growth).

Bacterial identification was done according to the standard procedures of the laboratory [START_REF] Tegtmeier | Pathological and microbiological studies on pneumonic lungs from Danish calves[END_REF]. For M. haemolytica, the identification was based on the methods given by [START_REF] Angen | Phenotypic and genotypic characterization of Mannheimia (Pasteurella) haemolytica-like strains isolated from diseased animal in Denmark[END_REF]. The identification of H. somni was performed by PCR as described by [START_REF] Angen | Development of a PCR test for identification of Haemophilus somnus in pure and mixed cultures[END_REF].

Cultivation and identification of Mycoplasma spp. were performed according to the procedures described by [START_REF] Friis | Isolation of mycoplasmas from Danish cattle[END_REF]. The samples had been stored at -20 ºC for 2 years before cultivation for mycoplasmas was initiated.

The collection of samples in the herds was performed in connection with herd diagnosis by a trained veterinary surgeon according to the Law for veterinarians in Denmark and is thereby accepted by the Danish Animal Experiments Inspectorate.

PCR detection

DNA was extracted by addition of 200 μl PrepMan™ Ultra (Applied Biosystems) to the sedimented trans-tracheal sample according to the manufacturer's recommendations. The DNA preparation was stored at -20C until used for PCR analysis. Species specific detection using previously published A c c e p t e d M a n u s c r i p t 6 methods was performed for H. somni [START_REF] Angen | Development of a PCR test for identification of Haemophilus somnus in pure and mixed cultures[END_REF], P. multocida [START_REF] Miflin | Development of a 23S rRNA-based PCR assay for the identification of Pasteurella multocida[END_REF], M. dispar and M. bovirhinis [START_REF] Miles | Rapid detection of Mycoplasma dispar and M. bovirhinis using allele specific polymerase chain reaction protocols[END_REF], and M. bovis [START_REF] Subramaniam | Species identification of Mycoplasma bovis and Mycoplasma agalactiae based on the urvC genes by PCR[END_REF] using 2 l of the extracted DNA per reaction.

For M. haemolytica, the lktA-artJ intergenic region was used for PCR amplification. From pure cultures, one bacterial colony was resuspended in 100 μl distilled water and lysed by boiling for 10 min. One μl sample from lysed bacteria or 2 l of DNA extracted by PrepMan™ Ultra was added to respectively 49 l and 48 μl prepared reaction mixture containing 10 mM Tris/HCl pH 8.3, 50mM KCl, 2.5mM MgCl 2 , 100 μM of each dNTP, 65 pmol of each primer (forward primer: GTCCCTGTGTTTTCATTATAAG; reverse primer: CACTCGATAATTATTCTAAATTAG), and 0.5 U of Taq polymerase (Applied Biosystems, Foster City, CA, USA). The reaction mixture was covered with 50 μl of paraffin oil. Samples were subjected to an initial denaturation step at 94 C for 3 min followed by 35 cycles of denaturation at 94 C for 1 min, annealing at 58 C for 1 min, and extension at 72 C for 1 min in a thermal cycler.

Samples of PCR amplification products (10 μl) were subjected to electrophoresis in a 1.2% agarose gel in Tris/borate buffer according to standard protocols. DNA was visualized by UV fluorescence after staining with ethidium bromide. The PCR test was expected to give a specific amplicon of 385 bp. For evaluation of the species specificity of this test, see supplementary material.

Detection of viral antigens and antibodies to BRSV

The transtracheal aspirations were investigated for the presence of antigens from bovine respiratory syncytial virus (BRSV), parainfluenza-3 virus (PI-3) and corona virus using the ELISA tests described by Uttenthal et al. (1996). Blood samples from diseased calves were tested for IgG 1 antibodies against BRSV according to Uttenthal et al. (2000).

Measurement of acute phase proteins

Serum haptoglobin and serum amyloid A (SAA) were determined as earlier described [START_REF] Heegaard | The acute phase response of haptoglobin and serum amylod A (SAA) in cattle undergoing experimental infection with bovine respiratory syncytial virus[END_REF]. For these analyses, only the sera from diseased calves in herds 1, 3, and 5 were available in addition to the sera from clinically healthy calves in all herds.

Statistical methods

Differences in frequencies were evaluated using least-squares test statistics and the relationship between disease status and test results by a logistic regression model evaluated with the chi-square test using the statistical software package S-Plus 6.1 for Windows (Profession Release 1; Insightful). Data from the acute phase protein measurements were analyzed using a two-sided t-test.

The similarity of the different populations was tested using the Mann-Whitney non-parametric test as some of the sample sets proved to be not normally distributed.

Results

Clinically healthy animals

Among the 56 clinically healthy calves, potentially pathogenic bacteria were detected either by cultivation or PCR from 68% of the animals (Table 1), this number varied from 29% to 90% between the herds (Table 2). Bacteria were cultivated from 33 samples (59%). From 18 of the calves (32%), H. somni, P. multocida, M. haemolytica, or A. pyogenes were isolated in high numbers in pure culture or as the dominating flora in a mixed culture. From three of the calves only few P. multocida in pure culture was cultivated. M. dispar and M. bovirhinis were detected by PCR from 21% and 14% of the samples, respectively. M. bovis was not detected. Forty-one percent of the samples tested positive in one or more of the PCR tests applied. By cultivation or PCR, P.

Diseased animals

Among the 34 diseased animals, potentially pathogenic bacteria were isolated in high numbers in pure culture or as the dominant flora in a mixed culture from all animals (Table 1). P. multocida, H. somni, and M. haemolytica were cultivated or detected by PCR in 82%, 41%, and 29% of the calves, respectively. A. pyogenes was isolated from 2 of the calves and Moraxella sp. from one calf.

M. dispar and M. bovirhinis were both detected from 79% of the calves. M. bovis was not detected.

From 33 of the samples (97%) two or more bacterial species were detected. A higher percentage of the samples were positive for all bacterial species investigated in the group of diseased animals compared to the clinically healthy animals, however this difference was statistical significant only for M. dispar and M. bovirhinis (p<0.01 for both).

Herd prevalences

Some differences between the herds in the prevalence of the different microorganisms were observed (Table 2). P. multocida, M. haemolytica, M. dispar and M. bovirhinis were found in all 6 herds. H. somni was found in all herds except for herd 3. Coronavirus antigen was only detected in herds 1 and 5. BRSV antigens were detected in 60% and 100% of the calves from herds 1 and 3, respectively (Table 2). In herds 5 and 6, no BRSV antigens were detected. The test of paired sera from these two herds showed that seroconversion to BRSV had not taken place. M. bovis and PI-3 were not detected in any of the herds.

A c c e p t e d M a n u s c r i p t 9

PCR and cultivation

For H. somni and M. haemolytica, a significantly higher number of samples were found positive by the PCR tests than by cultivation (p=0.04 and p=0.01, respectively). For P. multocida, a higher number of samples were found positive by cultivation than by PCR but the difference was not statistically significant (p=0.10).

There was a significant correlation between detection of H. somni by cultivation and pneumonia (p<0.01) but no significant correlation between presence of pneumonia and detection of H. somni by PCR (p=0.05). For P. multocida a significant correlation (p<0.01) was found between disease status and both cultivation and PCR, whereas there were no significant correlations observed for detection of M. haemolytica and disease status.

Cultivation for mycoplasma was performed on 10 samples representing each sampling event per herd. M. dispar was isolated from five of these samples and M. bovirhinis from two samples.

Acute phase proteins

Comparing the three diseased versus healthy calf populations within each of the herds 1, 3 and 5, statistically significant differences were found for haptoglobin as well as SAA concentrations for all herds with the clear exception of SAA in herd 5 where no difference was found (Fig. 1).

Haptoglobin concentrations also showed a less significant increase in herd 5 compared to herds 1 and 3. This increase was, however, significant for all 3 herds. The SAA concentrations of the healthy populations were generally closer to the values of the diseased calves than what was found for haptoglobin. Thus, for SAA both herd 3 and 5 diseased populations were not significantly different from the healthy population taken as a whole.

Discussion

In the present investigation, 68% of the clinically healthy calves were found to harbor potentially pathogenic bacteria in the lower respiratory tract. Only a limited number of reports have been published on the normal flora of the lower respiratory tract. Viso et al. (1982) sampled 23 healthy animals and found bacteria in 52% of the calves and 83% of these bacteria were identified as "Pasteurella sp.". [START_REF] Espinasse | Use of transtracheal aspiration to identify pathogens in pneumonic calves[END_REF] performed trans-tracheal aspiration on 49 healthy calves.

P. multocida and M. haemolytica were each found in 8 of these calves. Virtala et al. (1996) also performed trans-tracheal aspiration on 47 healthy calves and found 55% of these to harbor pathogenic bacteria, finding Mycoplasma spp. and P. multocida in 47% and 17% of the samples, respectively. [START_REF] Autio | Etiology of respiratory disease in non-vaccinated, non-medicated calves in rearing herds[END_REF] investigated a group of 144 healthy calves by tracheobronchial lavage using a double catheter and found pathogenic bacteria (excluding mycoplasmas), Mycoplasma spp., and pathogenic bacteria including mycoplasmas in 27%, 75%, and 83% of the samples, respectively. However, none of these investigations reported the quantity of bacteria isolated in the healthy animals. In the present investigation, a striking observation was that 32% of the healthy calves harbored bacteria in the lower respiratory tract in high numbers without showing clinical disease.

These observations underline the multi-factorial nature of calf pneumonia and supports earlier reports that bacteria seldom act as primary pathogens in relation to calf pneumonia [START_REF] Tegtmeier | Pathological and microbiological studies on pneumonic lungs from Danish calves[END_REF](Tegtmeier et al., , 2000a)). Both [START_REF] Baudet | A clinical study of the microbiology in respiratory diseases of young cattle[END_REF] and [START_REF] Babiuk | Viral-bacterial synergistic interaction in respiratory disease[END_REF] concluded that virus-infections precede 90% of all bacterial pneumonias. Samples from clinically diseased animals were only obtained from four of the six herds. Among the calves in herds 1 and 3, BRSV antigens could be detected in 83% of the diseased calves. In these herds, a viral infection is probably the primary cause for development of clinical [START_REF] Larsen | Diagnosis of enzootic pneumonia in Danish cattle: Reverse transcription -polymerase chain reaction assay for detection of bovine respiratory syncytial virus in naturally and experimentally infected cattle[END_REF]Uttenthal et al., 2000). On the other hand, no BRSV-antigens could be detected in herds 5 and 6 and the test of paired sera from these two herds showed that seroconversion to BRSV had not taken place.

Consequently, the etiology of pneumonic disease in these two herds was probably dependent on the presence of bacteria alone. However, as a number of different bacterial species were isolated from these herds and no autopsies were performed, firm conclusions about the etiology of the observed pneumonia cannot be drawn.

A general conclusion from the present study is that haptoglobin is the more sensitive indicator of disease in the herds investigated, while neither haptoglobin nor SAA levels were affected by the potentially harmful agents in the lower respiratory tract if animals were healthy.

SAA concentrations of the healthy calves were much closer to those of the diseased populations than what was found for haptoglobin. Of the three herds in which healthy and diseased populations were compared to each other, BRSV could be demonstrated in the diseased subpopulations of herds 1 and 3 but not in the diseased population of herd 5. SAA concentrations in herd 5 did not differ between the diseased and the normal subpopulation. This might indicate that SAA needs virus to be present in order to respond while haptoglobin is fully induced by bacterial infection alone. It has previously been shown that SAA seems to be a more sensitive marker for viral infections [START_REF] Heegaard | The acute phase response of haptoglobin and serum amylod A (SAA) in cattle undergoing experimental infection with bovine respiratory syncytial virus[END_REF] and acute inflammation (Alsemgeest et al., 1994;[START_REF] Horadagoda | Acute phase proteins in cattle: discrimination between acute and chronic inflammation[END_REF] compared to haptoglobin. In experimental bacterial infections (intratracheal inoculation of Mannheimia haemolytica), SAA was found to be more rapidly induced than haptoglobin [START_REF] Horadagoda | Immediate responses in serum TNF alpha and acute phase protein concentrations to infection with Pasteurella haemolytica A1 in calves[END_REF]. The results presented here show that even if SAA is more sensitive and rapidly reacting, haptoglobin might be preferable in the field, its bigger and more prolonged response giving rise to its higher sensitivity in detecting disease. In the present investigation, a higher percentage of the samples were found positive for P. multocida, M. dispar, M. bovirhinis and H. somni in the group of diseased animals compared to the clinically healthy animals (Table 1), this increase was however not statistically significant. In herds 3, 5, and 6, P. multocida was found in all diseased animals, indicating a possible role for this organism in the development of pneumonia. Autrio et al. (2007) also reported an association between P. multocida and clinical respiratory disease among calves in Finland, provided P. multocida was found together with other bacterial pathogens. This supports the common opinion that P. multocida should be regarded as an opportunistic pathogen [START_REF] Maheswaran | Current knowledge on the pathogenesis of lung injury caused by Mannheimia haemolytica and Pasteurella multocida in the bovine[END_REF]. On the other hand, [START_REF] Nikunen | Association of bovine respiratory disease with clinical status and acute phase proteins in calves[END_REF] in another Finnish study found strong indications for P. multocida having a pathogenic role, provided other known pathogens were absent. Virtala et al. (1996) found a significant association between clinical disease and increased isolation rate of P. multocida and Mycoplasma spp., alone or in combination.

In the present investigation we found a significantly higher detection rate of M. dispar and M. bovirhinis among clinically diseased calves although a high proportion of the healthy calves also harbored these organisms. On the other hand, in two Finnish studies, no association between clinical disease and the presence of Mycoplasma spp. was observed [START_REF] Autio | Etiology of respiratory disease in non-vaccinated, non-medicated calves in rearing herds[END_REF][START_REF] Nikunen | Association of bovine respiratory disease with clinical status and acute phase proteins in calves[END_REF].

M. bovis is in many countries regarded as one of the major causes of respiratory disease in cattle with reports of increasing prevalence (Nicolas and Ayling, 2003). M. bovis was not found in any of the 6 herds in the present investigation. Earlier studies in Denmark have only found a low prevalence of this organism [START_REF] Friis | Isolation of mycoplasmas from Danish cattle[END_REF][START_REF] Feenstra | A field study of Mycoplasma bovis infection in cattle[END_REF], so apparently M. bovis is still of low importance in connection with pneumonic disease in Denmark.

Cultivation of Mycoplasma spp. was attempted from ten PCR-positive samples.

However, due to the long storage time before analysis, isolation of Mycoplasma spp. was only For H. somni and M. haemolytica, a significantly higher number of samples were found positive by the PCR tests than by cultivation. For P. multocida, a higher number of samples were found positive by cultivation than by PCR, but the difference was not statistically significant.

The lower sensitivity of the P. multocida PCR compared to cultivation may be due to the large size of the amplicon produced by this method (1250 bp) compared to the H. somni and M. haemolytica PCRs (both approximately 400 bp). Detection of P. multocida by both cultivation and PCR was significantly associated with the disease status of the animal, whereas no such association was observed for M. haemolytica. A previous study on H. somni demonstrated a higher sensitivity of a species-specific PCR test (Tegtmeier et al., 2000b) compared to bacterial cultivation. However, the present investigation indicates that PCR nevertheless is less suited for prediction of H. somnirelated pneumonia compared to bacterial cultivation. These observations might be related to the fact that the use of a highly sensitive method such as PCR will also detect the presence of a very low number of bacteria that not necessarily have any correlation with disease. On the other hand, a higher diagnostic value of a PCR test could be obtained if real-time PCR tests were developed, whereby the infectious agents can be quantified and give a result which might better reflect the clinical status of the animal.

  aspirations from 56 apparently healthy calves and 34 calves with clinical signs of pneumonia were collected in six different herds during September and November 2002. The 90 samples were cultivated and investigated by PCR tests targeting the species Histophilus somni, Mannheimia haemolytica, Pasteurella multocida, Mycoplasma bovis, Mycoplasma dispar, and Mycoplasma bovirhinis. A PCR test amplifying the lktC-artJ intergenic region was evaluated and

  in 48%, H. somni in 20%, and M. haemolytica in 23% of the calves. A. pyogenes was isolated from 3 of the calves and Moraxella sp. from one calf. From 23 samples (41%) two or more bacterial species were detected. Only one of the samples contained virus antigens (corona virus).

  observations are in accordance with previous results, showing that BRSV plays an important role for development of calf pneumonia in Denmark

  of these samples. The cultivation nevertheless confirmed the presence of M. dispar and M. bovirhinis in these samples as found by PCR.

Figure legend

 legend Figure legend

  Figure 1

  Herd prevalences (%) of pathogenic microorganisms found in trans-tracheally aspirated bronchoalveolar fluid from clinically normal calves (N) and calves with pneumonia (P) from 6 Danish herds. disease not observed during study period. b : No seroconversion to BRSV detected in herds 5 and 6.

Table 1 .

 1 Bacteriological investigation of trans-tracheally aspirated bronchoalveolar fluid from clinically normal calves (n=56) and calves with pneumonia (n=34) from 6 Danish herds.

		Clinically normal calves	Diseased calves
			(% positive)			(% positive)
		Cultivation	Cultivation	PCR	Cultivation	Cultivation	PCR
		or PCR			or PCR
	Pasteurella multocida	48	43	32	82	82	74
	Histophilus somni	20	4	20	41	29	38
	Mannheimia haemolytica	23	16	23	29	6	29
	Arcanobacterium pyogenes	5	5	ND	6	6	ND
	Moraxella sp.	2	2	ND	3	3	ND
	Mycoplasma dispar	ND	ND	21	ND	ND	79
	Mycoplasma bovirhinis	ND	ND	14	ND	ND	79
	Mycoplasma bovis	ND	ND	0	ND	ND	0
	Total number of positive samples	68	59	41	100	100	97
	ND: not done
				1

Table 1

 1

Table 2

 2

Acknowledgements

The authors want to thank Birgitte Møller, Jannie Jensen, Tamara Plambeck, and Ivan Larsen for skilful technical assistance and Anders Stockmarr for help with the statistical analyses.


F Boyen

V Eeckhaut

F Van Immerseel

F Pasmans

R Ducatelle

F Haesebrouck

QUORUM SENSING IN VETERINARY PATHOGENS: MECHANISMS, CLINICAL IMPORTANCE AND FUTURE PERSPECTIVES

Keywords: quorum sensing, signal molecules, veterinary pathogens Manuscript

Under certain circumstances the individuals of a bacterial population may find advantages in acting together and making "collective decisions". This phenomenon is better known as quorum sensing. When the concentration of signal molecules produced by the surrounding bacteria exceeds a certain threshold, the bacterial population acts as a single organism, collectively expressing virulence genes, biofilm forming genes, etc. Several mechanisms of quorum sensing are discussed, each with its distinct signal molecules and respective receptors. Some of these mechanisms are restricted to sensing intraspecies signalling, but interspecies and even interkingdom signalling have also been described. Several veterinary pathogens such as Staphylococcus aureus, Staphylococcus pseudintermedius, Pseudomonas aeruginosa and Salmonella Typhimurium use quorum sensing as a means to optimize virulence gene expression and host colonization. Therefore, targeting of the QS mechanisms may provide a novel strategy for combating bacterial infections, also in veterinary medicine.

INTRODUCTION

The communication mechanism that enables bacteria to make 'collective decisions' is called 'quorum sensing'. For this purpose, small chemical signal molecules are synthesized and secreted into the environment. By using specific detection systems to sense the concentration of these signal molecules, the surrounding bacteria are aware of the bacterial cell density. When the signal molecule concentration reaches a critical threshold, the gene expression and, as a result, the behaviour of the total population is changed (Figure 1). Quorum sensing (QS) was first described in the 1970s, when Nealson and colleagues (1970) investigated the bioluminescence of Vibrio fischeri and Vibrio harveyi. Ever since, QS has been assigned a role in various mechanisms of gene regulation: biofilm formation [START_REF] Haesebrouck | Biofilms: Betekenis voor de behandeling en bestrijding van bacteriële infecties bij huisdieren[END_REF][START_REF] Irie | Quorum sensing and microbial biofilms[END_REF], virulence adaptation [START_REF] Salmond | The bacterial `enigma': cracking the code of cell-cell communication[END_REF][START_REF] Vendeville | Making 'sense' of metabolism: autoinducer-2, LuxS and pathogenic bacteria[END_REF], the production of antimicrobial substances [START_REF] Salmond | The bacterial `enigma': cracking the code of cell-cell communication[END_REF][START_REF] Kleerebezem | Quorum sensing control of lantibiotic production; nisin and subtilin autoregulate their own biosynthesis[END_REF], motility [START_REF] Bearson | The role of the QseC quorum-sensing sensor kinase in colonization and norepinephrine-enhanced motility of Salmonella enterica serovar Typhimurium[END_REF][START_REF] Atkinson | Functional interplay between the Yersinia pseudotuberculosis YpsRI and YtbRI quorum sensing systems modulates swimming motility by controlling expression of flhDC and fliA[END_REF], sporulation [START_REF] Lazazzera | Quorum sensing and starvation: signals for entry into stationary phase[END_REF], etc. Although these mechanisms have mostly been studied in nonpathogenic bacteria or in human pathogens, the last few years, more studies have been conducted focusing on bacterial species of veterinary importance.

In this manuscript, the different mechanisms of QS, some examples of the exploitation of QS by bacterial pathogens of veterinary importance, and possible applications in future veterinary medicine are reviewed.

Quorum sensing in Gram positive bacteria: autoinducing polypeptides

Gram positive bacteria mainly use post-translational modified oligopeptides as QS signal molecules [START_REF] Lyon | Peptide signaling in Staphylococcus aureus and other Grampositive bacteria[END_REF]. These autoinducing polypeptides (AIPs) are made up of 5 to 17 amino acids with side chain modifications consisting, for example, of isoprenyl groups (Bacillus spp.) or thiolactone rings (Staphylococcus spp.) [START_REF] Ji | Bacterial interference caused by autoinducing peptide variants[END_REF][START_REF] Lyon | Peptide signaling in Staphylococcus aureus and other Grampositive bacteria[END_REF]. The AIPs are synthesized in the cytoplasm as precursor peptides, and then cleaved, modified and transported to the extracellular environment [START_REF] Lyon | Peptide signaling in Staphylococcus aureus and other Grampositive bacteria[END_REF].

In the surroundings of the producing bacterial cell, the AIPs can be detected by membranebound two-component detection systems. Detection of the AIPs by the sensorial part of the two-component systems leads to phosphorylation and therefore activation of the response regulator protein [START_REF] Lyon | Peptide signaling in Staphylococcus aureus and other Grampositive bacteria[END_REF][START_REF] Waters | Quorum sensing: cell-to-cell communication in bacteria[END_REF]. This response protein interacts with the target promoter DNA and the expression of the QS regulated genes is switched on (Figure 2).

The structure of the signal peptide varies between different Gram positive species.

Since the binding efficiency on the receptors is highly dependent on the chemical structure of the AIPs, it is generally accepted that mainly Gram positive, but also some Gram negative bacteria use this system for intraspecies communication [START_REF] Goerke | Evaluation of intraspecies interference due to agr polymorphism in Staphylococcus aureus during infection and colonization[END_REF][START_REF] Dirix | Peptide signal molecules and bacteriocins in Gram-negative bacteria: a genome-wide in silico screening for peptides containing a double-glycine leader sequence and their cognate transporters[END_REF][START_REF] Novick | Quorum sensing in staphylococci[END_REF].

Quorum sensing in Gram negative bacteria: N-acylhomoserine lactones

In Gram negative bacteria, the signal molecules are N-acylhomoserine lactones (AHLs), which are also called autoinducer 1 (AI-1). They consist of a conserved homoserine lactone ring which is connected to an acyl side chain of variable length (4 to 18 carbon atoms) and variable extra modification. AHLs with a short acyl side chain can diffuse passively in and out the bacterial cell [START_REF] Moré | Enzymatic synthesis of a quorum-sensing autoinducer through use of defined substrates[END_REF][START_REF] Fuqua | Regulation of gene expression by cell-to-cell communication: acyl-homoserine lactone quorum sensing[END_REF]. This is in contrast with AHLs with a long acyl side chain and the AIPs, which need active transport mechanisms to cross the bacterial cell membrane [START_REF] Fuqua | Regulation of gene expression by cell-to-cell communication: acyl-homoserine lactone quorum sensing[END_REF][START_REF] Novick | Quorum sensing in staphylococci[END_REF].

LuxI homologous proteins play a major role in the synthesis of the AHLs, which increase in concentration as the cell density increases. At a critical concentration, LuxR homologues bind the signal molecules and subsequently regulate gene transcription [START_REF] Fuqua | Regulation of gene expression by cell-to-cell communication: acyl-homoserine lactone quorum sensing[END_REF][START_REF] Waters | Quorum sensing: cell-to-cell communication in bacteria[END_REF] (Figure 2). It is becoming more and more obvious, however, that there is a considerable diversity in the way in which the LuxI and LuxR homologues from different bacterial species operate [START_REF] Smith | Variations on a theme: diverse N-acyl homoserine lactone-mediated quorum sensing mechanisms in gramnegative bacteria[END_REF]. Since the LuxR type proteins only detect specific AHLs, this QS system is also mainly used for intraspecies communication. Nevertheless, LuxR type proteins that are able to detect several AHLs have been described. For example, SdiA of Salmonella is believed to be mainly involved in interspecies bacterial communication [START_REF] Walters | Quorum sensing in Escherichia coli and Salmonella[END_REF].

Quorum sensing in both Gram positive and Gram negative bacteria: autoinducer 2

In the late 1990s, a second QS system was discovered in various Gram negative bacteria [START_REF] Surette | Quorum sensing in Escherichia coli, Salmonella typhimurium, and Vibrio harveyi: a new family of genes responsible for autoinducer production[END_REF]. In this system, the autoinducer (AI-2) is a furanosyl borate ester that is formed by the LuxS protein [START_REF] Waters | Quorum sensing: cell-to-cell communication in bacteria[END_REF]. To date, AI-2 has been described in more than 50 different bacterial species, comprising both Gram positive and Gram negative bacteria [START_REF] Surette | Quorum sensing in Escherichia coli, Salmonella typhimurium, and Vibrio harveyi: a new family of genes responsible for autoinducer production[END_REF][START_REF] Schauder | The LuxS family of bacterial autoinducers: biosynthesis of a novel quorum-sensing signal molecule[END_REF][START_REF] Hardie | Establishing bacterial communities by 'word of mouth': LuxS and autoinducer 2 in biofilm development[END_REF]. In Vibrio harveyi, the receptor for the AI-2 molecules is the periplasmatic LuxP protein. The LuxP-AI-2 complex binds to a second protein, LuxQ, which is a two-component protein that contains both a sensor kinase domain and a response regulator domain [START_REF] Neiditch | Regulation of LuxPQ receptor activity by the quorum-sensing signal autoinducer-2[END_REF]. In low bacterial concentrations, and therefore in the absence of the AI-2, LuxQ phosphorylates LuxO with the aid of the LuxU intermediary protein, which in turn activates the transcription of a repressor protein blocking the transcription of the luciferase operon. At high bacterial densities, the AI-2 presence induces the phosphatase activity of LuxQ, which inactivates LuxO, resulting in the LuxR mediated transcription of the luciferase operon [START_REF] Neiditch | Regulation of LuxPQ receptor activity by the quorum-sensing signal autoinducer-2[END_REF] (Figure 2). The LuxR component of this system should not be confused with the LuxR protein of Vibrio fischeri (see above). The autoinducer AI-2 could serve as a 'universal signal' for interspecies communication [START_REF] Xavier | LuxS quorum sensing: more than just a numbers game[END_REF], though in most enteric bacteria the genes that are directly regulated by AI-2 code for an ABC transporter system and it has been suggested that the role of AI-2 is mainly metabolic [START_REF] Vendeville | Making 'sense' of metabolism: autoinducer-2, LuxS and pathogenic bacteria[END_REF][START_REF] Walters | Quorum sensing in Escherichia coli and Salmonella[END_REF][START_REF] Rezzonico | Lack of genomic evidence of AI-2 receptors suggests a nonquorum sensing role for luxS in most bacteria[END_REF].

Quorum sensing beyond bacterial borders: autoinducer 3

Enterohaemorrhagic Eschericia coli (EHEC) is the cause of various food-borne outbreaks of severe intestinal disease and the causative agent of the hemolytic uremic syndrome (HUS) and thrombotic thrombocytopenic purpura (TTP) in humans [START_REF] Moxley | Escherichia coli 0157:H7: an update on intestinal colonization and virulence mechanisms[END_REF][START_REF] Karch | Enterohaemorrhagic Escherichia coli in human medicine[END_REF]. EHEC motility and attaching and effacing virulence genes are regulated by a QS mechanism [START_REF] Moxley | Escherichia coli 0157:H7: an update on intestinal colonization and virulence mechanisms[END_REF]) that involves the poorly characterized auto inducer 3 (AI-3), which is produced by several enteric bacterial species [START_REF] Kendall | Quorum sensing by enteric pathogens[END_REF]. Since epinephrine/norepinephrine induce the same virulence gene expression in EHEC and since the effects of AI-3 can be inhibited by adrenergic receptor antagonists, AI-3 probably is structurally similar to epinephrine/norepinephrine (Sperandio et al., 2003). In addition, the presence of epinephrine/norepinephrine itself may serve as a QS signal. Apart from the EHEC QseE/F two-component system, QseB/C has been identified as the receptor/response regulator of both AI-3 and epinephrine/norepinephrine in a number of bacterial species (Clarke et al., 2006;[START_REF] Walters | Quorum sensing in Escherichia coli and Salmonella[END_REF] (Figure 2). Therefore, this adrenergic receptor is thought to play a role in interkingdom signaling [START_REF] Kendall | Quorum sensing by enteric pathogens[END_REF].

CLINICAL SIGNIFICANCE OF QUORUM SENSING IN VETERINARY PATHOGENS

Staphylococcus aureus

The agr (accessory gene regulator) QS system in Staphylococcus aureus (S. aureus) is one of the most studied QS mechanisms in Gram positive bacteria. Using sequence analysis, S. aureus can be divided into 4 different agr specific groups. The AIPs of one agr group inhibit the expression of the agr expression of the other groups [START_REF] Novick | Autoinduction and signal transduction in the regulation of staphylococcal virulence[END_REF]. Several haemolysins, enterotoxins, exfoliating toxins, enzymes and surface proteins are regulated by agr. Therefore agr has been assigned various functions in the pathogenesis of S. aureus infections in several animal species.

In rabbits, S. aureus infections lead to skin infections, mastitis and internal abscessation. Low virulent strains may occasionally infect an individual animal, while highly virulent strains are the cause of chronic problems and they spread rapidly throughout the rabbitry [START_REF] Vancraeynest | International dissemination of a high virulence rabbit Staphylococcus aureus clone[END_REF]. It has been shown that highly virulent strains are clonal and grouped into agr type IV [START_REF] Vancraeynest | International dissemination of a high virulence rabbit Staphylococcus aureus clone[END_REF]. In addition, it has been shown that agr deletion mutants are attenuated in their capacity to induce endocarditis, osteomyelitis and endophtalmitis in rabbits [START_REF] Cheung | Diminished virulence of a sar-/agr-mutant of Staphylococcus aureus in the rabbit model of endocarditis[END_REF][START_REF] Gillaspy | Role of the accessory gene regulator (agr) in pathogenesis of staphylococcal osteomyelitis[END_REF][START_REF] Booth | Accessory gene regulator controls Staphylococcus aureus virulence in endophthalmitis[END_REF].

S. aureus is worldwide the most frequently isolated bacterium from bovine intramammary infections. In contrast to highly virulent rabbit associated S. aureus isolates, strains from bovine mastitis cases are predominantly classified in agr group I and to a lesser extent in agr groups II and III [START_REF] Buzzola | Differential abilities of capsulated and noncapsulated Staphylococcus aureus isolates from diverse agr groups to invade mammary epithelial cells[END_REF][START_REF] Vautor | Genetic differences among Staphylococcus aureus isolates from dairy ruminant species: A single-dye DNA microarray approach[END_REF]. Bovine S. aureus strains belonging to agr group I show significantly increased capacities to be internalized in bovine mammary epithelial cells and persist in higher numbers in murine mammary glands [START_REF] Buzzola | Differential abilities of capsulated and noncapsulated Staphylococcus aureus isolates from diverse agr groups to invade mammary epithelial cells[END_REF], while agr group II strains might prefer the extracellular niche and therefore might be more dependent on biofilm formation [START_REF] Melchior | Biofilm formation and genotyping of Staphylococcus aureus bovine mastitis isolates: evidence for lack of penicillinresistance in Agr-type II strains[END_REF]. In addition, it has been suggested that penicillin resistance in bovine S. aureus strains is correlated to the presence of agr group I genes [START_REF] Melchior | Biofilm formation and genotyping of Staphylococcus aureus bovine mastitis isolates: evidence for lack of penicillinresistance in Agr-type II strains[END_REF]. In contrast, for example, to Pseudomonas aeruginosa, biofilm formation in S. aureus is negatively regulated by the main QS system. Agr dependent expression contributes to the detachment of biofilms and subsequent colonization of new sites [START_REF] Boles | Agr-mediated dispersal of Staphylococcus aureus biofilms[END_REF].

Therefore, although still speculative, the QS dependent detachment from biofilms may render some S. aureus strains more capable of spreading through a herd and causing chronic problems at the herd level (e.g. highly virulent rabbit strains). This may also explain why agr+ and agr-variants of S. aureus might have a cooperative interaction in certain types of infections [START_REF] Traber | Agr function in clinical Staphylococcus aureus isolates[END_REF].

Staphylococcus pseudintermedius

Staphylococcal strains formerly designated as Staphylococcus intermedius (S. intermedius) are currently assembled in the so-called S. intermedius group (SIG), consisting of S. intermedius, Staphylococcus pseudintermedius (S. pseudintermedius) and Staphylococcus delphini (S. delphini) [START_REF] Hajek | Staphylococcus intermedius, a new species isolated from animals[END_REF]Varaldo et al., 1988;[START_REF] Devriese | Staphylococcus pseudintermedius sp. nov., a coagulase-positive species from animals[END_REF][START_REF] Devriese | Staphylococcus pseudintermedius versus Staphylococcus intermedius[END_REF]. From a clinical point of view, S. pseudintermedius is probably the most significant species of the SIG, being an important canine opportunistc pathogen, often isolated from dermatitis, otitis and other secondary infections in dogs (Bannoehr et al., 2007). Since S. pseudintermedius produces toxins analogous to the agr dependend toxins of S. aureus, it has been hypothesized that the S. pseudintermedius toxins are regulated by a comparable QS mechanism (Sung et al., 2006). However, the Staphylococcus spp. of the SIG differ from all other Staphylococcus spp., including S. aureus, in the fact that their AIP contains a lactone in stead of a thiolactone ring [START_REF] Dufour | High genetic variability of the agr locus in Staphylococcus species[END_REF]Ji et al., 2005;[START_REF] Novick | Quorum sensing in staphylococci[END_REF]. This nonapeptide shows both auto-inducing and cross-inhibiting activities (Ji et al., 2005). DNA sequencing revealed the presence of four predicted AIP variants within the SIG (Bannoehr et al., 2007). [START_REF] Novick | Quorum sensing in staphylococci[END_REF] suggest that the divergence of the lactone containing AIP of the SIG from the thiolactone containing AIP of S. aureus, may be correlated to the divergence of S. pseudintermedius towards the canine host.

However, the fact that no association could be made between the AIP variants of the SIG and the different host species, disease status of the host, clinical or geographic origin of the strains might nuance this hypothesis (Bannoehr et al., 2007).

Pseudomonas aeruginosa

Pseudomonas aeruginosa (P. aeruginosa) is an important opportunistic pathogen in human and veterinary medicine. Pseudomonas infections are difficult to treat since this bacterium is resistant to several antimicrobial agents [START_REF] Rubin | Antimicrobial resistance and genetic characterization of fluoroquinolone resistance of Pseudomonas aeruginosa isolated from canine infections[END_REF][START_REF] Weese | A review of multidrug resistant surgical site infections[END_REF] and its antimicrobial susceptibility is even lower when it is present in biofilms [START_REF] Haesebrouck | Biofilms: Betekenis voor de behandeling en bestrijding van bacteriële infecties bij huisdieren[END_REF][START_REF] Girard | Central role of quorum sensing in regulating the production of pathogenicity factors in Pseudomonas aeruginosa[END_REF]. P. aeruginosa may cause various tissue infections in different animal species, which illustrates the adaptability of this bacterium. A multitude of virulence factors are regulated by at least one of the 2 different QS systems present in P. aeruginosa: Las and Rhl [START_REF] Whiteley | Identification of genes controlled by quorum sensing in Pseudomonas aeruginosa[END_REF][START_REF] Girard | Central role of quorum sensing in regulating the production of pathogenicity factors in Pseudomonas aeruginosa[END_REF].

Pseudomonas is notorious for its capability to form biofilms. Through the formation of a biofilm, the bacteria are able to colonize all kinds of animal tissues, plants and inert surfaces, thereby increasing environmental persistence [START_REF] Haesebrouck | Biofilms: Betekenis voor de behandeling en bestrijding van bacteriële infecties bij huisdieren[END_REF]. In addition, bacteria inside a biofilm are difficult to kill either by antimicrobial substances or by host defense mechanisms, and therefore often result in chronic infections that do not respond to antimicrobial therapy. Since P. aeruginosa is found ubiquitously in water, wet surfaces are predisposed to be colonized with a P. aeruginosa biofilm. In veterinary medicine, the biofilm producing capability of P. aeruginosa is most feared in cases of infections involving invasive devices or implants [START_REF] Weese | A review of multidrug resistant surgical site infections[END_REF]. P. aeruginosa is also a severe complicating factor in cases of equine ulcerative keratitis [START_REF] Keller | Bacterial isolates and antimicrobial susceptibilities in equine bacterial ulcerative keratitis (1993--2004)[END_REF], in dermatitis, otitis externa and urinary tract infections in small animals [START_REF] Rubin | Antimicrobial resistance and genetic characterization of fluoroquinolone resistance of Pseudomonas aeruginosa isolated from canine infections[END_REF], in fleece rot in sheep [START_REF] Kingsford | The occurrence of Pseudomonas aeruginosa in fleece washings from sheep affected and unaffected with fleece rot[END_REF], in bovine mastitis, which is often associated with contaminated udder washing water or contaminated intramammary dry-cow preparations [START_REF] Mclennan | Pseudomonas mastitis in a dairy herd[END_REF], and in hemorrhagic pneumonia in mink [START_REF] Hammer | Comparison of Pseudomonas aeruginosa isolates from mink by serotyping and pulsed-field gel electrophoresis[END_REF].

Even though QS often favors the expression of virulence genes, [START_REF] Tron | Pseudomonas aeruginosa from canine otitis externa exhibit a quorum sensing deficiency[END_REF] showed that P. aeruginosa canine otitis externa isolates share a deficiency of one or more components of the Rhl QS system, leading to the loss of the elastase virulence factor. The virulence factor that favors colonization of canine ears may be under the negative control of the QS system, or a factor that facilitates removal of the bacterium from the canine ears may be under the positive control of Rhl. The authors postulate that either this phenotype might be present before infection or else it might be acquired after infection in an effort to adapt to the novel environment. Recent research suggests an alternative explanation for the presence of QS deficient strains on sites where it can be expected that biofilm formation should be favourable for colonization. Evolutionary biologists describe the phenomenon of 'cheating' individuals in the population [START_REF] Dunny | Multicellular behavior in bacteria: communication, cooperation, competition and cheating[END_REF]. Such cheaters profit from the benefits of the cooperating population, without contributing to the community. Since these cheaters do not invest in the production of the biofilm, they have a competitive advantage relative to the biofilm producing individuals and, as a consequence, can become predominant in the community. However, when the proportion of cheaters becomes too large, this can affect the stability of the biofilm and the entire population may crash as a result [START_REF] Dunny | Multicellular behavior in bacteria: communication, cooperation, competition and cheating[END_REF].

Salmonella

Several QS systems have been described in Salmonella. Salmonella has a LuxR homologue, SdiA, but does not have a luxI homologue and therefore does not produce AHLs [START_REF] Walters | Quorum sensing in Escherichia coli and Salmonella[END_REF]. It has been suggested that SdiA senses AHLs produced by other bacterial species [START_REF] Michael | SdiA of Salmonella enterica is a LuxR homolog that detects mixed microbial communities[END_REF] and in this way senses the presence of the intestinal environment. SdiA regulates the expression of virulence genes with different functions, ranging from resistance to complement killing to the expression of fimbriae. In murine, poultry or bovine models, however, an sdiA mutant strain is not attenuated and, very recently, SdiA dependent gene expression was shown to be activated in the gut of turtles, but not in the intestines of several homoiothermic animal species [START_REF] Smith | SdiA, an Nacylhomoserine lactone receptor, becomes active during the transit of Salmonella enterica through the gastrointestinal tract of turtles[END_REF]. The role of SdiA in Salmonella pathogenesis therefore still remains largely unknown.

In Salmonella enterica subspecies enterica serotype Typhimurium (Salmonella Typhimurium), the luxS gene is directly involved in the AI-2 synthesis. It was recently demonstrated that luxS and the Salmonella AI-2, 4,5dihydroxy-2,3-pentanedione (DPD), are necessary for Salmonella virulence [START_REF] Choi | Implication of quorum sensing in Salmonella enterica serovar typhimurium virulence: the luxS gene is necessary for expression of genes in pathogenicity island 1[END_REF]. Deletion mutants lacking the luxS gene are severely attenuated in the expression of the virulence genes of the Salmonella Pathogenicity Island 1 (SPI-1). These genes are required for the efficient invasion of intestinal epithelial cells and are therefore crucial in the pathogenesis of Salmonella infections in a wide variety of animal hosts, including poultry, pigs and cattle [START_REF] Morgan | Identification of host-specific colonization factors of Salmonella enterica serovar Typhimurium[END_REF][START_REF] Bohez | Salmonella enterica serovar Enteritidis colonization of the chicken caecum requires the HilA regulatory protein[END_REF][START_REF] Boyen | Salmonella Typhimurium SPI-1 genes promote intestinal but not tonsillar colonization in pigs[END_REF]. This finding suggests that Salmonella might secure the initial infection process by producing SPI-1 virulence factors only when its population size attains a high enough level [START_REF] Choi | Implication of quorum sensing in Salmonella enterica serovar typhimurium virulence: the luxS gene is necessary for expression of genes in pathogenicity island 1[END_REF].

Eukaryotic cells communicate with each other using various hormones or hormonelike substances, for example epinephrine and norepinephrine. These signals can be misused, however, by bacterial pathogens, such as Salmonella, to colonize the host or to induce disease [START_REF] Walters | Quorum sensing in Escherichia coli and Salmonella[END_REF]. Catecholamines may stimulate Salmonella Typhimurium by two mechanisms: as a quorum sensing signal or as a supplier of iron [START_REF] Bearson | Iron regulated genes of Salmonella enterica serovar Typhimurium in response to norepinephrine and the requirement of fepDGC for norepinephrineenhanced growth[END_REF]. In Salmonella, the QseC AI-3 sensor, which is able to detect norepinephrine, has also been implicated in the regulation of virulence traits such as motility and in vivo competitive fitness in pigs [START_REF] Bearson | Iron regulated genes of Salmonella enterica serovar Typhimurium in response to norepinephrine and the requirement of fepDGC for norepinephrineenhanced growth[END_REF]. Even though the role of catecholamines or even other neurotransmittors or hormones in the pathogenesis of Salmonella infections is still in its infancy, the ability to respond to norepinephrine produced by the host has potential food safety consequences. The mechanism described above may be one of the factors causing the sudden burst of Salmonella excretion by stressed pigs, for example, during transport to the slaughter house [START_REF] Bearson | Iron regulated genes of Salmonella enterica serovar Typhimurium in response to norepinephrine and the requirement of fepDGC for norepinephrineenhanced growth[END_REF].

Other veterinary pathogens

It has been shown that several Pasteurellaceae, including Mannheimia haemolytica, Pasteurella multocida, Bibersteinia (formerly Pasteurella) trehalosi, Actinobacillus suis and Actinobacillus pleuropneumoniae, contain LuxS homologues in their genome and are able to produce AI-2-like molecules [START_REF] Malott | Studies on the production of quorum-sensing signal molecules in Mannheimia haemolytica A1 and other Pasteurellaceae species[END_REF]. Also in Histophilus somni, QS may contribute to pathogenesis [START_REF] Corbeil | Histophilus somni host-parasite relationships[END_REF]. A luxS deletion mutant in Mannheimia haemolytica did not show altered leukotoxin expression levels, but had increased expression levels of putative virulence genes such as a transferrin binding protein, an adhesin and capsule biosynthesis, and was outcompeted by the wild type parent strain in a calf challenge model [START_REF] Van Der Vinne | Construction and analysis of a Mannheimia haemolytica A1 luxS mutant[END_REF]. Very recently, a luxS deletion mutant of Actinobacillus pleuropneumoniae was shown to be significantly attenuated in an in vivo mouse model (Li et al., 2008). The down-regulation of the apxIIA toxin, a major virulence factor of Actinobacillus pleuropneumoniae, may contribute to the virulence attenuation of the luxS deletion mutant [START_REF] Li | Enhanced biofilm formation and reduced virulence of Actinobacillus pleuropneumoniae luxS mutant[END_REF].

There are numerous studies dealing with the role of QS in the pathogenesis of infections with EHEC (e.g. [START_REF] Kendall | Quorum sensing by enteric pathogens[END_REF], but studies of the importance of QS in infections with E. coli in veterinary medicine are scarce. Recently, a luxS deletion mutant of a rabbit enteropathogenic E. coli (EPEC) strain was found to be impaired in its virulence in vitro, but was not attenuated in experimentally infected rabbits [START_REF] Zhu | The possible influence of LuxS in the in vivo virulence of rabbit enteropathogenic Escherichia coli[END_REF].

Even though QS was initially described and thoroughly examined in several Vibrio spp., reports linking the QS mechanisms to virulence in fish are much more limited, both for Vibrio spp. [START_REF] Bruhn | Quorum sensing signal molecules (acylated homoserine lactones) in gram-negative fish pathogenic bacteria[END_REF][START_REF] Austin | Vibrio harveyi: a significant pathogen of marine vertebrates and invertebrates[END_REF][START_REF] Nelson | A novel lux operon in the cryptically bioluminescent fish pathogen Vibrio salmonicida is associated with virulence[END_REF][START_REF] Ye | Regulation of Vibrio alginolyticus virulence by the LuxS quorum-sensing system[END_REF] and non-Vibrio fish pathogens [START_REF] Morohoshi | Identification of quorumsensing signal molecules and the LuxRI homologs in fish pathogen Edwardsiella tarda[END_REF][START_REF] Bruhn | Quorum sensing signal molecules (acylated homoserine lactones) in gram-negative fish pathogenic bacteria[END_REF][START_REF] Atkinson | Quorum sensing and the lifestyle of Yersinia[END_REF][START_REF] Bi | Contribution of AhyR to virulence of Aeromonas hydrophila J-1[END_REF][START_REF] Rasch | Quorum sensing signals are produced by Aeromonas salmonicida and quorum sensing inhibitors can reduce production of a potential virulence factor[END_REF].

Quorum sensing has been described in various other veterinary or zoonotic pathogens, such as Clostridium perfringens [START_REF] Ohtani | The luxS gene is involved in cell-cell signalling for toxin production in Clostridium perfringens[END_REF], Yersinia pseudotuberculosis [START_REF] Atkinson | Functional interplay between the Yersinia pseudotuberculosis YpsRI and YtbRI quorum sensing systems modulates swimming motility by controlling expression of flhDC and fliA[END_REF] and Campylobacter jejuni [START_REF] Jeon | Promoter analysis of cytolethal distending toxin genes (cdtA , B, and C ) and effect of a luxS mutation on CDT production in Campylobacter jejuni[END_REF][START_REF] He | Analysis of AI-2/LuxS-dependent transcription in Campylobacter jejuni strain 81-176[END_REF], but a clear correlation between these QS mechanisms and virulence is yet to be demonstrated.

FUTURE PERSPECTIVES OF QUORUM SENSING IN VETERINARY MEDICINE

Quorum sensing signal molecule detection as a diagnostic tool? Since bacterial quorum sensing has been implicated in the onset of bacterial pathogenicity [START_REF] Wu | Detection of N-acylhomoserine lactones in lung tissues of mice infected with Pseudomonas aeruginosa[END_REF][START_REF] Kendall | Quorum sensing by enteric pathogens[END_REF], the signal molecules produced by the bacterial pathogens are potential biomarkers for the diagnosis and follow-up of bacterial infections [START_REF] Kumari | Detection of bacterial quorum sensing N-acyl homoserine lactones in clinical samples[END_REF]. Because the chemical structure of the various signal molecules differ greatly, different sensory systems for detecting these molecules have been described [START_REF] Kalkum | Detection of secreted peptides by using hypothesis-driven multistage mass spectrometry[END_REF]Steindler and Venturi, 2007;[START_REF] Kumari | Detection of bacterial quorum sensing N-acyl homoserine lactones in clinical samples[END_REF]. Most of the studies dealing with the detection of QS signal molecules, however, focus on the detection of AHLs, since these molecules are involved in the development of chronic bacterial infections with P. aeruginosa or Burkholderia cepacia in the lungs of cystic fibrosis patients in human medicine [START_REF] Kumari | Detection of bacterial quorum sensing N-acyl homoserine lactones in clinical samples[END_REF]. Despite the plethora of detection systems, only a fraction can be used to detect QS signal molecules in clinical samples [START_REF] Kumari | Detection of bacterial quorum sensing N-acyl homoserine lactones in clinical samples[END_REF].

AHLs of various Gram-negative bacterial pathogens have been detected in clinical samples of sputum, mucopurulent secretions of the respiratory tract, lung, liver and spleen homogenates and feces of humans and/or experimentally infected mice [START_REF] Middleton | Direct detection of N-acylhomoserine lactones in cystic fibrosis sputum[END_REF][START_REF] Jacobi | Detection of N-(3oxohexanoyl)-L-homoserine lactone in mice infected with Yersinia enterocolitica serotype O8[END_REF][START_REF] Kumari | Biosensing systems for the detection of bacterial quorum signaling molecules[END_REF][START_REF] Kumari | Detection of bacterial quorum sensing N-acyl homoserine lactones in clinical samples[END_REF]. In bronchoalveolar lavages (BALs) from transplanted human lungs, long-chain AHLs were detected, even in lungs that were clinically stable and showed no signs of bacterial infection [START_REF] Ward | Preliminary findings of quorum signal molecules in clinically stable lung allograft recipients[END_REF]. Therefore, the detection of QS signal molecules as a means to detect bacterial infections in a very early stage merits further attention. There are currently no reports of detections of QS signal molecules of veterinary bacterial pathogens in clinical samples of animal host species of veterinary importance.

Quorum sensing inhibition as a therapeutic tool?

The concept of QS inhibition, as used by a range of prokaryotic and eukaryotic organisms, is not new. For example, certain S. aureus strains produce auto-inducers that stimulate their own QS mechanisms, but have a cross-inhibitory effect on the QS systems of other strains, possibly excluding them from infection or colonization sites [START_REF] Ji | Bacterial interference caused by autoinducing peptide variants[END_REF].

Certain Bacillus sp. harbour the acyl-homoserine lactonase enzyme, which inactivates AHL activity by hydrolysing the lactone bond of AHLs of competing bacterial species [START_REF] Dong | Quenching quorum-sensing-dependent bacterial infection by an N-acyl homoserine lactonase[END_REF]. The fact that certain human cell lines are able to inactivate QS signal molecules suggests that mammalian host defences might have developed interfering QS systems against bacterial infections as well [START_REF] Chun | Inactivation of a Pseudomonas aeruginosa quorum-sensing signal by human airway epithelia[END_REF].

Increasing antimicrobial resistance in pathogenic bacteria has created the need for the development of novel therapeutic agents. It is clear that QS is a key regulatory system in the pathogenesis of various bacterial infections. Therefore, the targeting of the QS mechanisms provides a novel strategy for combating bacterial infections [START_REF] Bjarnsholt | Quorum-sensing blockade as a strategy for enhancing host defences against bacterial pathogens[END_REF].

These compounds ideally have no direct effect on bacterial growth, but attenuate bacterial pathogenicity, thereby increasing the susceptibility of the pathogen to the host defences. Even though the macrolides may show potential as QS inhibitors [START_REF] Tateda | Suppression of Pseudomonas aeruginosa quorum-sensing systems by macrolides: a promising strategy or an oriental mystery?[END_REF], the lack of an effect of "pure" QS inhibitors on bacterial growth could mean a strong advantage relative to the "classic" antimicrobial agents, since the use of these products may not exert a strong selective pressure towards the development of resistance.

Very recently, a small molecule, LED209, was discovered using a high-throughput screening which inhibits the QseC mediated activation of virulence gene expression and subsequent in vivo virulence of various bacterial pathogens, such as EHEC, Salmonella Typhimurium and Francisella tularensis [START_REF] Rasko | Targeting QseC signaling and virulence for antibiotic development[END_REF]. Being non-toxic for mammals, promising future [START_REF] Rasko | Targeting QseC signaling and virulence for antibiotic development[END_REF]. Inhibition of biofilm formation has been described using QS inhibitors in P. aeruginosa [START_REF] Bjarnsholt | Quorum-sensing blockade as a strategy for enhancing host defences against bacterial pathogens[END_REF], S. aureus [START_REF] Wright | Transient interference with staphylococcal quorum sensing blocks abscess formation[END_REF] and Salmonella Typhimurium [START_REF] Janssens | Interference with the quorum sensing systems of Salmonella enterica serovar typhimurium: possibilities and implications[END_REF].

Research aimed at the use of QS inhibitors in veterinary medicine is currently limited to their use in aquaculture. For the fish pathogens Aeromonas salmonicida and Vibrio (Listonella) anguillarum, AHL analogues reduced the virulence gene expression and the fish mortality rates, respectively [START_REF] Rasch | An inhibitor of bacterial quorum sensing reduces mortalities caused by Vibriosis in rainbow trout (Oncorhynchus mykiss, Walbaum)[END_REF][START_REF] Rasch | Quorum sensing signals are produced by Aeromonas salmonicida and quorum sensing inhibitors can reduce production of a potential virulence factor[END_REF].

CONCLUSION

Quorum sensing is a widespread phenomenon used by various bacterial pathogens of veterinary importance. Different QS mechanisms make use of diverse signal molecules, leading to intraspecies, interspecies and even interkingdom signalling. Since QS probably plays a role in the pathogenesis of many bacterial infections in animals, the detection of QS signal molecules at early stages of the infection and the use of QS inhibitors for treatment may provide new tools in future veterinary medicine.

Figure legend

Figure 1: The principle of quorum sensing is depicted. In situations where the bacterial density is low (left), the signal molecule concentration is also low and does not exceed the quorum sensing treshold. In case the bacterial density is high (right), the signal molecule concentration is also high and the quorum sensing treshold is exceeded. As a result, the signal molecules are bound to their receptors on the bacterial cells and the gene expression of the total population is changed and synchronised.

Figure 2 :

 2 Figure 2: Four simplified models of the described quorum sensing mechanisms.

Figure 1 Figure 2

 12 Figure 1


DVM W L A Loeffen
email: willie.loeffen@wur.nl

DVM W A Hunneman

J Quak

DVM J H M Verheijden

DVM J A Stegeman

POPULATION DYNAMICS OF SWINE INFLUENZA VIRUS IN FARROW-TO-FINISH AND SPECIALISED FINISHING HERDS IN THE NETHERLANDS

Keywords: swine influenza, H1N1, H3N2, H1N2, incidence, herd type, population dynamics

Influenza virus infections with subtypes H1N1, H3N2 and H1N2 are very common in domestic pigs in Europe. Data on possible differences of population dynamics in finishing pigs in farrow-to-finish herds and in specialised finishing herds are, however, scarce. The presence of sows and weaned piglets on the same premises may, however, affect the exposure of finishing pigs to influenza viruses. In a longitudinal study on 14 farrow-to-finish herds and 15 finishing herds, groups of pigs were followed by repeatedly testing the same animals for antibodies against all three influenza virus subtypes (H1N1, H3N2 and H1N2). At the end of the finishing period, the seroprevalences in farrow-to-finish and specialised finishing herds were 44.3% and 62.0% respectively for H1N1, 6.6% and 19.3% respectively for H3N2, and 57.2% and 25.6% respectively for H1N2. For all three subtypes, the incidence of influenza virus infections was highest at the beginning of the finishing period in farrow-to-finish herds, while the incidence of influenza virus infections was highest at the end of the finishing period in finishing herds. Respiratory disease, probably related to the influenza infections, was observed in five of these herds only, but also occurred at the beginning of the finishing period in farrow-to-finish herds and at the end of the finishing period in finishing herds. The observed differences of population dynamics of influenza virus may affect choice and timing of intervention measures.

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

Introduction

Influenza virus infections in swine are very common. In several studies in European countries, seroprevalences for the swine influenza strains H1N1 and H3N2 were found to be in the range of 20-80% in finishing pigs at the end of the finishing period and in sows [START_REF] Masurel | Prevalence of influenza viruses A-H1N1 and A-H3N2 in swine in the Netherlands[END_REF]Haesebrouck and Pensaert, 1986b;Yus et al., 1989;Elbers et al., A c c e p t e d M a n u s c r i p t 3 1990; [START_REF] Teuffert | Seroepidemiologische Untersuchungen mit dem HAHT zum Vorkommen porciner und humaner Infl-A-Viren bei Haus-und Wildschweinen in der ehemaligen DDR (in German)[END_REF][START_REF] Elbers | Variation in seropositivity for some respiratory disease agents in finishing pigs: epidemiological studies on some health parameters and farm and management conditions in the herds[END_REF][START_REF] Groschup | Serological studies on the potential synergism of porcine reproductive and respiratory syndrome virus and influenza-, corona-and paramyxoviruses in the induction of respiratory symptoms in swine[END_REF][START_REF] Ewald | Factors Associated with Influenza-A Virus-Infections in Finishing Pigs[END_REF]Brown et al., 1995b;[START_REF] Maes | Risk indicators for the seroprevalence of Mycoplasma hyopneumoniae, porcine influenza viruses and Aujeszky's disease virus in slaughter pigs from fattening pig herds[END_REF][START_REF] Maldonado | Evidence of the concurrent circulation of H1N2, H1N1 and H3N2 influenza A viruses in densely populated pig areas in Spain[END_REF]. A more recent subtype, H1N2, seems to originate from the UK where it was found for the first time in 1994 (Brown et al., 1995a). H1N2 was subsequently reported from Belgium [START_REF] Van Reeth | Isolations of H1N2 influenza A virus from pigs in Belgium[END_REF], Italy and France [START_REF] Marozin | Antigenic and genetic diversity among swine influenza A H1N1 and H1N2 viruses in Europe[END_REF], Germany [START_REF] Schrader | Genetic characterization of a porcine H1N2 influenza virus strain isolated in Germany[END_REF], and Spain [START_REF] Maldonado | Evidence of the concurrent circulation of H1N2, H1N1 and H3N2 influenza A viruses in densely populated pig areas in Spain[END_REF]. Many, if not all of these strains are related to the original H1N2 strain from the UK [START_REF] Marozin | Antigenic and genetic diversity among swine influenza A H1N1 and H1N2 viruses in Europe[END_REF]. Seroprevalence studies in Belgium [START_REF] Van Reeth | Isolations of H1N2 influenza A virus from pigs in Belgium[END_REF] and Spain [START_REF] Maldonado | Evidence of the concurrent circulation of H1N2, H1N1 and H3N2 influenza A viruses in densely populated pig areas in Spain[END_REF] resulted in high seroprevalences of approximately 70% and 50% respectively for H1N2, but both studies were carried out in sows.

Studies in finishing pigs so far give overall estimates of the seroprevalence at the end of the finishing period. However, it is not clear at what moment during the finishing period these infections take place, nor do they differentiate between finishing pigs in specialised finishing herds and finishing pigs in farrow-to-finish herds. The presence of sows and piglets on the same premises as the finishing pigs may, however, affect the population dynamics of swine influenza in these herd types, and thus also affect the choice and timing of intervention measures, like vaccination or zoosanitary measures.

Longitudinal studies were therefore carried out to be able to compare the seroprevalences and incidences of swine influenza virus infections at different ages in finishing pigs in both herd types. This allowed us also to determine whether there are differences in the population dynamics of swine influenza virus infections in finishing pigs in farrow-to-finish herds versus finishing pigs in specialised finishing herds.

Materials and methods

Selection of herds

In the Netherlands, there are three regions with a high pig density (figure 1). In these regions the average pig density is more than 600 pigs per square kilometre. In contrast, the In each category (farrow-to-finish and finishing herds) 64 herds were randomly selected.

A written request for participation in a longitudinal serological survey was sent to these farmers. Vaccination against swine influenza was not allowed. The first 15 in each category returning a positive response were included in the study. One farrow-to-finish herd that applied for the survey withdrew shortly before the survey actually started and could not be replaced on such short notice. Thus, 29 herds finally participated. In eleven of the finishing herds the investigated pigs originated from only one breeding herd, in three finishing herds the pigs originated from two breeding herds and in one finishing herd the pigs originated from three breeding herds.

Sampling

All farms were visited three (finishing herds) or four (farrow-to-finish herds) times during the months of January to May 1999. In each herd one compartment was followed in a longitudinal study and blood samples were collected during each visit.

In finishing herds one compartment where the piglets were 12 weeks old was sampled during the first visit. On average, these piglets arrived 2-3 weeks before on the farm. Pigs were tagged individually during the first sampling to allow for resampling of the same pigs during subsequent visits. The second blood samples were taken four weeks later (age of 16 weeks) and a final sample was taken within one week before the first pigs from that compartment were delivered to the slaughter house (on average at the age of 22 weeks).

Piglets within the sampled compartment always originated from one breeding herd. During the finishing period no other pigs were added to that compartment.

In farrow-to-finish herds one compartment where the piglets were 8 weeks old was sampled during the first visit. Pigs were tagged individually during the first sampling to allow A c c e p t e d M a n u s c r i p t 5 for resampling of the same pigs during subsequent visits. The second series of blood samples was taken four weeks later (age of 12 weeks), the third another four weeks later (age of 16 weeks) and the final series of samples was taken within one week before the first pigs from that compartment were delivered to the slaughter house (on average at the age of 22 weeks). Tagged piglets were kept together in the same compartment until slaughter, but while being transferred to the finishing facilities, piglets from two compartments were sometimes mixed. However, during the finishing period no other pigs were added to that compartment anymore.

The sample size for each compartment was calculated so that with an estimated seroprevalence of 50% and a confidence of 0.95 the margin of error was less than 20%. This resulted in sample sizes of 16-24 pigs per compartment using the formula for simple random sampling [START_REF] Thrusfield | Veterinary epidemiology[END_REF]. Within a compartment an equal number of pigs were sampled per pen, as far as the total number of samples allowed for this. Within each pen, pigs were selected at random (haphazardly).

All farmers were asked to record all clinical signs and medications in the compartment under study.

Serological examination

All sera were tested in a hemagglutination inhibition (HI) test [START_REF] Kendal | Concepts and procedures for laboratorybased influenza surveillance[END_REF] for antibodies against influenza virus strains A/swine/Neth/Best/96 (H1N1), A/swine/Neth/St Oedenrode/96 (H3N2) and A/swine/Gent/7625/99 (H1N2). Sera were pre-treated with chicken erythrocytes and cholera filtrate to remove non-specific hemagglutinating factors and non-specific inhibitors of hemagglutination. Four hemagglutinating units (HAU) were used in each test.

A/swine/Neth/Best/96 (H1N1) and A/swine/Neth/St Oedenrode/96 were isolated in the Netherlands and are representative for influenza strains circulating in the Netherlands [START_REF] Loeffen | Survey of infectious agents involved in acute respiratory disease in finishing pigs[END_REF]. A/swine/Gent/7625/99 (H1N2) is a Belgian strain, representative for H1N2 strains circulating among pigs in Belgium, which in turn is expected to be A c c e p t e d M a n u s c r i p t 6 representative for H1N2 strains all over Western Europe [START_REF] Marozin | Antigenic and genetic diversity among swine influenza A H1N1 and H1N2 viruses in Europe[END_REF]. Although H1N1 and H1N2 strains both possess a hemagglutinin H1, these are of different origin [START_REF] Brown | Multiple genetic reassortment of avian and human influenza A viruses in European pigs, resulting in the emergence of an H1N2 virus of novel genotype[END_REF] and little or no cross reactivity was found in serological tests after experimental infections [START_REF] Van Reeth | Isolations of H1N2 influenza A virus from pigs in Belgium[END_REF][START_REF] Van Reeth | Genetic relationships, serological cross-reaction and cross-protection between H1N2 and other influenza A virus subtypes endemic in European pigs[END_REF][START_REF] Van Reeth | Serological profiles after consecutive experimental infections of pigs with European H1N1, H3N2, and H1N2 swine influenza viruses[END_REF].

Sera were tested in serial twofold dilutions, starting at 1:9. Titres 18 were considered positive. A fourfold rise in titres in consecutive samples was considered evidence of an influenza virus infection somewhere during that period (taking into account approximately one week for titres to develop after an influenza virus infection). In the presence of maternal antibodies, titres of the second sera were not compared to the titres of the first sera, but to expected titres of the second sera if these were due to maternal antibodies only. To do this, the decay of maternal antibodies, having a half-life of approximately 12 days, was taken into consideration, extrapolating titres from the first sera [START_REF] Loeffen | Estimating the incidence of influenza-virus infections in Dutch weaned piglets using blood samples from a cross-sectional study[END_REF].

Statistical analysis

Statistical analyses for the differences between farrow-to-finish herds (FFH) and specialised finishing herds (FH) were performed per strain and per time interval. The incidence, cumulative incidence and prevalence data were expressed as fractions per farm.

The incidence being the number of seroconversions since the previous sampling, the cumulative incidence the total number of seroconversions until the moment of sampling, and the prevalence the fraction of seropositive pigs at each sampling. The test statistic (t) for the null hypothesis of no difference between the herd types was:

Seroprevalence

For H1N2, at the end of the finishing period the seroprevalences in farrow-to-finish and specialised finishing herds were 57.2% and 25.6% respectively (table 1). The seroprevalence was in all age groups higher in farrow-to-finish herds than in specialised finishing herds (P=0.01, P<0.01 and P=0.03 for 12, 16 and 22 weeks of age respectively).

For H1N1 and H3N2, at the end of the finishing period the seroprevalences in farrow-tofinish and specialised finishing herds were 44.3% and 62.0% respectively for H1N1 and 6.6% and 19.3% respectively for H3N2. The differences of the seroprevalences in farrow-tofinish herds and finishing herds were not statistically significant..

Incidence

For H1N1 the differences between both herd types are statistically significant between 16 and 22 weeks of age (P<0.01) and for H1N2 between 0 and 12 (P=0.05) and 12 and 16 (P=0.01) weeks of age (table 2). No significant differences (P<0.05) were seen for H3N2.

The cumulative incidences are shown in table 3. They are essentially comparable to the seroprevalence, however, corrected for antibodies due to maternal immunity or antibodies from an infection that declined to a level below the detection limit of the test.

Clinical symptoms of respiratory disease

Twenty-four farmers (from 11 farrow-to-finish herds and 13 finishing herds) kept a record of clinical signs and medications. In eleven of them, an episode of respiratory disease was recorded: five in farrow-to-finish herds and six in finishing herds. The severity of the symptoms was variable, ranging from only coughing for a few days up to a combination of coughing, laboured breathing and decrease of feed intake. Five of these episodes coincided with an incidence of 80-95% of the pigs for one of the influenza subtypes in the same period.

Two of these were in farrow-to-finish herds and both occurred between the age of 8 and 12

Discussion

In this study we found that the timing of influenza infections in finishing pigs is quite different in finishing pigs from farrow-to-finish herds compared to finishing pigs from specialised finishing herds. In farrow-to-finish herds the incidence of influenza virus infections was highest at the beginning of the finishing period, while in finishing herds the incidence of influenza virus infections was highest at the end of the finishing period. This pattern was observed for all three influenza virus subtypes. It can be speculated that the exposure of finishing pigs to influenza virus is different in both herd types.

Sows and especially weaned piglets may be a reservoir for continuous circulation of influenza viruses [START_REF] Loeffen | Estimating the incidence of influenza-virus infections in Dutch weaned piglets using blood samples from a cross-sectional study[END_REF]. Indirect exposure of finishing pigs to influenza virus from sows or weaned piglets will be more easily achieved in farrow-to-finish herds than in specialised finishing herds. Multiple virus introductions into a compartment of finishing pigs in a farrow-to-finish herd could thus result in a high incidence at the beginning of the finishing period. Large outbreaks are apparently rare at that time, possibly due to the combination of decreasing numbers of piglets with maternal antibodies and increasing numbers of piglets with antibodies due to an infection. Introductions later during the finishing period may then die out quickly due to the already high seroprevalence. A compartment of pigs in a finishing herd on the other hand may be subject to only a few virus introductions. On average this may result in populations with more susceptible animals in the second half of the finishing period, with any introduction at that time resulting in a large outbreak within that compartment.

The presence of finishing pigs in a herd might also affect influenza virus infections in sows and weaned piglets. Especially with subtype H1N2, at least 8.6% percent of the weaned piglets seroconverted before they were 8 weeks old. This could also explain the higher seroprevalence at the age of 12 weeks in farrow-to-finish herds, compared to A c c e p t e d M a n u s c r i p t 9 finishing herds. It may be that in the breeding herds that supplied the piglets to the specialised finishing herds, virus circulation in weaned piglets was lower than in weaned piglets on farrow-to-finish herds. This would suggest that the presence of finishing pigs may result in an additional exposure of weaned piglets to the virus and thus may affect virus circulation in weaned piglets. It's also possible that the relatively recent introduction of the H1N2 subtype in the population somehow causes this effect. This would, however, indicate that farrow-to-finish herds and specialised breeding herds are at a different risk for virus introduction. Given that the two herd types were not located in exactly the same place, these results have to interpreted with some care anyway.

Even though serological differentiation between H1N1 and H1N2 is possible in experimental sera [START_REF] Van Reeth | Genetic relationships, serological cross-reaction and cross-protection between H1N2 and other influenza A virus subtypes endemic in European pigs[END_REF][START_REF] Van Reeth | Serological profiles after consecutive experimental infections of pigs with European H1N1, H3N2, and H1N2 swine influenza viruses[END_REF], nothing is known about differentiation in field sera. In the field multiple infections can be expected, and variations occur in haemagglutinin, even within one subtype [START_REF] De | Antigenic and molecular heterogeneity in recent swine influenza A(H1N1) virus isolates with possible implications for vaccination policy[END_REF]. Seroprevalence and incidence of H1N1 and H1N2 might as a result be overestimated. However, if the serological results of these two subtypes are compared at the age of 22 weeks, the agreement beyond chance is very low (Kappa value 0.078). Under the assumption that both subtypes occur independently, this suggests a very low level of cross-reactivity.

The seroprevalence of H1N1 is comparable to the levels that were found in finishing pigs in 1980 (44%) [START_REF] Masurel | Prevalence of influenza viruses A-H1N1 and A-H3N2 in swine in the Netherlands[END_REF] and 1987 (62%) [START_REF] Elbers | Seroepidemiological screening of pig sera collected at the slaughterhouse to detect herds infected with Aujeszky's disease virus, porcine influenza virus and Actinobacillus (Haemophilus) pleuropneumoniae in the framework of an integrated quality control (IQC) system[END_REF] in the Netherlands.

The seroprevalence found for H3N2 is, however, much lower than the ones found in those same studies (68% in 1980; 33% in 1987). All other seroprevalence studies carried out in Europe in the past two decades also showed much higher seroprevalences (Haesebrouck and Pensaert, 1986b;Yus et al., 1989;[START_REF] Teuffert | Seroepidemiologische Untersuchungen mit dem HAHT zum Vorkommen porciner und humaner Infl-A-Viren bei Haus-und Wildschweinen in der ehemaligen DDR (in German)[END_REF][START_REF] Groschup | Serological studies on the potential synergism of porcine reproductive and respiratory syndrome virus and influenza-, corona-and paramyxoviruses in the induction of respiratory symptoms in swine[END_REF][START_REF] Ewald | Factors Associated with Influenza-A Virus-Infections in Finishing Pigs[END_REF]. The reason for the low seroprevalence therefore remains unknown and it remains to be seen whether this is a structural decrease or just an uncommonly low seroprevalence during a single season. The seroprevalence of H1N2 is not comparable to published results from other countries, because they were carried out in sows [START_REF] Van Reeth | Isolations of H1N2 influenza A virus from pigs in Belgium[END_REF][START_REF] Maldonado | Evidence of the concurrent circulation of H1N2, H1N1 and H3N2 influenza A viruses in densely populated pig areas in Spain[END_REF].

A c c e p t e d M a n u s c r i p t 10 While the population dynamics of influenza virus infections is different between farrow-tofinish and finishing herds, it would be equally important to determine whether the occurrence of clinical symptoms, and subsequent economic losses, are also different between both herd types. Unfortunately, also with respect to this, no information can be found in existing literature. Some studies that tried to determine the importance of influenza virus infections as a cause for respiratory disease, found that approximately 50% of the respiratory outbreaks were caused by influenza virus infections [START_REF] Loeffen | Survey of infectious agents involved in acute respiratory disease in finishing pigs[END_REF][START_REF] Barigazzi | Swine influenza in Italy[END_REF][START_REF] Choi | Retrospective analysis of etiologic agents associated with respiratory diseases in pigs[END_REF]. There was however no differentiation in herd type, or time of infection. In this study, eleven episodes of respiratory disease were noticed, almost equally distributed over both herd types. Only five of them coincided with a seroconversion against one of the influenza subtypes of at least 80% of the pigs in the same period, also equally distributed over both herd types. These five episodes are the ones most likely related to the influenza virus infections, which is also consistent with the 50% of respiratory disease caused by influenza virus infections [START_REF] Loeffen | Survey of infectious agents involved in acute respiratory disease in finishing pigs[END_REF]. Although this is not enough to allow for any statistical analysis, it is noticeable that the two episodes of respiratory disease in farrow-to-finish herds occurred between the age of 8 and 12 weeks, while the three episodes in finishing herds occurred between the age of 16 and 22 weeks. Finishing pigs in both herd types may be equally subject to respiratory disease, but at a different moment during the finishing period. All three subtypes may have been involved in the occurrence of clinical symptoms.

Differences in time of influenza virus infections in both herd types may ask for a different approach in preventive measures, whether these are zoosanitary measures or vaccination. If infections, and subsequent economic losses, in specialised finishing herds occur mainly at the end of the finishing period, it would be possible to wait with vaccination, until maternal antibodies have mostly disappeared. Interference of vaccination with maternal antibodies (Wesley and Lager, 2006) would thus not be a real problem and full and equal protection may be reached. It might even be considered to vaccinate only once at such a time. In general, single vaccinations are considered to be insufficient for full protection. However, A c c e p t e d M a n u s c r i p t 11 partial protection was shown after single vaccination, that may be enough to sufficiently reduce damages due to influenza virus infections under the right circumstances (Haesebrouck and Pensaert, 1986a).

In farrow-to-finish herds on the other hand, there seems to be no optimum with respect to the time of vaccination. Before all maternal antibodies disappear in a population, most of the influenza virus infections, and possibly also the economic losses due to the infection, already occur. Vaccination at a young age, and probably multiple vaccinations to circumvent the negative effect of maternal antibodies, may be indicated.

With respect to zoosanitary intervention measures, the possibilities for influenza seem somewhat limited. Zoosanitary measures may focus on reduction of damages (either due to the primary infection or due to secondary infections). Mainly climate control and general hygiene seem to be relevant zoosanitary measures for this purpose. Zoosanitary measures may also focus on preventing infection by keeping the virus out. Given the ease with which influenza viruses may spread, hygienic measures for this purpose probably have to be rather strict, involving air filtration and strict hygiene protocols for anything and anyone entering the premises. In farrow-to-finish herds this may be more difficult to achieve, also being more expensive because it involved a broader range of age groups, than in specialized finishing herds, given that most infections seem to occur at a later stage in the latter herd type.

While these conclusions and recommendations may apply to swine herds in general, variations in individual herds may occur. It is therefore advisable that, where possible, results from investigations in an individual herd are also taken into account before putting together a strategy of vaccination or any other kind of intervention.

  in the remaining part of the Netherlands is below 100 pigs per square kilometre. From the three pig dense regions all herds with >400 finishing pigs were selected for possible participation in the study. This selection included 27% of the Dutch swine herds, housing 69% of the Dutch finishing pig population.

  b are the fractions averaged over FFH and FH farms respectively. The nulldistribution was obtained by random permutation of the labels FFH and FH over the farms.

  H1N2 and 1x H3N2). The other three occurred in finishing herds, all of them between the age of 16 and 22 weeks (1x H1N1 and 2x H1N2).

  .D., Lager, K.M., 2006. Overcoming maternal antibody interference by vaccination with human adenovirus 5 recombinant viruses expressing the hemagglutinin and the nucleoprotein of swine influenza virus. Vet. Microbiol. 118, 67-75.Yus, E., Laviada, M.D., Moreno, L., Castro, J.M., Escribano, J.M., Simarro, I., 1989. The prevalence of antibodies to influenza virus and respiratory coronavirus in fattening pigs in Spain. Zentralbl. Veterinarmed.[B] 36, 551-556.

  for swine influenza subtypes H1N1, H3N2, and H1N2, in farrow-to-finish herds and finishing herds. Any significant (P<0.05) differences between farrow-to-finish and finishing herds (pair wise comparisons for the same influenza virus subtype over the same period) are indicated with a shaded background. Any trends towards significance (P<0.1) are indicated by a square. Table 2: Incidences for swine influenza subtypes H1N1, H3N2, and H1N2, in farrow-tofinish herds and finishing herds. Incidences were calculated based on the number of seroconversions per period; in farrow-to-finish herds the periods of 0-8 and 8-12 weeks are added to each other to be able to compare them to the period of 0-12 weeks in finishing pigs. Legend as in table 1.Table 3: Cumulative incidences for swine influenza subtypes H1N1, H3N2, and H1N2, in farrow-to-finish herds and finishing herds. Incidences were calculated based on the number of seroconversions per period. Legend as in table 1.

Figure 1 :

 1 Figure 1: Location of the three most pig-dense regions in the Netherlands and the

Table 1

 1

	Farrow-	8	14	290	21.7	11.1-32.3	290	21.7	12.9-30.6	290	53.8	38.4-69.2
	to-finish	12	14	287	20.9	5.9-35.9	287	15.7	2.4-29.0	287	48.8	28.9-68.7
		16	14	286	44.4	22.3-66.5	286	8.7	0.0-20.1	286	60.8	42.7-79.0
		22	14	287	44.3	23.1-65.4	287	6.6	0.0-15.7	283	57.2	38.2-76.3
	Finishing	12	15	322	22.0	8.1-36.0	322	35.7	19.7-51.8	322	18.6	6.6-30.6
		16	15	321	22.1	8.7-35.6	321	20.2	7.0-33.5	320	10.3	0.0-21.1
		22	15	321	62.0	44.6-79.4	321	19.3	3.7-34.9	320	25.6	7.6-43.7
	1

Acknowledgements

The authors thank the farmers for their willing cooperation and hospitality in this study.

The people at the lab are thanked for their dedication in testing all these samples. Bas Engel